Preface

*Salmonella*, a Gram-negative bacterium belonging to the *Enterobacteriaceae* family, can cause infections in humans and animals, and is one of the most common causes of bacterial gastroenteritis. According to the World Health Organization, more than 2 billion people worldwide suffer from diarrheal diseases annually and one of four of these diseases is caused by *Salmonella* each year.

Since the discovery of *Salmonella* in the late 1800s, great progress has been made in understanding its genetics, classification, pathogenesis, detection, prevention, control, and treatments. Numerous reviews and book chapters on *Salmonella* have been published. However, some gaps remain to be addressed. This book presents seven chapters that focus on low-cost prevention, control, and treatment of the salmonellosis in developing countries.

The nomenclature of *Salmonella* is complicated and constantly evolving. Chapter 1 provides an updated review of *Salmonella* nomenclature. Currently, it is commonly recognized that the genus *Salmonella* contains two species: *S. enterica* and *S. bongori* based on the genomic relatedness, with *S. enterica* containing six subspecies (subsp.), *enterica* (I), *salamae* (II), *arizonae* (IIIa), *diarizonae* (IIIb), *houtenae* (IV), *and indica* (VI). *Salmonella* isolates are further serotyped using the Kauffmann–White scheme with more than 2600 serovars identified. In addition, three pathogenic serotypes, *Paratyphi C*, Dublin, and Typhi, are identified based on a special subtype of heatsensitive K antigen at the bacterial capsular surface.

There are two types of human salmonellosis: (1) gastroenteritis (non-typhoid salmonellosis [NTS]), a localized infection due to ingestion of contaminated food or water, and (2) enteric fever (typhoid fever), a severe and life-threatening systemic infection. Only a small fraction of serovars is associated with human infections, mostly belonging to *S. enterica* subspecies *enterica*, which is composed of more than 1500 serovars. Two of these serovars, *S. typhimurium* and *S. enteritidis*, are responsible for more than 99% of NTS. Typhoid fever is caused mainly by S. typhi, and a clinically indistinguishable condition caused by S. Paratyphi A. Typhoid fever remains a global health problem, especially in the developing countries with substandard water supplies and poor sanitation. Certain *Salmonella* such as *Salmonella enterica* subsp. *salamae*, subsp. *arizonae*, subsp. *diarizonae, houtenae*, *S. enterica* subsp. *indica*, and *S. bongori* are usually isolated from cold-blooded animals and the environment with a potential to cause disease in humans.

Animals are major reservoirs of NTS. As reviewed in Chapters 1, 2, and 3, the transmission of NTS infection to humans can occur through the ingestion of food or water contaminated with infected animal waste, direct contact with infected domestic, wild, and companion animals, or consumption of infected animal food products, and direct contact with the contaminated environment. S. typhi causing typhoid fever can be transmitted through contaminated food and water or through close contact with

an infected person. The diversity of possible reservoirs of infection leads to significant challenges for public health authorities in controlling infections. Chapters 5 and 6 review low-cost measures to prevent and control *Salmonella* in water and animals using either solar disinfection for water or biocide for animals, which could be practical for low-income countries.

Most people recover from salmonellosis without any specific treatment. Antibiotics are typically used only to treat patients with severe illness caused by *Salmonella* infections. Since the report of the first incidence of *Salmonella* resistance to chloramphenicol in 1960s, the emergence of antimicrobial resistance towards single or multi drugs in *Salmonella* strains has become a serious health problem worldwide, particularly in Africa and Asia, as reviewed in Chapters 3 and 7. Interestingly, Chapter 7 also compares AMR in *Salmonella* with other microbes and introduces a new concept of AMR reversal using traditional Chinese medicine as alternatives for treatment, which could lead to new strategies for clinical treatment of bacterial infections.

Ensuring the safety of water and food, predominantly poultry, eggs, and dairy products, is the main strategy for eliminating possible transmission routes of typhoid *Salmonella* as well as NTS. Detecting and characterizing the isolates is crucial for epidemiology and prevention of salmonellosis. Chapter 4 reviews the research progress for understanding the roles played by CRISPR-Cas systems in *Salmonella* immune response, as well as genome editing and its potential for pathogen typing in diagnosis and surveillance.

We believe that the information provided in this book will encourage *Salmonella* researchers, medical professionals, and students to further enhance their own research and education, and encourage new researchers to include *Salmonella* in their future research initiatives. We are grateful to various researchers and scientists across the world who have contributed to this book and hope that the information provided will be well received in the *Salmonella* field, particularly in developing countries, and beyond.

**Hongsheng Huang and Sohail Naushad**

Canadian Food Inspection Agency, Ottawa Laboratory – Fallowfield, Ottawa, Ontario, Canada

**1**

pertaining to *Salmonella*.

**1. Introduction**

surveillance, prevention and control

**Chapter 1**

**Abstract**

*Salmonella*: A Brief Review

*Sohail Naushad, Dele Ogunremi and Hongsheng Huang*

*Salmonella* causes significant illness in humans and animals and is a major public health concern worldwide, contributing to an increased economic burden. *Salmonella* is usually transmitted through the consumption of contaminated food, such as raw or undercooked meat, poultry, eggs, and dairy products, and water or through contact with infected animals or their environment. The most common symptoms of salmonellosis, the illness caused by *Salmonella*, include diarrhea, fever, and abdominal cramps; in severe cases, the infection can lead to hospitalization and even death. The classification and taxonomy of *Salmonella* were historically controversial, but the genus is now widely accepted as composed of two species and over 2600 serovars. Some of these serovars infect a single host, that is, host-restricted, whereas others have a broad host range. Colonization of the host is complex and involves a series of interactions between the *Salmonella* and the host's immune system. *Salmonella* utilizes an array of over 300 virulence factors, mostly present in *Salmonella* pathogenicity islands (SPIs) to achieve adherence, invasion, immune evasion, and, occasionally, systemic infection. Once colonized, it secretes a number of toxins and inflammatory mediators that cause diarrhea and other symptoms of salmonellosis. The overuse and misuse of antibiotics in human and animal medicine and agriculture have contributed to the development of antimicrobial resistance (AMR) in *Salmonella*, making AMR strains more severe and difficult to treat and increasing the risk of morbidity and mortality. Various methods are used for the detection of *Salmonella*, including traditional culture methods, molecular methods such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP), and immunologicalbased assays. Because of its ubiquitous distribution, the prevention and control of *Salmonella* transmission remain a significant challenge. This chapter briefly covers the history, classification, transmission, pathogenesis and virulence factors, antimicrobial resistance genes, detection, diagnosis, surveillance, prevention, and control

**Keywords:** *Salmonella*, history, taxonomy, classification, transmission, pathogenesis, virulence factors, antimicrobial resistance genes, *Salmonella* detection, diagnosis,

*Salmonella* is a bacterial genus consisting of many closely related organisms, which remains a major cause of morbidity and mortality worldwide with significant public health implications, contributing to the economic burdens of both developed and

**Chapter 1**

## *Salmonella*: A Brief Review

*Sohail Naushad, Dele Ogunremi and Hongsheng Huang*

#### **Abstract**

*Salmonella* causes significant illness in humans and animals and is a major public health concern worldwide, contributing to an increased economic burden. *Salmonella* is usually transmitted through the consumption of contaminated food, such as raw or undercooked meat, poultry, eggs, and dairy products, and water or through contact with infected animals or their environment. The most common symptoms of salmonellosis, the illness caused by *Salmonella*, include diarrhea, fever, and abdominal cramps; in severe cases, the infection can lead to hospitalization and even death. The classification and taxonomy of *Salmonella* were historically controversial, but the genus is now widely accepted as composed of two species and over 2600 serovars. Some of these serovars infect a single host, that is, host-restricted, whereas others have a broad host range. Colonization of the host is complex and involves a series of interactions between the *Salmonella* and the host's immune system. *Salmonella* utilizes an array of over 300 virulence factors, mostly present in *Salmonella* pathogenicity islands (SPIs) to achieve adherence, invasion, immune evasion, and, occasionally, systemic infection. Once colonized, it secretes a number of toxins and inflammatory mediators that cause diarrhea and other symptoms of salmonellosis. The overuse and misuse of antibiotics in human and animal medicine and agriculture have contributed to the development of antimicrobial resistance (AMR) in *Salmonella*, making AMR strains more severe and difficult to treat and increasing the risk of morbidity and mortality. Various methods are used for the detection of *Salmonella*, including traditional culture methods, molecular methods such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP), and immunologicalbased assays. Because of its ubiquitous distribution, the prevention and control of *Salmonella* transmission remain a significant challenge. This chapter briefly covers the history, classification, transmission, pathogenesis and virulence factors, antimicrobial resistance genes, detection, diagnosis, surveillance, prevention, and control pertaining to *Salmonella*.

**Keywords:** *Salmonella*, history, taxonomy, classification, transmission, pathogenesis, virulence factors, antimicrobial resistance genes, *Salmonella* detection, diagnosis, surveillance, prevention and control

#### **1. Introduction**

*Salmonella* is a bacterial genus consisting of many closely related organisms, which remains a major cause of morbidity and mortality worldwide with significant public health implications, contributing to the economic burdens of both developed and

economically marginalized countries because of costs associated with monitoring, surveillance, prevention, and treatment of salmonellosis [1–7]. Karl Joseph Eberth of the University of Zurich, a physician and pathologist, described a bacillus in the abdominal lymph nodes and spleen of a patient who died of typhoid in 1879 [8]. At the time, the bacterium was referred to as Eberth's *Bacillus* [9, 10] and followed by the discovery of *Bacillus* as the cause of human typhoid fever by George Gaffky in 1884 [1, 8, 10]. Nevertheless, the genus "*Salmonella*" was named after Daniel Elmer Salmon, an American veterinary pathologist and head of the United States Department of Agriculture (USDA) Microorganism Research Program in the late 1800s [1]. Together with Theobald Smith, Salmon isolated *Salmonella* from the intestines of the pigs that succumbed to the disease known as hog cholera in 1884 [8, 11]. Historians and scientists studying past disease outbreaks have concluded that many catastrophic disease outbreaks of the early ages were likely caused by *Salmonella*, more specifically, typhoid infections [5, 12]. As early as 430 B.C., a plague, which is now believed to have been typhoid fever, wiped out a third of the population of Athens [1, 5].

Salmonellosis is a common cause of foodborne illness in the world [2, 3, 13]. Symptoms of salmonellosis can include fever, diarrhea, abdominal cramps, and vomiting and can last for several days. According to the World Health Organization (WHO), more than 2 billion people worldwide suffer from diarrheal diseases annually [8], and 1 of 4 of these diseases is caused by *Salmonella* [4, 7, 8, 14]. Depending on host factors and the serotype of *Salmonella,* as well as the presence of antimicrobial resistance (AMR) genes, 11–20 million cases of salmonellosis become severe and life-threatening, leading to 161,000 deaths annually [4, 7]. According to the Centers for Disease Control and Prevention (CDC), salmonellosis is one of the most common bacterial foodborne illnesses in the United States, with an estimated 1.35 million cases occurring annually [15]. The Public Health Agency of Canada (PHAC) estimates that there are about 87,500 cases of salmonellosis each year in Canada [16]. According to the European Centre for Disease Prevention and Control (ECDC) in 2021, salmonellosis was the second most common bacterial foodborne infection in Europe, with an estimated 60,050 cases occurring annually [15]. In developing countries, the burden of salmonellosis is usually higher because of the combination of many factors, such as poor hygiene and sanitation conditions, lack of access to safe water and proper food handling practices, lack of proper disease reporting structure, and limited resources for disease surveillance and response [4, 14, 17].

This chapter will briefly review the nomenclature, transmission, pathogenesis, diagnosis and detection, prevention, control, and treatment.

#### **2. The organism**

*Salmonella* is a rod-shaped, Gram-negative bacterium and consists of a cell wall, cell membrane, cytoplasm, ribosomes, plasmids, and nucleoid region. It has a diameter of around 0.7 to 1.5 μm, a length from 2 to 5 μm, and flagella, which allows for motility [18]. *Salmonella* is a chemoorganotroph, which means it obtains energy from the oxidation of the reduced organic compounds, and is a facultative anaerobe [18]. After colonizing the epithelium, *Salmonella* reproduces by binary fission, which begins with the replication and attachment of the DNA molecules to the cell membrane. Once the bacterium doubles its original size, the cell membrane begins to pinch inward and a cell wall forms between the two DNA molecules to divide the original

cell into two identical daughter cells [18, 19]. Once reproduced, the bacterium either stays within the intestine or enters the bloodstream or lymph tracts [19]. *Salmonella* can also survive for several weeks outside of a living host in a dry environment and several months in water and is often not destroyed by freezing temperatures. The bacteria will only be destroyed in temperatures above 75°C, which makes raw and undercooked food, together with improperly washed fruits and vegetables, a common source of transmission of the bacterium.

The genome of *Salmonella* is relatively small, with a size ranging from 4.7 to 5.3 million base pairs [20–22]. It is a single circular chromosome that encodes a wide range of proteins involved in various cellular processes, including metabolism, regulation, and pathogenicity [22–25]. Several studies have characterized the genomic features of *Salmonella* and identified genes that are important for its survival, virulence, and antimicrobial resistance [23, 25–29]. These include genes encoding toxins and other virulence factors that allow the bacteria to colonize and infect host tissues, as well as genes involved in the uptake and metabolism of nutrients. One of *Salmonella's* most well-known genomic features is the presence of prophages, which are bacteriophages (viruses that infect bacteria) that have integrated into the bacterial genome [30, 31]. Prophages can be activated under certain conditions, leading to the production of new phages that can potentially spread to other bacteria. Genomic studies of *Salmonella* have also identified a number of genes that are involved in antibiotic resistance [32–35]. These genes can be horizontally transferred among bacteria, leading to the spread of antibiotic resistance among different bacterial species. In recent years, the use of whole-genome sequencing (WGS) to study *Salmonella* has allowed for a deeper understanding of the organism's epidemiology, biology, evolution, and population structure [36–39]. WGS can accurately predict various characteristics and traits of a *Salmonella* isolate based on its genomic sequence, replacing the need for time-consuming and costly traditional methods [26, 30, 39].

#### **3. Classification**

*Salmonella* belongs to the Kingdom *Monera* or *Eubacteria,* phylum *Proteobacteria,* class *Gamma-Proteobacteria,* order *Enterobacteriales*, family *Enterobacteriaceae*, and the genus *Salmonella* [40, 41]. The nomenclature of the genus *Salmonella* has been confusing and controversial and has two systems of nomenclature widely used for the taxonomical assignments of *Salmonella*. One system, which does not conform to the rules of the Bacteriological Code but has wide acceptance, was proposed in the 1980s by Le Minor and Popoff in 1980 [18, 42], whereas the second system conforms to the rules of the Bacteriological Code and is not widely used [43]. To resolve the discrepancies in the taxonomical system of *Salmonella*, the Judicial Commission of the International Committee on the Systematics of Prokaryotes issued an Opinion (Opinion 80), with the intention that it should solve these discrepancies [41]. However, Opinion 80 was also limited to matters of nomenclature and meant to provide a clear presentation and interpretation of *Salmonella* taxonomy of the widely accepted division of the genus *Salmonella* into two species [41]. There are approximately 2000 similar species of *Salmonella*, which has caused much confusion in terms of classifying each species. In order to simplify this, the Center for Disease Control and Prevention (CDC) has agreed upon two species of *Salmonella*, *Salmonella enterica* and *Salmonella bongori*. *Salmonella enterica* is subdivided into six subspecies: *S. enterica* ssp. *enterica* (I), *S. enterica* ssp. *salamae* (II), *S. enterica* ssp. *arizonae* (IIIa), *S.* 

*enterica* ssp. *diarizonae* (IIIb), *S. enterica* ssp. *houtenae* (IV), and *S. enterica* ssp. *Indica* [44, 45]. These species and subspecies are further classified into multiple serotypes based on the White–Kauffmann–Le Minor scheme updated by the World Health Organization's Collaborating Centre for Reference and Research on *Salmonella* at the Pasteur Institute, Paris, France [45, 46]. The genus *Salmonella* is made up of approximately 2600 serovars based on antigenic polymorphisms of their somatic O antigens (lipopolysaccharide), H antigens (flagellar proteins), and Vi antigens (capsular polysaccharides; [41, 43]). Most of the serovars belong to *S. enterica* ssp. *enterica* (I), and the most common serogroups are A, B, C1, C2, D, and E [43].

To avoid confusion in writing names and differentiate between serovars designation and species-level designation, it is recommended to write them in Roman style starting with a capital letter [43]. The current convention used in scientific writing is to state first the genus name and then the species name, followed by the word "serovar" (which can be abbreviated as "ser."), and finally the actual name of the serovar [43]. An example, at first, is to write *Salmonella. enterica* subsp. *enterica* serovar (or ser.) Typhimurium. To simplify this long written convention and avoid the long nature of nomenclature, the name can be shortened by writing the genus name, followed directly by the serovar name starting with a capital letter; for example, *Salmonella. enterica* subsp. *enterica* serovar Typhimurium can be written as *Salmonella* Typhimurium.

*Salmonella* has a wide host range, which based on host adaptability, can be divided into three broad groups [47]. Group 1 *Salmonella* serovars are adapted to humans and higher primates such as *Salmonella* Typhi, *Salmonella* Paratyphi A, B, C, and *Salmonella* Sendai [47]. Group 2 *Salmonella* are largely adapted to specific animal hosts such as *Salmonella* Dublin in cattle, *Salmonella* Gallinarum in poultry, *Salmonella* Abortusequi in horses, *Salmonella* Abortusovis in sheep, and *Salmonella* Choleraesuis in pigs [47]. Group 3 *Salmonella* have a wide host range including humans, animals, and the environment, such as *Salmonella* Typhimurium and *Salmonella* Enteritidis, the two most common serotypes of *Salmonella* transmitted to humans in most parts of the world [47].

#### **4. Transmission**

*Salmonella* generally resides in the gut of animals, including birds, and is usually transmitted to humans by eating contaminated foods [7, 15]. These contaminated foods are typically from animal origin such as beef, poultry, milk, or eggs, but all food types including vegetables may be contaminated [15, 16]. The bacteria are commonly found in raw eggs and undercooked chicken and eggs. Person-to-person spread is possible in close contact, especially during the acute diarrheal phase of the illness [15, 16, 48]. *Salmonella* is transmitted by the consumption of raw food that is contaminated with the bacteria, such as vegetables that have not been cooked or washed properly, meat, or eggs. *Salmonella* can be transferred if the food handler or processor does not use gloves when dealing with food [15, 16]. It can also be transmitted by reptiles or rodents through their feces [49]. If the food is contaminated with a high concentration of *Salmonella*, the person is more likely to become infected. Children, elderly people, and HIV-positive people are more likely to become infected [7, 16, 49]. Once ingested, *Salmonella* embeds itself into the intestinal epithelium where it reproduces [50]. The liver, spleen, and especially the gall bladder have a high concentration of *Salmonella*.

If left untreated, the organism can travel through the bloodstream to joints, organs, placenta, and membranes around the brain [50]. The toxins released by the bacteria can damage various organs in the body [51, 52].

#### **5. Diseases**

The general term for infections caused by *Salmonella* is salmonellosis, which is generally is divided into two main types: typhoidal and non-typhoidal. Typhoidal salmonellosis or typhoid fever is caused mainly by *Salmonella* Typhi, characterized by symptoms such as fever, weakness, abdominal pain, and loss of appetite [1, 4, 53] and typically acquired through the consumption of contaminated food or water and more common in developing countries [4]. However, non-typhoidal salmonellosis is caused by a variety of *Salmonella* serotypes and mostly causes food poisoning symptoms, such as diarrhea, abdominal cramps, and fever and is more common in developed countries [14, 53, 54]. Most of the people infected with *Salmonella* will develop diarrhea, abdominal cramps, fever, and vomiting, which can last up to a week [1, 4, 55, 56]. Other symptoms caused by *Salmonella* infection include the enlargement of the spleen and lymph nodes, accumulation of fluid and blood in organs such as the lungs, and damage to the liver [1, 53, 55]. In chronic cases, arthritis may even occur, known as Reiter's Syndrome, and can last for months or even years [57, 58]. Different symptoms will occur in different mammals and birds.

#### **6. Pathogenesis and virulence factors**

*Salmonella* uses an array of virulence genes as part of its mechanism of pathogenesis [59]. These genes encode proteins that help the bacteria to evade the host's immune system, colonize and survive in host tissues, and cause inflammation and tissue damage [55, 60, 61]. Understanding the virulence genes of *Salmonella* can help researchers to develop strategies for preventing and treating infections caused by these bacteria. Some key virulence genes involved in each step of *Salmonella* pathogenesis include the following:


Many of these virulence genes are located on pathogenicity islands known as *Salmonella* pathogenicity islands (SPIs), which are thought to be acquired by horizontal gene transfer [68, 69]. SPIs are regions of bacterial DNA found in some strains of *Salmonella* and believed to play a role in the bacteria's ability to cause diseases [68, 69]. SPIs are typically composed of several genes, including virulence genes that encode proteins involved in the bacterium's ability to invade host cells, evade the immune system, and survive in different environments [69]. There are a total of 24 SPIs (1–24) recognized in *Salmonella* so far [70]. Each SPI is believed to have a specific function in the pathogenesis of *Salmonella* infections [68, 69]. For example, SPI-1 is involved in the bacterium's ability to invade and replicate within host cells [70], whereas SPI-2 is involved in the production of a toxin that can cause inflammation in the intestinal tract [69]. SPI-1 is a large and complex region of DNA, comprising approximately 40 genes [69]. Many of these genes are involved in the production of proteins called effectors, which are secreted by the bacteria into host cells and function to alter host cell function [70]. For example, some effectors can disrupt the normal functioning of the host cell's cytoskeleton, enabling the bacteria to move within the host tissue and evade immune cells [69, 70]. Other effectors can interfere with the host cell's signaling pathways, helping the bacteria to evade detection by the host's immune system. The type III secretion system (T3SS) encoded by SPI-1 is considered to be the most important virulence factor for *Salmonella* [68–70]. SPI-2 is another 40 kb long region of DNA found in certain strains of *Salmonella* bacteria, which has two distinct regions encoding proteins required to establish and maintain *Salmonella*-containing vacuole essential for *Salmonella* replication [71]. SPI-2 encodes a second T3SS, implicated in systemic pathogenesis [72]. The two regions of SPI-2 have unique species-specific distribution; for example, the larger 25 kb region is exclusive to *S*. *enterica*, whereas a second 15 kb long region is identified in *S*. *bongori* [69]. Like SPI-1, it contains a number of genes that contribute to the pathogenicity of the bacteria; SPI-2 is a smaller and less complex region of DNA than SPI-1 [69].

SPI-3 is a 17 kb long chromosomal DNA region that encodes many proteins involved in adhesion, such as MisL protein, which is vital for the long-term persistence of *Salmonella* [69]. SPI-3 is thought to be conserved in *S*. Typhi and *S*. Typhimurium [69, 73]. Similarly, SPIs 4–24 are involved in various aspects of pathogenesis and functions, all of which are not fully understood yet [69]. However, understanding the role of SPIs in *Salmonella* pathogenesis is important for the development of vaccines and therapies against the bacteria. Researchers are currently studying the mechanisms by which *Salmonella* utilizes SPIs to cause diseases, with the goal of finding new ways to prevent or treat infections caused by this bacterium.

Various *Salmonella* strains also contain plasmids, which have virulence and AMR genes [74–77]. *Salmonella* plasmids are usually small, circular pieces of DNA that are found in some strains of *Salmonella*. However, some strains also carry large *Salmonella* virulence plasmids [22, 77, 78]. Plasmids are separate from the bacterial chromosome and can carry a variety of genes, including those that confer antibiotic resistance or other traits that can help the bacteria survive and thrive in different environments [22, 79–81]. Some *Salmonella* plasmids carry virulence genes, which are responsible for the bacteria's ability to cause illness in humans and animals. *Salmonella* plasmids can be transmitted from one bacterium to another through horizontal gene transfer and can contribute to the evolution of new pathogenic strains [74, 82, 83]. Plasmids are an important tool in molecular biology and are often used to introduce new genes into bacterial cells for research or biotechnology purposes. There are several types of

#### Salmonella*: A Brief Review DOI: http://dx.doi.org/10.5772/intechopen.112948*

*Salmonella* plasmids that vary in size from 2 to more than 200 kb, which have been identified and characterized. Some of these include the following:


### **7. Antimicrobial resistance genes in** *Salmonella*

Some strains of *Salmonella* have developed resistance to certain antibiotics, which can make it more difficult to treat infections [90–92]. These are known as antibioticresistant *Salmonella* or AMR *Salmonella*. AMR is a growing global health concern because it can make it more difficult to effectively treat bacterial infections, including those caused by *Salmonella* [92–94]. The overuse and misuse of antibiotics are major contributing factors to the development of AMR in bacteria [93, 95]. Antibiotic resistance in *Salmonella* has a long history [96]. *Salmonella* have been known to cause illness for over a century, and antibiotics have been used to treat *Salmonella* infections since the 1940s [96, 97]. However, as with many other types of bacteria, *Salmonella* has developed resistance to many of the antibiotics that have been used for clinical treatment [98]. One of the first reported cases of antibiotic resistance in *Salmonella* was in the 1950s, when strains of *Salmonella* that were resistant to streptomycin were identified [96, 97]. Since then, *Salmonella's* resistance to other antibiotics, such as tetracycline and ampicillin, has also been reported [55, 99], and some strains are now resistant to multiple antimicrobial drugs or antibiotics.

Some common AMR genes found in *Salmonella* include the following:

1.*blaTEM* gene encodes for beta-lactamase, an enzyme that hydrolyzes beta-lactams (e.g., ampicillin, penicillins, and cephalosporins etc.; [100]).


The presence of an AMR gene does not necessarily mean that the bacterium will be resistant to the use of the antimicrobial drug [109, 110]. The ability of bacteria to survive antimicrobial treatment depends on many factors, including the specific strain of bacteria, the type and dosage of the drug, and the presence of other AMR genes [95, 110].

Recently, extensively drug-resistant (XDR) or more commonly known as multiple-drug resistant (MDR) *Salmonella* types, that is, *Salmonella* resistant to a wide range of antimicrobial drugs including many antibiotics that are typically used to treat *Salmonella* infections, have been on the rise, especially in developing countries [95, 111–113]. XDR *Salmonella* is of particular concern because it can be more difficult to treat and may lead to more severe or even fatal infections [95, 112]. XDR *Salmonella* can be transmitted through contaminated food, water, or surfaces, as well as through contact with infected animals or people. XDR phenotype in *Salmonella* arises through the acquisition of multiple AMR genes, which enables the bacteria to survive exposure to multiple drugs [109, 114]. The specific AMR genes present in XDR *Salmonella* can vary, but they may include genes that confer resistance to antibiotics such as ciprofloxacin, amoxicillin, and ceftriaxone. China has recently reported the first case of a waterborne outbreak caused by XDR *S.* Typhi in Beijing [113]. Similarly, the World Health Organization (WHO) recorded about 5274 cases of XDR typhoid fever in Pakistan from November 2016 to December 2018 [115, 116]. The prevalence of AMR in *Salmonella* can vary significantly by region, with some areas having higher rates of AMR than others. For example, studies have shown that the prevalence of AMR in *Salmonella* isolates from animals and food in the United States is generally low, with most isolates being susceptible to a range of antimicrobial drugs [7].

However, the prevalence of AMR *Salmonella* isolates from humans in the United States is higher, with some studies reporting resistance rates as high as 30–40% [93]. In other parts of the world, the prevalence of AMR *Salmonella* may be higher. For example, studies have shown that the prevalence of AMR *Salmonella* isolates from humans in some European countries is as high as 50–60% [117]. The distribution of AMR *Salmonella* in developing countries can vary significantly depending on the specific country and region. However, in general, the prevalence of AMR in *Salmonella* in developing countries tends to be higher than in developed countries [95]. There are several factors that may contribute to the higher prevalence of AMR in *Salmonella* in developing countries including the following [7]:


Overall, the frequency distribution of AMR in *Salmonella* among developing countries can vary significantly, but it is generally considered a major public health concern.

#### **8. Detection and diagnosis**

Accurate, sensitive, and specific detection of *Salmonella* is critical for food safety worldwide. Over the last decade, various other detection methods and techniques such as immunology, molecular biology, mass spectrometry, spectroscopy, optical analysis, and biosensor-based methods have been developed [34, 118, 119]. Generally, these methods can be divided into many categories as follows:

1.Culture methods: One of the most common methods for diagnosing *Salmonella* is through the use of traditional culture-based techniques, which are usually slow, labor-intensive, and not suitable for on-location or high-volume testing. However, these methods are considered gold standard and are in use since the discovery of enteric fever and have been standardized by the International Organization of Standards [120] for *Salmonella* detection, which is being used by many regulatory bodies all over the world [118, 121]. Similar standards have been published by FDA's Bacteriological Analytical Manual (BAM). The first stage in traditional culture methods for most food samples involves pre-enrichment in a nonselective liquid medium, such as buffered peptone water, which is then subcultured into two selective enrichment media, such as Rappaport Vasiliadis Soy broth (RVS) and Muller-Kauffmann Tetrathionate-Novobiocin (MKTTn) broth that inhibit background flora. This is followed by the inoculation on at least two selective differential agar media, such as Brilliant Green Sulfa (BGS), Bismuth Sulfite (BS), BrillianceTM *Salmonella* Agar, Xylose Lysine Deoxycholate (XLD), Xylose Lysine Tergitol-4 (XLT-4), and others, to allow the growth of *Salmonella* and distinguish them from other background microbial flora [118]. The last step in traditional culture methods includes the confirmation of presumptive positive *Salmonella* colonies.


Multiple materials have been tested, which provide varying degrees of selective advantages and have their own limitations. These include magnetic nanoparticles (MNPs)-based electrochemical biosensors, carbon nanoparticles-based electrochemical biosensors, metallic nanoparticles-based electrochemical biosensors, amperometric biosensors, potentiometric biosensors, conductometric biosensors, microfluidics-based biosensing platforms, Internet of Things (IOT)-supported

sensing of *Salmonella*, and clustered regularly interspaced short palindromic repeats (CRISPR)-based electrochemical sensors.

Overall, the choice of diagnostic method for *Salmonella* will depend on the specific circumstances and resources available, as well as the specific goals of the diagnosis such as identifying the specific strain of *Salmonella* or determining the severity of the infection.

