**4.1.1** *Salmonella* **in feedlot cattle**

The study by Tabe et al (2010a, 2010b) reported *Salmonella* prevalence of 12.7% in fecal samples tested. A larger study (Dargatz et al 2003) that evaluated presence of *Salmonella* in fecal samples from cattle in US feedlots (73 feedlots in 12 states during the period from October 1999 to September 2000) had earlier reported a lower overall *Salmonella* prevalence of 6.3%. However, *Salmonella* prevalence at pen and feedlot level was higher. In that study (Dargatz et al 2003) although overall individual animal prevalence was 6.3% (654/10,417), 22.2% (94/422) of pens and 50.7% (37/73) of feedlots had one or more positive samples. Samples collected during the period of April to June (6.8%, 209/3054) and July to September (11.4%, 286/2500) were more likely to be positive than those collected during October to December (4.0%, 73/1838) and January to March (2.8%, 86/3025). The study by Tabe et al (2010a, 2010b) was conducted from October 2006 to March 26, 2007.

An understanding of the genetic diversity of *Salmonella* isolated from cattle could help determine if contamination at a feedlot is due to bacteria that are transient or resident (Galland et al., 2001) in their gut. Transient bacteria can be introduced into the feedlot by arriving cattle, in ingredients for cattle rations such as legume hay, from contaminated water sources, or by other animals (wild or domestic), motor vehicles, and employees (Galland et al., 2001). In the study by Tabe et al (2010a, 2010b), the isolation of *S*. Typhimurium vars Copenhagen as the major *Salmonella* serovar 95% of the time supported previous reports (Hegde et al., 2005; Khaitsa et al., 2007a) of the existence of common genotypes circulating among the steers. *Salmonella* Typhimurium vars Copenhagen which was primarily reported to be found in pigeons is now frequently isolated from cattle, swine,

Antimicrobial Drug Resistance and Molecular Characterization

gene cassettes encoding antibiotic resistance (Zhang et al, 2004).

dissemination of antimicrobial drug resistance in S. enterica.

of *Salmonella* Isolated from Domestic Animals, Humans and Meat Products 237

study missed some large amplicons and most especially integron 2, which contains some

The emergence and dissemination of MDR among *Salmonella* isolates from health cattle may have potential adverse implication in public health. Since the first description of class 1 integron by Stokes and Hall (Stokes, H.W., and R.M. Hall. 1989), integron-mediated resistance has been reported in clinical isolates of various organisms including *K. pneumoniae, K. oxytoka, Pseudomonas aeroginosa, E. coli, C. fruedii* and *V. cholerae* (Orman,et al 2002; Sallen *et al, 1995*)*.* It has been reported (Collis, et al, 2002) that classes 1 and 2 are most common in resistant bacteria, and the mobility of these integrons was undoubtedly important in facilitating their spread into many different bacterial species. A study (Krauland et al, 2009) reported that Salmonella enterica bacteria have become increasingly resistant to antimicrobial agents, partly as a result of genes carried on integrons, and that clonal expansion and horizontal gene transfer may contribute to the spread of antimicrobial drug-resistance integrons in these organisms. Krauland et al (2009) investigated this resistance and integron carriage among 90 isolates with the ACSSuT phenotype (resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline) in a global collection of S. enterica isolates. Four integrons, dfrA12/orfF/aadA2, dfrA1/aadA1, dfrA7, and arr2/blaOXA30/cmlA5/aadA2, were found in genetically unrelated isolates from 8 countries on 4 continents, which supports a role for horizontal gene transfer in the global dissemination of S. enterica multidrug resistance. Serovar Typhimurium isolates containing identical integrons with the gene cassettes blaPSE1 and aadA2 were found in 4 countries on 3 continents, which supports the role of clonal expansion. The study by Krauland et al (2009) demonstrated that clonal expansion and horizontal gene transfer contribute to the global

