Virulence potential of antimicrobial-resistant extraintestinal pathogenic Escherichia coli from retail poultry meat in a Caenorhabditis elegans model.

Healthy poultry can be a reservoir for extraintestinal pathogenic Escherichia coli (ExPEC), some of which could be multidrug resistant to antimicrobials. These ExPEC strains could contaminate the environment and/or food chain representing thus, food safety and human health risk. However, few studies have shown the virulence of poultry-source antimicrobial-resistant (AMR) ExPEC in humans. This study characterized AMR ExPEC and investigated the virulence potential of some of their isolates in a Caenorhabditis elegans infection model. A total of 46 E. coli isolates from poultry (chicken, n = 29; turkey, n = 12) retail meats and chicken feces (n = 4), or humans (n = 1) were sequenced and identified as ExPEC. Except eight, all remaining 38 ExPEC isolates were resistant to at least one antibiotic and carried corresponding antimicrobial resistance genes (ARGs). About 27 of the 46 ExPEC isolates were multidrug-resistant (≥3 antibiotic classes). Seven ExPEC isolates from chicken or turkey meats were of serotype O25:H4 and sequence type (ST) 131 which clustered with an isolate from a human urinary tract infection (UTI) case having the same serotype and ST. The C. elegans challenge model using eight of studied ExPEC isolates harboring various ARGs and virulence genes (VGs) showed that regardless of their ARG or VG numbers in tested poultry meat and feces, ExPEC significantly reduced the life span of the nematode (P < 0.05) similarly to a human UTI isolate. This study indicated the pathogenic potential of AMR ExPEC from retail poultry meat or feces, but more studies are warranted to establish their virulence in poultry and human. Furthermore, relationships between specific resistance profiles and/or VGs in these E. coli isolates for their pathogenicity deserve investigations.

Epidemiology of antimicrobial resistant Campylobacter spp. isolated from retail meats in Canada.

Campylobacter is an important zoonotic pathogen found in livestock and can cause illness in humans following consumption of raw and undercooked meat products. The objectives of this study were to determine the prevalence of Campylobacter spp. in retail meat (poultry, turkey, pork and beef) purchased in Alberta, Canada and to assess antimicrobial resistance and genetic relatedness of recovered Campylobacter strains with previously isolated strains from clinical and environmental sources. A Comparative Genomic Fingerprinting (CGF) method was used for assessing genetic relatedness of isolates. A total of 606 samples comprising 204, 110, 145 and 147 samples of retail chicken, turkey, ground beef and pork, respectively, were obtained. Campylobacter was isolated from 23.5% (48/204) of chicken samples and 14.2% (8/110) of turkey samples. Pork and beef samples were negative for Campylobacter. Campylobacter jejuni was the most common (94.6%) spp. found followed by C. coli (5.4%). Resistance to tetracycline was found in 48.1% of isolates, followed by resistance to ciprofloxacin (5.5%), nalidixic acid (5.5%), azithromycin (1.78%), and erythromycin (1.78%). All isolates were susceptible to clindamycin, florfenicol, gentamicin and telithromycin. Tetracycline resistance was attributable to the presence of the tetO gene. CGF analysis showed that Campylobacter isolated from poultry meat in this study were genetically related to clinical isolates recovered from human infections and to those isolated from animals and the environment.

Prevalence and antimicrobial resistance of Salmonella isolated from two pork processing plants in Alberta, Canada.