#### **9. Surveillance, prevention, and control**

Many socio-economic factors contribute to the spread of *Salmonella*. The main factors are poverty and lack of education [7]. Poor environmental conditions contribute to poor hygiene, which ultimately helps spread the disease. Some *Salmonella* strains can cause serious and sometimes life-threatening infections, particularly in people with compromised immune systems. Different countries have developed regulatory framework for the testing and early detection of *Salmonella* in food. The US Centers for Disease Control and Prevention (CDC) conducts surveillance for *Salmonella* in the United States through the National *Salmonella* Surveillance System. This system tracks cases of *Salmonella* infection through laboratory testing and reporting by state health departments. CDC has developed a comprehensive national *Salmonella* surveillance program in the U.S. CDC has several systems for obtaining information about *Salmonella*, each of which has different purpose and provides information on various features of the organism's epidemiology, such as the number of outbreaks, antimicrobial-resistant infections, and subtypes. These programs include Laboratory-based Enteric Disease Surveillance (LEDS), National Notifiable Diseases Surveillance System (NNDSS), Foodborne Disease Active Surveillance Network (FoodNet), National Molecular Subtyping Network for Foodborne Disease Surveillance (PulseNet), National Antimicrobial Resistance Monitoring System enteric bacteria (NARMS), and Foodborne Disease Outbreak Surveillance System (FDOSS; https://www.cdc.gov/*Salmonella*/reportspubs/surveillance.html). Similarly, in Canada, the surveillance of *Salmonella* is conducted by the Public Health Agency of Canada (PHAC), which monitors and tracks cases of *Salmonella* in people through its integrated *Salmonella* surveillance system, collecting data from the provinces and territories under the National Enteric Surveillance Program (NESP), FoodNet Canada, and Canadian Notifiable Disease Surveillance System (https://www.canada. ca/en/public-health/services/diseases/salmonellosis-*Salmonella*/surveillance.html). In Europe, the European Centre for Disease Prevention and Control (ECDC) and the European Food Safety Authority (EFSA) are responsible for the surveillance of *Salmonella*. European Centre for Disease Prevention and Control has framed and adopted Regulation (EC) No 2160/2003 on protecting human health against *Salmonella* and other specified foodborne zoonotic agents, with the goal of controlling *Salmonella* at every stage of food production and in animal feed to reduce its prevalence and the risk to public health (https://leap.unep.org/countries/no/nationallegislation/regulation-no-1703-control-*Salmonella*-and-other-food-borne). The ECDC is responsible for the surveillance of *Salmonella* infections across the European Union (EU). The organization collects data on *Salmonella* infections in humans from the EU Member States through the EU Surveillance Network for Communicable Diseases (TESSy) system. This allows the ECDC to track the number of cases, identify outbreaks, and monitor trends in *Salmonella* infections across the EU. The EFSA, on the other hand, is responsible for food safety and conducts surveillance of

*Salmonella* in food products. It collects data on *Salmonella* in food from EU Member States and also conducts its own risk assessments on specific food products. The EFSA also provides scientific advice and support to the European Commission and EU Member States on food safety issues, including *Salmonella* control in the food chain. Additionally, the European Union Reference Laboratory for *Salmonella* (EURL-*Salmonella*) also plays an important role in the surveillance of *Salmonella* in Europe. This laboratory is responsible for coordinating the network of national reference laboratories for *Salmonella* and providing scientific and technical support for the detection and control of *Salmonella* in food and animal feed.

Surveillance of *Salmonella* in the developing world varies from one country to another, but in general, it is less robust and comprehensive compared with that in developed countries [7, 128, 129]. In developing countries, the surveillance of *Salmonella* is conducted by the national public health department or ministry of health [7, 128]. However, the capacity for laboratory testing and data collection is limited because of the lack of resources and infrastructure. In addition, lack of awareness and education on food safety and good hygiene practices among the population, inadequate sanitation, and poor infrastructure exacerbate the spread of *Salmonella* in developing countries. However, efforts to improve them through international collaboration and aid programs, education, and capacity building are essential to curb the spread of these bacteria. To control salmonellosis, it is important to follow good hygiene practices, such as washing hands thoroughly with soap and water before handling food and cooking food to a safe temperature to kill *Salmonella* that may be present. It is also important to store food properly and avoid cross-contamination, for example, by using separate cutting boards and utensils for raw and cooked foods. In addition to these measures, it is important to control the spread of *Salmonella* in food-producing animals, as they can be a source of contamination. To be successful, *Salmonella* control requires a focus on the sources of the organism and the means of transmission to humans, which is best achieved through the One Health approach, with adequate attention paid to animal and food sources and the environment that harbors organisms and provides avenues of transmission to humans. Specific measures such as proper animal feeding and husbandry practices, effective disinfection of animal housing and equipment, monitoring wildlife sources especially avian species, and effective food safety practices are required.

#### **10. Conclusion**

In conclusion, *Salmonella* is one of the leading causes of food poisoning in humans. It is commonly found in raw or undercooked meat, poultry, eggs, and dairy products, as well as in fruits and vegetables that have come into contact with contaminated water or soil. *Salmonella* is a pathogen of great concer, which can cause severe illness and leads to death in some cases. The virulence of *Salmonella* is determined by a variety of factors including the serotype, presence of specific virulence genes, and the host's immune response. In addition, the emergence of antibiotic-resistant *Salmonella* is a significant concern in the field of food safety. *Salmonella* can acquire AMR genes through horizontal gene transfer, and the presence of these genes makes the treatment of infections more difficult and complicated. Additionally, the emergence of extensively drug-resistant (XDR) *Salmonella* has become a great public health concern because of its ability to resist multiple classes of antibiotics, leading to a significant public health concern because of prolonged illness and increased

Salmonella*: A Brief Review DOI: http://dx.doi.org/10.5772/intechopen.112948*

health care costs. It is important to limit the use of antibiotics to decrease the risk of antibiotic resistance and implement strategies to prevent the spread of resistant strains of *Salmonella*, such as proper food handling and sanitation practices. It is also important to develop advanced, fast, and efficient methods to monitor the emergence of antibiotic-resistant *Salmonella* to quickly detect and respond to outbreaks.

### **Author details**

Sohail Naushad\*, Dele Ogunremi and Hongsheng Huang Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency, Ottawa, Ontario, Canada

\*Address all correspondence to: sohail.naushad@inspection.gc.ca

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 2** Salmonellosis in Food and Companion Animals and Its Public Health Importance

*Joseph K.N. Kuria*

#### **Abstract**

Salmonellosis in animals is caused by typhoidal and non-typhoidal *Salmonella* organisms. Non-typhoidal salmonellosis is a zoonosis of major public health concern occasioning over 155, 000 mortalities yearly worldwide. The majority of the human infections are mainly acquired directly through consumption of contaminated foods of animal origin, particularly poultry, eggs and dairy products or consumption of contaminated fruits. Rodents and will birds are the main reservoirs of non-typhoidal salmonellosis. Salmonellosis has a great economic and health impact occasioned by the cost of surveillance, investigation, treatment, and prevention in both animals and humans. Non-typhoidal salmonellosis is further complicated by the wide host range and the emergence of multidrug resistant *Salmonella* strains due to intensification of livestock production and uncontrolled antimicrobial drug use. There is a need for more innovative prevention and control measures to safeguard losses in animals and human health. This chapter will discuss salmonellosis in food and companion animals, the public health importance, and the challenges facing its control.

**Keywords:** salmonellosis, animals, public health, control, salmonellosis transmission

#### **1. Introduction**

Salmonellosis is caused by bacterial species in the genus *Salmonella*, a member of the family *Enterobacteriaceae*, comprising about 63 genera. *Salmonella* has a wide host range, occurring in mammals, birds, reptiles, amphibians, fish and invertebrates. The genus has two taxonomic species, based on differences in their 16S rRNA sequence analysis, namely *Salmonella* enterica and *Salmonella bongori* [1, 2]. *S. enterica* has six subspecies namely subspecies *enterica*, *salamae*, *arizonae*, *diarizonae*, *houtenae*, and *indica* [3]*. S. enterica* subsp. *enterica* is the most common subspecies and predominantly infects warm-blooded animals. *S. bongori* species is usually found in cold-blooded animals and the environment but some are occasionally associated with human disease. *Salmonella* subspecies are further classified antigenically into serotypes, or serovars, of which there are currently close to 2700 [4]. Some *Salmonella* serovars are host adapted but the majority are not and can cause disease in a broad range of hosts.

In animals, host-adapted or typhoidal *Salmonella* serovars cause severe disease in the specific hosts, characterized by septicemia but generally pose no threat to other species including humans. The non-host -adapted serovars are generally carried asymptomatically in animals although in some cases they cause disease characterized by diarrhea. These serovars are zoonotic or potentially zoonotic. In humans, they cause non-typhoid salmonellosis (NTS) the 4th most important cause of gastroenteritis [1]. It is also one of the most important bacterial zoonotic diseases, estimated to cause, 155,000 deaths yearly worldwide [5]. Non-typhoidal salmonellosis is therefore not only a major public health concern worldwide but has great negative economic impact due to the cost of surveillance, investigation, treatment and prevention of illness [6]. It is transmitted to humans through the feco-oral route, mainly by consumption of contaminated raw or improperly cooked animal products. The major sources of infection are poultry, eggs and dairy products but contaminated fresh fruits and vegetables have also been recognized as vehicles of transmission [7, 8]. Other common sources of infection include pigs products and contact with companion animals, particularly dogs, cats and horses, as well as pet reptiles such as snakes and tortoises. Contamination of animal products with *Salmonella* can also negatively impact on food trade by limiting market access NTS organisms occur widely in the environment but the main reservoirs are rodents, reptiles and wild birds [9]. The wide host range further makes control of NTS challenging. Emergence of multidrug resistant *S. enterica* strains in animals due to misuse and over use of antimicrobial agents is an added complication. Since the majority of the human infections are acquired through the consumption of contaminated foods of animal origin, NTS from animals is likely to continue to be a threat to human health. This chapter will discuss salmonellosis in food and companion animals, the associated public health risks, the challenges facing its control and future research needs. The discussion material is derived from existing literature as well as personal experience. It is hoped the chapter will be found useful by students, researchers, practitioners and managers of animal and public health.

#### **2. History of salmonellosis**

Salmonellosis has been around for centuries. It has been determined, through recent technological development, that typhoid fever was implicated in a plague which wiped out a third of the population in the city Athens, around 430 B.C. [10]. The organism *Salmonella* is named after Daniel E. Salmon, an American veterinarian. It was named in his honor by his research assistant, Theobald Smith, who isolated the first known strain of *Salmonella* from a case of hog cholera, which he named *Salmonella choleraesuis,* in 1885 [11]. The first study of *Salmonella* in humans was conducted by Karl Joseph Eberth, a German pathologist and bacteriologist, when he described a bacillus that he suspected was the cause of typhoid. The findings were later confirmed by pathologist Georg Theodor August Gaffky and the organism name "Gaffky-Eberth bacillus", which today is known as *S. enterica* serovar Typhi [12].

Notable personalities thought to have died from *Salmonella* infection include a US president, William Henry Harrison [13] and one of the famous Wright brothers, Wilbur Wright. The importance of typhoid as a scourge in human health is exemplified by the story of "typhoid Mary "(Mary Mallon), the domestic worker who transmitted *Salmonella* to at least eight households [14].

#### **3. Etiology of salmonellosis in animals**

#### **3.1 Classification and nomenclature**

*Salmonella* genus is classified under the family *Enterobacteriaceae* comprising about 63 genera. Their natural habitat is the intestinal tract of animals, of which about 25, such *Shigella, Salmonella, Enterobacter, Klebsiella, Serratia, Proteus* as are clinically significant. Others, such as *Escherichia coli,* are considered part of the normal intestinal microbiota and cause disease only incidentally. Phylogenically, there are only two species in this genus, *S. enterica and bongori bongori* [1, 2], based on differences in their 16S rRNA sequence analysis. *S. enterica* has six subspecies (subspecies *enterica*, *salamae*, *arizonae*, *diarizonae*, *houtenae*, and *indica* [3].

#### **3.2 Antigenic classification**

*Salmonella* are further classified antigenically by the Kauffman and White classification system, which classifies the organism into serotypes on the basis of a common somatic (O), flagella (H) and capsular (K), antigens [15]. The (O) antigen is present in all serotypes and is a heat-stable component of the lipopolysaccharide (LPS) located in the outer cell membrane present in all Gram negative bacteria. The heat-labile H antigens are part of the flagella protein, flagellin, present in all motile *Salmonella* spp. Two different genes code for the flagella proteins and either or both may occur in a serovar. Only one gene is expressed at a time, hence a serovar may possess only one protein at a time and the cells are thus diphasic. The two proteins are designated as phase I and Phase II. Phase I antigens are specific to a serotype and confer serological identity whereas phase II antigens are non-specific [16]. The K antigens are heat-sensitive polysaccharides located in the bacterial capsule, which is rare among *Salmonella* serotypes. The human-restricted serovar Typhi and serovar Paratyphi C produce a variant of the K antigen, known as the virulence (Vi) antigen [17].

The antigenic structure of *Salmonella* is useful in identification of serovars. It is also a useful epidemiological tool in determining sources of infection and mode of spread [18]. In nomenclature of *Salmonella* serotypes, subspecies name is usually omitted. For instance, *S. enterica* subspecies *enterica* serotype *gallinarum* is shortened to *Salmonella* ser. *Gallinarum* or *Salmonella gallinarum* [1, 11]. There are about 2700 serotypes (serovars) so far identified [4], each having a unique combination of somatic O and flagella phase I and Phase II antigens. Over 50% of these serotypes belong to the *S. enterica* subspecies [11].

#### **3.3 Cellular, cultural and biochemical characteristics**

*Salmonella* species are Gram-negative non-spore forming large rods measuring 0.7–1.5 by 2.0–5.0 μm. They are motile by peritrichous flagellation with the exception of *S*. Gallinarum and *S*. Purollum. Capsulation in *salmonella* is limited to a few serovars such as *Salmonella typhi*. *Salmonella* are aerobic, facultatively anaerobic in gaseous requirements. Nutritional requirement is non-fastidious and they can be cultivated in simple media such as nutrient agar. The majority of *Salmonella* are lactose fermenters. Utilizing this characteristic, selective and differential media have been formulated for isolation, and identification. Such media include MacConkey agar, *Salmonella*-Shigella agar, brilliant green agar xylose lysine deoxycholate agar and Hektoen enteric [19].

*Salmonella* species have the ability of to utilize tetrathionate (S4O6 2−) as an alternative electron acceptor in anaerobic respiration. This confers the organism a selective growth advantage, a property that used for non-selective enrichment in cultures containing competitive bacteria [20]. Common to *Enterobacteria*, *Salmonella* are oxidase negative, catalase positive, nitrate positive and they metabolize glucose fermentative, often with gas production. Other biochemical properties used for identification of *Salmonella* include hydrogen sulfide production (except few serovars such as *Salmonella* paratyphi A, and *S*. *choleraesuis*), the ability to utilize citrate as a sole carbon source, decarboxylation of lysine and fermentation of dulcitol. *Salmonella* are negative for production of urease and indole, deanimation of phenylalanine or tryptophan and Voges–Proskauer reaction [21, 22].

#### **3.4 Pathogenicity and virulence factors**

Many virulence factors play a variety of roles in the pathogenesis of *Salmonella* infections. These factors enable the organism adhere to and colonize its host, invade host cell, survive and multiply in macrophages, secret toxins and evade or bypass host's defense mechanisms. The factors include capsule, flagella, fimbriae, adhesins, invasins, hemagglutinins, exotoxins and endotoxins [16]. The various virulence factors are encoded by gene clusters, referred to as *Salmonella* pathogenicity islands (SPIs), located in chromosomes, plasmids and transporons [23–25].

The polysaccharide capsular O and Vi antigens in no-typhoidal and typhoidal *Salmonella* are known to aid the organism evade host's defense by modifying the cell surface in order to inhibit host's cellular response [26, 27]. Flagella are possessed by majority of *Salmonella* serovars and are known to confer pathogenicity in addition to motility. Certain *Salmonella* serovars are able to evade or minimize the host immune response by antigenic variation of flagella antigens, from one phase to the other [24, 25]. Fimbriae are the most common adhesion factors in *Enterobacteria.* They facilitate adhesion of *Salmonella* not only to hosts' cells, thus enabling colonization, but also to surfaces and foods. They are also implicated in a variety of other roles such as biofilm formation [28], which serves to shield the organism from attack by host's defense systems.

Endotoxin or lipopolysaccharide (LPS) is located in the outer membrane of Gram negative bacteria. It is heat stable and is released only upon bacterial cell lysis. It plays a role in pathogenesis of *Salmonella* infection by evoking pyrexia, activating complement system and depressing lymphocyte function among others. Endotoxin also plays a part in septic shock that can occur in systemic infections [29, 30].

Exotoxins comprise of cytotoxins and the enterotoxins. Cytotoxins are associated with killing of the mammalian cells in vitro and probably play a role in nonesecretory diarrhea [31]. There is limited information regarding the mode of action of enterotoxins of *Salmonella* but they are antigenically related to the cholera family of enterotoxins. They are associated with diarrhea disease, probably through stimulation of intestinal secretion [16].

Certain *Salmonella* strains are hemolytic, another important virulence factor mediated by HylE protein, a product of hylE gene, thought to play a role in the pathogenesis of systemic salmonellosis. The protein produces hemolysis in blood agar made from the blood of a range of animals, including humans, with certain blood types [16].

*Salmonellosis in Food and Companion Animals and Its Public Health Importance DOI: http://dx.doi.org/10.5772/intechopen.109324*

#### **3.5 Host range**

*Salmonell*a and salmonellosis occur worldwide in mammals, birds, reptiles, amphibians, fish and invertebrates. Majority of *Salmonella* are not host specific and can cause disease in a broad range of hosts but some are host restricted. *S. enterica* subsp. *enterica* is the most common and predominantly infects warm-blooded animals. In the subspecies *enterica*, serovars *typhi*, *paratyphi* and *hirschfeldii* are restricted to humans and cause typhoid and paratyphoid fever respectively. They have no significant animal or environment reservoirs. Serovars *pullorum* and *gallinarum* are restricted to poultry; *abortusovis* to sheep; *choleraesuis* to pigs; and *dublin t*o cattle [32, 33]. The rest of the serovars, referred to as non-typhoidal *Salmonella*, are zoonotic or potentially zoonotic, the most common being serovars *typhimurium* and *enteritidis* [1].

The other five *S. enterica* subspecies (*salamae, arizonae, diarizonae, houtenae* and *indica*) and *S. bongori* are usually found in cold-blooded animals and the environment but some are occasionally associated with human disease. All animal species are susceptible to *Salmonella* infection but clinical disease occurs more commonly in some and not others. Among domestic animals, poultry, cattle, pigs, poultry and horses show clinical disease but cats and dogs commonly do not [34, 35].

#### **3.6 Isolation and identification**

Isolation of *Salmonella* from samples with competing microbes involves an initial non-selective pre-enrichment followed by a selective enrichment. Selenite (SeO3 2−) is inhibitory to coliforms and certain other microbial species such as fecal streptococci and is used for selective enrichment of *Salmonella spp* from both clinical and food samples. Selective enrichment is followed by plating onto selective agars, followed by biochemical and serological confirmation of suspect presumptive colonies [36]. Serogrouping by somatic and flagella antigens, can be achieved by using monovalent specific 'O', 'H' and 'Vi' antisera. Phage typing, immunomagnetic separation and ELISA-based assays are some of the screening methods developed to produce rapid results, especially from food and environmental samples [37]. Several PCR assays targeting various genes have also been developed for identification of *Salmonella.* These include the 16S rRNA, invA, agfA, viaB, hilA, sirA, ttr, bcfD and phoP genes, among others [38–40].

#### **4. Salmonellosis in animals**

*Salmonell*a has a wide host range that includes mammals, birds, reptiles, amphibians, fish and invertebrates. It is a major cause of morbidity and mortality in animals and also a major cause of economic loss in livestock. The main importance of nontyphoidal salmonellosis in animals is however its zoonosis, causing a major health, social and economic impact due to cost of surveillance, investigation and treatment. The major source of direct human infections is consumption of contaminated or infected foods of animal origin, particularly meat, eggs and dairy products, and direct contact with animals, particularly companion animals, mainly dogs, cats and horses. Rodents and wild birds are the main reservoirs of non-typhoidal salmonellosis for animals. This chapter wills therefore limit the discussion on salmonellosis to livestock food animals, companion animals, rodents and wild birds.

#### **4.1 Salmonellosis in poultry**

#### *4.1.1 Etiology and transmission*

Salmonellosis in poultry and other avian species is caused by serovars in the subspecies *enterica*. Two of the serovar, *S. pullorum* and *S. gallinarum* are avian hostspecific and cause typhoidal salmonellosis, while other serovars cause non-typhoidal infections, the most important being S*. typhimurium* and *Salmonella* enteritidis [35, 41, 42] *S. enterica* subsp. *arizonae* is also recognized as a cause of paratyphoid but mainly in turkeys [43]. Although *S. pullorum* and *S. gallinarum* can infect a wide range of avian species, clinical signs are observed in a few, which include chickens, turkeys and wild birds such as quails and pheasants [42].

Transmission is horizontal via fecal-oral route and vertically via infected embrocated eggs. Transovarian infection in the egg results in subsequent infection in chicks or poults and is one of the most important modes of transmission of these two diseases. Some infected hens become asymptomatic carriers and continually transmit it to their progeny. This mode of transmission is particularly critical in hatcheries since it can result in widespread dissemination of the diseases. Transmission by cannibalism and through the respiratory tract has been reported. Humans constitute a big potential of disease introduction through mobility and duties. They can track infections on vehicles, footwear, clothing, hands and contaminated equipment. Mammals, particularly dogs, cats, rodents as well as insects can also act as mechanical transmitters [42, 44, 45].

Similar to typhoidal salmonellosis, non-typhoid salmonellosis in poultry is transmitted vertically or horizontally*. S. enteritidis* serovar has a particular preference for vertical transmission. Horizontal transmission occurs through fecal contamination of feed and drinking water and by penetration of microorganisms into the egg subsequent to fecal contamination. Infection can be introduced into a farm by humans through clothing, footwear, equipment and vehicles. Rodents and wild birds are a notable reservoir of paratyphoid *Salmonella*. They are attracted into poultry houses by left-over feed and contaminate the feed by fecal material [42, 46, 47]. Dogs and cats can also track *Salmonella* infections over long distances to contaminate farms.

#### *4.1.2 Clinical signs*

Both *S.* g*allinarum* and *S*. *pullorum* cause systemic disease but whereas the former affects birds of all ages, *S. pullorum* affects primarily young ones. Birds hatched from infected eggs may be found dead in the hatching trays. Young birds may die soon after hatching without any observable signs and most acute outbreaks occur in birds under three weeks of age. In mature birds, infection is manifested by decreased egg production, fertility, hatchability and by anorexia. Diarrhea, which is usually white or yellow, watery to mucoid, is common, with fecal pasting seen around the vent.

#### *4.1.3 Post mortem lesions*

Lesions from *S. gallinarum* and *S. pullorum* infections are characterized by septicemia, with inflammation of all internal organs, including intestines, and notably liver and spleen, which show classic gray granulomatous nodules [46]. Infected ovaries

#### *Salmonellosis in Food and Companion Animals and Its Public Health Importance DOI: http://dx.doi.org/10.5772/intechopen.109324*

may be misshapen and/or shrunken and follicles are often pedunculated, being attached to the ovary by fibrous stalks, while the abnormal ova may contain caseous material [45]. Impaction of oviducts, resulting in egg peritonitis, is also common [48]. Recently hatched chicks show signs of septicemia and omphalitis, a condition characterized by infected yolk sacs, often accompanied by unhealed navels. The yolk sacs usually contain creamy or greenish, caseous material [49–51].

In non- typhoidal salmonellosis, the highest morbidity and death rates are usually observed during the first 2 weeks after hatching. Infected adult birds are asymptomatic and do not present signs of the disease and main importance is human infection, through consumption of contaminated meat and eggs. In young birds, it may however cause enteritis with dissemination toward the spleen, lungs, liver, spleen, and kidneys [43]. An enlarged, friable liver, with necrotic foci, is common. Chicks infected transovariary will show signs and lesions similar to those in typhoidal salmonellosis.

#### *4.1.4 Diagnosis, treatment and control*

Diagnosis of salmonellosis in poultry is achieved through significant clinical signs, necropsy finding as well as isolation and identification of the organism. *S*. *gallinarum* and *S*. *pullorum* can be differentiated with biochemical, serological tests and PCR.

*S. gallinarum* and *S. pullorum* may survive for a long time, months or even years in the environment, which makes it difficult to eliminate them in infected poultry houses. Once a flock is infected, the amount of *Salmonella* can be reduced, but not completely eliminated and depopulation is usually the only option. Pullorum disease and fowl typhoid are notifiable disease in many countries under OIE guidelines. Both diseases can be controlled and eradicated by use of serological testing and elimination of positive birds but vaccines may be used to control the disease. The diseases have largely been eradicated from commercial poultry in developed countries. Various antibiotics can be used to treat clinical cases, but they do not eliminate the organisms from the flock. The two serovars are highly adapted to the host species, and therefore are of little public health significance [45, 51].

#### **4.2 Salmonellosis in cattle**

#### *4.2.1 Etiology and transmission*

Salmonellosis in cattle is caused mainly by *S. enterica* ser *dublin*. The serovar is adapted to cattle but can also cause infection in other species including human. It causes economic losses in cattle production and is also a threat to human health [52]. Other serovars can also infect cattle and indeed, majority of *Salmonella* isolated from cattle are the non–host specific [53]. *Salmonella* infection in cattle is most commonly acquired by ingestion of feed or water contaminated by fecal matter from other livestock, rodents and wild birds or by contaminated animal by-products. *Salmonella* are shed by clinically infected animals and contaminate feed, water, yards, and equipment. The bacterium is also shed in saliva, nasal secretions, urine and milk in cases of systemic illness. Aerosol transmission between animals is considered possible in closely confined production systems [53–55]. Probability of vertical transmission from a dam to fetus, with calves born already infected, has been proposed [56]. The outcome of infection is determined

by virulence of the serotype, dose of inoculum, degree of immunity and other stress factors.

#### *4.2.2 Clinical signs*

Salmonellosis in cattle affects all age groups causing both intestinal and systemic infection but is most severe in the young. Clinical presentations are highly variable and the differential diagnosis list is considerable [53]. Acute disease is characterized by fever, anorexia and diarrhea of varying degree. The feces may be foul smelling, and may contain varying amounts of blood, mucus, and shreds of intestinal lining. Lactation drops suddenly in dairy cows. Clinical signs may last up to a week and death is due to dehydration and toxemia. In newborn calves, the disease most commonly affects those that receive inadequate or no colostrum and signs may include central nervous system (CNS) signs or pneumonia, and death may occur in 1–2 days. Those calves that survive longer may develop complications such as polyarthritis, or gangrene of the extremities of limbs, ears and tail [57]. Pregnant cows may abort, either with or without other clinical signs [54, 57, 58]. Subacute disease is seen mainly in adult animals and signs may include mild fever, anorexia, diarrhea dehydration and weight loss. Chronic disease in manifested by low intermittent fever and anorexia. There may be watery diarrhea resulting in progressive dehydration and weight loss. The feces are usually normal or contain mucus or blood. Sick cows that recover may become carriers that shed *Salmonella* for varying periods of time and cause continuous new infections in the herd [59, 60].

#### *4.2.3 Post mortem lesions*

In animals that die peracutely due to septicemia, there may be no gross lesions other than extensive submucosal and subserosal petechial hemorrhages. In acute enteritis, seen mainly in calves, the small intestines typically shows a diffuse mucoid or mucohemorrhagic enteritis and the mesenteric lymph nodes are edematous, congested and greatly enlarged [58]. In adult cattle, chronic infection is characterized muco/necrotic enteritis, especially of the ileum, caecum and colon. The wall is thickened and covered with yellow-gray necrotic material overlying a red, granular surface. Characteristic "button" ulcers may be seen in the colon [61] and the mesenteric lymph nodes and spleen may be enlarged.

#### *4.2.4 Diagnosis, treatment and control*

Clinical signs of salmonellosis are indicative of infection but definitive diagnosis of infection involves isolation and identification of the organism. Response to antibiotic treatment is usually poor. Animals that recover from infection can remain carriers and shed bacteria intermittently or continuously for long, especially during stress periods such as transportation or calving. The carrier status can even progress to full blown clinical disease.

Control involves sourcing animals from disease-free herds in order to ensure a clean herd. New animals should be put on quarantine for at least 4 weeks. Continuous serological tests and fecal culture is recommended and positive animals culled. Control of rodents and wild birds, particularly in feeding troughs, is important. Routine disinfection of premises should be considered and aborting animals should be considered suspect, isolated, tested and culled. Vaccines are available as part of a prevention or control tool.

#### **4.3 Salmonellosis in pigs**

#### *4.3.1 Etiology and transmission*

Salmonellosis in swine occurs in form of two clinical disease entities, typhoidal and non-typhoidal. Typhoidal salmonellosis is mainly caused *S. choleraesuis.* This serovar is adapted to swine and do not commonly affect other animals including, humans. Nontyphoidal infections are caused by *S. typhimurium* and is the most commonly found serotype in pigs and a common source of food poisoning in humans Other serotypes that commonly infect pigs are *e*nteritidis, agona, d*erby, hadar* and h*eidelberg* [62, 63].

The main route of transmission is feco-oral, which is exacerbated by poor hygiene and overstocking [45]. Pigs start shedding the bacteria shortly after infection and can continue to shed up to 5 months after recovery from the illness. Feed ingredients of animal origin, are another important source of infection for pigs. Mechanical transmission can be effected by humans through tracking of infections on vehicles, footwear, clothing, hands and contaminated equipment [64]. *Salmonella* also localizes in the tonsils and can lead to nose-to-nose transmission [65, 66]. Piglets can also get infected by the sow through milk, although rarely [67]. In addition, transmission of non-typhoidal salmonellosis can occur indirectly through contamination of feed and water by infections carried in the intestinal tract of wild birds and rodents.

#### *4.3.2 Clinical signs*

*S. choleraesuis* infections may occur at any age, but are more frequent in growing pigs, between 8 weeks and 5 months old. Outbreaks are frequently associated with stress conditions such as overcrowding, transportation, weather, concurrent infectious diseases such as parasitism, and poor management [68]. The disease is manifested as an acute septicemia characterized by fever, depression and anorexia. Sudden death is quite common in the acute phase of the disease, with pigs showing signs of cyanosis on the extremities such as ears, nose and tail, due to septicemia [69]. Pigs that survive the acute phase will show signs of yellow diarrhea and coughing. The diarrhea is foul- smelling and may contain blood and mucus. The bacterium may cross the blood-brain barrier during the septicemia phase and cause meningitis and nervous signs may be observed, but rarely. Arthritis may also be observed subsequent to localization of the organism in joints. Sick pregnant sows may abort.

Morbidity in *S. choleraesuis* infection is usually low (less than 10%) but mortality is high. The organism may localize in the mesenteric lymph nodes and such subclinical carriers intermittently or continuously shed the organism in feces, particularly under stress conditions [70].

