Characterization of Virulence-Associated Genes,Antimicrobial Resistance Genes,and Class 1 Integrons in Salmonella enterica serovar Typhimurium Isolates from Chicken Meat and Humans in Egypt

Introduction

Salmonellosis is a worldwide health problem of great concern, with an estimate of 93.8 million human cases and 155,000 deaths each year (Hendriksen et al., 2011). Consumption of contaminated food, particularly of poultry origin, is the main route of acquiring infection with Salmonella enterica species (Thorns, 2000). Salmonella Typhimurium and Salmonella Enteritidis are the serovars commonly causing human infection, with Salmonella Typhimurium mostly associated with poultry and bovine meat consumption (Capuano et al., 2013).

The virulence potential of bacterial pathogens is influenced by the presence of both virulence-associated and antimicrobial resistance-associated genes (Capuano et al., 2013). The invasion gene invA is located in Salmonella pathogenicity islands coding for the production of proteins responsible for the invasion of the bacteria into the host cells (Valdez et al., 2009). Plasmid-encoded fimbria (pef) locus has a role in bacterial adherence to the intestinal epithelial cells (Friedrich et al., 1993), while the fimbrial gene bcfC has a role in cell invasion (Huehn et al., 2010). Induction of cell apoptosis to limit the host's inflammatory responses is mediated by the avrA gene (Ben-Barak et al., 2006; Borges et al., 2013). Moreover, Salmonella outer proteins (sops) have a role in the invasion of the bacteria through deformation of membranes and rearrangement of the host cells' cytoskeleton (Galan and Zhou, 2000; Borges et al., 2013). The mgtC gene is required for the intracellular survival of Salmonella species (Blanc-Potard and Groisman, 1997). Another virulence-associated gene is stn gene that mediates enterotoxin production and was reported to be associated with causing acute gastroenteritis (Zou et al., 2012).

The uncontrolled usage of antimicrobials in the veterinary and medical sectors has resulted in the emergence of multiple drug-resistant Salmonella species in the food production continuum (Chuanchuen et al., 2010). A variety of antimicrobial resistance genes are detected on mobile genetic elements, for instance, class 1 integrons that play an essential role in the dissemination of resistance genes due to the association with conjugative plasmids (Chen et al., 2004; Miko et al., 2005; Chuanchuen et al., 2010). Integron-associated gene cassettes can be mobilized by site-specific recombination mechanisms catalyzed by the integron-encoded integrase (intI) (Partridge et al., 2009). In addition, several nonintegron-associated resistance genes, such as chloramphenicol and tetracycline resistance determinants, have also been reported (Chen et al., 2004; Miko et al., 2005).

In Egypt, the misuse of antibiotics in humans and animals is not uncommon, resulting in the emergence of multidrug-resistant Salmonella serotypes. Therefore, the current study aimed to investigate the prevalence of virulence-associated genes and antimicrobial resistance profiles of Salmonella Typhimurium isolated from retail chicken meat and humans at Mansoura, Egypt. In addition, characterization of class 1 integron gene cassettes and other resistance-associated genes was carried out.

Materials and Methods

Results

Discussion

Salmonellosis is generally a self-limiting disease with no need for antimicrobial therapy. However, infection caused by antimicrobial-resistant strains has an impact on public health (Zou et al., 2012).

The variety of virulence factors among Salmonella serovars has resulted in differences in their pathogenicity (Fluit, 2005). The detection of invA gene in all the examined isolates is in agreement with previous reports in Egypt (Osman et al., 2013, 2014a, 2014b) and worldwide (Chuanchuen et al., 2010; Campioni et al., 2012; Borges et al., 2013; Capuano et al., 2013; Rowlands et al., 2014). These studies described this gene as a marker for the molecular detection of Salmonella serotypes by PCR. The avrA gene was detected also in 100% of the isolates. The high frequency of this gene is only observed in serovars that have a potential to cause severe salmonellosis in humans (Ben-Barak et al., 2006; Borges et al., 2013).

Our results revealed that only 6.7% of the isolates were positive for pef gene. In accordance, Chuanchuen et al. (2010) reported very low prevalence of the plasmid-associated pef gene in Salmonella isolates. In contrast, the pef operon was previously reported with high frequency in Salmonella isolates from diseased birds (Hudson et al., 2000).