This study investigated the frequency of Salmonella serovars on pig carcasses at various processing steps in two commercial pork processing plants in Alberta, Canada and characterized phenotypic and genotypic antimicrobial resistance (AMR) and PFGE patterns of the Salmonella isolates. Over a one year period, 1000 swab samples were collected from randomly selected pigs at two slaughter plants. Sampling points were: carcass swabs after bleeding (CSAB), carcass swabs after de-hairing (CSAD, plant A) or skinning (CSASk, plant B), carcass swabs after evisceration (CSAE), carcass swabs after pasteurization (CSAP, plant A) or washing (CSAW, plants B) and retail pork (RP). For plant A, 87% of CSAB and 8% of CSAE were positive for Salmonella while at plant B, Salmonella was recovered from 94% of CSAB and 10% of CSAE. Salmonella was not recovered from the RP samples at either plant, indicating that the plants used effective control measures. Salmonella enterica serovar Derby was the most common serotype (23%, 29/127) recovered in plant A and plant B (61%, 76/124). For plant A, 35% (45/127) of isolates were resistant to at least one antimicrobial. Five isolates (3.9%), 4 serovar Ohio strains and one serovar I:Rough-O:I,v:-, strain were simultaneously resistant to antimicrobials of very high (Category I), high (Category II), and medium (Category III) importance to human medicine. The 4 S. Ohio isolates were recovered from 3 different steps of pork processing on the same sampling day and displayed resistance to 5-7 antimicrobials, with all of them displaying resistance to ceftiofur and ceftriaxone (Category I). An I:Rough-O:l,v:- isolate, recovered on a different sampling day, was resistant to 7 antimicrobials that included resistance to ampicillin/clavulanic acid, ceftiofur and ceftriaxone (Category I). Salmonella strains isolated from plant A harbored 12 different AMR genes. The most prevalent genes were sul1, sul2, tet(A), tet(B), aadA, strA/strB, aac(3)IV and aphA1. For Salmonella isolates from plant B, 7 resistance genes were identified alone or in combination where tet(B) was found in 77 (62.3%) of the isolates. For plant A, 19 different PFGE subtypes of Salmonella isolates that displayed phenotypic and/or genotypic resistance were observed while 13 different PFGE subtypes were observed for plant B. The lack of detection of Salmonella on the surfaces of RP suggests that current pork processing practices can dramatically reduce Salmonella. Salmonella isolates from pig carcasses at various steps displayed multidrug resistance, including to those of very high importance in human medicine, which represent a public health concern.

Virulence characterization and molecular subtyping of typical and atypical Escherichia coli O157:H7 and O157:H(-) isolated from fecal samples and beef carcasses in Mexico.

The objective of the study was to characterize virulence genes and subtype Escherichia coli O157:H7 and O157:H( 2 ) isolates obtained from a vertically integrated feedlot slaughter plant in Mexico. A total of 1,695 samples were collected from feedlots, holding pens, colon contents, hides, and carcasses. E. coli O157:H7 detection and confirmation was carried out using conventional microbiology techniques, immunomagnetic separation, latex agglutination, and the BAX system. A total of 97 E. coli O157 strains were recovered and screened for key virulence and metabolic genes using multiplex and conventional PCR. Eighty-eight (91.72%) of the strains carried stx2, eae, and ehxA genes. Ten isolates (8.25%) were atypical sorbitol-fermenting strains, and nine were negative for the flicH7 gene and lacked eae, stx1, stx2, and ehxA. One sorbitol-positive strain carried stx2, eae, tir, toxB, and iha genes but was negative for stx1 and ehxA. Pulsed-field gel electrophoresis (PFGE) analysis yielded 49 different PFGE subtypes, showing a high genetic diversity; however, the majority of the typical isolates were closely related (80 to 90% cutoff). Atypical O157 isolates were not closely related within them or to typical E. coli O157:H7 isolates. Identical PFGE subtypes were found in samples obtained from colon contents, feedlots, holding pens, and carcasses. Isolation of a sorbitolfermenting E. coli O157 positive for a number of virulence genes is a novel finding in Mexico. These data showed that genetically similar strains of E. coli O157:H7 can be found at various stages of beef production and highlights the importance of preventing cross-contamination at the pre- and postharvest stages of processing.

Characterization of Extraintestinal Pathogenic Escherichia coli isolated from retail poultry meats from Alberta, Canada.