Clinical signs of *S. typhimurium* are not common in well managed commercial herds but can occur in stressed and immuno-compromised ones. The main symptoms are fever, anorexia, yellowish diarrhea, dehydration, prostration, and mortality [71]. Affected pigs may recover in a period of one week but re-infection is common within the next three to four weeks. Mortality is rare, but those animals that survive can remain carriers, and therefore a source of continuous infection, for up to five months after recovery.

#### *4.3.3 Post mortem lesions*

In pigs that die suddenly from *S. choleraesuis* infection*,* the most common lesion is skin cyanosis, particularly on the ears, feet and abdomen, accompanied by swelling of the gallbladder, lymph nodes, spleen and liver. There may be necrotic foci in the liver, as well as icterus. Consolidative bacterial pneumonia will be observed in pigs that show coughing. In Pigs that show signs of diarrhea, intestinal lesions, mainly pseudomembranous inflammation of the ileum and button ulcers in the colon will be observed.

The most common macroscopic lesion in *S. typhimurium* infection in pigs is inflammation of the ileum, the caecum and colon. The inflammation is characterized the presence of yellowish necrotic pseudomembranes. Mesenteric lymph nodes may be inflamed and enlarged. Characteristic "button' ulcers may be observed in the colon [72]. Some cases of rectal strictures have been reported after clinical salmonellosis. In these cases, pigs cannot defecate and intestinal contents remain trapped in the intestines, creating severe distension.

#### *4.3.4 Diagnosis, treatment, and control*

Clinical signs and lesions found during necropsy can be indicative of salmonellosis but not diagnostic. A definitive diagnosis is achieved by isolation and identification of the organism from suitable samples such as lung, liver, spleen, kidney, or lymph nodes [73]. Isolation from the intestine or feces is often unsuccessful.

Clinical disease can be controlled my antimicrobial therapy early in the onset of the disease but this will not eliminate the pathogen. The prophylactic use of antimicrobial agents is also not recommended because of expense, and promotion of antimicrobial resistance. Vaccines are available for preventing infection but their efficacy is often disappointing [74]. However, good management and husbandry is the best method of preventing clinical disease. This involves, but is not limited to, proper cleaning and disinfection. All-in-all-out pig flow and rodent control should be part of management procedures.

#### **4.4 Salmonellosis in companion animals**

#### *4.4.1 Salmonellosis in dogs and cats*

#### *4.4.1.1 Etiology and transmission*

Numerous *Salmonella* serovars have been isolated from dogs and cats with *S*. *typhimurium* and *S*. *enteritidis* being the most common serovars. There are no hostadapted serovars identified in dogs or cats [75–77]. Most dogs and cats are asymptomatic carriers and prevalence of *Salmonella* in dogs is associated with raw feed diets and contaminated feed, due to indiscriminate feeding habits, including scavenging [76, 78]. Fecal shedding of *salmonellae* by dogs is also a possible source of infection for other dogs as well as humans [79]. Cats may get infection from eating birds and rodents [80].

#### *4.4.1.2 Clinical signs*

These are rare although some dogs and cats may manifest signs of septicemia, particularly in puppies and kittens or in adults stressed by debilitating concurrent diseases [76]. Acute gastroenteritis is the most common symptom. The signs include fever, anorexia, diarrhea and vomiting. The diarrhea may contain blood. Other syndromes may include pneumonia, pelvic limb paresis, or conjunctivitis. As enteritis progresses, abortion may occur in pregnant dogs and cats or they may give birth to

weak puppies or kittens. Recovered animals can continue to shed the pathogen in their feces and saliva due to localization of the organism in the lymph nodes.

#### *4.4.1.3 Postmortem lesions*

Description of post mortem lesions in dogs and cats is scarce but the most common is enterocolitis [81]. Other recorded lesions include liver necrosis [82], pyonephrosis [83], cholecystitis [84], hemorrhagic gastroenteritis [85] and pneumonia [57].

#### *4.4.1.4 Diagnosis, treatment and control*

Diagnosis is based on isolation of the organism in conjunction with significant clinical signs. A diagnosis is conclusive if the organism is isolated from a normally sterile site, such as blood or synovial fluid in a live animal or from tissues samples from postmortem examination. Isolation of *Salmonella* may not necessarily be a definitive diagnosis in healthy animals.

Treatment for a *Salmonella* infection is primarily supportive, to compensate for the fluid lost through vomiting and diarrhea. Depending on the extent of the infection, antibiotics may be required for septic cases to prevent shock. Control of fecal contamination is of primary importance. Dogs and cats should be fed uncontaminated and properly cooked food.

#### *4.4.2 Salmonellosis in horses*

#### *4.4.2.1 Etiology and transmission*

*Salmonella* a*bortusequi* is an equine-adapted serovar and is associated with abortion in mares, neonatal septicemia, polyarthritis and testicular lesions in males [24, 25, 86]. Infections are common in Asia and African but rare in the rest of the world [87]. However, the most common serovar isolated from horses is S. *typhimurium* [88].

*Salmonella* a*bortusequi* transmission is oral or venereal. Infection may result from ingestion of feed contaminated by uterine discharges from mares that have recently aborted or from carrier mares. Transmission from stallions to mare during mating is also thought to occur [89]. The infection may localize in the uterus and cause repeated abortion or infection of subsequent foals.

Transmission of *S. typhimurium* is primarily fecal-oral. Feed, water and environment are contaminated by organism excreted through feces of sick or carrier horses, birds and rodents. Acutely ill animals excrete large amounts of bacteria. Risk factors for development of disease include stress due to transportation, overcrowding, changes in feed, intense physical activity, deprivation of feed and water and surgical treatment. Antibiotic treatment has also been found to increase risk for symptomless carriers. Another source of infection is eating manure, especially in foals.

#### *4.4.2.2 Clinical signs*

Serovar *abortusequi* primarily affects the reproductive system. In mares, the main clinical sign is abortion, with no other evidence of illness. Abortion usually occurs at about the seventh or eighth month of pregnancy. Retained placenta and metritis are common sequel of abortion. Foals from infected mares may develop an acute septicemia soon after birth while those that survive longer may develop polyarthritis. Sign

of infection in the stallion include fever, swelling of the prepuce and scrotum, and arthritis. Epididymitis, orchitis and testicular atrophy are other abnormalities associated with infection [89].

Equine salmonellosis caused by *S. tyhimurium* can be asymptomatic, but is commonly associated with fever and. Diarrhea that can progress to septicemia in young animals [90, 91]. Infected foals are more prone to clinical disease than adult horses. Diarrhea, often severe and watery, is the most common symptom. Other symptoms include fever, colic and poor condition. The infection is often self-limiting but some conditions may progress to septicemia, resulting in death. Septicemia leads to polyarthritis, and/or pneumonia. Laminitis is a possible complication of salmonellosis in horses, and is attributed to bacterial endotoxins.

#### *4.4.2.3 Post mortem findings*

Necropsy findings in cases of *S.* a*bortusequi* include placentitis manifested by edema, hemorrhages and areas of necrosis. Foals dying soon after birth will have nonspecific changes of acute septicemia. Polyarthritis is found in those dying at a later stage.

The main lesions in cases of *S. tyhimurium* infection in horses includes fibrinonecrotic or necrohemorrhagic enteritis, mainly in the large intestine (large colon and cecum) [90]. Other lesions reported are enterocolitis and meningoencephalomyelitis in foals [65].

#### *4.4.2.4 Diagnosis, treatment and control*

*Salmonella* a*bortusequi* can be isolated from the placenta, uterine discharges, aborted foals, and the joints of foals with polyarthritis. Serological diagnosis is possible since a high titer of anti- *Salmonella* agglutinins develop in mares about 2 weeks after abortion. *S. tyhimurium* may be isolated from fecal material but this is not reliable due to intermittent shedding of the bacteria.

Antimicrobial drugs recommended in the treatment of salmonellosis should also be effective against *S*. a*bortusequi* infection. However, antibiotics use may promote latent carrier state following recovery [92]. Isolation of infected mares and disposal of aborted material should be practiced to avoid spread of the infection and infected stallions should not be used for breeding. In areas where the disease is common, vaccination is also used as a control measure. The widespread use of vaccines is credited with the almost complete eradication of the disease in developed countries.

Antibiotic treatment of equine *S. tyhi*murium infection is not recommended, especially in cases of uncomplicated diarrhea, due to the risk of worsening symptoms, as a result of disruption of the normal intestinal microflora by the antibiotics. Instead, supportive treatment is recommended if necessary. A major problem in control is the long-term survival of the organism in the environment. Manure should be disposed of frequently and animals with diarrhea should be isolated. Rodents and wild birds control is advisable.

#### **5.** *Salmonella* **from rodents and wild birds**

"Typhimurium" comes from "murine" Latin for mouse, a rodent of the subfamily Murinae. Rodents and wild birds are the main reservoir for *Salmonella* in the environment. They carry the organism in their intestines, mostly asymptomatically,

#### *Salmonellosis in Food and Companion Animals and Its Public Health Importance DOI: http://dx.doi.org/10.5772/intechopen.109324*

which they transmit to food animals in the farm environment [16, 93]. Rodents are attracted to feed and shelter around livestock farms, particularly in intensive production systems [94, 95]. Apart from *Salmonella*, rodents are carriers of a variety of other diseases such as leptospirosis and plague [96]. The source for infection is rodents' droppings which contaminate feed and water but mice and rats can also carry diseasecausing organisms on their feet and hair [97]. Chicken can also get infection from eating dead mice and rats [94].

Salmonellosis in wild birds can be asymptomatic or it can be a fatal disease [98]. Asymptomatic birds may disseminate *Salmonella* to susceptible individuals through fecal shedding, shared environments, and via direct contact [99]. Birds can also transmit *Salmonella* to food animals with their feet [100]. Wild birds are particularly hazardous since they can transmit infections over long distances through migration. The most frequent serovar isolated from wild birds is *S. typhimurium* [101].

#### **6. Public health importance of salmonellosis in animals**

Non-typhoidal salmonellosis is one of the four major global causes of diarrheal diseases in human, alongside *E. coli*, Cholera and *Campylobacter* [1]. It is also one of the most important bacterial zoonotic diseases, estimated to cause, 155,000 deaths yearly worldwide [5]. Non-typhoidal salmonellosis in humans is therefore not only are major public health concerns worldwide but great negative economic impacts due to the cost of surveillance, investigation, treatment and prevention of illness [6]. It is transmitted from animals by the fecal-oral route in several ways:


#### **6.1 Transmission from poultry**

Poultry meat and eggs are the most common vehicles of salmonellosis to humans [7, 8] and *S. enteritidis* is one of the most commonly identified serovars in association with human infection [103]. Contamination of poultry products can occur at multiple points in the production chain. This includes during rearing, live birds transportation, slaughter, dressing and packaging [104]. During slaughter, fecal contamination of carcasses can occur from gut contents. In retail outlets, including butcheries and supermarkets, poultry meat can get contaminated or cross contaminate other products [105–107]. Leaking poultry packages can contaminate ready-to-eat foodstuffs in supermarket refrigerators and in the kitchen, poultry meat can cross-contaminate other foodstuffs during meal preparation [108], particularly, foodstuffs that are eaten raw such as fruits and salads. Eggs are important sources of *Salmonella* for humans. Eggs become contaminated either by fecal contamination of the eggshell or through transovarian transmission from infected hens [109, 110], and this can lead to human

disease after consumption of the contaminated eggs. Another potential source of food contamination is poultry manure which can contaminate vegetables in the field [111].

#### **6.2 Transmission from cattle, goats and sheep**

Milk and dairy products are the second most important source of *Salmonella* infections for humans. Salmonellosis from dairy products is usually related to consumption of raw or inadequately pasteurized milk although *Salmonella* may contaminate dairy products after the pasteurization process. Milk may be contaminated by cow fecal material or manure during milking. The pathogen is shed in the feces of cows and can be present in or on the udders of cows and contaminate their milk. Unpasteurised milk and products made from it such as ice-cream, cheese, milk powder and infant formulae have been associated with *Salmonella* outbreaks [112, 113]. A variety of *Salmonella* serotypes have been isolated from these products. S*. dublin*, which is highly adapted to cattle as the primary host, has been associated with systemic form of salmonellosis in humans [52].

Goat meat, mutton, beef and beef products are recognized as important sources of human salmonellosis [114–116] Infections in most cases are associated with the consumption of raw meet, contaminated cooked meat or as a result of inadequate cooking. Organs and carcasses become contaminated with intestinal contents during slaughter and this is considered one of the important sources of infection [11]. Untreated manure can also contaminate vegetables at production stage [1, 117].

#### **6.3 Transmission from pigs**

Pork is ranked as the third most common source of human salmonellosis and *Salmonella* is the most common zoonotic pathogen affecting swine associated to human gastroenteritis [118, 119]. Many *Salmonella* serotypes are present in pigs, but the most commonly associated with foodborne illness in human is *Salmonella typhimurium*. One serotype, *S*. *choleraesuis* is adapted to swine as the primary host but also causes severe systemic illness in man [120], although it is not commonly isolated from pork. The most common cause of infection is eating improperly prepared or stored pork products that are contaminated with *Salmonella.*

#### **6.4 Transmission from companion animals**

Close contact between dogs and cats and their owners or those working with dogs can also be a potential source of *Salmonella* infections for humans [121, 122]. Organisms shed in the animal's feces can contaminate human food or hands. *Salmonella* shedding by dogs and cats has been incriminated in infections in humans living in the same household with the shedding pet, with children accounting for a high proportion of cases. Other persons that are particularly vulnerable are the aged and the immuno-compromised. Transmission of *Salmonella* from horses to humans in contact has also been documented [123].

#### **7. Conclusion**

Microorganism will always be with us [124], and in absence of effective control, salmonellosis in animals will continue to be a major economic and public health concern for several reasons:


Control of salmonellosis must therefore be addressed from these perspectives. Salmonellosis in farms is spread by contact between animals, from the environment and from reservoirs, particularly rodents. Since the primary infection with *Salmonella* occurs at the farm level, on-farm control of *Salmonella* is critical in reducing transmission during production, thereby minimizing contamination of meet during slaughter and processing and therefore reducing food safety risks [125]. Design and implementation of innovative biosafety practices are needed. Although cleaning and disinfection are the main hygiene practices in livestock production, they are less effective in the presence of rodents. A central part of hygiene practice should therefore include rodent control. This should include design of farm structures so as to eliminate rodent breeding sites and to prevent entry of the pests into animal houses. It has been shown that even the smallest population of rodents on farms presents a hazard [94]. Innovative, safe and efficient methods of rodents control in farm structures, including use of natural predators such as barn owls, are need.

Vaccination is the most cost-effective method for prevention and control of animal diseases and the most widely used tool in veterinary medicine. It can play an important role in prevention of salmonellosis in food animals Although vaccines against *Salmonella* in various animal species are in use worldwide, their efficacy is limited probably due to the diversity and complexity of pathogenesis of *Salmonella* infections. There is need for research into more efficacious vaccines against *Salmonella.*

The close contact between companion animals and people constitutes a risk for transmission of salmonellosis particularly for children, the aged and the immunocompromised. Studies are required to determine the extent of human salmonellosis attributable to companion animals and to identify risk factors for transmission. Sensitization of animal owners, caretakers and animal and human medical practitioners on risks associated with companion animals is important.

The wide host range of NTS implies that the risk of infection for any host is high. Measures to prevent disease in animals and humans must therefore be directed at all *Salmonella* serovars. Surveillance systems designed to map the spread and identify sources of infection, particularly in humans will be of great value in control of infections.

*Salmonella* is a complex genus that has evolved intricate virulence and antimicrobial resistance mechanisms and uncontrolled and indiscriminate use of antibiotics has increased the isolation frequency of *Salmonella* serovars resistant to one or more antibiotics globally [6]. Non-therapeutic use of antibiotics in farms is a threat

to human and animal health since majority of the human infections are acquired through the consumption of contaminated foods of animal origin. It has been demonstrated that sub-therapeutic use of antibiotics in animals may even trigger the spread *Salmonella* infection throughout a herd [125]. Whereas the global movement toward barn of antibiotics use in animals is encouraging, one of the major causes of uncontrolled antibiotic use is the commercialization of manufacture, distribution and retail of antibiotics. A significant misuse of antibiotics in humans is therefore likely to continue in absence of stringent regulation supported by surveillance data. Ongoing research on methods of blocking development of antibiotic resistance in bacteria by preventing mutation, is encouraging.

### **Author details**

Joseph K.N. Kuria Faculty of Veterinary Medicine, Department of Veterinary Pathology and Microbiology, University of Nairobi, Kenya

\*Address all correspondence to: jknkuria@uonbi.ac.ke

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Salmonellosis in Food and Companion Animals and Its Public Health Importance DOI: http://dx.doi.org/10.5772/intechopen.109324*

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#### **Chapter 3**

## *Salmonella enterica* Transmission and Antimicrobial Resistance Dynamics across One-Health Sector

*Leonard I. Uzairue and Olufunke B. Shittu*

#### **Abstract**

From human infection to animal production and the environment, *Salmonella* enterica has become a global-threat. The pathogen's dynamics have been determined by its transfer from sector to sector. Antibiotic-resistant bacteria can survive and proliferate in antibiotics. Misuse of antibiotics has made certain *S. enterica* resistant. The One-Health sector has antibiotic-resistant *Salmonella* (an approach that recognizes that human health is closely connected to the health of animals and the shared environment). According to certain studies, most animal and environmental *S. enterica* have virulence genes needed for human infections. *S. enterica* antibiotic resistance patterns have varied over the decades, resulting in pan-drug-resistant-strains. Plasmid-mediated fluoroquinolone resistance genes are found in One-Health *Salmonella* species. The *S. enterica* subspecies Typhi has been found to be extensively drug-resistant (XDR) in some areas. Cephalosporinresistant *S. enterica* subspecies Typhi is a severe problem that underscores the need for Vi-conjugat-vaccines. New diagnostics for resistant-*Salmonella* in food, animal, environment, and human sectors are needed to control the spread of these deadly infections. Also, hygiene is essential as reduced transmissions have been recorded in developed countries due to improved hygienic practices. This chapter aims to discuss the transmission and antimicrobial resistance dynamics of *S. enterica* across the One-Health sector.

**Keywords:** *Salmonella*, transmission, one-health, resistance, detection of *Salmonella*

#### **1. Introduction**

Antibiotic-resistant *Salmonella* are pathogens that antibiotics cannot control or kill. They may survive and even increase in the presence of an antibiotic*. Salmonella* is the causative agent of salmonellosis, an intestinal illness affecting humans and animals [1]. Salmonellosis is a fairly prevalent disease that is transmitted around the world. *Salmonella* is a leading agent in the development of acute and chronic diarrhea. Some species have been linked to systemic infection and sepsis that led to the deaths of various animals and humans. Salmonellosis is significant in the One-Health strategy and is consequently of major relevance to public health [2]. Recently, resistant

*Salmonella* has been found in humans, animals, and the environment (One-Health sector) [3]. One-Health sector is a concept used to recognize the interrelation of human health, animal health, and the shared environment and how diseases and pathogens move across the three sectors. *Salmonella* infection has been shown to produce severe systemic illness, which is responsible for large economic losses to the commercial chicken sector due to morbidity, mortality, and decreased egg production [4, 5] as reported by the Food and Agriculture Organization (FAO). The transmission has been a subject of argument by several writers [6, 7].

The selection pressure induced by antimicrobials *Salmonella* is a driving factor behind the emergence and spread of resistant bacteria, including *Salmonella enterica* pathogens, which were genetically encoded, transmitted by successive offspring, and in some instances could be transferred horizontally to distantly related bacteria [4]. Also, employing antimicrobials in food animal husbandry has increased the emergence of resistant *S. enterica* from food-producing animals [8]. Antibiotic-resistant *Salmonella* infections have grown in recent decades, making treatment more challenging. Because antimicrobial resistance is passed from one generation of bacteria through vertical transmission, resistant bacteria, in this case *S. enterica*, keep striving. Antibiotic-resistant *Salmonella* has risen for numerous causes. *Salmonella* infections require new antibacterial classes [9]. Some scientists hypothesize that *Salmonella* antimicrobial resistance is linked to *invA* expression and other mechanisms by which Gram-negative bacteria develop resistance [10, 11]. Some *S. enterica* subspecies Typhi strains with reduced ciprofloxacin sensitivity have emerged in the Indian subcontinent, southern Asia, and sub-Saharan Africa, leading to treatment failure [9, 12, 13]. *S. enterica* subspecies Typhi has resistant to first-line antibiotics such as chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole [13, 14]. Ceftriaxone, cefotaxime, and cefixime are also used to treat enteric fever, including nalidixic- and fluoroquinolone-resistant forms [10, 11].

Continuous abuse regarding the overuse of fluoroquinolone and certain cephalosporins in the management of *Salmonella* infection is underscored by the lack of effective antimicrobial stewardship programs, which has impacted antimicrobial resistance issues. Horizontal transfer of resistance genes via genetic elements like plasmids, transposons, and integrons has also impacted antimicrobial resistance issues, rendering those previously susceptible to becoming non-susceptible. Thus, the observed resistance or reduced susceptibility of *Salmonellae* to fluoroquinolones and some cephalosporins could result from genetic modification due to gene transfer. The resistant genes in *Salmonella* are embedded in the *Salmonella* pathogenic islands (SPIs). Studies have identified several SPIs, particularly the presence of mobile genetic elements (MGEs), which caused the rapid spread of resistant genes due to the high transmissible MCEs from one bacterium to the other [15, 16]. *S. enterica* are highly associated with multiple MGEs; these MGEs are in the SPIs, which are the center of virulence of *S. enterica* [17]. This book chapter aims to discuss the transmission dynamic of antibiotic-resistant *S. enterica* across humans, environments, and animals. The chapter discusses the genus *Salmonella*; the host adaptability of *Salmonella*; the virulence determinants of *Salmonella*; the transmission of antibiotic-resistant *S. enterica* in humans, animals, and the environment; and the detection of *S. enterica* and its antimicrobial resistance.

#### **2. The genus** *Salmonella*

Dr. Daniel Salmon, a veterinary bacteriologist who worked for the United States Department of Agriculture (USDA), was honored by having his name bestowed

Salmonella enterica *Transmission and Antimicrobial Resistance Dynamics across One-Health… DOI: http://dx.doi.org/10.5772/intechopen.109229*

on the genus *Salmonella* [18]. *Salmonella* is non-sporulating short Gram-negative bacilli [19], and most move with the help of peritrichous flagella. However, some serotypes of *Salmonella*, such as *Salmonella pullorum* and *Salmonella gallinarum*, are not motile [20]. They may be aerobic or facultative anaerobic, and the optimal temperature range for growth is between 5 and 45 degrees Celsius. At a temperature of 37 degrees Celsius, growth is most optimal. The optimum pH for reproduction is 7; however, *Salmonella* may live in environments with pH values ranging from 4 to 9 [21]. They grow in culture media designed for *enterobacteriaceae* and in blood agar. In addition, they grow in specialized media such as *Salmonella*-Shigella Agar and some ChromoAgar. Their colonies range from 2 to 4 millimeters in diameter and have smooth and round edges. They are slightly raised in a medium that contains carbon and nitrogen [22, 23]. When preserved in variable media such as peptone broth, colonies have the potential to maintain their viability for a significant amount of time [23, 24]. *Salmonella* strains have the biochemical capacity to catabolize nutrients, D-glucose, and other carbohydrates, except for lactose and sucrose, resulting in the generation of acid and gas [25]. *Salmonella* can utilize citrate as their only source of carbon, reduce nitrate to nitrite, and have the potential to produce hydrogen sulfide [19]. They are catalase positive and oxidase negative. They neither ferment malonate nor hydrolyze urea, and they do not produce indole. The bacterium has a coating of mucus around it, which helps to protect it from being digested by phagocytes, and it also has a fringe of fimbria placed around its outer surface, which helps it adhere to cells [19, 25, 26].

*Salmonella* is a member of the family of bacteria known as *Enterobacteriaceae,* which, along with other major pathogens in this group, are frequently implicated in causing illness in the small intestine. However, once the bacteria establish a foothold in the small intestine, they can move throughout the body and cause full-blown systemic disease [25]. *S. enterica* and *Salmonella bongori* are the two taxonomic species that make up the genus *Salmonella*. There are six subspecies of *S. enterica*: *S. enterica* subspecies *enterica*, *S. enterica* subspecies salamae (II), *S. enterica* subspecies arizonae (IIIa), *S. enterica* subspecies diarizonae (IIIb), *S. enterica* subspecies *houtenae* (IV), and *S. enterica* subspecies indica (VI) [25]. *S. bongori* and most of the subspecies of *S. enterica* populate the environments of cold-blooded animals, and in certain instances, *S. enterica* may cause sickness in these animals [27]. However, *S. enterica* subspecies *enterica* is the most biomedically significant subspecies. This is because these subspecies' serovars have a particularly important clinical importance in both veterinary and human disorders. Based on the structures of their flagellar (H) antigens and lipopolysaccharide (LPS) (O) antigens, *S. enterica* subspecies *enterica* may be further subdivided into approximately 2500 different serovars [25, 28].

#### **2.1 Host adaptability of** *S. enterica*

Despite their genetic connection, *Salmonella* strains may be distinguished from one another by their virulence, host adaptability, and host specificity [24, 25]. There is a wealth of epidemiologic information about *S. enterica* subspecies serovar host specificity. Certain serovars have a preference for certain hosts but are not exclusive to those hosts. Serovars Typhi, Paratyphi A, Gallinarum, and Pullorum are only transmitted from one host to another. Serovar Typhi is responsible for causing typhoid fever in humans [13, 29], while serovar Paratyphi A causes paratyphoid in humans [30]. The serovar Pullorum is responsible for causing disease in poultry, known as systemic pullorum disease [31–33], which is associated with high mortality and intestinal inflammation [34]. In contrast, serovar Gallinarum is responsible for causing severe systemic fowl [35, 36].

Both *Salmonella typhimurium* and *Salmonella enteritidis* can infect a wide variety of animal hosts. Interestingly, they are responsible for transmitting distinct illnesses in various animal species [3, 33–35]. In humans, the serovar Dublin is sometimes known to induce septicemia and gastrointestinal illness [32, 33]. The serovar Typhimurium and, less often, Enteritidis may cause enterocolitis in calves, which can lead to death from dehydration [37]. The serovars Enteritidis and Typhimurium that cause systemic illness and diarrhea in freshly born chicks are carried by older hens, who are asymptomatic carriers [4, 38]. Serovars Enteritidis and Typhimurium induce a localized, self-limiting form of enterocolitis in humans with healthy immune systems. However, immunocompromised people are more likely to develop a systemic form of the illness [29, 39]. In mouse strains that are sensitive to the illness, serovars Enteritidis and Typhimurium may induce a systemic fever similar to typhoid, although, in other investigations, they do not cause diarrhea [36, 40, 41].

#### **2.2 Virulence determinants of** *Salmonella*

*Salmonella* displays a wide range of virulence factors that make the bacterium harmful [9, 42]. Polymorphic surface carbohydrates, an abundance of fimbrial adhesins, phase-variable flagella, and well-structured invasion and survival mechanisms in host macrophages and other cells are likely examples of these traits [43, 44]. Nearly 200 genes on the chromosomes of *Salmonella* are crucial for the pathogenicity of the bacterium [36]. These genes are located in the five SPI-1 to SPI-5 chromosomal pathogenicity islands. A genetic component called a pathogenicity island may also be found on the chromosome [45]. This part of the chromosome exists as a separate and distinct entity from the rest of it. All pathogenicity islands have a few traits, including the inability to be identified in closely related, nonpathogenic reference species or strains and the frequent encapsulation of substantial areas of DNA (10–200 kB), containing genes that typically impart virulence to bacteria [46–51].

In most cases, they are also connected to elements like inverted repeats, transposases, and integrases [52]. Two of the type III secretion systems that allow *Salmonella* species to colonize new environments more easily are encoded by SPI-1 and SPI-2 [53]. *Salmonella* SPI-2 is only found in *Salmonella* species and is conserved across all of these species [43]. Even though SPI-2 is the sole gene unique to *Salmonella*, SPI-2 is necessary to establish bacterial invasion and internalization. At the same time, SPI-1 is necessary for developing systemic infection and intracellular replication [54–57]. The SPI-1 protein is necessary for bacterial invasion and internalization [58]. Therefore, an SPI1 gene that is both present and functioning is required for *Salmonella* species to be able to cause sickness [58]. On centisome 63 of *Salmonella* pathogenicity island-1, a 40 kb region carries a significant portion of the genes required for intestinal penetration and invasion of host cells [58]. The *Salmonella* pathogenicity island-1 includes this region [59]. Environmental isolates of *Salmonella* that had naturally occurring deletions in the SPI-1 region were unable to enter mammalian cells, according to research by Ginocchio and associates [60]. *Salmonella* isolates have the potential to colonize and infiltrate intestinal epithelial cells as well as transfer pathogenic effector proteins from the bacteria into the cytosol of the host cell due to the presence of at least 37 genes in the SPI-1. *Salmonella* isolates may also transfer harmful effector proteins into the host cell's cytoplasm. Numerous parts of the type III secretion systems (T3SSs) [61], as well as their regulators and

Salmonella enterica *Transmission and Antimicrobial Resistance Dynamics across One-Health… DOI: http://dx.doi.org/10.5772/intechopen.109229*

secreted effectors, are encoded by these genes [62]. SPI-1 is included inside the *Salmonella* pathogenicity island 1. After invaders seize control of host cells, the SPI-2 genes express themselves. These genes, which are required for intracellular life, are only present in *Salmonella* for survival within epithelial cells and macrophages [63]. Mutants' pathogenicity was much diminished because they could not colonize the infected individuals' spleens and lacked SPI-2 genes [58, 63]. The effector proteins *sipA*, *sipB*, *sipC*, *sifA*, *hilA*, *hilC*, and *hilD*, as well as *invA*, *spiC*, and *invF*, are among those secreted [61]. These chromosomal clusters of virulence genes can only be found in *Salmonella* and are unique to those species.

#### **2.3 Transmission of antibiotic-resistant** *S. enterica* **in humans, animals, and the environment**

Antibiotic resistance mechanisms in *S. enterica* include resistance to aminoglycosides (e.g., alleles of *aacC*, *aadA*, *aadB*, *ant, aphA*, and *StrAB*), B-lactams (e.g., *blaCMY-2*, *TEM-1*, and *PSE-1*), chloramphenicol (e.g., *floR*, *cmlA*, and *StrAB*), and other antibiotics [64]. In some strains of *Salmonella*, multidrug resistance mechanisms were shown to be associated with integrons or mobile genetic elements (MGEs) such as *IncA/C* plasmids [65, 66]. *Salmonella* that is resistant to antibiotics may be transmitted from animals raised for food to people; in this case, there will be similarities in the resistant patterns and genes present [67]. *Salmonella* strains that are resistant to antibiotics have been found in humans, and some of these strains have antibiotic-resistant components that are the same as those found in *Salmonella* isolated from food animals [5, 68, 69]. This suggests that these strains may have come from the same source, which is an evidence of cross-transmission.

