**Abstract**

Salmonellosis is a disease of great relevance in terms of public health given the economic and social impact that causes both in developing and highly industrialized countries. Due to its transmission mechanism, it affects hundreds or thousands of people every year and is considered an acute disease of worldwide distribution. Causative agent of salmonellosis is *salmonella* specie which are small gram-negative bacilli and facultative intracellular pathogen of the Enterobacteriaceae family. Multidrug resistance is reported more frequently in strains of *salmonella*, raising the necessity of new strategies to combat its spread and to treat the disease. Natural products (NPs) derived from traditional medicine knowledge have become an important resource to this end. In this chapter, we present a summary of information published from 2010 to 2020, as a sample of the potentiality of NPs as agents for Salmonellosis. This search was not exhaustive, rather, we aim to obtain a random sample of information using the simplest terms on the matter of natural products for salmonellosis, hopefully, as a reference source for interested researchers.

**Keywords:** *salmonella*, antibacterial activity, natural products, anti-salmonella, Salmonellosis

## **1. Introduction**

Salmonellosis is a disease of great relevance in terms of public health given the economic and social impact that causes both in developing and highly industrialized countries. Due to its transmission mechanism, it affects hundreds or thousands of people every year and is considered an acute disease of worldwide distribution [1] with variations in the frequency of serotypes from one country to another [2], being notably more frequent in areas that have not reached adequate sanitation and hygiene conditions or that do not have enough resources and public health infrastructure. There is no distinction in the occurrence of salmonellosis by sex, age, or social and economic status with high incidence at the extremes of life, being the most vulnerable groups, children under 5yo, adults over 60 years of age and immunocompromised individuals [3, 4]. On the other hand, it is also a seasonal disease, so incidence is higher on periods of increased environmental temperature like spring and summer, showing a decrease in autumn and winter [5].

The raise in salmonellosis at any part of the world is of maximum relevance. For example, an incidence of 0.78–3.8 million cases per year has been estimated in the United States. Natural reservoir is made up of domestic animals (dogs and cats), wild animals (reptiles such as iguanas and turtles) as well as humans (carriers, convalescent). Transmission is through food (with or without manufacture) and water contaminated with human or animal feces and from individual to individual. Salmonellosis presents as sporadic cases or as outbreaks with variable affectations. Incidence rate is dose-dependent in function of the disseminated serotype and is determined by incubation period, symptoms and severity.

Causative agent of salmonellosis are *salmonella* species, numerous disease outbreaks are related to the consumption of eggs, chicken meat and other raw products (mainly dairy). For instance, in an outbreak of enteric salmonellosis serotype Typhimurium (n = 99) induced by consumption of roast porcine meat in an institution for the mentally ill in Konagua it was shown that the incubation period was between 10–12 hours and that the supply of antibiotics prolonged excreta periods. Salmonellosis due to *Salmonella enterica* serotype Enteritidis was detected in an interstate outbreak in the United States in the early 90's, produced by the consumption of ice cream (224,000 cases) and in Canada due to the consumption of commercial packaged cheese (800 cases). Salmonella Javiana (n = 66) has been reported to produce outbreaks as in Boston due to the consumption of chicken sandwiches [6].

## **2.** *Salmonella*

*Salmonella* belongs to the Enterobacteriaceae family, which are small gramnegative bacilli varying in sizes ranging in average from 2–3 μm in length and 0.4– 0.6 μm in width. These bacilli do not form spores and possess peritrichous flagella hence are mobile microorganisms, although some genera, such as Klebsiella and Shigella, are lacking on these organelles and so on mobility. Traditional grouping classification is carried out using primary biochemical characteristics that allows a further sorting into subgroups based on antigenic structure determinants or using bacteriophage reactions. Currently, with the advances in molecular biology, the differentiation of groups and subgroups can be made using PCR technique for identification, diagnostic and epidemiological purposes.

Regarding its metabolic characteristics, *salmonella* grows in simple synthetic media and can use unique carbon sources, such as glucose in a fermentative way with the subsequent formation of acids and/or gases, reducing nitrates and nitrites, rendering oxidase negative reaction. *Salmonella* also tests positive for methyl red, hydrogen sulfide, indole-ornithine motility (MIO medium), lysine decarboxylase, arginine dihydrolase, ornithine decarboxylase, gas from glucose, and fermentation of numerous carbohydrates such as rhamnose, arabinose, mannitol, etc.

Most enteric microorganisms are resistant to inhibition by the action of certain bacteriostatic dyes, the selective media containing these compounds facilitate considerably isolation from fecal samples, *salmonella* is less sensitive than coliform microorganisms against citrate inhibition action; for instance, SS (Salmonella-Shigella) agar containing both citrate and bile salts is therefore used as a selective medium for the culture of pathogenic species [7, 8].

