**1. Introduction**

214 Salmonella – A Dangerous Foodborne Pathogen

http://www.cdc.gov/salmonella/outbreaks.html

www.cdc.gov/salmonella/saintpaul/jalapeno/map.html www.ers.usda.gov/briefing/vegetables/tomatoes.htm

> Infections with nontyphoid *Salmonella enterica* serovars represent an important public health problem worldwide (Zhao et al. 2003) and an economic burden in many parts of the world today (Gomez et al 1997; Vugia et al, 2004). In the United States (US), *Salmonella* is the second most common identifiable cause of illness, and the leading cause of hospitalizations and deaths, due to food-borne bacterial infection (Mead et al, 1999). Each year, 31 major known pathogens acquired in the US caused an estimated 9.4 million episodes of foodborne illness (Scallan et al, 2011), and an estimated 38.4 million episodes of domestically acquired foodborne illness were caused by unspecified agents, resulting in 71,878 hospitalizations and 1,686 deaths (Scallan et al, 2011). The annual economic cost due to foodborne *Salmonella* infections in the US alone is estimated at \$2.4 billion (http://www.ers.usda.gov) with an estimated 1.4 million cases of salmonellosis and over 500 deaths annually (Arshad et al. 2007). In 2004 for instance, among 3686 *Salmonella* isolates serotyped, 862 (23%) were serotype Typhimurium, 565 (15%) Enteritidis, 399 (11%) Newport and 248 (7%) Heidelberg (CDC, 2005). Similarly, the same *Salmonella enterica* serovars were reported as major causes of salmonellosis in humans in another study (Oloya et al. 2007). The predominance of *S. Typhimurium* and *S. Newport* in both domestic animals and human case reports further highlights their role in causing cross infections (Arshad et al. 2007; Bacon et al. 2002; Besser et al. 2000).

> Although human salmonellosis has been associated with exposure to other vehicles of transmission (e.g. pets, reptiles, and contaminated water), about 95% of human infections have been found to be associated with ingestion of contaminated foods; namely animal products (Gaul et al. 2007; McLaughlin et al. 2006; Padungtod and Kaneene 2006), poultry products (Plym and Wierup 2006; Mead et al. 1999), sea foods (Duran and Marshall 2005; Ozogul et al. 2007; Shabarinath et al. 2007) and fresh produce (Johnston et al. 2006; Puohiniemi et al. 1997). Direct contact with companion and food animals has also been documented as another important route of *Salmonella* transmission to humans (Coburn et al. 2006; Doyle and Erickson 2006; Gorman and Adley 2004; Mead et al. 1999; Padungtod and Kaneene 2006). Consumption of raw or undercooked ground beef and lack of safe food handling practices to prevent cross contamination are considered critical in infections at household levels (Ling et al. 2001). These reports highlight the possibility of increased

Antimicrobial Drug Resistance and Molecular Characterization

technique (Swaminathan, et al., 2001).

from food animals to humans (Fey et al, 2000).

bacterial infection in the US (Mead et al, 1999).

**1.1 Aim of chapter** 

of *Salmonella* Isolated from Domestic Animals, Humans and Meat Products 217

including non-typhoidal *Salmonella* serotypes has been using standardized PFGE

Most people who suffer from *Salmonella* infections usually present with temporary gastroenteritis that usually does not require treatment. However, when infection becomes invasive, antimicrobial treatment is mandatory (Winokur et al, 2000). As a result, traditionally ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole were used to treat such severe cases. However, the increasing number of antimicrobial-resistant *Salmonella* strains has led to a decrease in the efficacy of these treatments (Angulo et al, 2000). Additionally, the frequency of isolation of *Salmonella* strains resistant to one or more antimicrobial agents has risen in the US (Fey et al, 2000), and elsewhere in the world (Al-Tawfiq, 2007). Fluoroquinolones and broad-spectrum cephalosporins have been employed most recently, as the preferred drugs for treatment of adults and children, respectively, due to the low number of *Salmonella* isolates showing resistance to these drugs (Angulo et al, 2000; Chiappini et al, 2002*)*. However, the viability of these drugs may be diminishing as *Salmonella* strains producing β-lactamases conferring resistance to broad-spectrum cephalosporins have been isolated from clinical patients (Dunne et al, 2000; Winokur et al, 2000), some of which have been acquired from cattle (Fey et al, 2000). The situation is reported to be more complex and difficult in developing countries where there is a widespread misuse of antimicrobials both in human and veterinary medicine practices (Okeke et al, 2005). Furthermore, resistance to combinations of several classes of antimicrobials has led to the emergence of multidrug-resistant (MDR) strains that may pass