Extraintestinal Pathogenic Escherichia coli (ExPEC) have the potential to spread through fecal waste resulting in the contamination of both farm workers and retail poultry meat in the processing plants or environment. The objective of this study was to characterize ExPEC from retail poultry meats purchased from Alberta, Canada and to compare them with 12 human ExPEC representatives from major ExPEC lineages. Fifty-four virulence genes were screened by a set of multiplex PCRs in 700 E. coli from retail poultry meat samples. ExPEC was defined as the detection of at least two of the following virulence genes: papA/papC, sfa, kpsMT II and iutA. Genetic relationships between isolates were determined using pulsed field gel electrophoresis (PFGE). Fifty-nine (8.4%) of the 700 poultry meat isolates were identified as ExPEC and were equally distributed among the phylogenetic groups A, B1, B2 and D. Isolates of phylogenetic group A possessed up to 12 virulence genes compared to 24 and 18 genes in phylogenetic groups B2 and D, respectively. E. coli identified as ExPEC and recovered from poultry harbored as many virulence genes as those of human isolates. In addition to the iutA gene, siderophore-related iroN and fyuA were detected in combination with other virulence genes including those genes encoding for adhesion, protectin and toxin while the fimH, ompT, traT, uidA and vat were commonly detected in poultry ExPEC. The hemF, iss and cvaC genes were found in 40% of poultry ExPEC. All human ExPEC isolates harbored concnf (cytotoxic necrotizing factor 1 altering cytoskeleton and causing necrosis) and hlyD (hemolysin transport) genes which were not found in poultry ExPEC. PFGE analysis showed that a few poultry ExPEC isolates clustered with human ExPEC isolates at 55-70% similarity level. Comparing ExPEC isolated from retail poultry meats provides insight into their virulence potential and suggests that poultry associated ExPEC may be important for retail meat safety. Investigations into the ability of our poultry ExPEC to cause human infections are warranted.

Antibiotic resistance and diversity of Salmonella enterica serovars associated with broiler chickens.

The objective of this study was to analyze the antibiotic resistance phenotype and genotype of Salmonella isolated from broiler production facilities. A total of 193 Salmonella isolates recovered from commercial farms in British Columbia, Canada, were evaluated. Susceptibility to antibiotics was determined with the Sensititre system. Virulence and antibiotic resistance genes were detected by PCR assay. Genetic diversity was determined by pulse-field gel electrophoresis (PFGE) typing. Seventeen serovars of Salmonella were identified. The most prevalent Salmonella serovars were Kentucky (29.0% of isolates), Typhimurium (23.8%), Enteritidis (13.5%), and Hadar (11.9%); serovars Heidelberg, Brandenburg, and Thompson were identified in 7.7, 4.1, and 3.6% of isolates, respectively. More than 43% of the isolates were simultaneously resistant to ampicillin, amoxicillin-clavulanic acid, ceftiofur, cefoxitim, and ceftriaxone. This ß-lactam resistance pattern was observed in 33 (58.9%) of the Salmonella Kentucky isolates; 2 of these isolates were also resistant to chloramphenicol, streptomycin, sulfisoxazole, and tetracycline. Genes associated with resistance to aminoglycosides (aadA1, aadA2, and strA), ß-lactams (blaCMY-2, blaSHV, and blaTEM), tetracycline (tetA and tetB), and sulfonamide (sul1) were detected among corresponding resistant isolates. The invasin gene (invA) and the Salmonella plasmid virulence gene (spvC) were found in 97.9 and 25.9% of the isolates, respectively, with 33 (71.7%) of the 46 Salmonella Typhimurium isolates and 17 (65.4%) of the 26 Salmonella Enteritidis isolates carrying both invA and spvC. PGFE typing revealed that the antibiotic-resistant serovars were genetically diverse. These data confirm that broiler chickens can be colonized by genetically diverse antibiotic-resistant Salmonella isolates harboring virulence determinants. The presence of such strains is highly relevant to food safety and public health.

Characterization of antimicrobial resistance and virulence genotypes of Enterococcus faecalis recovered from a pork processing plant.