The sopB gene associated with prophages was found in 96.7% of the examined isolates. Different studies have also reported the detection of the gene in almost all the Salmonella isolates of food and human origin (Dione et al., 2011; Campioni et al., 2012; Borges et al., 2013; Capuano et al., 2013). The bcfC fimbrial gene was present in all Salmonella Typhimurium isolates in the present study. Likewise, Huehn et al. (2010) reported 100% prevalence of bcfC, mgtC, and sopB genes in Salmonella Typhimurium isolates related to human health in Europe. Finally, the stn gene coding for the production of enterotoxins was reported in 100% of the isolates. In accordance, the gene was previously reported to be widely distributed among different Salmonella serovars (Murugkar et al., 2003; Zou et al., 2012).

Emergence of multidrug-resistant Salmonella species has a great impact on human health directly through interference with treatment or indirectly through dissemination of resistance elements to other pathogens (Frye and Jackson, 2013).

The current study showed high frequency of antimicrobial resistance among Salmonella Typhimurium isolates of chicken and human origin. The high resistance to AMP, TET, and TMP observed in this study was consistent with that reported in Malaysia (22–49%), Thailand (41–92%),* and Vietnam (14–62%) (Van et al., 2012). In Egypt, Ahmed et al. (2014) reported high resistance to ampicillin (95.9%), streptomycin (91.5%), and sulfonamides (91.5%). The high prevalence of TMP associated with Salmonella Typhimurium could be attributed to the increased sulfonamide usage in food-producing animals (Antunes et al., 2006).

Resistance to CHL was found to be high, although the use of such drug in food-producing animals has been banned in Egypt. Thus suggesting that the use of other drugs, such as florfenicol, a fluorinated derivative of chloramphenicol, might maintain CHL resistance (Van et al., 2012). Resistance frequency to AMP (53.3%) in this study was nearly similar to 49% reported in Thailand from poultry and swine (Khemtong and Chuanchuen, 2008).

Similar to the results in our study, a low resistance rate to CRO (9%) was reported in Salmonella isolates from retail meat in the United States (Chen et al., 2004). The low resistance rate to CRO is of concern due to the use of the drug in treatment of salmonellosis in children.

The resistance-associated genes folR, aadA2, and sul1 were detected in 73.3%, 63.3%, and 96.7% of the isolates. In accordance, high prevalence of aadA2, folR, and sul1 genes was also reported among Salmonella enterica isolates from food in Germany (Miko et al., 2005).

Significant correlation was observed among all the isolates showing phenotypic resistance to ampicillin and the existence of blaTEM gene (p < 0.05; Supplementary Table 2). This finding concurs with previously published data, which describe the association between the phenotypic resistance to penicillin class of antimicrobials and the production of TEM lactamase enzymes (Hur et al., 2011; Zou et al., 2012). The wide use of ampicillin has led to a high selective pressure for Salmonella resistance and consequently the emergence of β lactamases in pathogens (Sow et al., 2007).

Tetracycline resistance was mainly mediated by tetA(A); in our study, 60% of the examined isolates contained tetA(A) showing significant correlation, while few isolates (20%) were found to harbor tetA(B). These results are in agreement with Zou et al. (2012) who reported that tetracycline resistance was encoded by the tet(A) (60%) followed by tet(B) (20%). The significant positive correlation between the presence of resistance genes and the corresponding phenotypes indicated the expression of the identified resistance genes (Randall et al., 2004).

The gene responsible for CRO resistance is blaCMY, however, none of the phenotypically resistant isolates harbored the gene. Likewise, occasions in which phenotypic resistance to certain antimicrobials was observed without the identification of the gene conferring resistance (Randall et al., 2004) indicated the existence of other resistance mechanisms (Chuanchuen et al., 2010).

Resistance to sulfonamides is mainly mediated by sul1, which is mainly found in connection with integrons (Aarestrup et al., 2003). Thus explaining the high resistance to TMP and the existence of sul1 gene in almost all the examined isolates. It has been reported that antibiotic resistance genes could be silent in Salmonella (Randall et al., 2004), and such assumption was supported by the current findings where nine of the examined isolates were susceptible to TMP although the presence of sul1 gene.