The spread of antibiotic resistance among bacteria have been associated with mobile genetic elements such as plasmids, transposons (Zhang et al, 2004) and integrons (Miko et al, 2005). Notably, MDR has been frequently linked with microbial genomic elements known as integrons, which have the ability to distribute genes encoding resistance to a number of antimicrobial drugs (Miko et al, 2005). Integrons do have specific structures and can capture genes notably those encoding antimicrobial resistance by a site-specific recombination system and have been located in both chromosomal and extra chromosomal DNA (Bennet, 1999; Hall and Collis 1995). The main classes of integrons are found in the family *Enterobacteriaceae* with class 1 integrons being the most extensively studied. Class 1 integrons are characterized by presence of two conserved segments, the 5′ -conserved segment (5′ -CS) and 3'-conserved segment (3'-CS) (Bennet, 1999), and are defined by an *intI* gene encoding integrase, a recombinant site *attI,* and a strong promoter*.* Previous studies (Zhang et al, 2004; Zhao et al, 2005) on integrons and associated antimicrobial resistance genes in *Salmonella* revealed a predominance of gene cassettes that confer resistance to aminoglycosides and trimethoprim, with *aadA* genes carried by all the integrons-containing *Salmonella* serovars. The investigation of multi-drug-resistance in foodborne pathogens in general and *Salmonella* in particular is essential for the proper understanding of the epidemiology of emerging multidrug resistance in *Salmonella* serovars (Winokur et al, 2000). The implications of therapeutic failure in public health due to multidrug resistance is particularly important given that *Salmonella* is the leading cause of hospitalizations and deaths, due to food-borne

This chapter will 1) describe prevalence, antimicrobial drug resistance (AMR) and molecular characterization of *Salmonella* commonly isolated from domestic animals, humans and meat

transmission of these organisms to humans through the food chain (Zhao et al. 2003). Understanding the association between human salmonellosis cases, animal sources and the environment is an important epidemiological factor needed to successfully control the spread of the infection within communities (Ling et al. 2001).

Recently, emergence of resistant and multi-resistant bacteria has become an important worldwide sanitary problem, impacting both veterinary medicine and public health through the potential for therapeutic failures (Lathers, 2001). Antimicrobial resistance among bacterial isolates from animals is also of concern because of the potential for these organisms to be food-borne or zoonotic pathogens or to be donors of resistance genes to human pathogens (Lathers, 2001). For instance, multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104, resistant to ampicillin, chloramphenicol/florfenicol, streptomycin, sulfonamides, and tetracycline, has disseminated worldwide (Mulvey et al, 2006). The resistance genes reside on the 43-kb Salmonella genomic island 1 (SGI1), which is transferable. Drug-resistant variants of SGI1 have been identified in numerous serotypes. Strains harboring SGI1 may be more virulent and have a tendency to rapidly disseminate (Mulvey et al, 2006).

International agencies, such as the World Health Organization (WHO) have recommended improving resistance surveillance studies in not only human but also animal origin strains (WHO, 2005). Because of its ubiquitous characteristics and zoonotic nature, *Salmonella spp*. can be used as a good indicator microorganism for resistance surveillance studies (Usera, et al, 2002). Yet there is little information available on *Salmonella* isolates from healthy animals on farms across a wide geographic area that uses various production practices (Dargatz, et al, 2002). This chapter will examine the genotypic relatedness of *Salmonella* serovars commonly isolated from domestic animals raised under different production systems, meat products and humans in order to quantify their role in causing human infection. Antimicrobial drug resistance (AMR) and genetic profiles of *Salmonella* will be used to assess their role in transferring drug resistance to humans.