Humans are the principal reservoir for *Salmonella* serovars Typhi and other humanspecific serovars. In contrast, other animal species are the key reservoirs for nontyphoidal *Salmonella* (NTS), which has been linked to human illnesses and infections in other animal species [13]. *Salmonella* may be found in the feces of practically every animal species; as a result, the zoonotic transmission of *Salmonella* is not restricted to animals raised for human consumption alone [70, 71]. Foods produced from poultry are the primary cause of *Salmonella* infections in humans, namely, in eggs, egg products, and chicken meat. Veterinarians and public health officials have identified the shedding of *Salmonella* as a source of infections for dog handlers, dog owners, and the communities in which they live [18, 22]. This suggests that pets, and particularly dogs in close contact with humans, may be responsible for the transmission of *Salmonella*. Infected dogs may continue to be carriers of the disease and feces shedders, making them a source of *Salmonella* for humans and other animals. Although these sources are not often responsible for big outbreaks, they may be responsible for isolated occurrences [70], which is why contact with ill cattle is a systematic way for farm workers to be exposed to diseases. The Centers for Disease Control and Prevention (CDC) reported several outbreaks of multidrug-resistant *S. typhimurium* infection associated with veterinary facilities. In areas with poor sanitation and contaminated water, fecal–oral transmission from person to person is the route for enteric or typhoid fever [71]. *Salmonella typhi* is only known to be carried by humans, not any other animals. *S. enterica* serovars, which have a wide host range, are common in the populations of warm-blooded animals that contribute to the human food supply.

Bacterial transmission typically occurs through the consumption of raw or undercooked food products [63], with poultry being one of the most important reservoirs of *Salmonella* species [13, 37]. *Salmonella* strains of many different serotypes have

been identified from their natural environments and food sources around the globe [29, 72]. According to Fazl and colleagues' research [45], hens are the primary vector for the vertical spread of *Salmonella*, which occurs via the ingestion of chicken eggs. *Salmonella* spreads quickly from breeding flocks to broiler and commercial egg-laying flocks. *Salmonella* spreads horizontally between birds through the fecal–oral pathway. The bacteria persist in the environment and have been isolated from poultry litter and dust [73]. The CDC reported in August 2018 about an outbreak of *Salmonella* Infantis from chicken products, which had also been reported previously [71, 74, 75].

Animal diseases are often brought on by ingesting contaminated food or water. To infiltrate the intestinal epithelium and colonize the mesenteric lymph nodes and other internal organs in the case of a systemic infection. *Salmonella* bacteria must withstand the challenging circumstances of the digestive tract [67]. Both humans and animals may get *Salmonella* infections when exposed. The ability of *Salmonella* to link with host cells and trigger its internalization has been studied [13]. These are essential for *Salmonella* to survive in the host environment and enter non-phagocytic cells. Animal waste commonly allows *Salmonella* to enter agricultural environments [76]. Plants and surface water used for irrigation or as a diluent for pesticides or fertilizers may be directly contaminated by animal feces [77]. There has been an increase in recent years in the number of reports that show a link between foodborne disease and the eating of fresh produce contaminated with *Salmonella* [78]. *Salmonella* can adapt to various environmental conditions, including those with a low pH or high temperature, allowing it to survive outside the host organism. *Salmonella* may adhere to plant surfaces and attach before actively infecting a variety of plant interiors. *Salmonella* that originates in plants retains its virulence when infecting animals [79]. Plants may thus act as a secondary host for *Salmonella* infections and contribute to spreading the bacteria to animals and humans.

#### **2.4 Antimicrobial resistance of** *S. enterica* **from humans, animals, and the environment**

The mechanisms of antibiotic resistance fall into three categories: (1) inactivation of the antimicrobial, (2) efflux or changes in permeability or transport of the resistance pathogen, or (3) modification or replacement of the antimicrobial target [80, 81]. Resistance is genetically encoded and may result from mutations in endogenous genes, horizontal gene transfer via plasmids, or horizontal acquisition of alien resistance genes [81, 82]. Both horizontally acquired genes and point mutations may contribute to resistance encoding. Promoter or operator point mutations might be the root cause of overexpression of endogenous genes like the *AmpC-*lactamase gene or the mar locus [83]. Some antimicrobial target genes, like the gyrase gene, are susceptible to point mutations that may turn them into resistant targets. Exogenous resistance genes encoded on plasmids, integrons, phage, and transposons can be horizontally propagated via the processes of transformation, conjugation, and transduction [84]. This includes genes that code for enzymes that render the antimicrobial inactive, such as lactamases that cleave the four-membered ring in lactams; efflux systems, such as *tet (A)*; altered versions of the enzymes the antimicrobial is intended to inhibit, such as *dfrA*; or enzymes that alter the antimicrobial target, such as ribosomal RNA methylase [85–87].

Additionally, by researching the mechanisms of resistance, one may discover the genetic link between animal and human resistance [88, 89]. Suppose the antibiotic resistances seen in human bacterial isolates are closely related to those seen in animal Salmonella enterica *Transmission and Antimicrobial Resistance Dynamics across One-Health… DOI: http://dx.doi.org/10.5772/intechopen.109229*

isolates. In that case, it may be possible to identify animal sources of resistant bacteria in human infections that can be targeted to reduce human disease [76, 90, 91]. This can be done by determining if the resistances seen in human bacterial isolates are similar to those seen in animal isolates [92]. This is possible due to the diversity of genetic factors contributing to antibiotic resistance.

Antibiotic resistance among *Salmonella* strains is increasing, which is a major cause for worry in protecting public health worldwide [93]. At the beginning of the 1960s, it was revealed that *Salmonella* had first developed resistance to a single antibiotic [88]. Since then, more *Salmonella* strains resistant to one or more antimicrobial medications have been isolated in various countries, including developed nations [94]. This trend has been seen in several countries. Traditional antibiotic therapies for *Salmonella* infections include penicillin, chloramphenicol, and trimethoprim-sulfamethoxazole, which are just a few available options. These treatments are believed to be the earliest lines of defense against *Salmonella*. *Salmonella* strains resistant to many antibiotics are referred to as multidrug-resistant *Salmonella*. The MDR phenotypic characteristic was extensively dispersed throughout *S. enterica* over an extended time, particularly in *S. typhi* and, to a lesser degree, in *Salmonella paratyphi* [68, 95]. Asia and Africa are two continents with a substantial incidence of *S. enterica* strains with the MDR feature [96]. During a surveillance investigation carried out in several nations in Asia and Africa, a significant number of *S. enterica* MDR isolates were identified [97]. The research particularly pointed to Pakistan, India, Nepal, and Vietnam, where extensive drug-resistant *Salmonella* was discovered. Because of the widespread use of fluoroquinolones and extended-spectrum cephalosporins, which were used to treat MDR *S. enterica*, there has been an increase in *S. enterica* that are capable of producing beta-lactamases [17, 98]. This is because traditional antibiotics have become less effective due to the widespread use of drugs like fluoroquinolones and extended-spectrum cephalosporins. Despite this, some evidence suggests that an increasing number of typhoid *Salmonella* are acquiring resistance to fluoroquinolones. Isolates from various nations have been reported to be resistant to nalidixic acid, which suggests that they have diminished sensitivity to ciprofloxacin and other fluoroquinolones [93, 99]. The rise in resistant non-typhoidal *Salmonella* (NTS), particularly in animals used in food production, has made controlling the spread of *S. enterica* strains resistant to antibiotics more difficult. This is true in particular for animals that are reared for their meat. According to the investigation findings, the MDR phenotype was present in most NTS clinical isolates. Public health officers have voiced their worries over treating ailment and prevention due to this phenomenon.

The use of antibiotics in animal feed to stimulate the development of food animals and in veterinary care to treat bacterial illnesses in those animals is the primary factor that contributes to the establishment of *Salmonella* with antimicrobial resistance [67]. This is a high risk of zoonotic illness due to the transfer of MDR *Salmonella* strains from animals to people via the intake of food or water contaminated with the feces of the animals, through direct contact or by the consumption of diseased food animals. Additionally, multidrug-resistant *Salmonella* strains were discovered in the aquatic habitat of some exotic pet animals, such as tortoises and turtles [100]. This might lead to an increased risk of zoonotic infections in people via direct contact with these animals [74, 76, 90].

#### **2.5 Detection of** *S. enterica* **and its antimicrobial resistance**

Several methods are used to detect *Salmonella* and its resistance patterns and genes. There are conventional or culture, serological, and molecular techniques,

including polymerase chain reaction and sequencing. Confirming infection with *Salmonella* is required before treatment [101–103]. A diagnosis may be confirmed by culture and isolation. *Salmonella* isolates may be differentiated in various ways, and the number of *Salmonella* species is continually expanding [104, 105]. *Salmonella* is typed using complex procedures in addition to serotyping based on antigens to track individual isolates and explain pathogenicity [58, 86]. It is essential from an epidemiological standpoint to distinguish *Salmonella* isolates because definitive typing may assist in locating the source of an epidemic and tracking changes in antibiotic resistance [105].

Pre-enrichment, selective enrichment and culturing, isolation, biochemical characterization, serological characterization, and final identification are the steps that are included in the standard approach for detecting *Salmonella* [106, 107]. This method needs at least 4 days to get a negative result, and it takes between six and 7 days to identify and confirm positive samples.

Antibiotic sensitivity testing (AST) measured by inhibition zones is determined by the disk diffusion method, and it is proportional to the susceptibility of the bacteria to the antibiotic on the disk [108]. This depends on the antibiotic disk's potency and infusing ability. It may not take much modification to use disk diffusion for testing antimicrobial disks [109]. It is used to screen many isolates to choose a subset for further testing, such as MIC determinations. Antimicrobial types must include interpretation criteria (susceptible, intermediate, and resistant) based on standards, guidelines, and quality control reference organisms. Approaches to AST are selected based on their user-friendliness, versatility, adaptability to automated or semiautomated systems, cost-effectiveness, dependability, and accuracy. Conventional *Salmonella* serotyping is most typically done [110].

*S. enterica* serotyping is conducted on a global scale, which has enabled improvements in the monitoring and detection outbreaks on a global scale. The O (somatic), H (flagellar), and Vi (capsular) antigens from the lipopolysaccharide (LPS) layer of the cell wall are used for serotyping *Salmonella* isolates [111, 112]. *Salmonella* may spontaneously and reversibly change between these two stages of flagellar antigen synthesis, each containing a unique set of H antigens. This phenomenon is known as diphasic flagellar antigen production. In the first phase, also known as the specific phase, the various antigens are denoted by lowercase letters; in the second phase, also known as the group phase, the antigens found initially are given numbers [113, 114]. Traditional serotyping, which uses the autoagglutination method, has some important drawbacks. One is the inability to identify non-typeable Vi antigens and strains [75, 115]. It takes a lot of time, a lot of different chemicals, and a lot of experienced laboratory workers to do this [4].

Latex agglutination, enzyme immunoassay (EIA), and enzyme-linked immunosorbent assay (ELISA) are three examples of the types of immunological tests that have been developed to identify and confirm *Salmonella* [29, 39, 98] quickly.

DNA hybridization and PCR are two more methods that may be used to identify *S. enterica* [116, 117]. Amplification and analysis of strain variation may be accomplished by using gene-specific primers in PCR testing. It can improve the detection and characterization of pathogenic bacteria by targeting species-specific DNA regions and specific pathogenicity traits, such as genes that code for toxins, virulence factors, or major antigens [84, 118, 119]. This makes it possible for it to improve the detection of pathogenic bacteria. Other *Salmonella* strain typing methods include utilizing antibiotic resistance genes as epidemiological markers using multilocus sequence typing [5, 7, 111].

Salmonella enterica *Transmission and Antimicrobial Resistance Dynamics across One-Health… DOI: http://dx.doi.org/10.5772/intechopen.109229*

These methods examine the DNA sequences of a series of housekeeping, ribosomal, and virulent genes, and therefore making isolates distinction based on the molecular analysis. This uses short sequence repeat motifs as a target to type isolates.

The polymerase chain reaction (PCR) and real-time PCR are being investigated as potential diagnostic tools for enteric fever [112]. In theory, nucleic acid amplification tests (NAATs) might amplify DNA from bacteria that are either dead or incapable of being cultured, hence rectifying low culture positives caused by antibiotic pretreatment [113]. According to research [114], the test sensitivity limitations for a PCR technique are the same as those for a culture approach. Culture and PCR are combined in some methodologies. The adoption of NAATs in developing countries is expected to be hampered because of the high cost and lack of laboratory infrastructure [120]. The effectiveness of NAATs for the diagnosis of enteric fever has been the subject of some research. The flagellin genes (*fliC-d* for *S. Typhi* and *fliC-a* for *S. Paratyphi* A) are most often targeted by PCR [121, 122]. In a study of blood PCR testing for enteric fever, researchers found that although all tests were 100 percent specific, their sensitivities differed [83, 123]. The sensitivity is considerable in most studies to be more than 90% [20] in persons with positive blood culture, but it is lower (3–13%) in those without clinical symptoms [14].

Additionally, PCR tests focusing on fliC have been applied to urine, and the findings have been favorable [124]. The primary benefit of PCR over other identification methods, like culture and conventional methods, is that it produces findings much more quickly [124]. PCR requires specialist laboratory equipment, which might be difficult in regions where typhoid fever is prevalent [14, 39]. Whole-genome sequencing (WGS) has revolutionized how antimicrobial resistance is studied [125, 126]. It has enabled the detection of resistance genes even before they are expressed and has also played a very important role in epidemiological studies of antimicrobial resistance *Salmonella* [127]. These techniques have helped develop newer diagnostics and are being explored in vaccine candidate development for several *Salmonella* species other than Typhi [128].

Both phenotypic and genotypic methods detect resistance genes or resistance mechanisms in bacteria, specifically in *S. enterica* [17]. The phenotypic method explores this bacteria's expression of certain traits to detect a resistance mechanism [122]. For example, the resistance by *Salmonella* to third-generation cephalosporin is an indication of possible possession of extended-spectrum beta-lactamases (ESBLs) [129]. *Salmonella* resistance to Meropenem is an indication to been carbapenemaseproducing [130]. Genotypic techniques are employed to confirm the phenotypic detection of an antibiotic-resistance mechanism [131]. Some of these genotypic methods for antibiotic resistance genes include whole genome sequencing and polymerase chain reaction application. Whole genome sequencing is expensive, and as such, it is not routinely used to detect resistance genes. Polymerase chain reaction (PCR) is the method of choice for laboratories with the capacity [132]. Quantitative and conventional PCR is used. Quantitative PCR uses specific primers and probes to detect the resistance or virulence gene of interest. Conventional PCR, which is mostly available and more cost-effective, uses specific primers, and the products are visualized in a gel documentation system after amplification [133].

#### **2.6 Prevention and control of resistant antibiotics and virulent** *S. enterica* **across one-health sectors**

The prevention of *S. enterica* infection involves proper co-ordination of preventive measures across the humans and their activities, that is, agriculture, animal rearing for food, the use of animals as companions or pets, and management of environment sector to detect and eliminate any threat [100, 134] of *Salmonella* [100, 134]. The different levels of prevention of *S. enterica* pathogens include: prevention of *S. enterica* pathogens from farms to human via food or through the contaminated environment via poor waste management [120, 135]. The proper management of farms to eliminate pathogenic bacteria from the animal facilities is one effective way of managing *Salmonella* outbreaks. Also, prosper handling of food processing and animal products contributed to reduced outbreaks [10, 136]. Some keys *Salmonella* infection outbreaks were associated with transmission from processed food animals like chickens, pork, and other meat products. The use of animal wastes as fertilizers has also been associated with *S. enterica* outbreaks [76]. Full implementation of good hygiene practices across all sectors, including house hygiene practices and deployment of WASH in all sectors, will eliminate *S. enterica* from the food chain and all possible transmission avenues.

By consuming contaminated food or drink, enteric fever is most often spread from person to person [134]. In the past, enteric fever was common in Western Europe and the United States [75]. Despite this, pasteurization of milk and other dairy products, the removal of human feces in the food-manufacturing process, and good food and water cleanliness have all contributed to a considerable decline in the prevalence of *Salmonella* infection [137]. There was a decrease in the number of *Salmonella* illnesses reported in Latin America simultaneously as sanitary techniques were implemented [137]. Giving access to clean water and food, maintaining proper sanitation, and administering typhoid vaccinations are the best ways to avoid enteric fever. Making and ensuring that water meant for human consumption is safe is the main goal of eliminating possible vectors for the transmission of typhoid *Salmonella* and non-typhoid *Salmonella* (NTS). This important objective has been easily achieved in wealthy nations like Europe and the United States but not in developing or underdeveloped countries [138]. In addition to water, a variety of foods may include *Salmonella* species. However, they are often found in poultry, eggs, and dairy products [139]. The adoption of proper food handling and cooking practices has been proposed to prevent bacterial contamination of food. Due to its efficacy in reducing the risk of food contamination, food irradiation has attracted considerable interest and support in several countries. Several public health agencies, including the WHO and the CDC, have approved irradiating food. Still, due to the risk posed by radioactivity, it is only partially used in certain parts of Europe and the United States [103]. Vaccination is one of the best methods to protect against enteric fever [140]. The inactive parenteral and oral live attenuated vaccines are the two immunization types that may presently be utilized to prevent enteric fever. However, these authorized immunizations are exclusively used in infants and are ineffective at preventing diseases by *S. enterica* subspecies Paratyphi and NTS. Limiting the erroneous use of antibiotics in food animals and the feed they ingest is one approach that is good for NTS.

Hazard analysis and critical control points (HACCP) are advantageous since it is an efficient strategy for minimizing risk and maximizing product security [141]. The HACCP is employed at various stages of the One-Health sector. This is important to avoid cross-contamination or transfer of pathogenic *S. enterica* from the environment to processed food, humans, and vice versa. One way of implementing HACCP in the animal sector is to ensure that *Salmonella* pathogens are not released into the environment [142]. And everyone involved in the processing steps of food are tested for *S. enterica* to avoid shedding in processed food [143]. Implementing HACCP has several advantages, including eliminating prejudice and providing a framework for prioritizing choices. HACCP helps ensure that only those with the necessary knowledge, skills, Salmonella enterica *Transmission and Antimicrobial Resistance Dynamics across One-Health… DOI: http://dx.doi.org/10.5772/intechopen.109229*

and experience are responsible for food safety. With HACCP in place, there is concrete proof of your food safety management, which will be useful in court in the case of any litigation. After the initial investment in implementing HACCP, the system may be very cost-efficient. As a result of HACCP, food manufacturers may fulfill their mandated duty to create healthy, wholesome fares in compliance with applicable regulations [144]. Applying HACCP's procedures and guidelines almost guarantees better results every time. This is mostly attributable to people's heightened sensitivity to risks and the fact that they come from all walks of the operation. The HACCP principles and the requisite support mechanisms for a robust food safety program form the basis of the Global Food Safety Initiative (GFSI) by ensuring the absence of *Salmonella* pathogens in processed food, poultry farms, and the food chain [142, 145].

#### **3. Conclusion**

The distribution of *Salmonella* subspecies capable of causing infections has been found in humans, animals, and environments. *Salmonella* genes such as *sipA*, *sipB*, *sipC*, *sifA*, *hilA*, *hilC*, *hilD*, as well as *invA, spiC*, and *invF* have been linked to epidemiologic of virulent *S. enterica*. Some of the *Salmonella* from all these sources tested positive for the beta-lactamase *TEM* enzyme. To detect *S. enterica*, *invA* has been found valuable in detecting *S. enterica* contamination in food products and the environment. The *invA* gene has been made into devices and diagnostics for diagnosing infections in the bloodstream, environmental contamination, and waterprocessing plants. These factors have also been utilized in investigating outbreaks and infection tracing and tracking, especially in food-processing industries. The detection of this genes even without viable growth of *S. enterica* has helped control and contain outbreaks. Genes for resistance to fluoroquinolones mediated by plasmids has also been widely found in *Salmonella* species across the One-Health sector. According to research findings, most *Salmonella*e are obtained from animals, and the environment carries the virulence genes essential to induce infections in humans. Extensively drug-resistant (XDR) *Salmonella typhi* is now a serious problem in some countries, multidrug-resistant (MDR) has grown in prevalence, and *S. enterica* has evolved resistance to an increasing number of antibiotic classes. Extensively drug-resistant (XDR) *S. typhi*, so designated due to its exhibited resistance to the recommended drugs for typhoid fever, including third-generation cephalosporin, has become a serious issue that highlights the urgency in deploying the Vi-conjugate vaccines.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Leonard I. Uzairue1,2\* and Olufunke B. Shittu1

1 Department of Microbiology, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria

2 Department of Medical Laboratory Sciences, Federal University Oye Ekiti, Ekiti State, Nigeria

\*Address all correspondence to: leonard.uzairue@fuoye.edu.ng; uzairue.leonard@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Salmonella enterica *Transmission and Antimicrobial Resistance Dynamics across One-Health… DOI: http://dx.doi.org/10.5772/intechopen.109229*

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#### **Chapter 4**

## Involvement of CRISPR-Cas Systems in *Salmonella* Immune Response, Genome Editing, and Pathogen Typing in Diagnosis and Surveillance

*Ruimin Gao and Jasmine Rae Frost*

#### **Abstract**

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated *cas* genes (CRISPR-Cas) provide acquired immunity in prokaryotes and protect microbial cells against infection by foreign organisms. CRISPR regions are found in bacterial genomes including *Salmonella* which is one of the primary causes of bacterial foodborne illness worldwide. The CRISPR array is composed of a succession duplicate sequences (repeats) which are separated by similar sized variable sequences (spacers). This chapter will first focus on the CRISPR-Cas involved in *Salmonella* immune response. With the emergence of whole genome sequencing (WGS) in recent years, more *Salmonella* genome sequences are available, and various genomic tools for CRISPR arrays identification have been developed. Second, through the analysis of 115 *Salmonella* isolates with complete genome sequences, significant diversity of spacer profiles in CRISPR arrays. Finally, some applications of CRISPR-Cas systems in *Salmonella* are illustrated, which mainly includes genome editing, CRISPR closely relating to antimicrobial resistance (AMR), CRISPR typing and subtyping as improved laboratory diagnostic tools. In summary, this chapter provides a brief review of the CRISPR-Cas system in *Salmonella*, which enhances the current knowledge of *Salmonella* genomics, and hold promise for developing new diagnostics methods in improving laboratory diagnosis and surveillance endeavors in food safety.

**Keywords:** *Salmonella*, CRISPR-Cas, WGS, CRISPR typing, immune response, genome editing, AMR, surveillance

#### **1. Introduction**

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated *cas* genes (CRISPR-Cas) are a family of DNA sequences, as an adaptive immune system, which protects microbial cells against infection by foreign nucleotide elements including plasmids and phages [1]. CRISPR are widespread in prokaryotes,

and found in approximately 50% bacterial genomes including *Salmonella* belonging to the family of *Enterobacteriaceae* [2], which is a primary cause of bacterial foodborne illness worldwide.

Through a computational analysis of CRISPR-Cas systems, a classification system was determined based on the gene or genes encoding the effector molecules [3]. This analysis determined that CRISPR-Cas systems can fall into two classes; class 1 systems (types I, III and IV) which use a complex of multiple proteins to degrade foreign nucleic acids, and class 2 systems (types II, V, and VI) which only require a single large Cas protein (**Figure 1**) [4]. The six types of systems are further divided into 36 subtypes (**Figure 1**) [3, 5–10]. Fully functional CRISPR-Cas systems consist of CRISPR array, Cas proteins and AT-rich leader sequences. The phylogeny of CRISPR and associated *cas* genes could reflect different evolutionary histories [11, 12]. The CRISPR array is composed of a succession of highly conserved direct repeats (DR) of 24–47 bp separated by similar sized unique sequences (spacers) [13]. The *cas* genes are usually located near the CRISPR locus but can also be located elsewhere on the genomes. Cas proteins perform many functions, for instance, destroying foreign genomes, mediating foreign sequences acquisition into CRISPR array, and assisting the mature CRISPR RNAs (crRNAs) production [14–17]. CRISPR-Cas systems adapt by acquiring new spacers at the leader proximal end [1]. The units (DR+spacer) may target an invading piece of DNA and result in its degradation via a proposed mechanism similar to RNA interference. The distribution of CRISPR-Cas loci in different *Enterobacteriaceae* families showed that *Eshcerichia* and *Salmonella* are the top two genera containing type I-E subtypes [2]. CRISPR are reported in two pairs of loci in *Escherichia*, and one single pair in *Salmonella*, with each pair loci showing similar repeat sequences and putative linkage to common *cas* genes [11].

#### **Figure 1.**

*General classification of CRISPR-Cas systems. Two classes—indicated by the red and blue colouring—cover six types. A total of 36 subtypes are further divided under the six types with the known signature proteins listed.*

#### *Involvement of CRISPR-Cas Systems in* Salmonella *Immune Response, Genome Editing... DOI: http://dx.doi.org/10.5772/intechopen.109712*

It has been shown that CRISPR spacer DNA sequences are molecular signatures used for pathogen subtyping [18] and CRISPR content correlates with the pathogenic potential of bacteria as CRISPR-Cas limits acquisition of foreign nucleotides in bacteria [19]. It has been demonstrated that there is a negative correlation between the amount of CRISPR units and pathogenicity traits, i.e. a higher number of virulence factors with lower CRIPSR repeats [19]. Based on the specific spacers, CRISPR array based quantitative PCR can be used to detect the presence of different serotypes in both *Escherichia* and *Salmonella*, with prominent sensitivity and specificity [20, 21]. Hence, the CRISPRs represent a promising genetic marker and diagnostic tool for comparative and evolutionary analysis of closely related bacterial strains [2]. Furthermore, the recognition of CRISPR-Cas9 by the Nobel Prize in Chemistry in 2020 [22] reflected its outstanding impact in genome editing field. Originated from bacteria, the CRISPR technology has already been broadly applied to fungus, yeast, insects, plants, and animals [23]. This technology has also demonstrated to functionally inactivate genes in human cell lines and cells. For instance, in 2019, CRISPR was used to treat a 34-year-old patient with sickle cell disease which is a blood genetic disorder disease [24]; and in 2020, CIRSPR-modified virus was injected into a patient's eye to treat Leber congenital amaurosis [25]. In this chapter, we will mainly focus on a foodborne pathogen *Salmonella* which is a primary cause of bacterial gastroenteritis worldwide.

*Salmonella enterica* is a tremendously diverse species comprising six subspecies and over 2600 serovars. *S. enterica* subsp. *enterica* accounts for the majority of clinical cases of salmonellosis and the majority of serovar diversity. Serovars of Enteritidis, Typhimurium, and Heidelberg are three main ones causing human illness. This book chapter will mainly focus on CRISPR-Cas in the immune response system of *Salmonella*, as well as its application in genome editing, pathogen typing, diagnosis and surveillance.

#### **2. CRISPR-Cas systems of** *Salmonella* **in comparison with other bacteria**

#### **2.1 Immune function of CRISPR-Cas systems**

In prokaryotes, bacterial CRISPR-Cas systems are unique in providing adaptive immunity against exogenous nucleotides elements, by utilizing sequence-specific RNA-guided nucleases to defend against bacteriophage infection. Bacteriophages (phages) are viruses infecting bacteria, and they are the most abundant life forms on earth. Generally, three major steps are involved in the CRISPR immune functional process: (1) new spacer acquisition—Cas proteins integrate short sequences of invading DNA into the CRISPR array; (2) CRISPR expression—CRISPR arrays are transcribed and processed to produce small crRNA; (3) CRISPR interference—crRNA along with Cas nucleases target the spacer sequence, resulting in degradation of the invader's nucleotides (DNA or RNA) [26, 27].

#### **2.2 Characterization of CRISPR loci and** *cas* **genes**

Like many other bacteria, the *Salmonella* genome also contains CRISPR loci. It usually contains two CRISPR loci, CRISPR1 and CRISPR2, both found on the minus strand. These two loci are separated by ~16 kb and share the same consensus direct repeat sequence (29 nt). Each CRISPR loci is fairly conserved in *Salmonella*, with the CRISPR1 locus being more conserved than CRISPR2. There are eight *cas* genes—*cas3*, *cse1*, *cse2*, *cas7*, *cas5*, *cas6*, *cas1* and *cas2*, which are located upstream of CRISPR1. Among these eight genes, *cas1* and *cas2* are universal and both are present in all CRISPR-Cas systems; *cas3* is a signature gene in the type I system; the remaining *cas* genes are type I-E dependent [28]. Furthermore, the *cse2*, *cas5*, *cas6e*, *cas1*, *cas2* and *cas3* genes are crucial for the expression of a master porin regulator named OmpR which is a two-component system regulator inducing the synthesis of OmpC, MmpF, and OmpS2 portins [29]. By taking advantages of whole genome sequencing (WGS), in 2014, researchers have demonstrated two distinct *cas* gene profiles and a high diversity of length for both CRISPR arrays, among the analysis of 64 *Salmonella* serovars [30].

#### **2.3 CRISPR and anti-CRISPR**

To combat bacterial CRISPR-Cas system, numerous phages are well known to produce proteins which can block the function of CRISPR-Cas systems, i.e. anti-CRISPR function [31]. For class 1 CRISPR system, the first discovered phage-encoded anti-CRISPR protein (Acr) was from type I-F and I-E CRISPR-Cas systems in *Pseudomonas aeruginosa*; these anti-proteins encode distinct, small proteins (50–150 aa) with different sequences and structures [32, 33]. Furthermore, these anti-CRISPRs are produced from prophages (phage sequences that have integrated into bacterial genomes) and inactivate the host (bacterial) CRISPR-Cas systems [32]. For class 2 CRISPR, four unique type II CRISPR-Cas9 inhibitor proteins have been discovered from the prophage sequences integrated into another foodborne pathogen *Listeria monocytogenes* genomes, which have type II-A CRISPR-Cas systems and their spacers have been identified by various virulent, temperate phages [34, 35]. Given more than half of *L. monocytogenes* strains with cas9 contain at least one prophage-encoded inhibitor, this suggests the possibility of widespread CRIPSR-Cas9 inactivation. Two of the discovered inhibitors in *L. monocytogenes* are also able to block the *Streptococcus pyogenes* Cas9 when analyzed in *Escherichia coli* and human cells. Similarly, in *Streptococcus thermophiles*, AcrIIA6 acts as an allosteric inhibitor and induced Cas9 dimerization [36]. Thus, the concept of natural Cas9-specific "anti-CRISPRs" presents a tool which can be used to regulate the genome engineering activities of CRISPR-Cas9 [31]. Similar to *L. monocytogenes*, in different *Salmonella* serovars, they all contain various types of prophage sequences [37]. To date there is no reported anti-CRISPR proteins in *Salmonella*, though this could change as more studies are carried out.