The objective of this study was to assess the antimicrobial resistance and virulence genotypes of Enterococcus faecalis isolated from samples obtained from a commercial pork processing plant. A total of 200 samples were randomly obtained from carcasses after bleeding (BC; 50 samples) and pasteurization (PC; 100 samples) and from retail pork products (RP; 50 samples). One isolate from each E. faecalis -positive sample was analyzed for antimicrobial susceptibility and characterized using a enterococcal microarray for analysis of resistance and virulence genes. E. faecalis was isolated from 79.5% of BC samples, 2% of PC samples, and 72.7% of RP samples. Resistance to the clinically important drugs ciprofloxacin (one isolate each from BC and RP samples) and daptomycin (one isolate each from PC and RP samples) was found. Multiresistance (to five or more antimicrobials) was more common in E. faecalis isolates from BC (77.4% of isolates) samples than those from PC (25%) and RP (37.6%) samples. Resistance to kanamycin (43.5%) and streptomycin (69.2%) was noted mostly in E. faecalis from BC samples. The most common resistance genes (>5% prevalence) found in E. faecalis were those for aminoglycosides (aac(6), aphA3, and aadE), macrolides-lincosamide (ermB, ermA, sat(4), and linB), and tetracyclines (tetL, tetM, and tetO ). The virulence genes expressing adhesion (ace, efaAfs, and agrBfs), gelatinase (gelE), and pheromone (cAM, ccF10, cob, and cpd1) factors were found in the majority of isolates. Significant associations were found between resistance and virulence genes, suggesting their possible relationship. These data suggest that carcasses entering the final product processing area are mostly free of E. faecalis but are recontaminated with antimicrobial-resistant strains during processing. The source of these contaminants remains to be identified; however, these results underscore the importance of E. faecalis as a reservoir of resistance and virulence genes.

Phenotypic and genetic characterization of antimicrobial resistance in Salmonella serovars isolated from retail meats in Alberta, Canada.

This study determined the prevalence of Salmonella serovars, antimicrobial resistance (AMR) and resistance genes in Salmonella isolated from retail meats purchased in Alberta, Canada. Samples were collected during one year period (May 2007-April 2008) on weekly basis from 19 census divisions in Alberta. A total of 564 samples including chicken (n = 206), turkey (n = 91), beef (n = 134) and pork (n = 133) were purchased. Salmonella were recovered from chicken (40%), turkey (27%) and pork (2%) samples and was not found in ground beef. A total of 21, 8, and 3 different serovars were recovered from chicken, turkey and pork meats, respectively. Salmonella Hadar was most common in chicken whereas S. Heidelberg was common in turkey meat. Overall 29% (32/110) of isolates were susceptible to tested antimicrobials and resistance to ciprofloxacin, amikacin and nalidixic acid was not found in any isolate. Multiresistance (≥2 antimicrobials) was found in 56% of isolates. Resistance to amoxicillin-clavulanic acid (AMC), ceftiofur (TIO), and ceftriaxone (CRO) was found in about 21% of chicken and 25% of turkey isolates. Resistance to either of tetracycline (TET), streptomycin (STR) or ampicillin (AMP) was unconditionally associated with S. Hadar but resistance to either of TET, AMP, AMC, TIO, CRO or cefoxitin was associated with S. Heidelberg. The strA/B (42% isolates), tet(A) (28% isolates), bla(CMY-2) (21% isolates) and bla(TEM) (17% isolates) were the most common resistance genes found. The bla(CMY-2) and bla(TEM) genes were unconditionally associated with S. Heidelberg; tet(A) and strA/B with S. Hadar and tet(B) gene with S. Kentucky. The strA/B genes were not associated with S. Heidelberg. Our data suggests that the prevalence of Salmonella serovars varied by the meat type and that AMR and resistance genes varied by the Salmonella serovars.

Antimicrobial resistance and resistance genes in Escherichia coli isolated from retail meat purchased in Alberta, Canada.