The 58 isolates of *Salmonella* Typhimurium var. Copenhagen reported by Tabe et al (2010a) belonged to nine PFGE profiles. Multiple genotypes were frequently observed among *Salmonella* isolated within and between pens sampled in one feedlot in this study (Tabe et al, 2010a). A similar result was reported by a previous study (Edrington et al., 2004) which highlighted the genotypic variation in *Salmonella* isolated from cattle within a farm and among four farms. Another study (Alam et al 2009) that investigated antimicrobial susceptibility profiles of 530 Salmonella enterica serotypes recovered from pens of commercial feedlot cattle reported tremendous strain diversity and multidrug resistance (MDR) among *Salmonella* recovered. This study determined antimicrobial susceptibility profiles, serotype, and presence or absence of the integron-encoded intI1 gene for 530 Salmonella isolates recovered using composite rope (n = 335), feces (n = 59), and water (n = 136) samples from 21 pens in 3 feedlots. Most isolates (83.0%) of the 19 Salmonella serotypes identified were susceptible or intermediately susceptible to all the antimicrobials evaluated. Resistance to sulfisoxazole (14.9%), streptomycin (3.8%), and tetracycline (3.6%) were the most common. None of the isolates tested positive for a class 1 integron, and only 2.5% were resistant to multiple antimicrobials. All the MDR isolates, namely, serotypes Uganda (n = 9), Typhimurium (n = 2), and Give (n = 2), were resistant to at least five antimicrobials. Most MDR isolates (n = 11) were from two pens during 1 week within one feedlot. Overall, many Salmonella isolates collected within a pen were similar in terms of serotype and antimicrobial susceptibility regardless of sample type. However, MDR Salmonella and rare serotypes were not recovered frequently enough to suggest a general strategy for appropriate composite sampling of feedlot cattle populations for Salmonella detection and monitoring. This observation offers an insight into the complexity of the population

and other animals (Frech et al., 2003). Another study (NARMS-EB, 2003) reported Typhimurium variant Copenhagen as the most predominant serotype accounting for 16.9% of the total number of isolates examined by U.S. Department of Agriculture's National Animal Health Monitoring System for Enteric Bacteria and reported over a 7-year period (1997 to 2003).

The study by Tabe et al (2010a), reported widespread AMR among the *Salmonella* isolated; all but two of the *Salmonella* isolates were resistant to more than two of the antimicrobials tested with 96.6% of the isolates showing multidrug resistant antibiograms. The widespread AMR of *Salmonella* isolated from cattle in North Dakota had been reported before (Oloya, et al, 2009*)* with most animal strains showing more multidrug resistance compared to human *Salmonella* isolates possibly due to a difference in antimicrobial selection pressure exerted to the microorganisms in the two populations. Isolation of *S*. Typhimurium vars Copenhagen as the major *Salmonella* serovar 95% of the time supports previous reports of the existence of common genotypes circulating among the steers. This similarity in clonal relationship and antimicrobial resistance of *S*. Typhimurium vars Copenhagen was reported in a study that characterized *Salmonella* isolates from feedlot cattle (Khaitsa et al, 2007a), humans, and ready to eat turkey produce (Oloya et al, 2007, 2009). This could possibly be responsible for the spread of such resistant genes among bacteria, a characteristic typical of gram negative bacteria. Surveillance of antibiotic resistance, especially of integrons distribution among bacteria is therefore critical. The genotypic variation in *Salmonella* isolated in healthy feedlot steers reported in this study plus variation in MDR antibiogram supports previous reports that not all MDR *salmonella* Typhimurium do carry a wide variety of resistance genes (Khaitsa et al, 2007a*;* White, 2005). Additionally, isolates with the same resistance phenotypes often had different resistance genotypes, a phenomenon that had been observed before by other studies (Frye and Fedorka-Cray, 2007).