Reliable and powerful typing methods are necessary in order to gain insight into the infection routes of pathogenic microorganisms. Traditionally, *Salmonella* serotyping combined with various molecular techniques such as phage typing, plasmid profiles, pulsed field gel electrophoresis (PFGE) (Gaul et al. 2007; Guerra et al. 2000; Pickard et al. 2008; Rabsch 2007; Trung et al. 2007) have been used to establish this association. The PFGE method particularly has been found to be very discriminatory and reproducible (Guerra et al. 2000; Tsen et al. 2002) and useful in epidemiological analysis of *Salmonella* infections (Refsum et al. 2002) to determine the relatedness of individual cases (Kim et al. 2007), detect and establish outbreaks (Puohiniemi et al. 1997; Xercavins et al. 1997) and determine linkage between human salmonellosis and consumption of foods of animal origin (McLaughlin et al. 2006). PFGE is increasingly being used as well to identify multidrug resistant strains (Bacon et al. 2002; Besser et al. 2000; McLaughlin et al. 2006; Santos et al. 2007). In fact, the method allows for the detection of DNA polymorphisms that were previously undetected by other techniques (Santos et al. 2007). Also, PFGE has been widely used to investigate the ecology of foodborne pathogens at various points along the food chain (Avery et al., 2002; Vali et al., 2005). This technique has also been used to evaluate the genetic diversity in *Salmonella* isolates from humans, animals, and the environment (Refsum et al., 2002; Gaul et al,2007), and from oysters (Brands et al., 2005). PFGE using XbaI restriction was used by Gaul et al (2007) for screening and identifying swine Salmonella serotypes. Additionally, in the US, molecular subtyping network for foodborne bacterial diseases

transmission of these organisms to humans through the food chain (Zhao et al. 2003). Understanding the association between human salmonellosis cases, animal sources and the environment is an important epidemiological factor needed to successfully control the

Recently, emergence of resistant and multi-resistant bacteria has become an important worldwide sanitary problem, impacting both veterinary medicine and public health through the potential for therapeutic failures (Lathers, 2001). Antimicrobial resistance among bacterial isolates from animals is also of concern because of the potential for these organisms to be food-borne or zoonotic pathogens or to be donors of resistance genes to human pathogens (Lathers, 2001). For instance, multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104, resistant to ampicillin, chloramphenicol/florfenicol, streptomycin, sulfonamides, and tetracycline, has disseminated worldwide (Mulvey et al, 2006). The resistance genes reside on the 43-kb Salmonella genomic island 1 (SGI1), which is transferable. Drug-resistant variants of SGI1 have been identified in numerous serotypes. Strains harboring SGI1 may be more virulent and have a tendency to rapidly disseminate

International agencies, such as the World Health Organization (WHO) have recommended improving resistance surveillance studies in not only human but also animal origin strains (WHO, 2005). Because of its ubiquitous characteristics and zoonotic nature, *Salmonella spp*. can be used as a good indicator microorganism for resistance surveillance studies (Usera, et al, 2002). Yet there is little information available on *Salmonella* isolates from healthy animals on farms across a wide geographic area that uses various production practices (Dargatz, et al, 2002). This chapter will examine the genotypic relatedness of *Salmonella* serovars commonly isolated from domestic animals raised under different production systems, meat products and humans in order to quantify their role in causing human infection. Antimicrobial drug resistance (AMR) and genetic profiles of *Salmonella* will be used to assess

Reliable and powerful typing methods are necessary in order to gain insight into the infection routes of pathogenic microorganisms. Traditionally, *Salmonella* serotyping combined with various molecular techniques such as phage typing, plasmid profiles, pulsed field gel electrophoresis (PFGE) (Gaul et al. 2007; Guerra et al. 2000; Pickard et al. 2008; Rabsch 2007; Trung et al. 2007) have been used to establish this association. The PFGE method particularly has been found to be very discriminatory and reproducible (Guerra et al. 2000; Tsen et al. 2002) and useful in epidemiological analysis of *Salmonella* infections (Refsum et al. 2002) to determine the relatedness of individual cases (Kim et al. 2007), detect and establish outbreaks (Puohiniemi et al. 1997; Xercavins et al. 1997) and determine linkage between human salmonellosis and consumption of foods of animal origin (McLaughlin et al. 2006). PFGE is increasingly being used as well to identify multidrug resistant strains (Bacon et al. 2002; Besser et al. 2000; McLaughlin et al. 2006; Santos et al. 2007). In fact, the method allows for the detection of DNA polymorphisms that were previously undetected by other techniques (Santos et al. 2007). Also, PFGE has been widely used to investigate the ecology of foodborne pathogens at various points along the food chain (Avery et al., 2002; Vali et al., 2005). This technique has also been used to evaluate the genetic diversity in *Salmonella* isolates from humans, animals, and the environment (Refsum et al., 2002; Gaul et al,2007), and from oysters (Brands et al., 2005). PFGE using XbaI restriction was used by Gaul et al (2007) for screening and identifying swine Salmonella serotypes. Additionally, in the US, molecular subtyping network for foodborne bacterial diseases

