Detection and Characterization of Shiga Toxin Producing Escherichia coli, Salmonella spp., and Yersinia Strains from Human, Animal, and Food Samples in San Luis, Argentina

Shiga toxin producing Escherichia coli (STEC), Salmonella spp., and Yersinia species was investigated in humans, animals, and foods in San Luis, Argentina. A total of 453 samples were analyzed by culture and PCR. The antimicrobial susceptibility of all the strains was studied, the genomic relationships among isolates of the same species were determined by PFGE, and the potencial virulence of Y. enterocolitica strains was analyzed. Yersinia species showed higher prevalence (9/453, 2.0%, 95% CI, 0.7–3.3%) than STEC (4/453, 0.9%, 95% CI, 0–1.8%) and Salmonella spp. (3/453, 0.7%, 95% CI, 0–1.5%). Y. enterocolitica and Y. intermedia were isolated from chicken carcasses (6/80, 7.5%, 95% CI, 1.5–13.5%) and porcine skin and bones (3/10, 30%, 95% CI, 0–65%). One STEC strain was recovered from human feces (1/70, 1.4%, 95% CI, 0–4.2%) and STEC stx1/stx2 genes were detected in bovine stools (3/129, 2.3%, 95% CI, 0–5.0%). S. Typhimurium was isolated from human feces (1/70, 1.4%, 95% CI, 0–4.2%) while one S. Newport and two S. Gaminara strains were recovered from one wild boar (1/3, 33%, 95% CI, 0–99%). The knowledge of prevalence and characteristics of these enteropathogens in our region would allow public health services to take adequate preventive measures.


Introduction
The detection and characterization of Shiga toxin producing Escherichia coli (STEC), Salmonella spp., and Yersinia enterocolitica strains in human patients, animal reservoirs, and foods of animal origin intended for human consumption are relevant to public health. These organisms are transmitted through contaminated drinking water and food and can cause intestinal and extraintestinal clinical manifestations in humans [1,2]. STEC is associated with hemorrhagic colitis and hemolytic uremic syndrome (HUS); its pathogenicity is attributed to virulence factors that facilitate effective colonization of the human gastrointestinal tract and subsequent release of Shiga toxins [3]. Argentina has the highest incidence of HUS in the world, with E. coli O157:H7 as the primary etiological agent [4]. Previous studies in our country International Journal of Microbiology the clinical laboratories of Argentina, a systematic epidemiological study of Y. enterocolitica in environment, reservoirs, foods, and humans is still pending.
For contributing to the knowledge of prevalence and distribution of these enteropathogens in patients, presumable animal reservoirs, and foods of our region, this study was aimed (i) to detect STEC, Salmonella spp., and Y. enterocolitica in human and animal feces and foods of animal origin intended for human consumption, (ii) to assess the pathogenic potential of Y. enterocolitica strains through phenotypic and molecular virulence markers, (iii) to test the antimicrobial susceptibility of all strains, and (iv) to determine possible genetic relationships among isolates of each species by subtyping using pulsed field gel electrophoresis (PFGE). In addition, counts of total coliforms were performed in samples of foods. Sor-/ glu-/E-Hly+/eae+, biotype C, producer of Stx1 and Stx2, was employed as positive control in PCR targeting STEC stx1/stx2 genes and Salmonella Braenderup H9812 was used as molecular size marker in PFGE. Y. enterocolitica B1A O:5 CLO229 and Y. enterocolitica B1A O:6,30 CLO225, local strains isolated from sausages, were utilized as positive controls in ystB gene PCR [8]. These strains were maintained at 4 ∘ C on trypticase soy agar slants (TSA, Merck, Darmstadt, Germany). For determining culture purity, isolations on Mac Conkey agar (MC, Merck), Sorbitol Mac Conkey agar (SMAC, Merck), and Salmonella Shigella agar (SS, Merck) for Y. enterocolitica, E. coli O157:H7, and S. Braenderup, respectively, were made prior to each experiment. Plates were incubated 48 h at 25 ∘ C for Y. enterocolitica and 24 h at 37 ∘ C for E. coli O157:H7 and S. Braenderup.