#### **3. Identification and characterization of CRISPR arrays**

Next generation sequencing (NGS) and especially WGS has emerged in recent years and has made it possible to sequence bacterial genomes within hours, a notable accomplishment that is revolutionizing the field of microbiology [38]. With the advent of microbial WGS, new light is shed on the nature of pathogens, for instance CRISPRs, and our understanding of the biology of *Salmonella* is steadily increasing as *Salmonella* genomes are generated at a rapid rate and are deposited in public database such as National Center for Biotechnology Information (NCBI). Based on the availability of genome sequences, various genomic and bioinformatics tools have been developed for identifying the potential CRISPR arrays in *Salmonella* genomes.

*Involvement of CRISPR-Cas Systems in* Salmonella *Immune Response, Genome Editing... DOI: http://dx.doi.org/10.5772/intechopen.109712*

#### **3.1** *In silico* **genomics based CRISPRs identification tools**

An example of how the field of CRISPRs has evolved can be seen in the work done with *in silico* analysis. *In silico* identification and analyses of CRISPRs started in 1995 [39], and several CRISPR software tools have been developed since then. In April 2007, the first specific stand-alone developed tool was CRISPRFinder, which was a web tool in identifying CRISPRs [40]. CRISPRFinder was able to define DRs and extract spacers; to get the flanking sequences and to determine the leader sequences; and then BLAST the identified spacers to check if the identified DR was present in other genomes [40]. Two months later in June 2007, in order to dissect and understand CRISPR structure and flanking sequences evolution, the same group created a public database named CRISPRdb, for which CRISPRFinder was used to analyze all the available prokaryotic genomes [41]. In the same month June 2007, a tool named CRISPR Recognition Tool (CRT) for automatic detection of the CRISPR arrays was also released [42]. CRT was demonstrated to be very reliable, with significant improvements in regards to performance in measures of precision, recall and quality, as compared to the previous existed detection tools Patscan and Pilercr [42]. In April 2008, a website based tool CRISPRcompar was created to compare CRISPRs that present a useful genetic marker for comparative analysis of closely related bacterial strains; this facilitated the development of CRISPR based pathogen typing processes [43]. More CRISPR-Cas related online tools can be found in CRISPR-Cas++, which are available at https://crisprcas.i2bc.paris-saclay.fr/. In 2018, an improved CRISPRs identification tool CRISPRCasViewer was released, which can predict CRISPR orientation, possess the latest classification scheme, and facilitate expert validation based on a rating system [44]. Alternatively, the public available "standalone" Unix/ Linux version of CRISPRCasViewer can also be downloaded and installed in highperformance computing cluster bioinformatics infrastructures (https://github.com/ dcouvin/CRISPRCasViewer). Thus, with the availability of all the genomic tools, the CRISPRs and *cas* genes present in each *Salmonella* isolate are able to be detected. Subsequently, comparative and evolutionary analysis can also be carried out to identify potential genetic markers, which will be useful for diagnosis and surveillance tools development in food safety.

Typically, the identified CRISPR arrays are represented by colored shapes based on nucleotide sequence identity. For facilitating and easy handling this process, Dion et al. [45] have introduced CRISPRStudio which is a user-friendly command-line tool to accelerate CRISPR analysis and standardize CRISPR array figures preparation. CRISPRStudio is able to compare nucleotide spacer sequences and then cluster them based on sequence similarity to assign a representative color; it also supports automatic sorting of CRISPR loci and highlighting shard spacers [45].

#### **3.2 CRISPR target**

In bacterial and archaeal adaptive immune systems, CRISPR-Cas targets specific protospacer nucleotide sequences in invading organisms, which requires nucleotide base pairing between processed crRNAs and target protospacer. Biswas et al. [46] have developed a flexible, interactive tool CRISPRTarget for the discovery of the target of crRNAs in diverse database. CRISPRTarget is available at http://crispr.otago. ac.nz/CRISPRTarget/crispr\_analysis.html, it can be used to discover targets from both genomic and metagenomics dataset in many pathogens, including the foodborne pathogen *Salmonella*.

#### **3.3 Conservation and diversity of** *Salmonella* **CRISPR arrays**

Similar to other genetic components, CRISPR sequences can be conserved throughout a pathogen family. Through genomic sequence analyses of four clinically relevant *Salmonella* serovars; Enteritidis, Typhimurium, Newport and Heidelberg, it was determined that both cas operons and leaders are conserved among these four serovars [28]. Furthermore, *Salmonella* seems to be lacking in spacer acquisition, and the majority of CRISPR allelic polymorphisms usually arise from deletion or duplication of direct repeat-spacer units [47–49].

With the development of NGS technology, more and more *Salmonella* isolates have complete genome sequences available. In order to eliminate the potential bias caused by incomplete genome sequences, a collection of 115 representative *Salmonella* isolates with complete genomes (size range 4,482,117–5,395,280 bp) were analyzed in this chapter (**Table 1**). Those selected isolates come from four different subspecies, with the subspecies *enterica* as the dominant one. For the isolates within these four



*Involvement of CRISPR-Cas Systems in* Salmonella *Immune Response, Genome Editing... DOI: http://dx.doi.org/10.5772/intechopen.109712*


*Involvement of CRISPR-Cas Systems in* Salmonella *Immune Response, Genome Editing... DOI: http://dx.doi.org/10.5772/intechopen.109712*


*\*These four Salmoenlla enterica isolates contain three CRISPR arrays, which is different from the common ones containing two.*

*\*\*This isolate has the longest CRISPR2 array.*

#### **Table 1.**

*Representative 115 Salmonella enterica isolates with complete genomes containing four known subspecies covering 90 serovars used for CRISPR arrays analysis in this study.*

subspecies, a total of 90 different *S. enterica* serovars were included in this analysis, the details of each isolate can be found in **Table 1**.

Briefly, all available *Salmonella* complete genomes were downloaded from the NCBI database using Bioinformatics Tools (bit) (https://github.com/AstrobioMike/ bit#bioinformatics-tools-bit) from GitHub and NCBI EDirect tools (https://astrobiomike.github.io/unix/ncbi\_eutils). By applying common NCBI BLAST keywords, the used commands for downloading those complete genomes were:

*"esearch -db assembly -query ' ("Salmonella"[Organism] OR Salmonella[All Fields]) AND (latest[filter] AND "complete genome"[filter] AND all[filter] NOT anomalous[filter])' | esummary | xtract -pattern DocumentSummary -element AssemblyAccession > Salmonella\_complete\_genome.txt";*

*"bit-dl-ncbi-assemblies -w Salmonella\_complete\_genome.txt -f fasta -j 12".*

Then a total of 115 representative genomes were manually selected and compiled by including as many serovars as possible.

To identify CRISPR arrays in those 115 representative *Salmonella* isolates, two main used software were CRISPRDetect\_2.2 (https://github.com/ambarishbiswas/ CRISPRDetect\_2.2) [50] and CRISPR\_Studio (https://github.com/moineaulab/ CRISPRStudio) [45]. Detailed procedures were described in the above two github links. Briefly, a specific python3 conda environment was created for this project, the used command for CRISPRDetect was: "*perl ../bin/CRISPRDetect\_2.2/CRISPRDetect. pl -f interested.fasta -o output\_file -array\_quality\_score\_cutoff 3 -T 0*". Subsequently, the CRISPRDetect produced "output\_file" containing detected CRISPR arrays was fed into and visualized using CRISPR\_Studio, and the used command was: "*python* 

#### **Figure 2.**

*Graphic representation of spacer profiles in three arrays of CRISPR1, CRISPR2 and CRIPSR3, detected from 115 Salmonella enterica isolates with complete genomes consisting of four known subspecies covering 90 serovars. The figure was created by CRISPRStudio. Each spacer is represented by a colored square and a geometric symbol. The earliest acquired spacer is shown on the right hand side and the newly acquired space is on the left hand side. Specifically, the four isolates containing CRISPR3 are: three S. enterica subsp. salamae isolates, and one S. enterica subsp. enterica serovar Gaminara, which are indicated as "\*" in the* **Table 1***. The identical CRISPR spacer profiles are grouped and indicated by red, blue, green, and orange dots.*

*CRISPR\_Studio\_1.0.py -i ../CRISPRDetect/output\_file*", with **Figure 2** presented in this chapter as final outputs.

Among the analyzed 115 *Salmonella* isolates, prominent diversity was observed in the detected CRIPSR array profiles. Unlike commonly reported knowledge that *Salmonella* usually contains two CRISPR loci [28], there were four isolates containing the 3rd loci, CRISPR3. Three of these isolates belonged to *S. enterica* subsp. *salamae* and the last one belonged to *S. enterica* subsp. *enterica* serovar Gaminara (**Table 1** and **Figure 2**). Additionally, there were five isolates that only contained CRISPR2 and 18 isolates that only had CRISPR1 (**Table 1** and **Figure 2**). Although prominent diversity was observed among isolates, respective identical CRISPR spacer profiles were observed for four groups. The first group (indicated by red dots) included a total of two serovars from the subspecies *enterica*, namely Nitra (CP019416) and Enteritidis (NC\_011294.1); these two serovars showed high similarities and are known to be difficult to distinguish in nature using different microbiological methods. In the second group (indicated by blue dots), one was *S. enterica* subsp. *enterica* serovar Dublin (CP001144.1), and the other one is unknown serovar from the same subspecies. The third group (indicated by green dots) consisted of three serovars of *S. enterica* subsp. *enterica*, namely India (CP022015.1), Panama (CP012346.1), and Koessen (CP019412.1). The last group (indicated by orange dots) has two serovars of

*Involvement of CRISPR-Cas Systems in* Salmonella *Immune Response, Genome Editing... DOI: http://dx.doi.org/10.5772/intechopen.109712*

Yovokome (CP019418.1) and Manhattan (CP022497.1) belonging to *S. enterica* subsp. *enterica* (**Figure 2**). The discovered CRIPSR arrays with certain similarities or dissimilarities might shed light on the phylogeny and evolutionary analysis of *Salmonella* isolates in the future.

#### **4. Major CRISPR applications of genome editing, AMR patterns and typing tools in** *Salmonella* **and other microorganisms**

CRISPR-Cas originates from bacteria and has also been broadly applied back in functionally studying bacteria. Here, CRISPR/Cas9 genome editing used in *Salmonella* host-pathogen interaction will be described, followed by the CRISPR-Cas diversity and its strong correlation with antimicrobial resistance (AMR) pattern studies will be introduced. The emerging application of CRISPR typing/subtyping will be explained. Finally, more advanced CRISPR-Cas related diagnosis and surveillance methods related to other microorganisms will also be demonstrated, as similar methods could be used as potential alternative methods for studying foodborne pathogens including *Salmonella*.

#### **4.1 CRISPR/Cas9 genome editing in** *Salmonella* **host-pathogen interaction studies**

The discovery of Cas9 has allowed for impressive advances in the field of genome editing. This protein can be utilized to modify the genome of interest, based on the segments in the CRISPR array. Cas9 endonuclease activity needs crRNAs to guarantee precise targeting, and an immediate downstream protospacer adjacent motif (PAM). With the aim of editing bacterial genomes, a vector encoding Cas9 and its guide RNAs, as well as recombination template containing required mutation are required [51]. For preventing the re-cleavage of Cas9 of the target genome, the spacer of PAM sequences will need to be modified [52, 53]. Using such approach, mutations have been introduced into the *sdiA* gene to study its effect on *S. enterica* pathogenesis. The introduced mutations affected *S. enterica* biofilm formation, cell adhesion and invasion [54]. CRISPR/Cas9 has also been used in generating macrophage knockout mice cell lines, which facilitates *S.* Typhimurium infection studies by determining the contribution of background contaminations in the phenotypes of primary macrophages from congenic mice [55]. It has also demonstrated that the CRISPR-Cas system is involved in the resistance to bile salts and biofilm formation in *S.* Typhi [29]. This demonstrated CRISPR/Cas9 genome editing based methods contribute significantly in carrying out functional studies of *Salmonella.*

#### **4.2 CRISPR/Cas diversity and its strong correlation with AMR pattern**

AMR is a global concern for human health and a World health organization global priority (https://www.who.int/news-room/fact-sheets/detail/antimicrobialresistance). WGS is replacing traditional phenotypic method such as disk diffusion method for routine testing of foodborne pathogens AMR. The tools of ResFinder [56, 57] or the Comprehensive Antibiotic Resistance Database (CARD) [58] detect the presence of AMR genes in an isolate by comparing its sequence against known genes cataloged in a reference database of known AMR determinants. Although

knowledge of the CRISPR-Cas systems has been applied in many research areas, there are not many studies in applying it to the analysis of antibiotic resistance in *Salmonella*. Recently, by using large-scale bioinformatics investigation of the 1059 isolates of *S.* Typhi CRISPR-Cas systems, 47 unique spacers and 15 unique DRs were identified, as well as unique conservation and clonality of the *S.* Typhi type I-E CRISPR-Cas system was observed [59]. The identified spacers and repeats showed specific patterns which demonstrated significant associations with AMR status, genotype, and demographic characteristics. This suggests they have the potential to be used as biomarkers to develop rapid and inexpensive diagnostics tests [59]. Similarly, on Chinese poultry farms, analysis of 75 *Salmonella isolates* consisting of 11 serovars, found that there were close correlations between CRISPR loci and AMRs, however, there was no close correlations between CRISPR loci and antibiotics [60].

#### **4.3 CRISPR typing and subtyping as improved laboratory diagnostic tool in**  *Salmonella*

Various molecular and phenotypic typing techniques have been developed to track bacterial origins, for instance, pulse-field gel electrophoresis (PFGE), phage typing, multi-locus sequence typing (MLST), multi-locus variable number tandem repeat (MLVA) and single nucleotide polymorphism (SNP) pipelines [61]. The above mentioned typing methods are limited in both speed and precision. In recent years, improved and innovative surveillance tools of CRISPR typing have been developed, which are used to gain knowledge in better understanding a variety of bacteria, such as *Salmonella* [47]. Serving as a complementary tool for the high-resolution core genome single nucleotide variant (cgSNV) method, CRISPR typing was useful for determining source attribution in foodborne *S.* Heidelberg outbreaks [62, 63]. CRISPR typing was also shown to facilitate further studies in understanding the virulence and global distribution of the *S.* Virchow serovar [64]. Furthermore, the combination of both MLVA and CRISPR (CRISPR-MLVA method) gave better genotyping results than using each one alone, when testing 171 *Salmonella* strains from nine serovars [48]. There are limitations to this method of typing, particularly in very closely related isolates. In these instances, it has been shown that using CRISPR typing in conjunction with a SNP analysis allows for better resolution, indicating the use of CRISPR typing still exhibits clear benefits [65]. A few CRISPR based typing tools are illustrated in details as below.

#### *4.3.1 Conventional CRISPR typing*

In conventional CRISPR typing (CCT), all spacer sequences in the two loci of CRISPR1 and CRISPR2 are extracted [48]. Then CRISPR1 and CRISPR2 spacer sequences profiles are analyzed and visualized using CRIPSRviz [66]. There are three main procedures: (1) CRISPR arrays are obtained by either directed wholegenome sequencing or PCR amplification of CRISPR loci using conservative sequences following by sequencing; (2) the identification and characterization of potential CRISPR arrays based on the previous sequencing results; (3) finally, clustering of analyzed isolates based on the absence or presence of analyzed CRISPR arrays.

*Involvement of CRISPR-Cas Systems in* Salmonella *Immune Response, Genome Editing... DOI: http://dx.doi.org/10.5772/intechopen.109712*

#### *4.3.2 CRISPR locus spacer pair typing*

CCT can be labor intensive to carry out. To increase the easy of typing, CRISPR locus spacer pair typing (CLSPT) was developed [67]. Instead of using all the obtained spacer sequences, only one spacer sequence in both CRISPR1 and CRISPR2 loci will be used for typing. This spacer sequence is the first one, found closest to the leader sequences. In this method, the first spacer sequence of the CRISPR1 leader sequences is combined with the first spacer sequence of the CRISPR2 leader sequences. Then these two spacer sequences were used as the total sequences for *Salmonella* strain typing.

#### *4.3.3 CRISPR locus three spacer sequences typing*

Usually, during the evolution of bacterial strains which contain CRISPR arrays, the first captured exogenous nucleotide sequence could display strain origins, and the spacers from the same serotype possess certain conservation. Thus, Li et al. have developed a *Salmonella* typing method, called CRISPR locus three spacer sequences typing (CLTSST) method, which could be used to distinguish different serotype clusters. They used three spacer sequences including the initial two spacer sequences (the first acquisitions or the ones with the furthest distance to the leader sequence) and latest spacer sequence close to the leader sequence are combined and used as the total analyzed sequences for strain typing [60].

#### *4.3.4 Conserved CRISPR arrays serving as quantitative PCR targets*

In addition to the sequences analyses of CRISPR loci typing in *Salmonella*, the conserved CRISPR arrays can also be used as targets for qPCR primers and probes design. It has been demonstrated that a *S.* Infantis-specific qPCR assay is able to detect the Infantis serovar from mixed cultures of *Salmonella* down to 0.1% of the population, and with the detection sensitivity of 10 colony forming units [21]. For the utility of this CRIPSR based qPCR molecular approach in improved surveillance system, two main parameters need to be met in regards to the CRISPR spacer sequences that are to be used for designing primers and probes: (1) the used spacers need to be specific for the tested serovar; (2) the selected spacers need to be conserved and present in all strains of that specific serovar.

#### *4.3.5 Other related applications of CRISPR-Cas*

It is well-known that efficient delivery of a CRISPR/Cas9 plasmid is critical for effective therapy in clinical settings. Other than a receipt of a plasmid carrying CRISPR/Cas, it has also been found that *Salmonella* can be used as a CRISPR/Cas9 plasmid carrier for *in vivo* therapy against virus-induced cancer [68]. It has been demonstrated that the usage of *Salmonella* in CRISPR system provides a simpler and more effective platform for *in vivo* therapy [68].

The CRISPR-Cas system can also be used for diagnostics by utilizing the properties of the proteins themselves. For example, the Cas9 protein has been used in detection assays to help increase the percentage of the genomic regions of interest that is present. During library preparation in NGS projects, a method known as Depletion

of Abundant Sequences by Hybridization uses recombinant Cas9 protein complexed with a library of guide RNAs to target and cleave unwanted DNA, leading to the increased yield of sequences of interest [69].

Additionally, the method Finding Low Abundance Sequences by Hybridization (FLASH) uses Cas9 and guide RNAs that allow sequences of interest to be cleaved into an ideal size for NGS sequencing, increasing the presence of reads that can be captured from the sequence of interest [70]. This can be used for example to detect antimicrobial resistant populations that may be present in low levels compared to the wild type. Recently, a modified Cas9 variant has been developed (SpCas9 named SpRY), that allows for the digestion of specific regions without the requirement of the PAM sequence [71].

Other diagnostic tools have also been developed with the CRISPR-Cas system. Although the most reported cas protein in *Salmonella* is related to Cas9, the other two most popular cas proteins related to diagnosis tests are *cas12a* and *cas13*. Different from Cas9 which cuts double stranded DNA relying on a precise location "T rich" PAM, both *cas12a* [72] and *cas13* [73] remain bound to the target and then cleave other DNA/RNA non-discriminately. This feature is recognized as collateral cleavage of trans-cleavage activity, which has been broadly applied in the development of various diagnostic technologies [74]. DNA endonuclease-targeted CRISPR trans reporter is a method developed using the Cas12a protein and its ability to degrade single-stranded DNA (ssDNA). Using this property, along with a ssDNA reporter, this system can be used to detect if specific pathogen types are present in a sample [72]. Another example can be seen from the global pandemic associated with betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For the detection of SARS-CoV-2 in clinical validations, specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), has been shown to be a promising alternative method to qPCR with regards to its visualization speed and experimental settings with limited resources [75–77]. These are just a few ways the CRISPR-Cas system can be alternatively used for diagnostic purposes. These diagnostic or detection methods could also be adapted and used as alternative methods for surveillance or typing in *Salmonella*.

#### **5. Conclusions**

The CRISPR-Cas system in *Salmonella* has been shown to be useful in differentiating between different strains. According to the WGS based genome analysis of 115 isolates, three *S. enterica* subsp. *salamae* isolates, and one *S. enterica* subsp. *enterica* serovar Gaminara possess three CRISPR loci, namely CRISPR1, CRISPR2 and CRISPR3, which differs from the commonly reported two CRIPR loci CRISPR1 and CRISPR2 in *Salmonella*. On the contrary, 18 isolates only had CRISPR1 and five isolates only had CRISPR2. With the emerging applications of CRISPR arrays in *Salmonella* genome editing, AMR studies, typing and subtyping in diagnosis and surveillance, a thorough investigation of the uses of CRISPR-Cas will facilitate better understanding its host-pathogen interaction, immune response and its usages in improving laboratory tests. Adapting the many advanced CRIPSR-based diagnostic tools such as SHERLOCK, and FLASH, will allow for faster detection and/or the ability for more detailed analyses to be carried out. This will allow for improved laboratory diagnosis and surveillance endeavors in food safety, as well as offer better tools for any future outbreak responses.

*Involvement of CRISPR-Cas Systems in* Salmonella *Immune Response, Genome Editing... DOI: http://dx.doi.org/10.5772/intechopen.109712*

#### **Acknowledgements**

This work was supported by the Public Health Agency of Canada and Canadian Food Inspection Agency.

#### **Conflict of interest**

The authors declares no conflict of interest.

### **Acronyms and abbreviations**


#### **Author details**

Ruimin Gao1,2\* and Jasmine Rae Frost1

1 National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada

2 Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency, Ottawa, Ontario, Canada

\*Address all correspondence to: ruimin.gao@phac-aspc.gc.ca

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Involvement of CRISPR-Cas Systems in* Salmonella *Immune Response, Genome Editing... DOI: http://dx.doi.org/10.5772/intechopen.109712*

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#### **Chapter 5**

## Perspective Chapter: Solar Disinfection – Managing Waterborne *Salmonella* Outbreaks in Resource-Poor Communities

*Cornelius Cano Ssemakalu*

#### **Abstract**

*Salmonella* outbreaks remain a significant problem in many resource-poor communities globally, especially in low and middle-income countries (LMICs). These communities cannot reliably access treated piped water, thus reverting to the use of environmental water for domestic and agricultural purposes. In most LMICs, the maintenance and expansion of the existing wastewater and water treatment infrastructure to meet the growing population are not considered. This results in regular wastewater and water treatment failures causing an increase in an assortment of waterborne pathogens, including *Salmonella*. Solving these problems would require the maintenance, expansion and construction of new wastewater and water treatment infrastructure. The implementation of such interventions would only occur over a long period. Unfortunately, time is not a luxury in communities experiencing the effects of such problems. However, highly disruptive household interventions such as solar disinfection (SODIS) could be implemented in communities experiencing endemic *Salmonella* outbreaks. SODIS has been shown to inactivate a variety of water-related pathogens. SODIS requires significantly less financial input to implement in comparison to other household-level interventions. Various studies have shown better health outcomes due to SODIS in communities that previously struggled with waterborne diseases, including *Salmonella*. The aim of this chapter is to share a perspective on the continued reliance on SODIS as for the control waterborne *Salmonella* in LMICs.

**Keywords:** SODIS, *Salmonella*, sanitation, hygiene, water treatment, disinfection, filtration, Coagulation, Flocculation, oxidation, water, Waterborne, LMIC

#### **1. Introduction**

The genus *Salmonella* consists of two species with over 2500 serovars. The serovars within the species *Salmonella enterica* are classified as either typhoidal or non-typhoidal. Although genetically similar these serovars elicit significantly different diseases. Typhoidal *Salmonella* serovars such as Typhi and Paratyphi A are human restricted and cause an invasive systemic typhoid fever that is life threating in both healthy and

immune compromised individuals [1]. Non typhoidal *Salmonella* (NTS) serovars such as Typhimurium and Enteritidis cause self-limiting gastroenteritis in either humans or animals. The gastroenteritis caused by NTS is often mild in healthy adults but severe in immune compromised individuals [2, 3].

*S. enterica* infections primarily those associated with serovars Typhimurium and Enteritidis [4, 5] remain a global burden especially in low and middle income countries in Africa and Asia affecting more than 93 million people and causing the deaths of over 1.2 million people globally [6, 7]. Most of these infections and deaths occurred in people living in resource-poor communities, especially those in Low- and Middle-Income Countries (LMICs) in Africa [4, 8]. This could be attributed to the high prevalence of malnutrition and immune compromising diseases such as malaria and AIDS. Infections due to *Salmonella* are not exclusive to LMICs. According to the Centre for Disease Control (CDC), more than 1 million people in the United States of America (USA) experience a *Salmonella* infection. This costs the USA more than \$ 3.7 billion US in medical costs [9]. Recently, the World Health Organisation (WHO) was alerted to a *Salmonella* outbreak associated with European food products for the European and Global markets [10]. The CDC estimates that 46% of foodborne diseases and 23% of deaths are linked to produce consumption [9]. In High-Income Countries (HIC), there is a more likelihood of acquiring a *Salmonella* infection through the consumption of food products as opposed to water [11, 12].

This chapter highlights the role played by water in the transmission of *Salmonella* especially in resource poor LMICs. Thereafter, an overview of how water and sanitation infrastructure is prioritised in Africa is provided. This is followed by an evaluation of water treatment approaches that could be used at a household level to reduce the burden of *Salmonella*. The chapter ends by providing reasons why SODIS is an ideal water treatment intervention at a household level.

#### **2. Water and** *Salmonella* **infections**

Environmental water resources play a critical role in food crop produce linked to *Salmonella* infections occurring in HICs [12, 13]. Environmental water bodies can harbour *Salmonella* for several months [13, 14]. This makes *Salmonella* a waterborne pathogen that could be introduced into a susceptible animal host when untreated environmental water is consumed or used for domestic and agricultural purposes. Previously, *Salmonella* infections were mainly associated with consuming contaminated animal products. However, in recent years, *Salmonella* outbreaks associated with consuming contaminated food crops such as fresh fruits, vegetables, spices, and nuts have increased [15, 16]. This is probably driven by the increased adoption of a vegetarian or vegan lifestyle [17]. The presence of *Salmonella* on food crops has been attributed to the microbiological quality of water used for irrigation [12, 16].

Water remains a key factor in the transmission of *S. enterica* in LMICs [14] and HICs [12, 13, 18]. Access to clean water is a fundamental human right. However, many resource-poor communities worldwide, especially LMICs, struggle to access clean water [14]. Currently, more than 2 billion people lack access to safely managed water, of which more than 700 million live without basic drinking water. Most of these live in Africa [19]. The current paradigm of *Salmonella* infection places poor sanitary habits and practices as critical contributors toward the reintroduction of *S. enterica* into the environmental water resources. Although an increase in global sanitation has been reported, more than 3.6 billion people lack access to well-managed sanitation, of

*Perspective Chapter: Solar Disinfection – Managing Waterborne* Salmonella *Outbreaks... DOI: http://dx.doi.org/10.5772/intechopen.108999*

which 1.7 billion still lack basic sanitation [19]. Therefore, people living in resourcepoor communities in LMICs contract *Salmonella* infections by consuming contaminated food and water [14]. But, if these communities had access to treated water, a reduction in waterborne *Salmonella* would occur as observed in HICs.

Furthermore, practicing proper sanitation and hygiene in tandem with the availability of treated water would reduce the prevalence of *S. enterica* in the environmental water resources. This would improve the microbiological quality of natural water resources for agricultural purposes. Providing resource-poor communities with clean water would require establishing effective sanitary and water treatment infrastructure.

#### **3. Investment in water and sanitation infrastructure with focus on Africa**

Sanitary and water treatment infrastructure availability is a major driver of economic development because it curbs health risks, enables education and other productive activities, and enhances the labour force's productivity [20, 21]. For instance, the lack of proper sanitation in South Asia results in financial and economic losses of up to 2 and 9 billion dollars, respectively, while adequate sanitation infrastructure in France enables tourism and sustains the jobs of more than 2000 people in the tourism sector [20].

The African continent consists of 53 member states with a combined population of more than 1.4 billion people [22]. Currently, the African continent has the highest population below the age of 15 [23]. By 2050 Africa will be home to more than half of the world's population, and 1.3 billion people will live in urbanised areas [24, 25]. About 56% of people living in urban areas in Africa have access to piped water compared to 67% in 2003, and just 11% can access a sewer connection [26]. This observation implies that the current infrastructure cannot support the increasing population and hence threatens social stability and may act as a driver of migration within and out of Africa [26]. Given the growing population, it is logical to prioritise the expansion of existing as well as the construction of new sanitary and water treatment infrastructure in Africa.

However, this is not reflected in the African infrastructural commitments. In 2017 the transport infrastructure sector received the highest commitment (\$ 34 billion, 41.7% of the total obligations) in comparison to the water infrastructure sector (\$ 13.2 billion, 16.2% of the total commitments) [27]. In the same year, the funding gap for transport infrastructure (8%) was lower than that of the water infrastructure (84%) sector [27]. Previous reports showed that the transport and water sectorial infrastructural commitments had increased by 30 and 8% between 2016 and 2017 [27]. Nevertheless, African states' water and sanitation infrastructure financing declined by 3% between 2016 and 2017 [27]. During that period, foreign aid commitments were made to finance water and sanitation infrastructural projects in many Low-Income Countries (LIC) in Africa. For instance, Italy committed \$ 69 million to a Mozambique water and sanitation project. China committed \$ 1.5 billion to construct the Gerbi Dam in Ethiopia to provide water to Addis Ababa [27].

Investment in sanitation and water treatment infrastructure should be prioritised because water remains a critical link between agriculture and energy. Therefore, robust sanitation and water treatment infrastructure provides and supports opportunities in the agriculture, manufacturing and energy sectors but to mention a few [21]. The African agriculture sector offers and supports the highest number of jobs compared to any other sector [17]. Therefore, there is a need to

assess and invest in agricultural water needs. The link between *Salmonella* infections and crop produce in HIC has been established [12]. This may make the export of African crop produce challenging based on the quality of water used for agricultural purposes. This justifies prioritising wastewater and sanitation infrastructure because of their positive impact on the quality of water used for agricultural purposes. Improving the quality of water used for agricultural purposes will enable the export of better-quality produce.

Investment in sanitation and water treatment infrastructure offers social and economic benefits. But why is it that African governments do not prioritise such critical infrastructure? Water infrastructure financing would require loans and hence a well-managed system of offering a paid water service [26]. Currently, the provision of paid water services remains a challenge. Thus, the servicing of water, wastewater, and sanitation infrastructure loans is associated with a high financial risk to the lender. Perhaps this is one of the reasons why social impact research and interventions have focused on point-of-use systems to ensure the availability of treated water for consumption at a household level. The only challenge with this approach is that the sanitation aspect may not receive the attention it deserves.