spread of the infection within communities (Ling et al. 2001).

their role in transferring drug resistance to humans.

(Mulvey et al, 2006).

including non-typhoidal *Salmonella* serotypes has been using standardized PFGE technique (Swaminathan, et al., 2001).

Most people who suffer from *Salmonella* infections usually present with temporary gastroenteritis that usually does not require treatment. However, when infection becomes invasive, antimicrobial treatment is mandatory (Winokur et al, 2000). As a result, traditionally ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole were used to treat such severe cases. However, the increasing number of antimicrobial-resistant *Salmonella* strains has led to a decrease in the efficacy of these treatments (Angulo et al, 2000). Additionally, the frequency of isolation of *Salmonella* strains resistant to one or more antimicrobial agents has risen in the US (Fey et al, 2000), and elsewhere in the world (Al-Tawfiq, 2007). Fluoroquinolones and broad-spectrum cephalosporins have been employed most recently, as the preferred drugs for treatment of adults and children, respectively, due to the low number of *Salmonella* isolates showing resistance to these drugs (Angulo et al, 2000; Chiappini et al, 2002*)*. However, the viability of these drugs may be diminishing as *Salmonella* strains producing β-lactamases conferring resistance to broad-spectrum cephalosporins have been isolated from clinical patients (Dunne et al, 2000; Winokur et al, 2000), some of which have been acquired from cattle (Fey et al, 2000). The situation is reported to be more complex and difficult in developing countries where there is a widespread misuse of antimicrobials both in human and veterinary medicine practices (Okeke et al, 2005). Furthermore, resistance to combinations of several classes of antimicrobials has led to the emergence of multidrug-resistant (MDR) strains that may pass from food animals to humans (Fey et al, 2000).

The spread of antibiotic resistance among bacteria have been associated with mobile genetic elements such as plasmids, transposons (Zhang et al, 2004) and integrons (Miko et al, 2005). Notably, MDR has been frequently linked with microbial genomic elements known as integrons, which have the ability to distribute genes encoding resistance to a number of antimicrobial drugs (Miko et al, 2005). Integrons do have specific structures and can capture genes notably those encoding antimicrobial resistance by a site-specific recombination system and have been located in both chromosomal and extra chromosomal DNA (Bennet, 1999; Hall and Collis 1995). The main classes of integrons are found in the family *Enterobacteriaceae* with class 1 integrons being the most extensively studied. Class 1 integrons are characterized by presence of two conserved segments, the 5′ -conserved segment (5′ -CS) and 3'-conserved segment (3'-CS) (Bennet, 1999), and are defined by an *intI* gene encoding integrase, a recombinant site *attI,* and a strong promoter*.* Previous studies (Zhang et al, 2004; Zhao et al, 2005) on integrons and associated antimicrobial resistance genes in *Salmonella* revealed a predominance of gene cassettes that confer resistance to aminoglycosides and trimethoprim, with *aadA* genes carried by all the integrons-containing *Salmonella* serovars. The investigation of multi-drug-resistance in foodborne pathogens in general and *Salmonella* in particular is essential for the proper understanding of the epidemiology of emerging multidrug resistance in *Salmonella* serovars (Winokur et al, 2000). The implications of therapeutic failure in public health due to multidrug resistance is particularly important given that *Salmonella* is the leading cause of hospitalizations and deaths, due to food-borne bacterial infection in the US (Mead et al, 1999).