Samples.
A total of 453 samples from human and animal sources in San Luis city and upcoming rural areas, Argentina, were analyzed during the period June 2008-November 2011. They included 70 samples of human stools from patients with enterocolitis symptoms attending a local clinical laboratory; 167 stool samples obtained from feedlot bovines ( = 61), grazing bovines ( = 68), porcine ( = 20), ovine ( = 10), goats ( = 6), and equines ( = 2); and 216 samples of animal origin intended for human consumption such as chicken carcasses ( = 80), porcine skin and bones ( = 10), goat cheeses ( = 30), fresh sausages "chorizos" ( = 90), and six samples of three wild boars, tongues ( = 3), and tonsils ( = 3). Samples of animal stools were randomly collected in regional cattle markets and farms immediately after defecation. Samples of wild boars were hunt products from our region. Samples of foods of animal origin intended for human consumption were purchased at five retail markets in San Luis city. All samples were packed in individual sterile plastic bags and stored at 4 ∘ C for up to 6 h before processing.

Investigation of Total Coliforms.
The presence of total coliforms was investigated in samples of animal origin intended for human consumption, except tonsils and tongues of wild boars. Decimal dilutions of each sample were prepared in 0.1% peptone water pH 7.2 (PW, Merck); one milliliter of each dilution was seeded in violet-lactose-neutral red-bile agar (VLRB, Merck) and incubated at 37 ∘ C for 24 h. Counts of characteristic colonies were performed and results were expressed as log 10 CFU/g. DNA extraction was performed as described [9]. A nested-PCR targeted to Y. enterocolitica yadA gene [11] was applied using two sets of primers. Five microliters of the template was used for the first PCR and 2 L of the product obtained in this step was used as template for the second PCR. The reaction mixture (50 mL) contained 1X PCR buffer, 1.

2.8.2.
Simple ystB PCR. DNA extraction was performed by the boiling method [9]. The reaction mixture (25 L) contained PCR buffer 1X, 200 M of each dNTP, 1.5 mM MgCl 2 , 0.08 U/ L of Taq DNA-polymerase, and 1 pmol/ L of each primer (Productos Biológicos). It used the primer pair constituted by YstB-F 5 GTACATTAGGCCAAGAGACG 3 and YstB-R 5 GCAACATACCTCACAACACC 3 for amplifying a 146 bp fragment [12]. The PCR product was electrophoresed at 80 V for 40 min in a 2% agarose gel stained with GelRed and visualized in an UV transiluminator (UVP). The molecular mass of amplicons was determined as described above.
A sample was considered "STEC positive" when E. coli colonies carrying stx1/stx2 genes could be recovered by culturing, or "presumptive STEC positive" when only stx1/stx2 signals were detected by PCR on DNA extracted from confluent growth on SMAC. Thus, four samples were positive: one stool sample from a pediatric patient with diarrhea (1/70, 1.4%, 95% CI, 0-4.2%) yielded one E. coli O157:H7 strain by culture on SMAC which was characterized as stx2 + by PCR and three samples of bovine stools (3/129, 2.3%, 95% CI, 0-5.0%) that amplified stx genes from DNA extracted   International Journal of Microbiology from confluent growth (two samples were stx1 + /stx2 + and the third one was stx2 + ). Individual STEC colonies could not be isolated from these samples.

PFGE.
XbaI-restricted DNA polymorphisms of Salmonella isolates are observed in Figure 1. Two major clusters, A and B, with a 65% similarity were obtained. Even though six S. Newport isolates from tonsil and five S. Gaminara isolates from tonsil and tongue were initially recovered from one wild boar, the analysis of their DNA restriction profiles by PFGE showed that all S. Newport strains grouped in cluster A while all S. Gaminara ones grouped in the genotype GTB1 within cluster B. Since identical DNA band patterns between isolates of the same serovar were observed, only three Salmonella strains were reported ( Table 2). In dendrogram, S. Typhimurium of human source was included in GTB2 within cluster B, showing 68% similarity with GTB1.
Although fifteen Yersinia isolates were originally recovered from nine positive samples, the analysis of DNA restriction profiles observed by PFGE allowed to conclude that some isolates were replicates of the same strain. Therefore, bacterial isolates were grouped into two major clusters, A and B (63% similarity), according to Yersinia species (Figure 2