In the study by Tabe et al (2010a), although the prevalence of class 1 and 2 integrons were 50% (29/58) and 35% (2/58), respectively, more than 90% of the isolates were multidrug resistant to Amoxicillin/clavulanic acid, Ampicillin, Chloramphenicol, Streptomycin, Sulfizoxazole, and Tetracycline. The lower frequency of class 2 integron relative to class 1 as seen in this study could probably result from lower exposure to selective pressure of antibiotics among the isolates (Zhao et al, 2005). Additionally, two isolates positive for integron 1 had integron 2. These isolates belonged to genotypes I and IV and showed only about 67% genomic similarity (Figure 1). Additionally, these isolates were recovered from different sampling periods (sampling time one and two respectively). It is important to note that, all 29 isolates with integron 1, were susceptible to Amikacin, Cefoxitin, Cetriaxone, Ciprofloxacin, Gentamycin, Kanamycin, Nalidixic acid, and Trimethoprim-sulfamethoxazole possibly due to the presence of defective resistant genes or the presence of quiescent integrons as reported in a previous study (Khaitsa et al, 2008). The fact that integrons 1 and 2 were not detected in some of the isolates (n=29), 93% (27/29) which were resistant to two or more of the antibiotics, with patterns similar to the positive integron isolates, may be an indication that integrons may play a sufficient but not a necessary role in antibiotic resistance in bacteria. This observation is similar to what has been reported in a previous study where class 1 integron was not always involved in the resistance of *E. coli* isolates to antimicrobial agents (Khaitsa et al, 2008). However integrons have been often associated with broad antibiotic resistance, even if they do not encode multiple drug resistant determinants (Zhang et al, 2004). This was also evident in our study as not all integron bearing strains expressed resistance to antibiotics. Additionally, it is possible that our PCR analysis as designed in this

and other animals (Frech et al., 2003). Another study (NARMS-EB, 2003) reported Typhimurium variant Copenhagen as the most predominant serotype accounting for 16.9% of the total number of isolates examined by U.S. Department of Agriculture's National Animal Health Monitoring System for Enteric Bacteria and reported over a 7-year period

The study by Tabe et al (2010a), reported widespread AMR among the *Salmonella* isolated; all but two of the *Salmonella* isolates were resistant to more than two of the antimicrobials tested with 96.6% of the isolates showing multidrug resistant antibiograms. The widespread AMR of *Salmonella* isolated from cattle in North Dakota had been reported before (Oloya, et al, 2009*)* with most animal strains showing more multidrug resistance compared to human *Salmonella* isolates possibly due to a difference in antimicrobial selection pressure exerted to the microorganisms in the two populations. Isolation of *S*. Typhimurium vars Copenhagen as the major *Salmonella* serovar 95% of the time supports previous reports of the existence of common genotypes circulating among the steers. This similarity in clonal relationship and antimicrobial resistance of *S*. Typhimurium vars Copenhagen was reported in a study that characterized *Salmonella* isolates from feedlot cattle (Khaitsa et al, 2007a), humans, and ready to eat turkey produce (Oloya et al, 2007, 2009). This could possibly be responsible for the spread of such resistant genes among bacteria, a characteristic typical of gram negative bacteria. Surveillance of antibiotic resistance, especially of integrons distribution among bacteria is therefore critical. The genotypic variation in *Salmonella* isolated in healthy feedlot steers reported in this study plus variation in MDR antibiogram supports previous reports that not all MDR *salmonella* Typhimurium do carry a wide variety of resistance genes (Khaitsa et al, 2007a*;* White, 2005). Additionally, isolates with the same resistance phenotypes often had different resistance genotypes, a phenomenon that had been observed

In the study by Tabe et al (2010a), although the prevalence of class 1 and 2 integrons were 50% (29/58) and 35% (2/58), respectively, more than 90% of the isolates were multidrug resistant to Amoxicillin/clavulanic acid, Ampicillin, Chloramphenicol, Streptomycin, Sulfizoxazole, and Tetracycline. The lower frequency of class 2 integron relative to class 1 as seen in this study could probably result from lower exposure to selective pressure of antibiotics among the isolates (Zhao et al, 2005). Additionally, two isolates positive for integron 1 had integron 2. These isolates belonged to genotypes I and IV and showed only about 67% genomic similarity (Figure 1). Additionally, these isolates were recovered from different sampling periods (sampling time one and two respectively). It is important to note that, all 29 isolates with integron 1, were susceptible to Amikacin, Cefoxitin, Cetriaxone, Ciprofloxacin, Gentamycin, Kanamycin, Nalidixic acid, and Trimethoprim-sulfamethoxazole possibly due to the presence of defective resistant genes or the presence of quiescent integrons as reported in a previous study (Khaitsa et al, 2008). The fact that integrons 1 and 2 were not detected in some of the isolates (n=29), 93% (27/29) which were resistant to two or more of the antibiotics, with patterns similar to the positive integron isolates, may be an indication that integrons may play a sufficient but not a necessary role in antibiotic resistance in bacteria. This observation is similar to what has been reported in a previous study where class 1 integron was not always involved in the resistance of *E. coli* isolates to antimicrobial agents (Khaitsa et al, 2008). However integrons have been often associated with broad antibiotic resistance, even if they do not encode multiple drug resistant determinants (Zhang et al, 2004). This was also evident in our study as not all integron bearing strains expressed resistance to antibiotics. Additionally, it is possible that our PCR analysis as designed in this

before by other studies (Frye and Fedorka-Cray, 2007).