#### **3. Classification**

Although controversial and evolving, there is a *salmonella* nomenclature used by the Centers for Disease Control and Prevention (CDC) and recommended by the

Collaborating Center of the World Health Organization (WHO), which according to the differences in their 16S rRNA sequence analysis classifies this genus into two species, *Salmonella enterica* and *Salmonella bongori*. *S. enterica* can also be further classified into six subspecies mainly found in mammals and is responsible for 99% of infections in humans and warm-blooded animals. On the other hand, *S. bongori* is predominantly environmental and on cold-blooded animals [9].

#### **3.1 Classification according to Kauffmann-White**

Since decades ago, classification of *salmonella* finalizing at the species level are based on its antigenic structure. Although certain strains that have the same antigenic activity could present different metabolic reactions (biotype variants or serotypes), this sorting method is generally accepted and is actively in use.

Surface antigen studies are based on H, O, K and Vi antigens. H is denominated surface or flagellar antigen and participates in host immune response, O (aka somatic antigen) is a lipopolysaccharide located in the cell membrane, K is a capsular antigen and Vi antigen is a subtype of K antigen associated to virulence [9] and the obtention of antisera containing antibodies against all these fractions allows the identification of *salmonella* species. More than 2,500 serotypes have been identified related to the H, O, K and Vi antigens [10] as a result of the numerous absorption tests and cross-reactions studies carried out in Denmark and England by Kauffman and White. Currently, large centers in Copenhagen, London, and Atlanta have the necessary collections of specific antisera for salmonellas typing. In most testing and diagnostic laboratories, *salmonella* strains are identified and classified by their fermentative characteristics and agglutination reactions using group-specific antisera.

In Mexico, for example, a study for the classification and identification of *salmonella* serotypes at public and private health centers and hospitals analyzed 24,394 *salmonella* strains isolated from different sources, 15,843 (64.9%) of human origin and 8,551 (35.1%) non-human demonstrating the usefulness of Kauffmann-White scheme and using antisera produced at the National Institute of Diagnosis and Reference (INDRE) in accordance with the Center for Disease Control and Prevention, Atlanta (GA), showing that most frequent serotypes both in human and nonhuman samples were *S.* Typhimurium*, S.* Enteritidis*, S.* Derby*, S.* Agona and *S.* Anatum. From the epidemiological point of view, it is interesting to identify which are the circulating and emerging serotypes to implement prevention strategies [8].

## **4. Pathogenicity**

*Salmonella* spp. is a highly pathogenic microorganism that presents different pathogenicity mechanisms including adherence, invasiveness, colonization and growth, toxicity and tissue damage [11]. It is a facultative intracellular pathogen causing moderate to severe infections, or even compromising systemic infections risking patients' lives, depending on the serotype, virulence, inoculum and immunological state of involved host, and all of this using only a mixture of toxins and other virulence factors.

Clinical manifestations in humans include enteric fevers, acute gastroenteritis and septicemia in extreme cases. Prototypical enteric fevers are caused by *Salmonella* Typhi, this is also known as typhoid fever, after its incubation period (7– 14 days), symptoms such as anorexia, headache, followed by general malaise and fever may occur. The interaction patient-causative agent is essential for the progression of the disease, *salmonella* must find a microhabitat suitable for its establishment, multiplication and virulence factors expression.

*Salmonella* produces at least three toxins: enterotoxin, lipopolysaccharide endotoxin (LPS), and cytotoxin. Enterotoxigenicity, which is a property present in many serotypes of this microorganism, including S. Typhi, is expressed a few hours after contact with the host cell. The pathogenicity mechanisms by which *salmonella* induces diarrhea and septicemia have not yet been clearly elucidated, but it appears to be a complex phenomenon involving numerous virulence factors such as those mentioned above.

The specific virulence factors are encoded by a group of genes for the formation of pathogenicity islands (SPI), with G + C percentages differing from the average of the bacterial genome. Direct repeats are present at the filament ends, carrying genes that encode mobility factors such as integrases, transposases or insertion sequences and are frequently inserted on tRNA. This suggests that they have been obtained from other species by horizontal transfer or by plasmids. There are numerous genes that participate in the invasion and that are present in salmonellas, genes that code for the synthesis of proteins related to the translocation of effector molecules within the cytoplasm of the host cell. Today, it is known that *salmonella* has five islands of pathogenicity: SPI-1, SPI-2, SPI-3, SPI-4 and SPI-5 [10].