Discussion
Total coliforms are considered indicators of hygienic quality and their presence in foods may correlate with the presence of pathogenic bacteria. The low total coliform counts observed in porcine skin and bones, and goat cheeses might be attributed to the effects of thermal treatments applied to pig carcasses during slaughtering, and pasteurization and preservation of dairy products, respectively. No microbiological specifications for porcine skin and bones are included in the Argentinean Alimentary Code (AAC, http://www.anmat.gov .ar/alimentos/normativas alimentos caa.asp, accessed November 2013). On the other hand, values up to 500 total coliforms per gram at 45 ∘ C are allowed for cheeses with 36 to 46% moisture (AAC). Thus, low coliform counts for this food International Journal of Microbiology 7 would be consistent with good practices of manufacture. In contrast, low microbiological quality of ingredients or poor hygiene could explain coliform counts higher than 10 3 MPN/ g which is the maximal limit established by AAC for fresh sausages. Although no microbiological standards for chicken carcasses are addressed by AAC, counts of coliforms in this work were higher than 2.7 log 10 CFU/g observed by Capita et al. [14] in Spain. Clearly, contamination is possible at any stage of the production process, from defeathering, evisceration, and washing to storage by cooling or freezing.
Regarding the search of enteropathogens, the human E. coli O157:H7 strain was isolated by culture and characterized as stx2 + by PCR. On the contrary, no STEC strain could be isolated from positive stx1/stx2 cattle stools, probably because they were viable but noncultivable strains. Concurrently, Jure et al. [15] identified the stx2 gene in seven samples of meat in Argentina; however, only one E. coli O157:H7 strain could be isolated. The low detection of STEC from cattle in San Luis contrasts with reports of 4 to 39% STEC isolates recovered from calves by Meichtri et al. [16] in our country, who enriched stools and rectal swabs in TSB added with antibiotics and then performed screening of stx genes by conventional PCR in DNA extracted from confluent bacterial growth on SMAC. If amplified, PCR was repeated on individual colonies. Similarly, Sanz et al. [17] recovered 44% STEC from bovines for slaughtering in other Argentina regions. A wide range of protocols have been described for detection or isolation of STEC since that all serotypes cannot be detected by one method [18]. Trypticase soy broth, E. coli broth, buffered peptone water, and brain heart infusion broth added with selective agents have been recommended for STEC enrichment. In the present study, samples were enriched in EC broth without antibiotics which may be advisable when stressed or injured STEC cells are cultured [19]. In addition, a comparative study of enrichment protocols by Vimont et al. [20] showed that the initial level of E. coli O157 was not greatly influenced by the enrichment protocol tested, whereas the initial level of background microflora appeared to decrease when EC broth was used. Other techniques have been recommended for improving the sensitivity of detection methods. The immunomagnetic separation (IMS) can be used after enrichment and prior to plating for the selective concentration of STEC cells, and it is well established for the detection of E. coli O157 in foods, yielding detection limits as low as 1-2 CFU/25 g [18]. While IMS was not used in this study, subsequent STEC researches in our laboratory will include this procedure. Otherwise, molecular methods such as conventional PCR and real-time PCR are very sensitive and provide results in shorter times than cultures. Thus, the ISO/TS 13136:2012 standard is based on the sample enrichment followed by a real-time PCR targeted to the detection of the stx and eae virulence genes, and the determination of O157, O111, O26, O103, and O145 STEC serogroups in foods and animal foodstuffs. When genes are detected, the STEC strain should be isolated for confirmation [18]. Also, immunoassay-based methods such as an available EIA for testing Shiga toxins 1 and 2 have been used in the STEC detection from human stools [21]. Differences in STEC carriage have been observed between grass-fed and feedlot cattle [22]; in the present study, two positive stx1/stx2 samples corresponded to feedlot animal and the other one came from a grazing animal. Although STEC detection and/or recovery were negative in other samples studied here, Ojo et al. [23] demonstrated STEC in feces of cattle (15.2%), sheep (10.7%), goats (7.5%), and pigs (5.6%) as well as in beef (3.8%), goat-meat (1.7%), and pork (4.0%).