(1997 to 2003).

study missed some large amplicons and most especially integron 2, which contains some gene cassettes encoding antibiotic resistance (Zhang et al, 2004).

The emergence and dissemination of MDR among *Salmonella* isolates from health cattle may have potential adverse implication in public health. Since the first description of class 1 integron by Stokes and Hall (Stokes, H.W., and R.M. Hall. 1989), integron-mediated resistance has been reported in clinical isolates of various organisms including *K. pneumoniae, K. oxytoka, Pseudomonas aeroginosa, E. coli, C. fruedii* and *V. cholerae* (Orman,et al 2002; Sallen *et al, 1995*)*.* It has been reported (Collis, et al, 2002) that classes 1 and 2 are most common in resistant bacteria, and the mobility of these integrons was undoubtedly important in facilitating their spread into many different bacterial species. A study (Krauland et al, 2009) reported that Salmonella enterica bacteria have become increasingly resistant to antimicrobial agents, partly as a result of genes carried on integrons, and that clonal expansion and horizontal gene transfer may contribute to the spread of antimicrobial drug-resistance integrons in these organisms. Krauland et al (2009) investigated this resistance and integron carriage among 90 isolates with the ACSSuT phenotype (resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline) in a global collection of S. enterica isolates. Four integrons, dfrA12/orfF/aadA2, dfrA1/aadA1, dfrA7, and arr2/blaOXA30/cmlA5/aadA2, were found in genetically unrelated isolates from 8 countries on 4 continents, which supports a role for horizontal gene transfer in the global dissemination of S. enterica multidrug resistance. Serovar Typhimurium isolates containing identical integrons with the gene cassettes blaPSE1 and aadA2 were found in 4 countries on 3 continents, which supports the role of clonal expansion. The study by Krauland et al (2009) demonstrated that clonal expansion and horizontal gene transfer contribute to the global dissemination of antimicrobial drug resistance in S. enterica.

The 58 isolates of *Salmonella* Typhimurium var. Copenhagen reported by Tabe et al (2010a) belonged to nine PFGE profiles. Multiple genotypes were frequently observed among *Salmonella* isolated within and between pens sampled in one feedlot in this study (Tabe et al, 2010a). A similar result was reported by a previous study (Edrington et al., 2004) which highlighted the genotypic variation in *Salmonella* isolated from cattle within a farm and among four farms. Another study (Alam et al 2009) that investigated antimicrobial susceptibility profiles of 530 Salmonella enterica serotypes recovered from pens of commercial feedlot cattle reported tremendous strain diversity and multidrug resistance (MDR) among *Salmonella* recovered. This study determined antimicrobial susceptibility profiles, serotype, and presence or absence of the integron-encoded intI1 gene for 530 Salmonella isolates recovered using composite rope (n = 335), feces (n = 59), and water (n = 136) samples from 21 pens in 3 feedlots. Most isolates (83.0%) of the 19 Salmonella serotypes identified were susceptible or intermediately susceptible to all the antimicrobials evaluated. Resistance to sulfisoxazole (14.9%), streptomycin (3.8%), and tetracycline (3.6%) were the most common. None of the isolates tested positive for a class 1 integron, and only 2.5% were resistant to multiple antimicrobials. All the MDR isolates, namely, serotypes Uganda (n = 9), Typhimurium (n = 2), and Give (n = 2), were resistant to at least five antimicrobials. Most MDR isolates (n = 11) were from two pens during 1 week within one feedlot. Overall, many Salmonella isolates collected within a pen were similar in terms of serotype and antimicrobial susceptibility regardless of sample type. However, MDR Salmonella and rare serotypes were not recovered frequently enough to suggest a general strategy for appropriate composite sampling of feedlot cattle populations for Salmonella detection and monitoring. This observation offers an insight into the complexity of the population

Antimicrobial Drug Resistance and Molecular Characterization

with no significant trends noted.