#### **5. Mechanisms of resistance**

Drug resistance and worldwide incidence of *salmonella* infections has been increasingly reported. For example, it has been observed a high incidence among humans, livestock and poultry of *Salmonella enterica* serotype [4, [5],12:i:-], with variants ranging from sensitive- to multi-drug resistant, since the 1990s. Other examples include a strain of *Salmonella enterica* discovered on 2015 that was provided with the gene mcr-1 of plasmid-mediated colistin resistance and clinical isolates from Portugal, China and United Kingdom observed in 2016 with this same gene [12].

Several types of *salmonella* with multi-drug resistance (MDR) are capable of generating diverse types of plasmids, with gene cassettes that provide the property of resistance against antibiotics such as chloramphenicol, tetracycline, ampicillin, and streptomycin [13, 14]. The chromosomal mutation in the regions that determine the resistance to quinolones of the gyrA gene are responsible for the appearance of *salmonella* serotypes with little susceptibility to ciprofloxacin [15]. On the other hand, the mutated genes that code for extended spectrum β-lactamases, are responsible for the serotypes that have begun to develop resistance to cephalosporins [16].

Resistance not only by *salmonella*, but by other microorganisms are currently a public health problem worldwide, which threatens the prevention, control and treatment of innumerable infectious diseases, having as expected consequences in terms of health and economic impact. This problem was recognized by the World Health Organization and in 2001 this organization published the Global Strategy for the Containment of Antimicrobial Resistance, publicizing interventionist actions to delay the appearance and to reduce the spread of resistant microorganisms [17]. For 2012, WHO proposed a series of actions such as strengthening health services and epidemiological surveillance, regulated use of antimicrobials in hospitals and in communities, promoting the development of new drugs and appropriate vaccines, among others [18]. This problem is one of the reasons for the development of new alternatives, being natural products derived from traditional medicine, one of the most used resources.

#### **6. Traditional medicine and natural products**

The origin of Natural and Traditional Medicine is indisputably linked both to human history and to its fight for survival [19]. Written evidence on plants being used as remedies for disease is as ancient as Mesopotamian tablets, and from there, a nearly endless number of registers in all cultures, supports its essential role on human well-being. Currently, traditional medicine has been delineated as the use of products of natural origin for health preservation, having the so-called Natural Products (NPs) at its focus.

NPs are broadly defined as small molecules produced by a living organism. This definition comprises a wide variety of compounds including the synthesized during basic metabolism (primary metabolites) or as by-products of it (secondary metabolites). Lipids, carbohydrates, proteins and nucleic acids are part of the first kind of NPs, while smaller molecules such as alkaloids, tannins, saponins and flavonoids are examples of secondary metabolites. Many of the latter does not seem to have a metabolic or evolutionary function for the parental organism, but regardless to that, its utility as drugs, preservatives, dyes, food additives and/or antibiotics is undeniable. Its application to counteract the pathogenic microorganisms affecting our specie, alongside side-effects and resistance to antibacterial drugs, is undoubtedly enough motivation for the current formalization and systematization of traditional knowledge, with methodological studies being carried out very frequently nowadays.

There has been an important upturn in the study of compounds of natural origin during the last decade, supported on ethnopharmacological information, folkloric reputation, traditional uses and the existence of previous evidence, and also based on NPs chemical composition and its chemotaxonomic classification. This explosion of information has been enriched primarily through the obtention and separation of crude extracts, essential oils, and/or other types of preparations that are subsequently analyzed for possible biological activities of metabolites or secondary products. Modern experimental strategies have included bioassays (mainly in vitro), development of NP libraries, production of active compounds in cell or tissue cultures, genetic manipulation of organisms, natural combinatorial chemistry, etc. [20]. NPs, being originated in living organisms, are essentially complex mixtures contained within cellular structures, hence the first step into the study of its properties is the separation of such structures. This first step is called extraction, and is generally carried out by liquid solvents at room temperature and atmospheric pressure, along with other well-known and widely used techniques such as steam distillation and the use of supercritical fluids or pressurized gases [21]. The proper choice of an extraction step is necessarily based on the nature, origin and composition of the product to be studied, taking into account the characteristics of the possible solvents (innokenty, reactivity, etc.), toxicity of secondary products, product sufficiency needs and evaluation methods to be followed afterwards, as a whole this step should result suitable to fulfill the objective of a research. Second and third steps are the setting of an adequate model for biological efficacy assessment and the elucidation of individual bioactive components.