The isolation of S. Typhimurium from stools of a patient was consistent with studies reporting this one as the most frequently isolated serovar from humans in Argentina since 2006 [24]. We report the isolation of S. Newport and S. Gaminara from wild boars for the first time in our region. These Salmonella serotypes have been previously isolated from clinical samples during an outbreak caused by consumption of unpasteurized orange juice in USA [25] and recovered from patients with diarrhea in Caribbean zone of Colombia [26]. Environmental factors and seasonal variations as well as different supply sources of samples might have influenced in the low recovery of Salmonella from animal samples in our study.
The Yersinia prevalence observed in this work was lower than 5.5% from pork and beef sausages and minced meat obtained by Lucero Estrada et al. [8] who detected Y. enterocolitica B1A (O:5 and O:6,30), B2 O:9, and Y. intermedia in our region. Previously, Floccari et al. [27] isolated 10% Y. enterocolitica B1A O:5, Y. intermedia, and Y. frederiksenii from 70 chicken carcasses in Argentina. AAC establishes no Y. enterocolitica limits in relation to any of the foods here analyzed, but the absence of this pathogen is desirable. Although Y. enterocolitica was not detected in human and animal stools investigated in the present study, this microorganism has been isolated from human diarrheic feces [28] and animal stools [29] in our country.
In this study, virulence phenotypic tests for Y. enterocolitica B1A strains produced negative results excepting autoagglutination at 37 ∘ C; however, the yadA gene was not detected by PCR. Lack of correlation between Y. enterocolitica phenotypic and genotypic virulence markers such as the above mentioned has been reported by Zheng et al. [30]. These authors found that some Y. enterocolitica strains contain other unknown virulence markers that interact with each other and play an important role in the pathogenesis. In this regard, the chromosomal gene ystB had been strongly linked to the production of diarrhea by B1A strains. Opportunely, among 115 Y. enterocolitica isolates of pig origin analyzed by Bonardi et al. [31], 75.7% corresponded to B1A with ystB as the most common virulence gene (72.4%). In our study, this gene was demonstrated in one Y. enterocolitica B1A O:7,8-8-8,10 strain (1/7, 14%, 95% CI, 0-42%) isolated from porcine skin and bones. The presence of Y. enterocolitica B1A and Y. intermedia in chicken carcasses and porcine skin and bones could be the result of cross-contamination during processing of these products or carriage by slaughtered pigs, respectively.
Related to STEC antimicrobial susceptibility, since antimicrobials can injure the bacterial membrane causing an acute release of preformed Shiga toxin [32], the treatment of HUS in patients is mostly supportive with adequate corporal fluid and electrolyte management, control of the haematological complications, antihypertensive and analgesic therapy, mechanical ventilation, and dialysis when necessary [33], avoiding antibiotic administration. In our region, STEC strains isolated from patients with diarrhea have demonstrated in vitro susceptibility to antibiotics commonly used in the treatment of infections triggered by other enterobacteria.
Contrary to the antibiotic sensitivity demonstrated by our Salmonella isolates, Ibar et al. [34] observed multidrug resistance in different Salmonella serotypes isolated from porcine in Argentina against antimicrobials commonly used in veterinary medicine. Regarding Y. enterocolitica antimicrobial susceptibility, our results matched those reported by Lucero Estrada et al. [8] and Bonardi et al. [31] who observed resistance to cephalotin and ampicillin.

Conclusions
A low prevalence of STEC, Salmonella spp., and Yersinia species was observed in human, animal, and food samples in this region of Argentina. The low number of STEC found in this study, one E. coli O157:H7 from human stool, as compared to other works, might be attributed to the detection methods used. Otherwise, the detection of stx1/stx2 genes in cattle stools highlights the risk of exposure to STEC animal carriers and reinforces the requirement of the good practices of hygiene during slaughtering and meat processing. On the other hand, the high Salmonella frequency observed in the small number of wild boar samples emphasizes the need of further studies in these animals whose byproducts are manufactured and marketed at retail. Lastly, bio/serotypes and virulence traits characterizing our Y. enterocolitica isolates were related to null or low pathogenicity for humans; however, a wide field of knowledge remains unexplored about Y. enterocolitica B1A virulence. Our results suggest that a close microbiological monitoring might contribute to the knowledge of prevalence and distribution of these enteropathogens in patients, presumable animal reservoirs, and foods in our region, which would allow public health services to take preventive measures.