#### **4. Water treatment at the household level**

The chronic lack of sanitation and water treatment infrastructure in resource-poor communities, especially those in LMICs, makes the people living in these communities vulnerable to *Salmonella* infections. Point of Use (POU) water treatment systems have been suggested as a short to mid-term intervention to protect human health. The currently available POU water treatment systems work based on coagulationflocculation, filtration, and disinfection [28].

#### **4.1 Coagulation: Flocculation**

Coagulation – flocculation-based systems offer a reliable, low, energy means of reducing the particulate matter in water, leaving it clearer than before. This approach would require using coagulants such as aluminium sulphate, lime, polyelectrolyte and iron salts (ferric chloride and ferric sulphate). Also, biopolymers, especially natural gums and bio-flocculants, have been investigated for their ability to serve as effective coagulants and flocculants [29]. However, the coagulation-flocculation treatments reduce turbidity the offer the added advantage of reducing the microbial burden of turbid water [29, 30]. Flocculation follows the addition of a coagulant. The flocculation process is often facilitated by gentle mixing to enable the formation of flocs. Mixing increases the collisions and interaction between the flocs and coagulant, thus increasing the size of the flocs resulting in them settling at the bottom by sedimentation. Coagulation – flocculation can be accessed at the POU using either the PUR or Poly Glu sachet manufactured by Procter & Gamble Co or Poly Glu International Co, respectively [28]. Both PUR and Poly are accessible worldwide. Nonetheless, extra measures are needed to ensure that these approaches are supplemented with either a disinfection method (PUR sachet) or proper hygiene handling of treated water to avoid recontamination (Poly Glu sachet) [28]. It should be noted that coagulationflocculation-based POU solutions are often single-use and hence may be costly for some communities in the long run.

*Perspective Chapter: Solar Disinfection – Managing Waterborne* Salmonella *Outbreaks... DOI: http://dx.doi.org/10.5772/intechopen.108999*

#### **4.2 Filtration**

Filtration systems offer a simple means of removing colloids, suspended solids, and microorganisms from water. Size exclusion is the basic principle behind the filtration process. As such, a properly configured filtration system can remove not only *Salmonella* or related bacteria from water but also viruses, toxins, and chemicals. This depends on the size of the pores on the filter membrane or biosand configuration. Membrane filtration systems used at the POU would ideally require one to consider the quality of the influent water and an external driving force relative to the membrane's pore size. These filters require maintenance because, with time, a foulant layer forms on them. This makes membrane-based filtration systems an option for communities that may have access to piped water that is not sufficiently treated. But inaccessible to those people in communities with no access to piped water.

Furthermore, membrane filtration is costly to maintain and would require technical skills to do so [28]. Sand-based filtration systems offer a more viable solution for those living in communities without access to treated piped water. Sand filters are easy to manufacture because the required raw materials are readily available. More than 500,000 people worldwide use biosand filters to meet their needs for potable water [28]. Biosand filters have been shown to reduce the turbidity of water. They have also been reported to reduce microbial contaminants but not to the level that meets the WHO water guidelines. Although the material to make biosand filters is easily accessible, the manufacturing process requires some technical skills. For instance, a correct balance between the flow rate and retention time is needed during manufacturing. These two variables have an inversely proportional relationship that influences the effectiveness of the removal of microbial contaminants such as *Salmonella* [28]. Also, the filter's depth needs to be considered to remove viruses.

#### **4.3 Disinfection**

Disinfection is an approach to enable the availability of safe water that relies on the destruction of the water contaminating microorganisms. Currently, two methods have been used to achieve disinfection: nanotechnology and Solar Disinfection (SODIS). Nanotechnology-based POU water treatment approach uses either titanium dioxide (TiO2) or silver (Ag) nanoparticles for disinfection. The TiO2 method requires a source of UV–vis light which facilitates the generation of hydroxyl radicals and hydrogen peroxide [31] that oxidise the organics, thus inactivating the microorganisms. TiO2 is not depleted during this process, so the reaction continues. TiO2 has been used to reduce biofouling on membranes used for water treatment and enhance the SODIS process [32]. TiO2 has been used to develop a POU product, the Solarbag produced by Paralytics.

Ag nanoparticles are toxic to microorganisms. They bring about the death of microorganisms by either permeabilising the cell membranes or bioaccumulating causing irreversible damage to the DNA [33]. Ag nanoparticles have been used to coat ceramic [34] and polyurethane filters [35] to improve microbial log reduction. Currently, Ag nanoparticles are used in POU products such as Tata Swach and Folia filters to disinfect water. Although TiO2 and Ag nanoparticles improve water microbiological quality, the long-term effects of these nanoparticles are not understood. At elevated levels, these nanoparticles are harmful to aquatic life [36, 37].

Furthermore, a high concentration of Ag nanoparticles has been shown to reduce mammalian cell vitality and mitochondrial function and cause cell membrane leakages [38]. This means that the use of nanoparticles to improve the quality of the water needs to be supplemented with proper disposal of damaged, unusable systems and accumulated waste. Besides the use of nanoparticles, natural sunlight could be used to sterilise the water before its consumption.

#### **5. Solar disinfection of water**

SODIS of water is an affordable and easy method of treating microbiologically contaminated water before its consumption. As such this section will focus on SODIS as opposed to the other approaches. During SODIS, microbiologically contaminated water in a transparent clear vessel is exposed to natural sunlight for approximately 6 hours on a sunny day with clear skies and on two rainy days overcast days. A detailed manual on the application of SODIS in the field has been developed and is accessible via the Swiss Federal Institute of Aquatic Science and Technology [39].

Effective bacterial inactivation is judged by the inability of the microorganisms to form colonies after SODIS treatment [40]. Downes and Blunt [41] were the first to present empirical evidence of the bactericidal effect of sunlight; however, its use to sanitise water can be traced as far back as 2000 BC. Presently, Downes and Blunts [41] observations on the bactericidal effect of natural sunshine are continuously confirmed by various research teams with consequent successful application in many countries globally. Studies by Acra et al. [42] and Conroy et al. [43] hypothesise that the observed bactericidal effect following sunshine exposure is due to the ultraviolet component of sunlight.

The harmful effects on the microbial population during SODIS are due to solar ultraviolet radiation (SUVR), which comprises wavelengths shorter than 400 nm. Natural sunlight reaching the earth's surface contains 6% of SUVR [44]. The UV wavelength is subdivided into three wavebands categorised as UVA (400–320 nm), UVB (320–280 nm) and UVC (280–100 nm) [45]. Of these three wavebands, UVA is the most abundant (95%) form of SUVR reaching the earth's surface, followed by UVB; UVC rarely reaches the earth's surface because the stratospheric ozone layer absorbs it. The amount of Solar Ultra-Violet Radiation (SUVR) reaching a given location on the earth's surface is influenced by geographical, meteorological and temporal factors such as the latitude, elevation, cloud cover, atmospheric conditions and ground reflection [45, 46]. The closer the exposure point is to the equator, the higher the levels of SUVR [46, 47]. However, due to the sun's elevation in the sky, 75% of the daily SUVR is received between 0900 and 1500H, irrespective of the exposure point [45].

#### **5.1 Factors influencing solar disinfection of water**

Although SODIS may seem like an ideal means of sanitising microbiologically contaminated water, it is influenced by several factors. One key factor to consider when using SODIS is the weather conditions. Cloud cover affects the amount of SUVR received on the earth's surface. It has been observed and reported that the amount of SUVR reaching the earth's surface is less when the sky is cloudy than in a cloudless sky. However, the enhancement of SODIS technology by incorporating compound parabolic concentrators could provide efficient inactivation within a short time during cloudy days [48]. In the absence of SUVR enhancers, it is advisable to establish guidelines on the duration required to achieve the necessary solar radiation intensity (500 W/m2 ) [49].

#### *Perspective Chapter: Solar Disinfection – Managing Waterborne* Salmonella *Outbreaks... DOI: http://dx.doi.org/10.5772/intechopen.108999*

Besides the weather conditions, water turbidity has a significant influence on SODIS. Turbid waters have been shown to reduce the efficacy of the SODIS process [50, 51] and thus protect microbes from inactivation. According to the recommendation by EAWAG, water turbidity higher than 30 Nephelometric turbidity units needs to be pretreated before SODIS treatment [52]. This could be achieved through filtration or simple settling. Turbidity can also be reduced by flocculation using minerals like Alum (potassium sulfate) and seeds of plants like Moringa oleifera. The ability of both these flocculants to clarify water before SODIS treatment has been tested and shown promising results [53]. However, consideration must be given to the fact that adding any form of pre-treatment step elongates the overall time required for disinfection and may have cost implications.

The amount of oxygen present in the water before SODIS significantly influences the outcome. Oxygen plays a key role in forming highly reactive forms of oxygen (oxygen free radicals and hydrogen peroxides) during solar irradiation. These reactive molecules react with cell structures and kill pathogens [54]. SODIS is more effective in water containing high oxygen levels [52]. Therefore, the guidelines recommend vigorously hand-shaking the vessel to dissolve oxygen in the water [52].

The material from which the vessel to be solar irradiated is made significantly influences the outcome of SODIS. Different types of transparent plastic materials made from either polyethene terephthalate (PET) or polyvinylchloride (PVC) are good transmitters of light in the UV-A and visible range of the solar spectrum [55, 56]. Transparent clear bottles such as empty soda and water bottles made from PET and PVC could be used for SODIS. There have been some concerns regarding the leaching of chemicals from the plastic bottles used for SODIS, but this threat is negligible [57, 58].

The temperature has been reported synergies with SUVR to enhance the SODIS water process [50]. Giannakis et al. [59] showed that SODIS carried at temperatures between 50 and 60°C increased inactivation efficiency. Several approaches to enhance the thermal rate of microbial inactivation have been investigated, and these include (i) circulating water over a black surface in an enclosed casing that was transparent to UV-A light [60], (ii) painting sections of the bottles with black paint, and (iii) using a solar collector attached to a double glass envelope container [61]—increasing the temperature past the optimum growth temperature results in the destabilisation of the core structures of most proteins through denaturation. Denatured proteins cannot carry out their critical biological tasks, and as a result, the death of the affected microorganism may result. The increase in the water temperature has been attributed to infrared radiation from the sun.

#### **5.2 The effect of SUVR on biological systems**

UV's bactericidal effect involves thermal and optical processes [62]. Exposure of biological systems to SUVR results in wavelength-dependent outcomes [47, 63]. The observed physical effects are based on the absorbing molecules' action spectrum [47]. An action spectrum can be defined as a plot showing the relative effectiveness of radiations of different wavelengths to produce a given biological effect [47]. Therefore, the action spectrum leading to the formation of a particular photoproduct would be similar to the absorption spectrum of the molecules responsible for forming that photoproduct [47]. The damaging effects of SUVR on microorganisms are demonstrated by reduced exoenzymatic activity that often results in reduced DNA and protein synthesis, reduced amino acid uptake, reduced oxygen consumption and

a decrease in bacterial abundance [64, 65]. Other biological entities, such as biofilms, greatly reduce the amount of SUVR absorption [66].

SUVR enables the formation of reactive oxygen species such as superoxide radicals, hydroxyl radicals, hydrogen peroxide and singlet oxygen. These reactive molecules, also known as photosensitisers, are formed through a process known as photo-oxidation [66–69]. During SODIS, the interaction between the photosensitisers and the actively growing microorganism results in irreversible damage to the microbial catalase systems rendering them susceptible to damage from peroxide formation [64, 70]. Furthermore, UVA, through photo-oxidation, blocks the electron transport chain (responsible for energy production), induces damage to the cell membrane, thus inactivating transport systems, and interferes with metabolic energy production, causing single-strand breaks in DNA [65, 71, 72]. Overall, UVA confers indirect multi-target damage to the microbial cellular components such as DNA, protein and lipids through the formation of photosensitisers [63].

Even though SUVR-exposed biological systems result in reduced functionality and destruction, protective cellular mechanisms are capable of reversing some of this damage. Several DNA repair mechanisms relevant to SUVR damage have been established, including photo reactivation repair, nucleotide excision repair (NER), post replication repair and SOS repair [47, 63, 73]. But these all depend on the dose of SUVR [53] and the exposure environment.

#### **5.3 Solar disinfection of water an ideal POU**

The efficacy of SODIS to inactivate a variety of pathogens such as *Vibrio cholera* [74], *Salmonella* Typhimurium [40], and *Shigella dysentriae* [40] has been demonstrated by various research teams. Millions of people in more than 50 countries, especially resource-poor communities, rely on SODIS-treated water [75, 76]. Input costs for low volume (< 5 litres) vessels are less than the other alternative approaches discussed in Section 4 above. Communities scale the process through the exposure of multiple vessels. Containers that can disinfect more than 5 litres significantly would require financial input. The Sustainable Sanitation and Water Management (SSWM) toolbox [44] offers a one stop hub for knowledge on SODIS where the SODIS manual [39] can also be accessed. The SSWM toolbox is an invaluable resource for organisations promoting access to clean water through the adoption of low-cost technologies such as SODIS.

#### **6. Conclusion**

*Salmonella* remains a critical pathogen of concern globally. This pathogen is responsible for the deaths of many children below the age of 5 and the fragile and elderly. Overcoming infections due to *Salmonella* would require that sanitary and water treatment infrastructure is prioritised, especially in LMIC. Resource-poor communities without access to sanitary or water treatment infrastructure could use a combination of coagulation-flocculation, filtration, and disinfection methods to access treated water at the POU. However, these methods do not address the frequent reintroduction of pathogens such as *Salmonella* into environmental waters. This requires the adoption of sanitary measures at a household level. The water treatment at the POU may reduce the burden of *Salmonella* transmitted through the consumption of contaminated water. However, *Salmonella* can be transmitted through the

*Perspective Chapter: Solar Disinfection – Managing Waterborne* Salmonella *Outbreaks... DOI: http://dx.doi.org/10.5772/intechopen.108999*

consumption of food of either animal or crop origin. Therefore, it is important to consider using some of these methods to treat agricultural water before its use. High water capacity SODIS interventions should be developed and evaluated for the provision of water for agricultural purposes. Perhaps this would require the combination of SODIS with other low-cost treatment approaches such as coagulation-flocculation.

#### **Acknowledgements**

I want to acknowledge the financial support provided by the Vaal University of Technology to support SODIS research.

#### **Conflict of interest**

The author declares no conflict of interest.

#### **Author details**

Cornelius Cano Ssemakalu Faculty of Applied and Computer Sciences, Department of Biotechnology and Chemistry, Vaal University of Technology, Vanderbijlpark, South Africa

\*Address all correspondence to: corneliuss@vut.ac.za

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 6**

## Biocide Use for the Control of Non-Typhoidal *Salmonella* in the Food-Producing Animal Scenario: A Primary Food Production to Fork Perspective

*João Bettencourt Cota, Madalena Vieira-Pinto and Manuela Oliveira*

#### **Abstract**

Biocides are a group of substances commonly used in food production settings to destroy or control a wide range of microorganisms, which can be present in food of animal origin, since contamination can occur in the several steps of the food production chains. In order to achieve the desired results, the users of biocides must first understand the diverse characteristics of such compounds, mainly the usage requirements, limitations, and the factors affecting the activity of biocides. Food-producing animals and their products, namely meat and eggs, represent a major source of non-typhoidal *Salmonella* for humans and are associated with foodborne outbreaks worldwide. The prevention of cross-contamination, which can occur in any step of the food production chain, is essential for the ultimate objective of producing safe food products. The correct use of biocides, along with good hygiene and manufacturing practices, is one of the pillars of *Salmonella* spp. control and should be implemented in all steps of the food production chain. The present chapter reviews the accumulated knowledge on the use of biocides to control non-typhoidal *Salmonella*, from a farm to fork standpoint, along with the possible impacts on human health arising from improper use.

**Keywords:** biocides, non-typhoidal *Salmonella*, control, farm to fork, food safety, food production chain

#### **1. Introduction**

Biocides, from a broad point of view, are substances with the ability of killing living organisms, meaning that this is an all-embracing group, which includes numerous active substances with different targets, ranging from animals, plants, to microorganisms. The use of biocides specifically targeting microorganisms is widely spread in modern societies, mainly due to an increased alarm regarding microbial

environmental contamination of living spaces [1]. Regardless of the growing usage of such biocides, antimicrobial chemical substances have long been regarded as very useful for mankind, for medical, agricultural, and food safety purposes [2]. Unlike antibiotics, which are used to treat infections in humans and animals since they are suitable to be in contact with living tissues, antimicrobial biocides are applied on contaminated suspensions or surfaces reducing the numbers or eliminating microorganisms [1]. These substances are available in very diverse formulations and used not only at an industrial level, but also at the households of consumers, for multiple sanitation procedures. Likewise, these biocidal substances are also used to control the dissemination of microbial pathogens among animal populations and to prevent the leakage of such pathogens from farms [2]. The selection of the most appropriate antimicrobial biocide for a specific application is highly dependent on multiple factors, which can seriously affect its effectiveness [3]. Even with the growing concern regarding the possible effects of such a vast use of these substances in various sectors, antimicrobial biocides are considered to be indispensable for food safety assurance, as their use is imperative along the food production chains, from livestock production up to food industries and retailers [4].

Non-typhoidal *Salmonella* (NTS) is one of the most notorious and studied foodborne pathogens worldwide due to its impact on human health, with an estimated burden of 93.8 million cases of disease and 155.00 deaths per year globally, affecting populations of both developing and developed countries [5]. In humans, NTS infection cases are commonly restricted to a self-limiting gastroenteritis, characterized by nausea, vomiting, and diarrhea starting within a 6–48 hours interval after exposure; however, life-threatening complications can arise from the initial gastrointestinal tract infection in more susceptible groups, such as infants or immunosuppressed and HIV-positive individuals, among others [6, 7]. Despite not being considered necessary for uncomplicated human infections, empirical antimicrobial therapy should be considered in patients belonging to the increased risk groups and recommended whenever bloody diarrhea is present [8]. The upsurge of antimicrobial resistant NTS isolates seen over the past decades is therefore worrying, and this phenomenon has long been identified as a serious global public health concern [9]. As mentioned, NTS is generally considered to be a foodborne pathogen, though human infection cases can occur without the ingestion of contaminated food [6]. Nevertheless, the epidemiological role of food in NTS outbreaks is strikingly greater when comparing with other sources of infection, as direct animal contact or with animal environments [10, 11]. Additionally, food of animal origin has been largely implicated in NTS foodborne outbreaks when comparing with produce [12–14]. The major food vehicles of animal origin associated with outbreaks over the years have been eggs, poultry meat, pork, and to lesser extent, beef and dairy products [15]. Previous works have highlighted the public health impact of eggs [16], poultry and poultry meat [17], and pork [18, 19] in the salmonellosis scenario. There are several steps along the food production chains in which NTS can unintentionally taint food; therefore, complex strategies to avoid the presence of this foodborne pathogen in the final product must be adopted.

This chapter aims to provide a straightforward review of the most relevant available information regarding the use of antimicrobial biocides for the control of non-typhoidal *Salmonella* in the multiple points of the animal-origin food chains, and its possible implications, with a farm to fork perspective. A brief description concerning antimicrobial biocides and their main characteristics will be presented. Additionally, information regarding non-typhoidal *Salmonella* and its dissemination along the food chains will be reviewed. Finally, the use of biocides to control

non-typhoidal *Salmonella*, biocide resistance, and possible implications of biocide usage will be discussed.

#### **2. Biocides**

Generally, a biocide can be defined as an active substance, or a formulation containing at least one active substance, used with the intention of destroying or controlling the effect of any harmful organism to human or animal health by any means other than mere physical or mechanical action [20]. Since the term biocide encompasses a wide spectrum of substances with diverse applications, in the scientific literature it is common to be replaced by disinfectant or sanitizer when addressing chemical substances with antimicrobial activity, in part due to different classifications and legislations. Within the scope of this chapter, only biocides used mainly for disinfection purposes will be addressed.

The legislation and the agencies that regulate these chemical substances have suffered changes over passed decades, mainly in the European Union (EU) and in the United States of America (USA). According to the EU's legislation, biocides are divided in four main groups regarding their purpose: disinfectants, preservatives, pest control products, and other biocidal products [20]. The EU's Biocidal products regulation (Regulation (EU) No 528/2012) further divides biocides used for disinfection in five groups: human hygiene biocidal products, private area and public health area disinfectants, veterinary hygiene biocidal products, food and feed area disinfectants, and drinking water disinfectants.

A different classification is seen in the USA as biocides with antimicrobial activity are classified as public health antimicrobial pesticides and are under the authority of the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA). Within the US legislation, these antimicrobial pesticides are classified according to the degree of effectiveness as sterilants, disinfectants, and sanitizers. While sterilants destroy all forms of bacteria and fungi, including their spores, and even viruses, disinfectants destroy or irreversibly inactivate bacteria, fungi, and/or viruses but not their spores. Disinfectants are subdivided based on their efficacy as hospital, general or broadspectrum, and limited disinfectants. With the lowest efficacy of all the public health antimicrobial pesticides, sanitizers reduce, without necessarily eliminating microorganisms from inanimate environment, and are divided as non-food-contact sanitizers and food-contact sanitizers [21].

#### **2.1 Antimicrobial biocides**

In terms of disinfection purposes, there are several biocidal active substances deriving from different chemical categories [22]. Overall, disinfectants can basically be divided into two groups, the oxidizing and the nonoxidizing. Among oxidizing disinfectants are halogens such as chlorine, chlorine dioxide, iodine, and peroxides, mostly peracetic acid and hydrogen peroxide. Within the group of nonoxidizing disinfectants are quaternary ammonium compounds (QAC), amphoterics, aldehydes, phenolic compounds, biguanides, and acid anionic agents [23, 24]. Their activity, and ultimately the desired effect, can be influenced by different factors, mainly the initial concentration, length of time of contact, temperature, pH, the presence of organic matter, and the type of surface [25–27]. Together with external factors, the nature of the microorganisms, their number, location, and condition, namely the presence

of a biofilm, can also have an impact on the activity of biocides [27, 28]. When these factors are not considered, ineffective disinfection procedures are likely to occur [29]. The typical usage of antimicrobial biocides, the factors affecting their activity, their advantages and disadvantages have been summarized by different authors in previously published reviews [3, 29, 30].

The mechanisms of action of biocides are not fully understood, but generally they can be divided according to the cell structures in the bacterial cells where the interactions occur to produce an antimicrobial effect, specifically the outer cell components, the cytoplasmic membrane, or the cytoplasmic components [31]. In order to develop their antimicrobial activity, the biocidal substance must be transported to the bacterial cell surface, adsorb, diffuse, penetrate, and interact with its target, and all of these processes are time-dependent [32]. In fact, after biocide exposure, the bacterium expresses multiple mechanisms to reduce the amounts of biocidal substance and to repair damages. Consequently, if the exposure is short, the stress and damage induced by the biocide are reversible, but long exposures lead to cell death due to irreversible changes in membrane integrity, leakage of cytoplasmic constituents, and coagulation of intracellular materials [2].

Despite being used for the same reasons and aiming for similar outcomes, some of the characteristics of the biocides used in animal production settings are different when comparing to the ones used in food processing environments. Biocides used for disinfection of animal houses are usually strong, and on some occasions, such as contaminated surfaces, toxic biocidal chemicals are used; in contrast, biocides used in food processing premises are commonly of low toxicity and applied in higher dilutions [26].

Though precise information regarding the actual biocidal substances being used on farms is not readily available since there are several commercially available disinfectant formulations, among the most common are hydrogen peroxide, acetic acid, QACs, aldehydes such as glutaraldehyde, formaldehyde, and isopropanol [33]. In the food industry, the biocidal substances used in commercially available formulations include amphoteric surfactants, polymeric biguanides, QACs, chlorhexidine, chlorine and chlorine-based derivatives, acid anionic agents, hydrogen peroxide, and peracetic acid since these biocidal groups are suitable to be used on food-contact surfaces [4, 32].

These substances or products are extremely important and broadly used for cleaning and disinfection (C&D) procedures of surfaces and environments in the multiple steps of the food production chain, from farms to abattoirs and food processing and handling establishments and even at the households of consumers [30, 34]. As previously mentioned, NTS is a major foodborne illness hazard, thus controlling its movement and persistence across the food production chains is imperative to diminish its impact on human health.

#### **3. Non-typhoidal** *Salmonella*

Despite belonging to the same species (i.e., *Salmonella enterica*), non-typhoidal and typhoidal *Salmonella* serotypes have very distinct behaviors regarding the hosts. While typhoidal *Salmonella* serotypes, specifically Typhi and Paratyphi, are highly adapted to the human host, NTS serotypes can infect a broad range of hosts, including humans, though some NTS serotypes are also known to be species restricted [35]. This level of adaptation of each serotype to specific hosts has clinical, *Biocide Use for the Control of Non-Typhoidal* Salmonella *in the Food-Producing Animal… DOI: http://dx.doi.org/10.5772/intechopen.109038*

epidemiological, and public health impacts, since the degree of pathogenicity of the same serotype can vary among different hosts. As previously mentioned, *Salmonella* Typhi and Paratyphi, which are highly adapted serotypes to humans and are the etiological agents of typhoid and paratyphoid fevers, respectively, are not considered to be pathogenic to other animals. A similar scenario is observed regarding serotypes highly adapted to animal hosts, namely *Salmonella* Gallinarum responsible for fowl typhoid, which is not considered to be pathogenic to humans. On the other hand, ubiquitous or generalist serotypes, such as *Salmonella* Enteritidis or Typhimurium, can affect a broad range of hosts, including humans [36] and are among the most frequently implicated in NTS-associated foodborne illness cases [37, 38]. It is assumed that infections with generalist serotypes are mainly characterized by gastrointestinal manifestations, with high morbidity but with low mortality, and that diseases arising from host-restricted serotypes have low morbidity and high mortality [39]. Nevertheless, some exceptions to this host adaption/pathogenicity degree association are known to occur, for example, *Salmonella* Choleraesuis and Dublin, two serotypes that have as primary hosts pigs and cattle, respectively, which are also responsible for systemic disease in humans [36]. Within the scope of the present chapter, the use of NTS will be replaced simply by *Salmonella.*

#### **3.1 Food production chains and** *Salmonella*

The food production chains have evolved greatly since the past century. The world's most industrialized countries have seen a paradigm change on how food is produced, shifting from small-sized farms supplying local markets to international networks producing and supplying food to large amounts of consumers, though it is estimated that 50–70% of the global food is still produced by smallholder farmers [40]. With a projected world population of almost 10 billion by 2050, and an expected growth of the income in low and middle-income countries, a higher consumption of meat, fruits, and vegetables is foreseen, resulting in additional efforts in the production chains and on natural resources [41]. These circumstances highlight the global challenge of producing enough food to satisfy the needs of the world's growing population, but in order to do so, food safety systems will also have to adapt to the changing needs of both developed and developing countries, enabling global food security [42].

Many stakeholders take part in the food of animal-origin production chains, ranging from cereal producers, feed mills, animal farms, transport operators, abattoirs to food processing industries. These networks of stakeholders can be extremely intricate and highly dependent of international trade, with globalization having a very important role. Feed ingredients can, in some cases, originate from different continents, traveling long distances before being processed in feed mills. The role of feed as a source of *Salmonella* for animals and humans is well known, and all efforts should be made to avoid feed contamination. In the first place, it involves preventing the entry of *Salmonella* in the feed mill's facilities by obtaining uncontaminated feed ingredients and managing several other factors, including flow of personnel and the control of unwanted animals (rodents and wild birds), among others [43].

When comparing different animal species, namely poultry and pigs, some variations in the production cycles are found, with a stratified organization of animal farms, such as breeder, multiplier and finishing or fattening farms, and as such live animal transport is necessary within and between countries. In fact, one of the main challenges regarding the control of *Salmonella* is the prevalence levels among animal populations. In Europe, several countries have implemented strict *Salmonella* surveillance and control programs for poultry (broilers, turkeys, and laying hens) [44–46] and, to a lesser extent, for pigs [47] and cattle [48–51]. Generally, these programs rely on the collection of samples for *Salmonella* detection and on the implementation of restrictions on farms whenever positive results are found. Additionally, a big emphasis is put on the application of biosecurity measures in farms as an effort to avoid the entry of *Salmonella*. Some of the most relevant biosecurity measures are associated with correct cleaning and disinfection (C&D) procedures of the houses where animals are reared in and of the transport vehicles [52, 53]. Moreover, each step of the life cycle of a food-producing animal (birth, rearing, slaughtering) can take place in a different region of the same country or even in different countries.

Finally, before being available to consumers, food-animal products must be carried to food processing facilities and/or to retailers where cross-contamination can occur. As reviewed by Carrasco et al. (2012), there are multiple scenarios where *Salmonella* can contaminate food through food handlers, food-contact surfaces, equipment, and utensils emphasizing the importance of preventive control measures, namely adequate sanitation procedures in food processing and handling facilities but also the consumer's knowledge on good hygiene practices [54].

There has been an increase of the number of food business operators adopting the vertical integration structure, connecting its upstream suppliers with the downstream buyers. The ultimate goal of integrative growth is to increase the business profitability by controlling the most important related activities [55]. Vertical integration is also considered to be a part of the food business operator's private control strategies to tackle food safety hazards along with Hazard Analysis and Critical Control Point (HACCP) systems and third-party certifications [56]. On the other hand, non-integrated food business operators are more likely to be affected by both upstream and downstream operators, not only regarding safety issues but also economically since they are more dependent.

The poultry industry, specifically the broiler sector, was the first to adopt a vertically integrated organization after World War II, during the 1950s, in the USA. Vertical integration of the pig sector was only achieved much later, due to technical and husbandry issues [57]. Nevertheless, at the present time these are the two main animal species reared by large vertically integrated food business operators, especially in high income countries.

Eggs, poultry meat, and pork are the main sources of human salmonellosis cases through contaminated food, and as such, stronger efforts to control *Salmonella* must be put in place along the poultry and pig-associated food production chains, namely the correct use of antimicrobial biocides.

#### **4. Biocide use throughout the food production chain**

To control the spread of *Salmonella* along the food production chain, several measures must be put in place at different stages starting at feed mills to assure high food safety standards. An efficient control of *Salmonella* in feed mills is based on blocking the entry of this pathogen firstly, reducing the chances of *Salmonella* multiplication within the facilities, and by rendering the final product *Salmonella*-free by using thermal process or adding chemicals to feed [43].