This study analyzed antimicrobial resistance (AMR) and resistance genes in generic Escherichia coli isolated from retail meat samples purchased (2007-2008) in Alberta, Canada, and determined potential associations between resistance phenotypes and resistance genes with relation to the meat types. A total of 422 E. coli isolates from retail chicken, turkey, beef, and pork meats were tested for antimicrobial susceptibility. Multiplex PCRs were used to detect major resistance genes for tetracyclines [tet(A), tet(B), tet(C)], sulfonamides (sul1, sul2, sul3), aminoglycosides (strA/B, aadA, aadB, aac(3)IV, aphA1, aphA2), and ß-lactamase (bla(CMY-2), bla(TEM), bla(SHV), bla(PSE-1)). Resistance to ciprofloxacin was not found in any isolate. Overall resistances to clinically important antimicrobials amoxicillin-clavulanic acid (16.8% of isolates) and ceftriaxone (12.6% isolates) were observed. These resistances were observed more frequently (p<0.0001) in chicken-derived E. coli than those from the other meat types. Resistance to multiple antimicrobials (≥ 5) was found in more chicken derived E. coli (32%) than E. coli from other meat types. The ß-lactamase genes of clinical importance, including bla(CMY-2) and bla(TEM), were found in about 18% of poultry-derived E. coli and in only 5% of ground beef. The bla(CMY-2) gene was more likely to be found in E. coli from chicken than turkey, beef, or pork meats. The tet(A) gene was associated with bla(CMY-2), whereas bla(CMY-2) and bla(TEM) genes were associated with strA/B genes. Resistance genes for tetracycline, sulfonamides, and aminoglycosides were associated with the phenotypic expression of resistance to unrelated classes of antimicrobials. These data suggest the prevalence of AMR and select resistance genes were higher in poultry-derived E. coli. The multiple associations found between AMR phenotypes and resistance genes suggest a complex nature of resistance in E. coli from retail meat, and hence the use of a single antimicrobial could result in the selection of resistant E. coli not only to the drug being used but to other unrelated classes of antimicrobials as well.

Characterization of antimicrobial resistance and virulence genes in Enterococcus spp. isolated from retail meats in Alberta, Canada.

The objective of this study was to characterize antimicrobial resistance (AMR) and virulence genotypes of Enterococcus spp. particularly Enterococcus faecalis isolated from retail meats purchased (2007-2008) in Alberta, Canada. Unconditional statistical associations between AMR pheno- and genotypes and virulence genotypes were determined. A total of 532 enterococci comprising one isolate from each positive sample were analyzed for antimicrobial susceptibility. A customized enterococcal microarray was used for species identification and the detection of AMR and virulence genes. E. faecalis was found in >94% of poultry samples and in about 73% of beef and 86% of pork samples. Enterococcus faecium was not found in turkey meat and its prevalence was 2% in beef and pork and 4% in chicken samples. None of the enterococci isolates were resistant to the clinically important drugs ciprofloxacin, daptomycin, linezolid and vancomycin. Multiresistance (≥3 antimicrobials) was more common in E. faecalis (91%) isolated from chicken and turkey (91%) than those isolated from beef (14%) or pork (45%). Resistance to aminoglycosides was also noted at varying degrees. The most common resistance genes found in E. faecalis were aminoglycosides (aac, aphA3, aadE, sat4, aadA), macrolides (ermB, ermA), tetracyclines (tetM, tetL, tetO), streptogramin (vatE), bacitracin (bcrR) and lincosamide (linB). Virulence genes expressing aggregation substances (agg) and cytolysin (cylA, cylB, cylL, cylM) were found more frequently in poultry E. faecalis and were unconditionally associated with tetM, linB and bcrR resistance genes. Other virulence genes coding for adhesion (ace, efaAfs), gelatinase (gelE) were also found in the majority of E. faecalis. Significant statistical associations were found between resistance and virulence genotypes, suggesting their possible physical link on a common genetic element. This study underscores the importance of E. faecalis as a reservoir of resistance and virulence genes and their potential transfer to humans through consumption of contaminated undercooked meat.

Characterization of antimicrobial resistance in Enterococcus spp. recovered from a commercial beef processing plant.