Salmonella Typhimurium has 1 and 1.2 kb class 1 integron in its chromosome for the dissemination of antimicrobial resistance from one generation to the other (Vo et al., 2010). The high prevalence of class 1 integrons (80%) was consistent with another study in Thailand that reported the presence of class 1 integrons in 60% of Salmonella isolates (Sinwat et al., 2015). In Egypt, 96.4% of Salmonella Typhimurium isolates from meat and dairy products were reported positive for class 1 integrons (Ahmed et al., 2014).

An integrase gene (intI1) is acquired by integrons to encode a site-specific recombinase that targets the attI site of the integron and the attC site of the resistance gene cassette (Fluit and Schmitz, 1999). The obtained results showed the identification of intI1 gene in the same isolates carrying class 1 integrons. Likewise, Glenn et al. (2011) reported the presence of the intI1 gene in the same isolates carrying class 1 integrons. However, 26% and 91% of Salmonella isolates in Thailand (Khemtong and Chuanchuen, 2008) and in Norwegian hospitals (Lindstedt et al., 2003) were intI1 positive, respectively.

Twelve isolates were shown to harbor 800 bp amplicon, which showed 99% similarity to dfrA15 gene coding for trimethoprim resistance. Similar results were also reported by Sow et al. (2007).

The analysis of PCR amplicons of 1000 bp size revealed the presence of aadA2 gene, which is responsible for resistance to streptomycin and spectinomycin. These results were in agreement with those reported in Thailand (Chuanchuen et al., 2010), Italy (Carattoli et al., 2002), Norway (Lindstedt et al., 2003; Miko et al., 2005), and in different Salmonella serovars isolated from human cases in Egypt (Osman et al., 2014a). Moreover, sequencing of 1500 bp amplicon revealed the presence of aadA1 gene, and these findings are in agreement with Sow et al. (2007).

Sequence analysis of 1900 bp amplicon revealed a gene cassette array of dfrA12-OrfF-aadA27. Such unusual gene cassette was previously reported in the Salmonella Typhimurium isolate from swine in Brazil (Lopes, 2014). The gene cassette constitutes trimethoprim resistance gene (dfrA12) that encodes DrfA12 enzyme, also it carries a reading frame coding for a new variant of aadA aminoglycoside adenyltransferase (aadA27). The more common dfrA12-OrfF-aadA2 gene cassette can be distinguished from dfrA12-OrfF-aadA27 by two amino acid substitutions within the aadA gene cassette forming the new variant of aminoglycoside adenyltransferase gene (Lopes, 2014). To the best of our knowledge, this is the first study to characterize the unusual gene cassette array dfrA12-OrfF-aadA27 from Salmonella Typhimurium isolates in Egypt.

According to the identified gene cassettes in class 1 integrons, seven integron profiles were defined (Table 1). The most prevalent profile was P-V (gene cassette dfrA15, aadA2). Khemtong and Chuanchuen (2008) also identified 11 integron profiles among Salmonella isolates and dfrA12-aadA2 was the most prevalent gene cassette. Moreover, a predominance of cassettes conferring resistance to aminoglycosides and trimethoprim in Salmonella enterica isolates from Portugal was reported (Antunes et al., 2006). Another study in Thailand defined six integron profiles from Salmonella enterica isolated from humans, chicken meat, and pork, with dfrA12-aadA2 the most prevalent cassette (Sinwat et al., 2015). The presence of the same integron profile in chicken meat and humans (P-V and P-VII) is of public health concern. Such observation suggests the circulation and horizontal transfer of integrons in the food chain (Sinwat et al., 2015).

In conclusion, the results of the current study provide evidence for the development of antimicrobial resistance in zoonotic Salmonella Typhimurium isolates from chicken meat and humans posing a public health risk to consumers. This could be linked to the uncontrolled use of antibiotics in the veterinary and medical sectors. The presence of both antimicrobial and virulence determinants makes these pathogenic strains potentially more hazardous. Routine monitoring of MDR Salmonella is essential for the assessment of their health risk. Further analysis of other virulence- and resistance-associated genes and mechanisms of dissemination of resistance determinants is recommended.