Despite the low-moisture environment found in feed mill facilities, which impairs bacterial multiplication, *Salmonella* persistence in such circumstances is known to

#### *Biocide Use for the Control of Non-Typhoidal* Salmonella *in the Food-Producing Animal… DOI: http://dx.doi.org/10.5772/intechopen.109038*

occur, and it is associated with biofilm-forming capability [58]. In these situations, chemical disinfection is necessary to eliminate this source of feed contamination. Despite being a crucial step of the C&D procedure, it seems that physical cleaning can also contribute for the dissemination of the bacterial contamination within the mill facilities [59].

The use of disinfectant formulations combining aldehydes, namely formaldehyde and glutaraldehyde and QACs, applied at high concentrations has been pointed out as the most appropriate against *Salmonella* on surfaces that are not easily cleaned [60]. A direct application of a 30% formaldehyde commercial solution is able to reduce *Salmonella* contamination down to undetectable levels in different types of surfaces, including stainless steel, plastic, polypropylene haul bags, rubber belts, and rubber tires [61]. However, a 70% ethanol-based disinfectant (P3- AlcoDes) and a peroxygen-based disinfectant (Virkon S) were reported to be the most effective when used on surfaces outperforming other disinfectants, even those with a QAC-aldehyde formulation, under laboratory conditions [62].

The specificities of feed mills must be considered by the business operators when choosing the biocidal formulations to be used for disinfection, specifically the need to maintain low levels of moisture. Once detected, *Salmonella* contaminations must be dealt with as soon as possible and rigorous monitoring after C&D should provide information regarding the effectiveness of the procedure. When comparing the legislation of different countries, the responsibility is placed upon the business operators as they must assure the production of safe compound feed. Besides, the economic costs of implementing controls to obtain *Salmonella*-free feed are considered to be limited and that the prevention of dissemination of this pathogen to animals through feed is economically achievable, supporting the implementation of *Salmonella*negative regulation [63].

The environments of the houses/farms where animals are raised in pose serious challenges when considering C&D procedures, mostly due to the amount of organic matter, construction materials used, and multiple fixtures. To obtain the best results possible, all animals should be moved out of the areas or houses before C&D can be started and new animals should only be moved in after C&D has been completed, a system commonly referred to as all in/all out.

There are multiple reports on the efficacy of C&D procedures for *Salmonella* control in poultry farms based on the application of different biocides, either from broiler [64–69], laying hen [70–73], or duck farms [74]. The most frequently used disinfectants were phenol-based, namely formaldehyde, glutaraldehyde, and QACs. Though the use of such substances is considered to result in effective C&D, the application of glutaraldehyde, formaldehyde, and peroxygen solutions at a concentration of 1% was unable to eliminate *Salmonella from a poultry house under experimental conditions* [75]. Wall and floor crevices, drinkers, feeders, and vents can be problematic since these areas/fixtures can promote bacterial persistence, mainly due to the accumulation of dust or organic matter protecting bacteria from the action of biocides [68, 69]. Incomplete disinfection of the houses or of the equipment, leading to *Salmonella* persistence, is likely to promote early *Salmonella* exposure to new laying hen flocks [71] and is considered to be one of the risk factors for the *Salmonella* status of broiler flocks at the end of the production cycle [67].

There are different types of disinfectant formulations, based on QACs, aldehydes, peroxygen or peracetic acid-based, iodine-based compounds or chlorocresols are available to be used on pig holdings for *Salmonella* control, though with diverse effectiveness levels [76]. Disinfectants based on sodium hypochlorite or QACs are believed to be able to eliminate *Salmonella* from pig houses when properly applied after a correct cleaning step [77]. Additionally, in pig housing settings, it seems that better results are achieved using concentrated phenolic disinfectants rather than peroxygenbased products [78]. Even though formulations using combinations of glutaraldehyde and QACs are more effective than iodine-based disinfectants, over-dilution of glutaraldehyde-QACs disinfectants affects its performance, leading to procedure failure and to *Salmonella* persistence in pig houses after C&D [76]. In pigs, as well as in poultry, the maintenance of *Salmonella* on the environment hinders the effects of all other biosecurity measures, such as feed or rodent control. The environment can be contaminated even though it looks clean or undergoes multiple C&D routines, contributing greatly for the transmission of *Salmonella* within pig farms [79].

Abattoirs are a paramount step for *Salmonella* cross-contamination control. Apparently healthy animals can be *Salmonella* carriers, which can easily contaminate the abattoir's facilities and/or equipment, transferring *Salmonella* to, or even infecting negative animals in the lairage area or transferring the pathogen to carcasses during the slaughtering processes. Due to the likely event of environment contamination, highly effective C&D procedures must be adopted. Disinfection in abattoirs can be carried out using one or more of the many formulations suitable to be used in the food industry premises including alcohols, chlorine-based compounds, QACs, oxidizing agents, persulfates, surfactants, and iodophors [80]. As an additional effort to reduce to possibility of cross-contamination, logistic slaughter should be implemented whenever the *Salmonella* status of the animals is known, meaning that *Salmonella*positive animals should only be slaughtered after negative animals. The effectiveness of this measure is strictly dependent of the absence of *Salmonella* from the environment and equipment of the abattoir [81].

In pig slaughterhouses, it has been shown that a main source of carcass contamination is the lairage environment rather than the gut or the lymph nodes of the slaughtered animals [82]. When comparing different protocols for *Salmonella* elimination in lairage pens, a procedure combining the use of detergent, followed by a chlorocresolbased disinfectant and a final drying step of 24 h, was the most effective [83]. Though not suitable for food-contact surfaces, chlorocresol can be used in lairage pens in abattoirs as these areas only receive live animals. *Salmonella*-free lairage pens are extremely important to reduce cross-contamination in the beginning of the process; nevertheless, the following steps also have a significant impact on the carcass hygiene. While some slaughtering processes can reduce *Salmonella* carcass contamination, namely scalding and singeing, others can promote carcass contamination, including inefficient scalding, dehairing, polishing, evisceration, and dressing activities [84]. Accordingly, not only should there be good hygiene and manufacturing practices during slaughter and carcass preparation, but also a special attention should be given to C&D of the slaughter line equipment avoiding the possibility of *Salmonella* biofilm formation and environmental persistence.

As for pigs, the poultry slaughterhouses are a decisive step for *Salmonella* contamination. The poultry abattoir scenario has some major differences when comparing with pigs: the animals are moved in crates or cages, and they are not placed in pens before slaughtering, also the slaughter line is almost entirely mechanized and the slaughtering procedures are automated allowing to process, in some broiler abattoirs, up to 15.000 birds per hour. The transport crates and the slaughter equipment have been pointed as possible sources for *Salmonella* contamination [85, 86]. Poultry should only be transported from the farms to abattoirs in clean and disinfected crates. Though C&D reduces the numbers of *Salmonella* present in crates, persistence can be

#### *Biocide Use for the Control of Non-Typhoidal* Salmonella *in the Food-Producing Animal… DOI: http://dx.doi.org/10.5772/intechopen.109038*

due to the presence of biofilms, improper application of biocides, recontamination, or even cross-contamination [87]. The slaughtering process of poultry encompasses different mechanized steps in intricate equipment, namely scalding, defeathering, evisceration, and chilling, which can ultimately increase the chances of *Salmonella* contamination [87]. The use of standard C&D protocols can in some cases fail to fully eliminate equipment contamination, namely from the plucking machine, after slaughtering *Salmonella*-positive flocks leading to the cross-contamination of *Salmonella*-free flocks slaughtered afterward [81].

Food safety is, and should always be, a top priority issue for food processing industries. Good hygiene and manufacturing practices along with a HACCP plan are essential for obtaining safe animal products. In order to maintain bacterial contamination levels, including *Salmonella*, in the working areas as low as possible C&D must be carried out routinely and effectively. The most relevant biocidal compounds used in the food industry are halogens, peroxygens, acids, and QACs [88]. Regarding egg packing centers, Wales, Taylor, and Davies have recently provided a review on the disinfectants allowed to be used on those facilities, namely QACs, amphoteric surfactants, non-ionic surfactants, sodium hypochlorite, and ancillary agents [89].

The persistence of *Salmonella* in food processing environments, mostly due to biofilm formation, specifically in food-contact surfaces and equipment, after C&D can be associated with insufficient procedures [88, 90]. Additionally, *Salmonella* biofilms in food processing facilities can be a serious problem as biofilms formed in foodcontact surfaces can turn out to be a continuous source of food contamination [91]. Despite the multiple reports available on the efficacy of different biocidal substances or formulations on *Salmonella* biofilms under laboratory conditions, studies focusing on the application of such biofilm treatments on food processing facilities are lacking.

Though not applicable in the EU, some countries allow the use of biocides on raw meat/carcasses for decontamination purposes, some examples are provided. In the USA, the use of sodium hypochlorite, peroxyacetic acid, cetylpyridinium chloride, trisodium phosphate, among others, during immersion chilling is preconized for antimicrobial treatment of poultry carcasses [92]. For pig carcasses, the possibility of chemical decontamination seems to be mainly limited to the use of organic acids, namely acetic and lactic acid [93].

The increase of the application of antimicrobial biocides along the food chain was mainly impelled on the one hand by the implementation of stricter food safety regulations and on the other by consumers' requirements. The possible impacts of such a change are still being studied, but some of the unintentional side effects are already clear.

#### **5. Possible implications of antimicrobial biocide usage**

As with any other biologically active substance, the application of antimicrobial biocides in multiple settings raises concerns due to the possible implications on human, animal, or environmental health. Subsequently, there are legal requirements enforcing an environmental impact assessment and an authorization by the competent authorities before issuing a license for marketing new biocides or biocide formulations [3, 32]. Nevertheless, the usage of antimicrobial biocides is not deprived of risks, namely their toxicity to humans or the tendency to allow the establishment of biocide resistance [94]. Some antimicrobial biocides can be highly reactive with other substances or can produce direct toxic effects or sensitization on users after

dermal or respiratory exposure [95–97]. Additionally, as part of their mechanism of action, these are non-selective compounds and thus can affect multiple organisms other than the intended but can also remain active in the environment after use since they are not easily biodegradable [25]. These characteristics are associated with the presence of biocides in aquatic ecosystems, posing an environmental threat [98]. Furthermore, the improper use of very aggressive antimicrobial biocides or the increase of their dosage to surpass resistance situations increases the possible negative impacts of biocide usage on public health [32]. In fact, the most commonly studied implication of antimicrobial usage is the upsurge of resistances either to antimicrobial biocides or cross-resistances with antibiotics. Any type of resistance to antimicrobial biocides or cross-resistances with antibiotics occurring in *Salmonella* must not be taken lightly, as these phenomena can hinder the previously effective C&D protocols and antibiotic therapeutics whenever necessary in severe salmonellosis cases in humans.

#### **5.1** *Salmonella* **resistance to biocides**

The effectiveness of C&D protocols to eliminate or reduce *Salmonella* is mainly based on the antimicrobial activity of biocidal substances; thus, resistance to biocides can render the disinfection step useless. A brief overview regarding *Salmonella* antimicrobial biocide resistance is provided along with the possibility of antimicrobial resistance co-selection.

In the literature, multiple definitions for biocide resistance can be found, though perhaps the simplest definition is resistance occurs whenever bacteria survive after biocidal exposure in practical use [99]. The use of other terms such as reduced tolerance or reduced susceptibility as a synonym for resistance is also frequent and is based on increases of the minimum inhibitory concentrations or the minimum bactericidal concentrations, which are assessed under laboratory conditions, and such changes might not have any practical significance [2]. In fact, the bacteria ability to survive is not only dependent on the conditions in which the disinfectant is applied, namely concentration and physical state, but also on bacterial characteristics and on environmental settings [100]. As reviewed by Maillard (2018), after biocide exposure, the stress induced in bacteria leads to the expression of different mechanisms in an attempt to avoid irreversible damage and cell death. These mechanisms include the decrease of the concentration of the biocide in bacteria, either by reducing its penetration, by means of efflux pumps or enzymatic degradation, by physiological or metabolic changes or due to mutations [2].

Apart from the presence of the outer membrane with the lipopolysaccharide layer, characteristic of all Gram-negative bacteria, which acts as a blockade to the entry of unwanted substances, it seems that the major mechanisms for *Salmonella* biocide resistance rely on efflux and enzymatic degradation of biocides as well on mutations on biocide targets and overexpression of target proteins [101]. Among the various mechanisms, the AcrAB-TolC efflux system is the best studied in *Salmonella* and has been associated with resistance in different studies under controlled laboratory conditions [102–104]. Still, biocide-resistant *Salmonella* isolates recovered from field studies are thought to be uncommon [101].

Some of the most conclusive reports on *Salmonella* biocide resistance originating from livestock have been reviewed by Wales and Davies, focusing not only on resistance to numerous biocides but also on the possible co-selection of antibiotic resistance arising from biocide exposure [105]. It is assumed that biocide use can

*Biocide Use for the Control of Non-Typhoidal* Salmonella *in the Food-Producing Animal… DOI: http://dx.doi.org/10.5772/intechopen.109038*

select antimicrobial resistant strains either by picking out biocide resistant bacteria with resistance determinants and mutations also responsible for antimicrobial resistance (cross-resistance) or by selecting bacteria with mobile genetic elements which encode several resistance determinants, simultaneously to biocides and antimicrobials (co-resistance) [34]. Despite the studies suggesting that such co-selection can occur [102, 106, 107], which can eventually have an impact in antimicrobial therapy, the conditions arranged in laboratories are supposed to be different from those observed in real-world practice and thus not accurate models to understand biocide interactions with bacteria in the environment [105]. The actual impact of biocide resistance is not fully understood, and it could be almost as important as antimicrobial resistance, making it a focus for future research [108].

#### **6. Conclusions**

The review presented has emphasized, in an uncomplicated manner, the usage of biocides to control *Salmonella* in the food of animal-origin production chains, mainly on poultry and pigs as the major sources, and the possible implications of using these antimicrobial biocides to control this foodborne pathogen, from feed to food or in other terms, from farm to fork.

The use of biocidal substances for disinfection purposes is critical for food safety purposes regarding the control of *Salmonella* along the complex food chains, which supply consumers nowadays. The correct implementation of C&D procedures must always take place in order to reduce the possibilities of *Salmonella* persistence in the environment, a major factor for cross-contamination. It is clear that, in most cases, failure to eliminate *Salmonella* is mainly associated with incorrect usage of biocides rather than a biocide resistance situation. The actual extent of biocide resistance in multiple bacterial pathogens from environmental and food samples should be studied, aiding for a rational usage of these substances or formulations. Nevertheless, with multiple biocidal formulations available in the market, there are several viable options to choose from, considering the different scenarios presented. Furthermore, the development of new biocide formulations, either based on phytochemicals or in nanoparticles ensuring an improved release of the antimicrobial active substances within the intricate structure of biofilms, seems to be promising. Whenever unsuccessful C&D is detected, all steps of the process must be revised, considering the possibilities of improper cleaning, human error on manipulation and application of the biocide, and finally, rotation of biocidal substances or formulations if needed.

Biocide use should not be looked as a panacea for *Salmonella-*associated food safety issues, but together with rigorous control and eradication programs at the herd level, good hygiene and manufacturing practices starting at feed mills up to the food processing industry, and even at the houses of consumers, the burden of salmonellosis in humans can be diminished. Likewise, the scientific community and the competent authorities should also raise the awareness of the consumers toward the possible impacts of the massive usage of household biocidal products as surrogates for good handling and hygiene practices.

This is a continuously growing field of knowledge to which multiple scientific areas are contributing. Further studies, both laboratory and field-based, are required so the most efficient, cost-effective, and safe disinfection protocols can be implemented in the several scenarios where they are irreplaceable.

### **Acknowledgements**

This work was supported by CIISA – Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Project UIDB/00276/2020, and AL4Animals – Associate Laboratory for Animal and Veterinary Sciences Project LA/P/0059/2020 – AL4AnimalS (Funded by FCT – Fundação para a Ciência e Tecnologia IP).

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

João Bettencourt Cota1 \*, Madalena Vieira-Pinto2 and Manuela Oliveira1

1 Faculty of Veterinary Medicine, CIISA—Centre for Interdisciplinary Research in Animal Health, Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), University of Lisbon, Lisbon, Portugal

2 Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), CECAV-Veterinary and Animal Research Centre, University of Trás-os-Montes and Alto Douro (UTAD), Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), Vila Real, Portugal

\*Address all correspondence to: joaobcota@fmv.ulisboa.pt

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Biocide Use for the Control of Non-Typhoidal* Salmonella *in the Food-Producing Animal… DOI: http://dx.doi.org/10.5772/intechopen.109038*

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### **Chapter 7**

Antimicrobial Resistance in *Salmonella*: Its Mechanisms in Comparison to Other Microbes, and The Reversal Effects of Traditional Chinese Medicine on Its Resistance

*Hongxia Zhao*

#### **Abstract**

*Salmonella* is one of the most notable pathogens leading to the outbreak of foodborne diseases worldwide. Antimicrobial chemotherapy with 3rd-generation cephalosporins or fluoroquinolones is often used for severe infections caused by *Salmonella.* Therefore, antibiotic or antimicrobial resistance (AMR) of *Salmonella* is a serious threat to human and animal health in China and worldwide. In order to better understand the current situation and development status of AMR in *Salmonella* isolates, this chapter will provide an overview of the following: 1. The history and development trend of AMR in *Salmonella*, and a comparison of its AMR with that of other major pathogenic bacteria in animals. 2. The AMR mechanisms of *Salmonella* to various antibiotics, with a particular focus on the commonly used antibiotics. 3. The mechanisms of the spread of AMR in *Salmonella*, including the AMR genes or mobile genetic elements carrying AMR genes among microbes, and among people, animal-derived foods, and the environment. 4. The elimination or reversal of AMR in *Salmonella* by using traditional Chinese medicine or the active ingredients in traditional Chinese medicine. 5. The development of detection technology for *Salmonella* serotypes, virulence, and AMR, and the improvement from conventional detection methods to more advanced biological detection methods and bioinformatics technology.

**Keywords:** antimicrobial resistance (AMR), *Salmonella*, salmonellosis in human and animals, comparison with other bacterial species, elimination and reversal of AMR, traditional Chinese medicine

#### **1. Introduction**

#### **1.1** *Salmonella* **and Salmonellosis**

*Salmonella* is a spore-free, capsule-free, gram-negative straight bacilli, which widely exists in human and animal intestines. Genus *Salmonella* currently has two species, *Salmonella enterica* and *Salmonella bongori*. The type species, *S. enterica*, can be further classified into six subspecies with Roman numerals based on their genomic relatedness and biochemical properties, namely, I, *S. enterica* subsp. *enterica*; II, *S. enterica* subsp. *salamae*; IIIa, *S. enterica* subsp. *arizonae*; IIIb, *S. enterica* subsp. *diarizonae*; IV, *S. enterica* subsp. *houtenae*; and VI, *S. enterica* subsp. *indica* [1–3]. So far, *S. bongori* (V) has 22 serotypes [4]., and *S. enterica* has approximately 2600 different serotypes or serovars [2, 5]. *Salmonella enterica* subsp*. enterica* (I) is present predominantly in mammals and contributes approximately 99% of *Salmonella* infections in humans and warm-blooded animals. The other five *Salmonella enterica* subspecies and *S. bongori* are mainly found in environment and cold-blooded animals [4, 6]. Among human isolates, *S*. Enteritidis is the most common serotype, accounting for 65% of all isolates, and *Salmonella enterica* serovar Typhimurium was reported most frequently among nonhuman isolates, although no serotype predominated [7].

People usually get salmonellosis by eating contaminated foods, particularly foods of animal origin, or by direct contact with infected animals. *Salmonella* infection causes diarrhea, fever, vomiting and abdominal cramps. Salmonellosis is a common zoonotic disease. It could not only cause serious economic losses in animal production, but also a serious threat to human health [8]. Infection with *Salmonella enterica* usually results in diarrhea, fever, and abdominal cramps, but some people become asymptomatic or chronic carrier as a source of infection for others.

*Salmonella* is one of the most notable pathogens leading to the outbreak of foodborne diseases worldwide [8–10]. In the United States and other developed countries, the annual incidence rate of *Salmonella* infection is as high as 15.4% [11], and the disease outbreak and hospitalization caused by *Salmonella* are higher than those caused by other foodborne bacteria [12]. In China, about 300 million people are infected with *Salmonella* every year [13]. Salmonellosis accounts for 70% - 80% of the total number of foodborne diseases every year in China, and seriously threatens food safety and human health. In one report, 88 *Salmonella* strains were collected from patients and asymptomatic people in Nantong city of China from 2017 to 2018 [14]. Among these strains, 20 serotypes belonging to 8 serogroups were identified. *Salmonella typhimurium* remained to be the predominant serotype in strains from both patients and asymptomatic people. Among the 27 strains from patients, *S*. *enteritidis* and *S*. Rissen were shown as the other two major serotypes, while *S*. London, *S*. Derby, and *S*. Meleagridis were demonstrated as the other significant serotypes among the 61 strains from asymptomatic people. AMR testing revealed that 84.1% of strains from both resources were multi-drug resistant. By comparing the characteristics of *Salmonella* strains from two different kinds of sources, effective strategies would be developed to control *Salmonella* infection in humans.

Typhoid fever caused by typhoid bacilli is a human acute intestinal infection transmitted between humans. Fowl typhoid is mainly caused by *S. typhimurium*. Salmonellosis in cattle is mainly caused by *S. typhimurium* and *S. dublin*. It mainly occurs in calves aged 10–30 days, and dysentery is the main symptom, so it is also called calf paratyphoid. It is reported that *Salmonella spp.* are among the most important foodborne pathogens and the third leading cause of human death among

#### *Antimicrobial Resistance in S*almonella: *Its Mechanisms in Comparison to Other Microbes… DOI: http://dx.doi.org/10.5772/intechopen.113376*

diarrheal diseases worldwide [15]. Animals are the primary source of this pathogen, and animal-based foods are the main transmission route to humans. Thus, understanding the global epidemiology of *Salmonella* serovars is key to controlling and monitoring this bacterium. The study conducted by Rafaela *et al*. evaluated the prevalence and diversity of *Salmonella* serovars in animal-based foods (beef, pork, poultry, and seafood) throughout the five continents (Africa, the Americas, Asia, Europe, and Oceania) [15]. The results showed *S. typhimurium* presented a cosmopolitan distribution in all four assessed matrices and continents. Poultry continues to play a central role in the dissemination of *S*. *enteritidis* serovar to humans, and *S*. Anatum and *S*. Weltevreden were the most frequently found in beef and seafood, respectively. Careful monitoring of certain serovars and the main vehicles for the transmission of this pathogen will promote the improvement of control programs to reduce the risk of this pathogen reaching humans.

The dominant serotypes of *Salmonella* from different countries and animals are different. The serotypes of *Salmonella* from American chickens are mainly from Kentucky [16]. The predominant serotype of *Salmonella* from cattle in Iran is *S*. *typhimurium* [17]. The serotypes of *Salmonella* from chickens in China are mainly *S*. Enteriditis, *S*. Pullorum, and *S*. *typhimurium* [18]. In terms of the serotyping of *Salmonella*, the conventional detection method is to determine the O antigen and H antigen by slide agglutination, and then determine the serotype according to the serum antigen table. Antibiotics have been used in clinical treatment for more than half a century. Antimicrobial therapy of infections based on the antibiotic susceptibility test results and type plays an important role in prevention and treatment of Salmonellosis.

#### **1.2 Antimicrobial-resistance of** *Salmonella* **and the effect of traditional Chinese medicines on antibiotic-resistant** *Salmonella*

The overall antibiotic or antimicrobial resistance (AMR) of *Salmonella* increased significantly from 20% ~ 30% in the early 1990s to 70% at the beginning of this century [8]. Different serotypes show different AMR to antibiotics, and the AMR rate to different antibiotics is also different [9–12]. In the past three decades, the drug resistance of *Salmonella* has been significantly enhanced, accompanied by the continuously widened spectrum of multiple AMRs. At present, the antibiotics used for *Salmonella* are mainly β-lactams, aminoglycosides, sulfonamides, macrolides, phenylpropanols, quinolones, and tetracyclines [19]. With the increasing dosage and abuse of antibiotics, the AMR of *Salmonella* is becoming more and more prominent. The irrational use of antibiotics has led to a gradual increase in AMR of animalderived pathogens. From the overall situation of China, China has become one of the countries with the most serious AMR of animal-derived bacteria in the world. The AMR is becoming more and more serious and leads to the effect of clinical treatment decreasing or failing. Multidrug-resistant strains are regionally prevalent and can be transmitted along the food chain, posing risks to food safety and human health.

Different serotypes show different AMR to antibiotics, and the drug resistance rate to different antibiotics is also different [9–12]. In recent years, *Salmonella* which has shown resistance to quinolones (ciprofloxacin) and the third-generation cephalosporins (ceftriaxone, cefotaxime) has been reported in China, France, and other countries and regions [20–23], indicating that with the wide clinical application, the therapeutic effect of ideal antibiotics is also declining. The AMR can be encoded by endogenous AMR genes, or generated by gene mutation or acquisition of exogenous

AMR genes carried by mobile genetic elements. Among them, the exogenous AMR genes carried by plasmids, Integron (In), bacteriophages, and Transposon (Tn) can be horizontally transferred through transformation, transduction, and conjugation, which is the main reason for the rapid spread of acquired AMR of bacteria [24].

Different serotypes of *Salmonella* have different AMR [25], and the rise of AMR levels also brings severe challenges to the prevention and treatment of salmonellosis [26]. Therefore, accurate and rapid serotype identification and AMR detection are of great significance for the prevention and control of salmonellosis [27, 28]. Therefore, how to quickly and efficiently identify the serotype and AMR of *Salmonella* has become an urgent practical problem, and the introduction of new detection methods is imperative.

Some traditional Chinese medicines have the following properties: anti-bacterial, anti-inflammatory, nourishing and improving immunity, low potential for building tolerance, and low toxicity and side effects. Some studies have shown that traditional Chinese medicine can eliminate AMR plasmids, have a reversal effect on bacterial resistance, and reduce the selection pressure of bacteria [29, 30]. Therefore, as an alternative to antimicrobial agents or a promoter of antimicrobial agents, it has become one of the research hotspots, which has important significance for the prevention and treatment of *Salmonella* infectious diseases.

#### **1.3 The objective of the chapter**

The AMR of *Salmonella* isolates from humans and animals is becoming more and more serious, which creates great difficulties in the prevention and control of infectious diseases caused by resistant *Salmonella* isolates. *Salmonella* with AMR can not only spread widely between animals but also through food to infect humans. Moreover, AMR can also be passed to humans, So it is a huge potential threat to human and animal health in China and worldwide. To make people pay more and more attention to the problem of AMR in *Salmonella,* this chapter will first review the history and developing trend of AMR in *Salmonella*. The occurrence and spread mechanisms of the AMR of *Salmonella* will be clarified, to provide a theoretical basis for searching a new efficacious antibiotic to eliminate and weaken its resistance and control of Salmonellosis caused by resistant *Salmonella.* In addition, the whole genome sequencing technology has high accuracy in predicting the serotype and AMR of *Salmonella*. The advanced biological detection methods and bioinformatics technology used in identifying *Salmonella* serotypes and AMR will be introduced in this chapter. They have broad application prospects in determining *salmonella* serotype and AMR and the results for prediction will play a very important part in providing strong guidance for the rational use of antibiotics in the clinic.

#### **2. The history and developmental trend of AMR in** *Salmonella***, and a comparison of its AMR with that of other major animal-derived pathogenic bacteria**

#### **2.1 Development trend of AMR in** *Salmonella*

*Salmonella* is one of the most common agents of gastrointestinal disease globally. In the United States, nontyphoidal *Salmonella* is the second most frequent bacterium causing foodborne illness and the first bacterial pathogen in terms of hospitalizations

#### *Antimicrobial Resistance in S*almonella: *Its Mechanisms in Comparison to Other Microbes… DOI: http://dx.doi.org/10.5772/intechopen.113376*

and deaths. For severe infections, antimicrobial chemotherapy with 3rd-generation cephalosporins or fluoroquinolones is recommended. Therefore, AMR in *Salmonella* is considered a serious public health threat. 22,102 genomes from public databases were analyzed to track AMR trends in nontyphoidal *Salmonella* in food animals in the United States. In 2018, genomes deposited in public databases carried genes conferring resistance, on average, to 2.08 antimicrobial classes in poultry, 1.74 in bovines, and 1.28 in swine. There was a decline in AMR of over 70% compared to the levels in 2000 in bovines and swine and an increase of 13% for poultry. Trends in resistance inferred from genomic data showed good agreement with U.S. phenotypic surveillance data. In 2018, resistance to 3rd-generation cephalosporins in bovines, swine, and poultry decreased to 9.97% on average, whereas in quinolones and 4th-generation cephalosporins, resistance increased to 12.53% and 3.87%, respectively.

At present, the antibiotics used for *Salmonella* are mainly β-lactams, aminoglycosides, sulfonamides, macrolides, phenylpropanols, quinolones, and tetracyclines. The β-lactam mainly includes penicillins (such as ampicillin, carbenicillin, etc.), β-lactam enzyme inhibitors (such as amoxicillin-clavulanic acid, ampicillin-sulbactam, etc.), and cephalosporins (such as ceftriaxone, cefoxitin, etc.). Other antibiotics mainly include aminoglycosides (such as gentamicin, kanamycin, etc.), sulfonamides (such as sulfamethoxazole, trimethoprim-sulfamethoxazole, etc.), macrolides (such as azithromycin, etc.), phenylpropanols (such as chloramphenicol, etc.), quinolones (such as nalidixic acid, ciprofloxacin, etc.) and tetracyclines (such as doxycycline, tetracycline) [18]. With the increasing dosage and abuse of antibiotics, the drug resistance of *Salmonella* is becoming more and more prominent. Such as the prevention and treatment of the decline, the emergence of new drug resistance genes, and multidrug resistance (MDR). *Salmonella* as a zoonosis, the enhancement of AMR is also seriously endangering human health and safety [21]. *Salmonella* resistance to a single antibiotic first appeared in the 1960s [22]. Subsequently, AMR of *Salmonella* emerged in different countries and regions of the world, and the isolation rate increased accordingly. In research by Khan *et al*. [23], the isolation rate of MDR of *Salmonella typhi* was higher in Asia and Africa. The results showed that the isolation rates in India, Pakistan, and Vietnam were significantly higher than those in Indonesia and China. Fluoroquinolones and cephalosporins are currently the preferred antibiotics for clinical prevention and control of *Salmonella* infection, but with the irregular use of fluoroquinolones and cephalosporins, the AMR spectrum of *Salmonella* is wider, and there is a large degree of cross-resistance. Hasan *et al*. [31] showed among MDR *Salmonella*, *S. paratyphi* showed a higher level of resistance to fluoroquinolones. *Salmonella* strains isolated from animal-derived foods have a high level of resistance to tetracycline. Generally, the resistance rate can reach 80%, and can even reach a high level of 85%. It shows a certain level of resistance to chloramphenicol, penicillin, nalidixic acid, and sulfonamide antibiotics. In addition, the problem of multi-drug resistance is also very serious. The resistance rate to two or more antibiotics can reach 75%, and the resistance rate to five or more antibiotics can reach 30% [24, 32, 33]. The resistance level of *Salmonella* differs between different studies and regions. Clinically isolated *Salmonella* strains showed a high level of resistance to nalidixic acid, ampicillin, chloramphenicol, and other antibiotics (65% -90%), and the resistance level to sulfonamides, tetracycline, streptomycin was around 50%, and the resistance to the second and third generation cephalosporins was lower, can reach 10% [34, 35].