The objective of this study was to characterize antimicrobial resistance in Enterococcus spp. recovered from a commercial beef processing plant. Samples were obtained from conveyers used for moving carcasses before the start of operation (CC), 2 h after operation has started (DC), and from ground beef (GB). Randomly selected isolates from each positive sample (13 from CC; 28 from DC; 26 from GB) were confirmed to genus and species levels using PCR and the API 20 Strep kit (BioMérieux Canada, Inc., St. Laurent, Canada). A total of 199 isolates comprising 39, 84, and 76 from CC, DC, and GB, respectively, were used for antimicrobial resistance testing, major resistance genes detection, and genetic analysis. Enterococcus faecalis (87%) was the most common species found followed by Enterococcus faecium (10%). The majority of enterococci were highly associated with DC samples. About 42% of E. faecium from DC samples were resistant to quinupristin-dalfopristin. Resistance to lincomycin was observed in >90% of E. faecalis from all the three sample sources. The tetracycline-resistant enterococci (52%) were significantly higher in DC samples. Intermediate resistance to erythromycin was significantly higher in enterococci from CC and DC samples. The tetracycline and quinupristin-dalfopristin resistance in enterococci was highly correlated with the presence of tet(M) and vat(E) genes. The erm(B) gene was found in about 50% of the E. faecium isolates from GB samples and was also present in >12% of the E. faecalis isolates from all the three sample sources. Enterococci from individual sample sources were genetically similar. A number of E. faecalis from CC, DC, and GB were clustered together at >85% similarity level. These findings suggest that antimicrobial-resistant Enterococcus spp. are prevalent during commercial beef processing and can transfer between various locations in the plant and that a pool of resistance genes can be found in these enterococci.

Antimicrobial resistance genes in Escherichia coli isolates recovered from a commercial beef processing plantt.

The goal of this study was to assess the distribution of antimicrobial resistance (AMR) genes in Escherichia coli isolates recovered from a commercial beef processing plant. A total of 123 antimicrobial-resistant E. coli isolates were used: 34 from animal hides, 10 from washed carcasses, 27 from conveyers for moving carcasses and meat, 26 from beef trimmings, and 26 from ground meat. The AMR genes for beta-lactamase (bla(CMY), bla(SHV), and bla(TEM), tetracycline (tet(A), tet(B), and tet(C)), sulfonamides (sul1, sul2, and sul3), and aminoglycoside (strA and strB) were detected by PCR assay. The distribution of tet(B), tet(C), sul1, bla(TEM), strA, and strB genes was significantly different among sample sources. E. coli isolates positive for the tet(B) gene and for both strA and strB genes together were significantly associated with hide, washed carcass, and ground meat samples, whereas sull gene was associated with washed carcass and beef trimming samples. The bla(TEM) gene was significantly associated with ground meat samples. About 50% of tetracycline-resistant E. coli isolates were positive for tet(A) (14%), tet(B) (15%), or tet(C) (21%) genes or both tet(B) and tet(C) genes together (3%). The sul2 gene or both sul1 and sul2 genes were found in 23% of sulfisoxazole-resistant E. coli isolates, whereas the sul3 gene was not found in any of the E. coli isolates tested. The majority of streptomycin-resistant E. coli isolates (76%) were positive for the strA and strB genes together. The bla(CMY), bla(TEM), and bla(SHV) genes were found in 12, 56, and 4%, respectively, of ampicillin-resistant E. coli isolates. These data suggest that E. coli isolates harboring AMR genes are widely distributed in meat processing environments and can create a pool of transferable resistance genes for pathogens. The results of this study underscore the need for effective hygienic and sanitation procedures in meat plants to reduce the risks of contamination with antimicrobial-resistant bacteria.

Genotype, serotype, and antibiotic resistance of sorbitol-negative Escherichia coli isolates from feedlot cattle.