In this chapter, we enlisted natural products frequently reported against *salmonella* from bacteria (**Table 1**), fungus (**Table 2**), animal (**Table 3**), plant (**Table 4**) or combined (**Table 5**) origin, organized on a chronologically descending order according to publishing date. To get a glimpse on the universe of information that NPs research has become, we made a fast search on two commonly used and easily accessible databases (PubMed and Google scholar) for the terms: *salmonella, antisalmonella, salmonellosis, natural product* and *antibacterial activity*, alone or in combinations. Search results without the terms *salmonella* or *salmonellosis* were excluded. From the remaining registers, we selected those corresponding to experimental reports where the extraction step was performed and thoroughly described by authors. Studies on isolated or synthetic NPs were not included and research on infection or tissue damage protection after *salmonella* colonization were also excluded. Review articles or abstracts were not considered, although we accounted


 *Summary of frequently reported natural products from bacteria origin against* salmonella.


**Table 2.**

*Summary of frequently reported natural products from fungi origin against* salmonella.


#### Salmonella *spp. - A Global Challenge*


**Table 3.**

 *Summary of frequently reported natural products from animal origin against* salmonella*.*




#### Salmonella *spp. - A Global Challenge*







#### Salmonella *spp. - A Global Challenge*







Salmonella *spp. - A Global Challenge*








**Table 4.**

*Summary of frequently*

 *reported natural products from plant origin against*

salmonella.

## *Natural Products for Salmonellosis: Last Decade Research DOI: http://dx.doi.org/10.5772/intechopen.96207*

**197**


#### **Table 5.**

*Summary of reported natural products of combined origins against* salmonella.

### Salmonella *spp. - A Global Challenge*

congress and meeting proceedings where useful data were present. NPs and bioactive principles were registered according to the molecules isolated by the authors and/or in contrast to the literature. This search was not exhaustive, rather, we aim to obtain a random sample of information using the simplest terms on the matter of natural products for salmonellosis.

All these works were developed on all continents, being Asia the most active, followed by Africa, America, Europe and Oceania (**Figure 1A** and **B**). It is noteworthy that much of the research was developed in equatorial locations where biodiversity is abundant. Country-wise, there is a remarkable number of publications from India and Indonesia, where incidence of *salmonella* is high. The map constructed for the distribution of publishing frequencies, in fact, resulted fairly similar to a previously reported *salmonella* incidence map (**Figure 1A** versus [9]). The number of articles per year showed an upward trend though it stabilizes in the last five years (**Figure 1C**).

The spectrum of biological activities evaluated are as diverse as the application to which they are oriented, from the study of antimutagenic, antioxidant,


#### **Table 6.**

*Checklist proposed for NPs research.*

**Figure 1.** *(A) Distribution map of publishing frequencies. (B) Continental frequency. (C) Publications per year.*

anticancer, anthelmintic, antiviral, antifungal activities to its antibacterial potential, being its activity against *salmonella* spp. one of the most studied activities.

The analysis of the last decade research render studies exploring the antibacterial activity against *salmonella* serovars of crude extracts and essential oils, from compounds of natural origin, as well as their components. A wide variety of these NPs have been evaluated from commercial formulations, products of animal origin such as honey, propolis, milk and chitosan, through complete plants and/or their components (roots, stem, leaves and flowers), up until products of microbial metabolism as crude protein extracts, membrane and cell wall glycosides, natural antibiotic peptides (nisin). Several chemical compounds such as water, ethanol, methanol, acetone, formaldehyde, hexane, ethyl acetate and chloroform were used as solvents by direct maceration extraction rather than vapor distillation or more complex methods.

Nonetheless, we believe the description of methodological conditions could further standardized with the inclusion of a fixed set of data. According to our observation, the list of items enlisted in **Table 6** could be a minimal checklist when performing NP research.

#### **7. Conclusions**

Salmonellosis, caused by *salmonella* serovars, is still an uneradicated disease both in industrialized and developing countries. Multidrug resistance is a phenomenon increasingly widespread and alternative tools for disease control are urgently necessary. Natural products research based on traditional medicine is nowadays a consolidated study field full of vitality, *salmonella* research in particular has an upward trend with work being develop worldwide. Authors cited within this chapter explored biological activities of local organisms for the solution of salmonellosis for their communities, although a minority showed interested in foreign resources or commercial formulations. We observed a higher number of active researches on countries with diverse and abundant natural resources coincidentally also with high salmonellosis incidence. Even though our search is a minimal sample from the whole work being published on NPs and salmonellosis, it reveals certain features of the field.

Most of the works displayed in here are initial screening in vitro studies, maybe due to the scarce number of sources for funding in vivo applications. In perspective, NPs studies for clinical applications is a potential goal in order to control this disease.

#### **Acknowledgements**

Authors acknowledge Hospital Juarez of Mexico for giving the facilities for writing this chapter. This work was developed with no funding.

#### **Conflict of interest**

Authors declare no conflict of interests.