of *Salmonella* Isolated from Domestic Animals, Humans and Meat Products 239

The United States National Animal Health Monitoring System's Dairy '96 study reported 54% of milk cows shed *Salmonella* and 275% of dairy operations had at least one cow shedding *Salmonella* [Wells et al, 1998; NAHMS, 1996]. *Salmonella* has been isolated from all ages of dairy cattle and throughout the production process. Mature dairy cattle typically appear asymptomatic while shedding this pathogen in their faeces (Richardson, 1975; McDonough, 1986; Edrington, 2004; Edrington et al, 2004) and while young calves are more susceptible to salmonellosis, cases in adult cattle have been reported (Gay and Hunsaker, 1993; Anderson, 1997; Sato, 2001). Previous research demonstrated significant variation in the prevalence of faecal *Salmonella* in healthy, lactating dairy cattle, not only among farms across the United States (Edrington et al, 2008) but also in farms within a small geographic area and in individual farms from season to season (Edrington et al, 2004 ) . Additional research examined production parameters (heifers *vs*. mature cows, lactation status, stage of lactation and heat stress) on *Salmonella* prevalence (Edrington, 2004; Fitzgerald et al, 2003). While minor differences were noted in *Salmonella* shedding, results were generally inconsistent

As part of a national study of US dairy operations, another study (Blau et al 2005) conducted between March and September 2002, in 97 dairy herds in 21 states reported an overall prevalence of 7.3% of fecal samples that were culture positive for *Salmonella.* In another study of dairy cattle (Warnick et al. 2003) , *Salmonella* was isolated from 9.3% of 4049 fecal samples collected from a 2 months study of 12 dairy herds originating from Michigan, Minnesota, New York and Wisconsin(Warnick et al, 2003). Also, Fossler et al (2004) sampled dairy cattle to describe the occurrence of fecal shedding, persistence of shedding over time, and serogroup classification of *Salmonella* spp on a large number of dairy farms of various sizes. The design was that of a longitudinal study and the sample population comprised 22,417 fecal samples from cattle and 4,570 samples from the farm environment on 110 organic and conventional dairy farms in Minnesota, Wisconsin, Michigan, and NewYork. Five visits were made to each farm at 2-month intervals from August 2000 to October 2001. Fecal samples from healthy cows, calves, and other targeted cattle groups and samples from bulk tank milk, milk line filters, water, feed sources, and pen floors were collected at each visit. *Salmonella* spp were isolated from 4.8% of fecal samples and 5.9% of environmental

Results from the various studies conducted indicated some variability in the prevalence of fecal shedding of *Salmonella* among the different cattle and production systems sampled possibly due to several factors such as state of origin, treatment with antimicrobials, herd size and season that have previously been reported (Fossler et al, 2005). The study by Fossler et al (2005) that investigated environmental sample-level factors associated with the presence of *Salmonella* in a multi-state study of conventional and organic dairy farms reported that State of origin was associated with the presence of *Salmonella* in samples from cattle and the farm environment; Midwestern states were more likely to have *Salmonella*positive samples compared to New York. Cattle treated with antimicrobials within 14 days of sampling were more likely to be *Salmonella*-negative compared with nontreated cattle (OR=2.0, 95% CI: 1.1, 3.4). Farms with at least 100 cows were more likely to have *Salmonella*positive cattle compared with smaller farms (OR=2.6, 95% CI: 1.4, 4.6). Season was associated with *Salmonella* shedding in cattle, and compared to the winter period, summer had the highest odds for shedding (OR=2.4, 95% CI: 1.5, 3.7), followed by fall (OR=1.9, 95% CI: 1.2, 3.1) and spring (OR=1.8, 95% CI: 1.2, 2.6). Environmental samples significantly more likely to be *Salmonella*-positive (compared to bulk tank milk) included, in descending order,

samples; 92.7% of farms had at least 1 *Salmonella*-positive sample.

dynamics of foodborne pathogens in food animals preharvest and demonstrates their variability in terms of shedding and environmental contamination (Edrington et al., 2004). In order to reduce the prevalence of foodborne pathogens in food animals at slaughter (which could produce significant reductions in the food supply; Hynes et al., 2000), a thorough understanding of the population dynamics of *Salmonella* at the farm level is crucial before implementation of pathogen reduction strategies can be expected to be successful (Edrington et al., 2004).