Rectal fecal samples from 80 steers receiving Rumensin, Revalor-S, and Liquamycin alone or in combination for growth promotion and disease prevention were examined for the presence of non-O157:H7 Shiga toxin-producing Escherichia coli. All isolates were identified with the API 20E test, virulence genes were detected with a PCR assay, and antibiotic susceptibilities were determined with the Sensititre system. Of the 153 E. coli isolates recovered 126 (82.3%) were sorbitol negative. Isolates were classified into 14 biochemical E. coli groups; 51.6% were negative for arginine dihydrolase, ornithine decarboxylase, sorbitol, and saccharose reactions but positive for lysine decarboxylase, indole production, and rhamnose reactions. Twenty-one O:H serotypes were detected in the 153 E. coli isolates. The most frequent serotypes were O2:H42 (49.7% of isolates), O49:NM (13.7%), O?:H25 (9.2%), and O10:NM (7.2%). One isolate of E. coli O172:H25 and one of E. coli O157: H39 were found. The stx1 gene was found in the two E. coli O98:H25 isolates. The eaeA and e-hlyA genes were detected in 21, 14, and 10 isolates of serotypes O49:NM, O?:H25, and O10:NM, respectively, and in each isolate of serotype O156:H25 and O172:H25. Four E. coli O132:H18 isolates were multiresistant to ampicillin, chloramphenicol, kanamycin, streptomycin, and sulfisoxazole. Tetracycline resistance due to the tet(B) gene was observed in 74 of the 76 E. coli O2:H42 isolates. Except for one isolate, all tetracycline-resistant isolates were negative for the virulence genes eaeA and e-hlyA or stx1. Pulsed-field gel electrophoresis typing revealed that the tetracycline-resistant serotypes were genetically diverse. Our data illustrate that cattle are a potential source of some atypical antibiotic-resistant E. coli isolates that harbor virulence genes.

Antimicrobial resistance and genetic profiling of Escherichia coli from a commercial beef packing plant.

The objective of this study was to investigate the extent of antimicrobial resistance and to genetically characterize resistant Escherichia coli recovered from a commercial beef packing plant. E. coli isolates were recovered by a hydrophobic grid membrane filtration method by direct plating on SD-39 medium. A total of 284 isolates comprising 71, 36, 55, 52, and 70 isolates from animal hides, washed carcasses, conveyers, beef trimmings, and ground beef, respectively, were analyzed. The susceptibility of E. coli isolates to 15 antimicrobial agents was evaluated with an automated broth microdilution system, and the genetic characterization of these isolates was performed by the random amplified polymorphic DNA (RAPD) method. Of the 284 E. coli isolates, 56% were sensitive to all 15 antimicrobial agents. Resistance to tetracycline, ampicillin, and streptomycin was observed in 38, 9, and 6% of the isolates, respectively. Resistance to one or more antimicrobial agents was observed in 51% of the E. coli isolates recovered from the hides but in only 25% of the E. coli from the washed carcasses. Resistance to one or more antimicrobial agents was observed in 49, 50, and 37% of the isolates recovered from conveyers, beef trimmings, and ground beef, respectively. The RAPD pattern data showed that the majority of resistant E. coli isolates were genetically diverse. Only a few RAPD types of resistant strains were shared among various sample sources. The results of this study suggest that antimicrobial-resistant E. coli isolates were prevalent during all stages of commercial beef processing and that considerably higher numbers of resistant E. coli were present on conveyers, beef trimmings, and ground beef than on dressed carcasses. This stresses the need for improving hygienic conditions during all stages of commercial beef processing and meatpacking to avoid the risks of transfer of antimicrobial-resistant bacteria to humans.

Origin of contamination and genetic diversity of Escherichia coli in beef cattle.

The possible origin of beef contamination and genetic diversity of Escherichia coli populations in beef cattle, on carcasses and ground beef, was examined by using random amplification of polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the fliC gene. E. coli was recovered from the feces of 10 beef cattle during pasture grazing and feedlot finishing and from hides, carcasses, and ground beef after slaughter. The 1,403 E. coli isolates (855 fecal, 320 hide, 153 carcass, and 75 ground beef) were grouped into 121 genetic subtypes by using the RAPD method. Some of the genetic subtypes in cattle feces were also recovered from hides, prechilled carcasses, chilled carcasses, and ground beef. E. coli genetic subtypes were shared among cattle at all sample times, but a number of transient types were unique to individual animals. The genetic diversity of the E. coli population changed over time within individual animals grazing on pasture and in the feedlot. Isolates from one animal (59 fecal, 30 hide, 19 carcass, and 12 ground beef) were characterized by the PCR-RFLP analysis of the fliC gene and were grouped into eight genotypes. There was good agreement between the results obtained with the RAPD and PCR-RFLP techniques. In conclusion, the E. coli contaminating meat can originate from cattle feces, and the E. coli population in beef cattle was highly diverse. Also, genetic subtypes can be shared among animals or can be unique to an animal, and they are constantly changing.