At present, the problem of AMR of pathogenic bacteria in veterinary clinics is becoming more and more serious [36]. To promote the growth of livestock and poultry, there will be a large number of antimicrobials used, and many veterinary surgeons in the clinical treatment of antibiotics for the irrational use of non-standard, resulting in a gradual increase in the level of *Salmonella* resistance, multi-drug resistance is becoming increasingly serious [37]. Changes in the resistance spectrum occur as *Salmonella* mutates in the natural environment and clinical treatments and are the result of bacterial evolution [38]. *Salmonella* isolates from clinical specimens have been increasing in recent years, and AMR rates are rising rapidly around the world [39]. With the introduction of new antibiotics into clinical use, the corresponding AMR strains will also be rapidly produced, and single AMR has gradually developed into multidrug resistance. The problem of AMR has become more and more serious, and the problem of bacterial resistance has been paid more and more attention [40, 41]. *Salmonella* resistance can not only spread widely between animals but also through food to infect humans, causing food poisoning. AMR can also be passed to humans, affecting human health [42]. The increasingly serious AMR of *Salmonella* has had a great impact on the efficacy of traditional antibiotics, and the increase in the resistance of *Salmonella* strains to new antibiotics has had a more adverse effect on clinical treatment.

#### *2.1.1 Resistance to tetracyclines*

Tetracycline antibiotics are broad-spectrum antibiotics produced by actinomycetes and contain a fused tetraphenyl ring structure [43]. They can be used to treat bacterial diseases caused by Gram-positive and Gram-negative bacteria. Tetracycline antibiotics are mainly divided into two categories: natural and semi-synthetic antibiotics, mainly chlortetracycline, oxytetracycline, methacycline, doxycycline, dimethylaminotetracycline, etc. Due to the characteristics of tetracycline antibiotics, livestock and poultry can only absorb part of them. Most antibiotics will enter the breeding environment in the form of antibiotics themselves or metabolites through the way of livestock and poultry excreta. In addition, livestock and poultry are closely related to human beings. With the continuous development of animal husbandry, bacterial diseases have become increasingly prominent in both intensive farming and free-range farming, and prevention and treatment are facing tremendous pressure. In the prevention or treatment of bacterial diseases, antibiotics are often used. However, when antibiotics are used, there is excessive use, misuse, and abuse, which leads to the specific selection of pathogenic microorganisms by antibiotics and the resistance of pathogenic microorganisms. Among these pathogenic microorganisms, *Salmonella* is more resistant to tetracycline antibiotics. The resistance of *Salmonella* to tetracycline antibiotics varies from country to country, which is related to the unreasonable use of tetracycline antibiotics.

Zhang [44] isolated and identified 34 strains of *Salmonella* from three breeding chicken farms in eastern Liaoning Province. After an antibiotic sensitivity test, 30 of them were resistant to tetracycline. Di *et al*. [45] found that the resistance rate of swine *Salmonella* to oxytetracycline was as high as 58.3%. Li *et al*. [46] found that the resistance rates to doxycycline and oxytetracycline in 247 strains of *Salmonella* isolated from pigs were as high as 89.77% and 94.88%, respectively. The strains showing resistance to doxycycline and oxytetracycline were as high as 89.3%. This shows that *Salmonella* is not only resistant to single tetracycline antibiotics but also resistant to two or more tetracycline antibiotics. The continuous emergence of high resistance rates indicates that tetracycline antibiotics are used too much and too frequently in the clinical treatment of avian salmonellosis. The use of tetracycline antibiotics should be appropriately reduced or replaced.

*Antimicrobial Resistance in S*almonella: *Its Mechanisms in Comparison to Other Microbes… DOI: http://dx.doi.org/10.5772/intechopen.113376*

#### *2.1.2 Resistance to quinolone*

Quinolone antibiotics, also known as pyruvic acid or pyridine copper acid antibiotics, are a class of synthetic antibiotics with 4-quinolone, which mainly inhibit gram-negative bacteria and mycoplasma. Quinolones have been used to treat human and animal infectious diseases and promote animal growth because of their broad antimicrobial spectrum, strong bactericidal effect, rapid action, lack of cross-resistance with other antibiotics, and few side effects [47]. The common quinolones in clinical treatment are enrofloxacin, ciprofloxacin, ofloxacin, sarafloxacin, difloxacin, and so on.

Yao *et al*. [48] found that the resistance rate of *Salmonella* isolated from Shanxi Province, China to the first-generation quinolones was the highest, reaching 56.93%. Zhang *et al*. [49] found that 2.33% (34 of 1523) of *Salmonella enteritidis* strains were resistant to ciprofloxacin. Among them, 11 strains had high resistance to ceftriaxone, and all ciprofloxacin-positive strains had resistance to at least 7 antibiotics. From 2013 to 2018, the resistance rate of *Salmonella enteritidis* to fluoroquinolone enrofloxacin (8.50% -16.30%) showed an increasing trend year by year. In 2012, Li *et al*. [50] conducted an antibiotic sensitivity test on 62 strains of *Salmonella* isolated from pigs. The results showed that the resistance rate of fluoroquinolones was 88.7%. As one of the main antibiotics for the treatment of *Salmonella*, quinolones still have an increasing resistance rate year by year, which has become the hardest hit area of *Salmonella* resistance.

#### *2.1.3 Resistance to aminoglycosides*

There are many kinds of aminoglycoside antibiotics. The earliest aminoglycoside antibiotic is streptomycin, followed by gentamicin, kanamycin, spectinomycin, neomycin, amikacin, netilmicin, and so on. Aminoglycoside antibiotics are mainly divided into two categories: natural and semi-synthetic. Natural aminoglycoside antibiotics include streptomycin, kanamycin, tobramycin, neomycin, spectinomycin, gentamicin, etc. Semi-synthetic aminoglycoside antibiotics include amikacin, netilmicin, etc. [51].

Because of their low price and remarkable effect, aminoglycoside antibiotics are widely used in the treatment and prevention of animal diseases in animal husbandry and aquaculture [52]. However, the use of aminoglycoside antibiotics is abused and abused, resulting in excessive antibiotic residues in animal bodies and AMR. Therefore, the use of aminoglycoside antibiotics has been limited by many countries [53]. Guan *et al*. [54] conducted an AMR test on 23 isolated and identified *Salmonella* strains*.* The results showed that the resistance rate to gentamicin was the highest, which was 66.7%. The resistance rate to spectinomycin was 33.3%, and the resistance rate to kanamycin and tobramycin was 16.7%. The 13 strains of *Salmonella* isolated by Zhang *et al*. [55] were tested for AMR to 10 commonly used antibiotics, all of which showed high AMR rates with resistance to more than two antibiotics. Some even achieved resistance to 8 of them, although sensitive to gentamicin and kanamycin. The AMR results varied among the 13 *Salmonella* isolates, possibly due to the changing breeding environment or AMR. Thus, in recent years, *Salmonella* resistance to aminoglycoside antibiotics has been very serious, and mostly multi-drug resistance.

#### *2.1.4 Resistance to amide alcohols*

Amide alcohol antibiotics are also called chloramphenicol antibiotics. They are a class of antibiotics with broad-spectrum antibacterial amide alcohol substances, which have

inhibitory effects on both Gram-positive and negative bacteria. In the field of agriculture in animal husbandry, aquaculture, and chemical industry in the cosmetics industry are widely used, mainly for the treatment of chicken, pig, cattle, and other animals respiratory disease infections. Amide alcohol antibiotics mainly include chloramphenicol, palm chloramphenicol, succinomycin, florfenicol, thiamphenicol, etc.

In 2019, China explicitly banned the continued use of chloramphenicol in foodborne animals. At present, thiamphenicol and florfenicol are widely used as substitutes for chloramphenicol in animal husbandry. With the wide application of amide alcohol antibiotics, the resistance of *Salmonella* to amide alcohol antibiotics has gradually increased. Huang *et al*. [56] conducted an antibiotic resistance or AMR test on 61 isolated *Salmonella* strains. The results showed that the resistance rate to florfenicol accounted for 19.67%. Mondal *et al*. [57] conducted an AMR test on 9 isolated *Salmonella* strains. The results showed that 9 *Salmonella* strains were highly sensitive to ciprofloxacin, kanamycin, nalidixic acid, cotrimoxazole, and ampicillin, but highly resistant to chloramphenicol. Li *et al*. [58] conducted an AMR test on 215 strains of *Salmonella* isolated in Henan Province in China. The results showed that the resistance rate to florfenicol was 92.56%, and the AMR was serious. With the extensive use of florfenicol, the number of strains resistant to florfenicol showed an increase. Since February 2022, *Salmonella* strains resistant to florfenicol mainly belong to *S. typhimurium*, *S.* Agona, and *S. paratyphi*.

#### **2.2 Comparison of AMR in** *Salmonella* **with other major animal-derived pathogens**

China has become the world's largest producer and consumer of livestock and poultry products [58]. The production of pork, poultry meat, and eggs has been the world's first for several consecutive years, and milk production is the third in the world. The rapid growth of China's aquaculture industry mainly depends on the expansion of the scale of aquaculture and the increase in the number of aquaculture facilities. The large-scale and intensive aquaculture industry continues to develop steadily. Veterinary antibiotics, especially antibiotics, play an important role. However, the irrational use of antibiotics has led to a gradual increase in AMR of animal-derived pathogens. The sensitivity of animal-derived pathogens to quinolones, β-lactams, and other important antibiotics is declining, and the AMR is getting higher and higher. Some clinical isolates of pathogens are resistant to more than 15–20 kinds of antimicrobial agents, leading to livestock and poultry disease prevention and control becoming increasingly close to the embarrassing situation of no antibiotic being available [59]. *Streptococcus*, *Haemophilus parasuis*, *Pasteurella multocida* and other important animal-borne pathogens of amoxicillin, enrofloxacin, and other antimicrobial resistance are becoming more and more serious with clinical treatment, losing effectiveness or failing. In the breeding industry, for a long time, widely through mixing, drinking water to livestock and poultry use of antimicrobial, healthy animal intestinal symbiotic *Escherichia coli*, *Enterococcus* resistance to commonly used antibiotics is also increasing year by year [59]. The AMR of *Salmonella* from livestock and poultry is developing continuously, and the antimicrobial resistance mechanism is becoming more and more complex [60]. Multidrug-resistant strains are regionally prevalent and can be transmitted along the food chain, posing risks to food safety and human health. The emergence and prevalence of five AMR *cfr* genes have brought great challenges to the clinical treatment of methicillin-resistant *Staphylococcus aureus* (MRSA), and vancomycin-resistant *Enterococcus* infection [58]. The detection rate of

*Antimicrobial Resistance in S*almonella: *Its Mechanisms in Comparison to Other Microbes… DOI: http://dx.doi.org/10.5772/intechopen.113376*

*S. aureus* clinical strains *cfr* in developed countries is less than 0.5%. The detection rate of *S. aureus* clinical strains *cfr* in China is much higher than that in developed countries by nearly 4%. This gene has even been found in animal-derived *Bacillus*, *Streptococcus*, *Enterococcus*, *Escherichia coli*, and *Proteus*, and is mostly located in plasmid DNA that can be horizontally transmitted [58].

Zhao *et al*. [59] isolated 4 main pathogenic bacteria from 260 cow endometritis samples in Inner Mongolia, including 126 strains of *E. coli* (48.5%), 84 strains of *Streptococcus* (32.3%), 53 strains of *S. aureus* (20.4%) and 21 strains of *Salmonella* (8.1%). The results of an antimicrobial susceptibility test showed that the resistance rate of *E. coli* to sulfonamides and benzylaminopyrimidines was more than 98%, and the resistance rate to ceftiofur was 13.7%. The resistance rate of *Streptococcus* to β-lactams, tetracycline, and kanamycin was more than 80%, and the resistance rate to vancomycin was 26.7%. The resistance rate of *S. aureus* to β-lactams ranged from 60–85%, to gentamicin and three combinations ranged from 7.5% to 1.2%, and was completely sensitive to vancomycin. The resistance rates of *Salmonella* to β-lactams, gentamicin, tetracyclines such as oxytetracycline and doxycycline were between 75% and 90%. *Salmonella* was sensitive to cefotaxime, and the resistance rate was 29%. The resistance rates to aminoglycosides such as tobramycin and amikacin were less than 10%. Four isolates were sensitive to fluoroquinolones and the resistance rates were less than 35%. Zhao *et al*. [60] isolated pathogenic bacteria from 40 samples of cow endometritis in Xinjiang mainly include *E. coli*, *Staphylococcus*, *Streptococcus*, *Bacillus cereus* and *Salmonella*, and the first three pathogens are the main pathogenic bacteria. The results of antibiotic sensitivity test showed that cefotaxime and amoxicillin had obvious antibacterial effect on *E. coli*, enrofloxacin and kanamycin had obvious antibacterial effect on *Staphylococcus*, and amoxicillin and ciprofloxacin had obvious antibacterial effects on *Streptococcus*. Almost all isolated bacteria were resistant to tetracycline and penicillin and were sensitive to quinolones and lactams. Based on the above studies, it seems that *Salmonella* showed different patterns of AMR to some commonly used antibiotics when compared with several other major animalderived pathogenic bacterial species. The underlying mechanisms are not clear. They could be due to different bacterial niches, different standards for antibiotic usage and animal breeds. Further research is needed to explore the mechanisms which could be important for designing strategies for migrations of AMR.

#### **3. The AMR mechanisms of** *Salmonella* **to various antibiotics, with a particular focus on the commonly used antibiotics**

The extensive use of antibiotics has inevitably improved the survival adaptability of pathogenic bacteria and the endogenous flora of humans and animals, and promoted the evolution of their genomes, thus leading to the emergence and spread of AMR strains. At the beginning of this century, the overall AMR of *Salmonella* increased significantly from 20% ~ 30% in the early 1990s to 70% [29]. Different serotypes show different AMR to antibiotics, and the AMR rate also varies between different antibiotics [30, 61–63]. In recent years, *Salmonella,* which has shown resistance to quinolones (ciprofloxacin) and the third generation of cephalosporins (ceftriaxone, cefotaxime) has been reported in China, France, and other countries and regions [64–67], indicating that with the wide clinical application, the therapeutic effect of ideal antibiotics is also declining. In addition, the emergence and global spread of multi-antibiotic resistant *Salmonella* make the situation of AMR

of *Salmonella* extremely severe. Therefore, the use of antibiotics should be further standardized and the AMR monitoring of *Salmonella* should be strengthened in the future.

The biochemical mechanisms of AMR can generally be classified into three categories [68–70]: 1) Produce inactivating enzymes to destroy antibacterial antibiotics through hydrolysis or modification, so that they can be converted into derivatives without antibacterial activity; 2) Reduce the permeability of the bacterial outer membrane, hinder the entry of antibacterial agents, or strengthen the efflux of active efflux pump to transport antibacterial agents out of the cell to reduce the antibiotic concentration in the cell; 3) To modify the action target of antibiotics or cause target mutation through gene mutation, thereby reducing the affinity of antibiotics to target proteins. The AMR can be encoded by endogenous AMR genes, or generated by gene mutation or acquisition of exogenous AMR genes carried by mobile genetic elements. Among them, the exogenous AMR genes carried by plasmids, Integron (In), bacteriophages, and Transposon (Tn) can be horizontally transferred through transformation, transduction, and conjugation, which is the major reason for the acquired AMR and rapid spread of bacteria [71].

Plasmids are extrachromosomal DNA molecules that can replicate autonomously and can confer host resistance to important antibiotics, including β-Lactamides, aminoglycosaminoamines, tetracyclines, chloramphenicols, sulfonamides, trimethoprims, macrolides and quinolones [72], and conjugated plasmids can transfer AMR to recipient bacteria through conjugation. Plasmids are closely related to the current situation of *Salmonella* resistance, and heavy metal resistance genes, disinfectant resistance genes, and virulence-related genes carried on plasmids have improved the survival adaptability of *Salmonella* to the environment [73].

*Salmonella* has a high level of resistance to quinolones, mainly due to the mutation of *gyrA*, *gyrB*, *parC* and *parE* genes in the quinolone resistance determining region (QRDR) on the bacterial chromosome, which makes the antibiotics lose their binding sites and efficacy [71]. The quinolone resistance genes *qnr, aac* (6′) *- Ib cr*, *qepA,* and *oqxAB* carried by plasmids can mediate low levels of quinolone resistance and accelerate the mutation of *gyrA*, *gyrB*, *parC,* and *parE* genes in QRDR, which is the main reason for the spread of quinolone resistance in *Salmonella* at present [67, 74].

The tolerance of *Salmonella* to β-lactam drugs is mainly due to the hydrolysis of antibacterial drugs β-lactamases, and most β-lactamase gene is carried by plasmid. Among them, plasmid-mediated ultra-broad spectrum β-lactamase genes *blaCTX-M*, *blaTEM,* and *blaSHV*, *AmpC* β-lactamase gene *blaCMY* and carbapenemase genes *blaKPC*, *blaVIM*, *blaIMP,* and *blaOXA* are prevalent worldwide [63, 71, 75].

In addition, the plasmid can also achieve the aggregation and transfer of antibiotic-resistant gene clusters by capturing mobile elements such as integrons or transposons. Integron is a natural cloning and expression system found in bacteria in recent years. Although the integron lacks the ability of autonomous movement, it often participates in the transfer as a component of the conjugated plasmid or transposon, thus promoting the diffusion of antibiotic-resistant genes [76]. Vo [77] detected *aadA1*, *aadA2*, *aadA5*, *blaPSE-1*, *blaOXA-30*, *dfrA1*, *dfrA12*, *dfrA17* and sat resistance gene cassettes in the type I integron carried by *Salmonella* isolates in Vietnam, forming nine different gene box arrays, and most of them are located on conjugative granules, which can transfer resistance to *E. coli* or *S. enteritidis* receptor bacteria.

*Antimicrobial Resistance in S*almonella: *Its Mechanisms in Comparison to Other Microbes… DOI: http://dx.doi.org/10.5772/intechopen.113376*

#### **4. The elimination or reversal of AMR in** *Salmonella* **by using traditional Chinese medicine or the active ingredients in traditional Chinese medicine**

Chinese herbal medicine is natural and has many advantages: low toxicity, and lower residual levels of toxic substances [78]. It plays an active role in modern infection prevention and control. Some traditional Chinese medicines have the following properties: anti-bacterial, anti-inflammatory, nourishing and improving immunity, low potential for building tolerance, and low toxicity and side effects. Some studies have shown that traditional Chinese medicine can eliminate AMR plasmids, have a reversal effect on bacterial resistance, and reduce the selection pressure of bacteria [78, 79]. Therefore, as an alternative to antimicrobial agents or a promoter of antimicrobial agents, it has become a research hotspot, which has important significance for the prevention and treatment of *Salmonella* infectious diseases.

Some studies have shown that Chinese herbal medicines have a bacteriostatic effect on *Salmonella* in calves, and the bacteriostatic intensity ranked from strongest to weakest as follows: gallnut, schisandra chinensis, wumei, chebula, *Ligustrum lucidu*m, pomegranate peel, sumu, and scutellaria. Among them, gallnut has the best bacteriostatic effect [79]. Wumei, coptis chinensis, and rhubarb have bacteriostatic effects on the intestinal *Salmonella* of dairy cows [80], among which, gallnut has good bacteriostatic effects on *S. typhimurium* and *S. cholera-suis* isolates from pigs [81]. Ma [82] found that the elimination rates of resistance to amoxicillin and tetracycline were 1% and 5%, respectively, in the resistant *Salmonella* treated with ebony. Cao [83] found the elimination effects of Galla Chinesis and Scutellaria on *Salmonella* AMR and with the highest removal rate of resistant strains 23.3%, 15.3% respectively by 20 hours, and 14.7%, 9.9% respectively by 48 hours. The Galla Chinesis and Scutellaria showed resistant plasmid removal rate of 15.6% and 10.8%, respectively.

#### **5. The development of detection technology for** *Salmonella* **serotypes, virulence, and AMR, and the change from conventional detection methods to more advanced biological detection methods and bioinformatics technology**

Different serotypes of *Salmonella* have different antimicrobial resistance [25], and the rise of AMR level also brings severe challenges to the prevention and treatment of salmonellosis [26]. Therefore, accurate and rapid serotype identification and AMR detection are of great significance for the prevention and control of salmonellosis [27, 28]. In terms of the serotyping of *Salmonella*, the conventional detection method is to determine the O antigen and H antigen by slide agglutination, and then determine the serotype according to the serum antigen table. This serotyping technique requires high serum quality, costs a lot, and takes a long time, and some agglutinations are not obvious and difficult to distinguish. In terms of AMR detection of *Salmonella*, the most commonly used method is the antibiotic sensitivity test recommended by the American Committee for Clinical Laboratory Standardization (CLSI) [84]. However, the accuracy of the experimental results of this method is easily affected by experimental materials, experimental conditions, and personnel operations. In terms of AMR gene detection, common PCR detection techniques cannot identify all AMR genes at once [85]. Therefore, how to quickly and efficiently identify the serotype and AMR of *Salmonella* has become an urgent practical problem, and the introduction of new detection methods is imperative.

With the increasing maturity of sequencing technology, rapid, low-cost, and costeffective whole genome sequencing technology (WGS) has been widely used in the research of bacterial epidemiology [86]. At the same time, the development of bioinformatics technology has also promoted the creation of a variety of public databases such as the serological typing of foodborne pathogens and antibiotic-resistant genes, such as the SeqSero serotype database and ResFinder AMR gene database. With the continuous updating and improvement of the databases, the accuracy of automatic data analysis will be higher and higher. Several studies have shown that WGS has broad application prospects in determining *Salmonella* serotype and AMR genotype, and may replace conventional laboratory methods in the future [87, 88]. At present, there is very limited research in this field in China.

The establishment of serotype databases promotes the application of WGS in *Salmonella* serotyping. The commonly used serotype databases include SeqSero and SISIR. At present, SeqSero has been updated to SeqSero2, which improves the accuracy of the serotype database. Compared with the SISTR database, SeqSero2 does not need the help of genome-wide multi-site sequence typing research, simplifying the operation process and making the application more convenient [89]. Xu *et al*. [90] selected 38 *Salmonella* strains from the American *Salmonella* surveillance system, and the coincidence rate between the WGS typing results and the original results was 100%. Zhang *et al*. [91] conducted molecular analysis on 308 known *Salmonella* serotypes through WGS, among which 304 strains were completely consistent in serotype, with a coincidence rate of 98.7%. Diep *et al*. [92] collected 100 *Salmonella* strains from the Netherlands, and the serotypes of 98 *Salmonella* strains predicted by WGS were consistent with the conventional typing results, with a coincidence rate of 98.0%. Robertson *et al*. [93] extracted *Salmonella* WGS data from the SPA public database, and the coincidence rate between the identified serotype and the original results was 95.0%.

In conclusion, WGS typing method has high accuracy in predicting common serotypes. Compared with the conventional serum typing method, WGS typing is faster. For rare serotypes that require different culture media and antisera to determine flagella (H1 and H2), WGS takes only a few minutes, while the conventional serum typing method may take several weeks, sometimes requiring multiple repetitions. Therefore, the typing method based on WGS opens a new door for the identification of *Salmonella* serotypes, which has great application value in *Salmonella* serotyping. With the improvement of sequencing technology and the improvement of the databases, WGS typing is expected to become a new standard for *Salmonella* serotyping [94, 95].

The emergence of AMR is closely related to the existence of AMR genes, and the expression of AMR genes determines bacterial AMR. Research shows that the ResFinder resistance gene database can detect more resistance genes in the prediction of resistance genes, and it is the preferred tool for AMR analysis [96]. Neuert *et al*. [97] compared the AMR of 3415 *Salmonella* strains to 15 kinds of antibacterial agents, and their genotypes, and found 97.8% correlation.

Zankari *et al*. [98] predicted the AMR of 49 strains of *S. typhimurium* to 17 kinds of antibiotics, which was completely consistent with the results of AMR phenotype. Among 189 *Salmonella* strains studied by Zhu *et al*. [99], the coincidence rates of WGS AMR prediction to sulfamethoxazole, ampicillin, and tetracycline with their AMR phenotypes were 97.8%, 94.6% and 85.7%, respectively.

For antibiotics whose AMR genotype is not clear or is still under study, the coincidence rate between the AMR phenotype predicted by WGS and the AMR genotype *Antimicrobial Resistance in S*almonella: *Its Mechanisms in Comparison to Other Microbes… DOI: http://dx.doi.org/10.5772/intechopen.113376*

is relatively low. The resistance mechanism of enrofloxacin and ceftiofur is mainly related to chromosome-mediated mutations. At present, WGS has only detected plasmid-mediated resistance genes, while the resistance genes generated by chromosome mutations have not been detected. This may be due to the low coverage of some regions in the genome sequencing process, preventing the detection of mutation sites, or the emergence of new resistance gene mutations [100].

Overall, the genome-based genotyping method avoids the influence of subjective judgment of conventional serotyping methods and has a high application prospect in serotyping. It is expected to replace conventional serotyping methods. The prediction of AMR by antibiotic resistant genotypes also provides a new perspective and method for clarifying AMR mechanisms and detecting AMR [101]. When new serotypes or AMR genes appear, they can be directly retrieved and analyzed through WGS data, without the need for routine bacterial culture and identification again, which provides a simpler method for the analysis of *Salmonella* serotypes and AMR. In addition, the application of WGS has also promoted research and development in other directions such as the genetic and variation characteristics of foodborne pathogens, AMR mechanisms [102], and will have an increasing impact on the analysis and research of the molecular biological characteristics of bacteria in different ecosystems and the substitution of traditional methods [103, 104]. With the development of whole gene sequencing technology and the reduction of its cost, rapid screening of antibioticresistant genes from genome data by bioinformatics methods has become a research hotspot.

#### **6. Conclusion**

The resistance of *Salmonella* to β-lactams, gentamicin, tetracyclines such as oxytetracycline and doxycycline was serious. However, *Salmonella* isolates were sensitive to fluoroquinolones, cefotaxime, and aminoglycosides such as tobramycin and amikacin. *Salmonella* has shown resistance to quinolones (ciprofloxacin) and the third generation cephalosporins (ceftriaxone, cefotaxime) in China, France, and other countries and regions. The resistance of *Salmonella* from livestock and poultry is developing continuously, and the AMR mechanism is becoming more and more complex. Multidrug-resistant *Salmonella* is regionally prevalent and can be transmitted along the food chain to human, which make the situation of AMR of *Salmonella* extremely severe. Therefore, the use of antibiotics should be further standardized and the AMR monitoring of *Salmonella* should be strengthened in the future.

The increasingly serious AMR of *Salmonella* has an adverse effect on the clinical treatment of salmonellosis. The biochemical AMR mechanisms of *Salmonella* are as follows: (1) Produce inactivating enzymes to destroy antibiotics; (2) Reduce the permeability of the bacterial outer membrane; (3) Strengthen the efflux of the active efflux pump to transport antibiotics out of the cell; (4) To modify the action target of antibiotics; (5) Target gene mutation. The serotypes or AMR genes can be retrieved and analyzed through the genome-based genotyping method and WGS data. The development of bioinformatics technology provides a new perspective and method for clarifying AMR mechanisms and detecting AMR.

To a certain degree, the AMR in *Salmonella* can be eliminated or reversed by traditional Chinese medicine or traditional Chinese medicine active ingredients. Some traditional Chinese medicines have good reversal effects on resistance of *Salmonella* isolates. By eliminating the resistant plasmids, Chinese herbal medicines can reduce AMR of *Salmonella* strains and reduce the selection pressure of bacteria. Therefore, some traditional Chinese medicines, as an alternative to antimicrobial agents or a promoter of antimicrobial agents have important significance for the prevention and treatment of *Salmonella* infectious diseases.

#### **Acknowledgements**

The authors would like to acknowledge Wei Mao and Weiguang Zhou, Professors of Veterinary Medicine of Inner Mongolia Agriculture University. They provided a large amount of information on the molecular epidemiology of zoonotic pathogens. In addition, JinShan Cao, the senior vice mayor of Tongliao City in the Inner Mongolia Autonomous Region is acknowledged for proposing the new design ideas before writing the manuscript. During the process of manuscript's revision, some valuable suggestions were given by Professor Cao. The authors also gratefully acknowledges the support from Inner Mongolia Science and Technology major project (2021ZD0013).

### **Author details**

Hongxia Zhao College of Veterinary Medicine, Inner Mongolia Agriculture University, Huhhot, Inner Mongolia, Peoples' Republic of China

\*Address all correspondence to: 18947199590@163.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Antimicrobial Resistance in S*almonella: *Its Mechanisms in Comparison to Other Microbes… DOI: http://dx.doi.org/10.5772/intechopen.113376*

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### *Edited by Hongsheng Huang and Sohail Naushad*

*Salmonella* is a Gram-negative bacterium and a member of the *Enterobacteriaceae* family that causes infections in humans and animals, making it one of the most common causes of bacterial gastroenteritis worldwide. Since its discovery in the late 1800s, significant progress has been made in the understanding of its genetics, classification, pathogenesis, detection, prevention, control, and treatment. Numerous reviews and chapters on *Salmonella* have been published, but some gaps remain to be addressed. This book includes seven chapters that focus on the low-cost prevention, control, and treatment of salmonellosis in developing countries. It begins with a brief review of *Salmonella*, followed by chapters on the transmission of the organism in food and companion animals relevant to the One Health approach, CRISPR-Cas systems in *Salmonella* for pathogen typing in diagnosis and surveillance, the low-cost control of *Salmonella* using solar disinfection of water in resource-limiting communities, and transmission and antimicrobial resistance (AMR) in *Salmonella* across the One Health sector. This book also introduces a new concept of AMR reversal using traditional Chinese medicine. The information provided in this book will encourage *Salmonella* researchers, medical professionals, and students to further enhance their own research and education as well as encourage new researchers to include *Salmonella* in their future research initiatives.

Published in London, UK © 2024 IntechOpen © vav63 / iStock

Salmonella - Perspectives for Low-Cost Prevention, Control and Treatment

*Salmonella*

Perspectives for Low-Cost Prevention,

Control and Treatment

*Edited by Hongsheng Huang and Sohail Naushad*