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.

Microbiological baseline study of beef and pork carcasses from provincially inspected abattoirs in Alberta, Canada.

In 2006 and 2007 beef and pork carcass swabs from provincially inspected abattoirs in Alberta, Canada were tested to determine the levels of total aerobic bacteria, coliform bacteria, and generic Escherichia coli, and the prevalence of Salmonella spp., Campylobacter spp., and Shiga toxin-producing E. coli (STEC). Swabs from beef and pork carcasses from 48 and 34 facilities, respectively, were analyzed. All samples tested were positive for aerobic bacteria with 99.8% of beef and 96.0% of pork samples, having total counts of ≤ 100 000 CFU/cm(2). Coliform bacteria were isolated from 22.4% and 42.0% of beef and pork carcass samples, respectively. Generic E. coli were recovered from 14.6% of beef and 33.7% of pork carcass samples. For beef carcasses, positive tests were obtained for 0.1% of 1036 samples tested for Salmonella spp., 1.5% of 1022 samples tested for Campylobacter spp. and 5.4% of 1018 samples tested for STEC. For pork carcasses, positive tests were obtained for 1.6 % of 1076 samples tested for Salmonella spp., 8.8% of 1070 samples tested for Campylobacter spp. and 4.8% of 1067 samples tested for STEC.

Antimicrobial drug use and antimicrobial resistance in enteric bacteria among cattle from Alberta feedlots.

The purpose of this study was to determine whether antimicrobial resistance (AMR) in foodborne pathogens (Escherichia coli O157, Salmonella, and Campylobacter) and non-type-specific E. coli obtained from fecal samples of feedlot cattle was associated with antimicrobial drug (AMD) use. A secondary objective was to determine if AMR in non-type-specific E. coli could be used as a predictor of AMR in foodborne pathogens. Fecal samples were collected from pen floors in 21 Alberta feedlots during March through December 2004, and resistance prevalence was estimated by season (Spring, Fall) and cattle type (fewest days-on-feed and closest to slaughter). AMD exposures were obtained by calculating therapeutic animal daily doses for each drug before sampling from feedlot records. Generalized linear mixed models were used to investigate the relationship between each AMR and AMD use. Non-type-specific E. coli was commonly recovered from fecal samples (88.62%), and the highest prevalence of resistance was found toward tetracycline (53%), streptomycin (28%), and sulfadiazine (48%). Campylobacter jejuni was recovered from 55.3% of the fecal samples, and resistance was generally less for the drugs that were evaluated (doxycycline 38.1%, ciprofloxacin 2.6%, nalidixic acid 1.64%, erythromycin 1.2%). E. coli O157 and Salmonella were recovered much less frequently (7% and 1% prevalence, respectively). The prevalence of recovery for the bacteria studied varied between seasons and cattle types, as did patterns of AMR. Among non-type-specific E. coli, resistance to tetracycline, streptomycin, and sulfadiazine was found to be positively associated with in-feed exposure as well as injectable tetracycline, but these differences were relatively small and of questionable practical relevance. Among C. jejuni isolates, cattle type was significantly associated with doxycycline resistance. Results suggested that resistance in non-type-specific E. coli to chloramphenicol, trimethoprim/sulfamethoxazole, and tetracycline might be used as predictors of resistance to these drugs in E. coli O157 recovered from the same fecal samples.

Evaluation of environmental sampling methods and rapid detection assays for recovery and identification of Listeria spp. from meat processing facilities.

Studies that isolated Listeria spp. from the environment of two meat processing facilities were conducted. Samples were collected in the processing environment of the facilities with three different sampling methods (cotton swab, sterile sponge, and composite-ply tissues) to evaluate their ability to recover Listeria spp. A total of 240 samples for each sampling method were collected and tested. The cotton swab method of sampling was significantly (P < 0.01) less efficient in recovery of Listeria spp. than the sterile-sponge and composite-ply tissue methods, which were similar (P > 0.05) in their ability to recover Listeria spp. The specificity and sensitivity of four detection methods (conventional culture, Petrifilm Environmental Listeria Plates [ELP], lateral-flow immunoprecipitation [LFI], and automated PCR) were evaluated for identification of Listeria spp. Facilities were visited until a minimum of 100 positive and 100 negative samples per detection method were collected. The LFI and PCR methods were highly sensitive (95.5 and 99.1%, respectively) and specific (100%) relative to the culture method. The ELP method was significantly less efficient (P < 0.01) than LFI and PCR in detection of Listeria spp., with lower sensitivity (50.6%) and specificity (91.5%). Kappa values indicated excellent agreement of the LFI and PCR assays and moderate agreement of the ELP method to the culture method. Overall, ELP was easy to use but less efficient in detection of Listeria spp. from environmental samples, while the LFI and PCR methods were found to be excellent alternatives to culture, considering performance and time and labor inputs.

Microbiological baseline study of poultry slaughtered in provincially inspected abattoirs in Alberta, Canada.

Studies to determine baseline levels of microbial contaminants and foodborne bacterial pathogens are needed to evaluate the effectiveness of Hazard Analysis Critical Control Point (HACCP) programs, Good Manufacturing/Production Practices, and various interventions. In 2004 and 2005 poultry carcass rinses from provincially inspected abattoirs in Alberta, Canada, were tested to determine the levels of aerobic plate count bacteria, coliform bacteria, and generic Escherichia coli, the prevalence and levels of Campylobacter spp., and the prevalence of Salmonella spp. and Shiga toxin-producing E. coli (STEC). Samples were collected from 3 high volume and 62 low volume abbatoirs. All samples (1296) were positive for aerobic plate count bacteria, with 98.8% of samples having counts of 100 000 or less colony forming units (CFU)/cm2. Coliform bacteria were isolated from 99.7% of the 1296 carcasses and were recovered at levels of < or = 1000 CFU/cm2 for 98.3% of the samples. Generic E. coli were recovered from 99.1% of the 1296 carcasses at levels of < or = 1000 CFU/cm2 for 98.6% of the samples. Seventy five percent of 1234 samples that were tested for Campylobacter were positive; 37.5% of 1295 samples that were tested for Salmonella were positive; and only 2 of 1296 samples tested for STEC were positive (0.15%).

Occurrence of foodborne bacteria in Alberta feedlots.

The occurrence of generic Escherichia coli, E. coli O157, Salmonella, and Campylobacter in cattle manure, beef carcasses, catch basin water, and soils receiving manure application was determined in 21 Alberta feedlots. In cattle manure, generic E. coli (98%, 2069/2100) and Campylobacter (76%, 1590/2100) were frequently detected; E. coli O157 (7%, 143/2100) and Salmonella (1%, 20/2100) were less frequently detected. Samples from beef carcasses in the cooler following Hazard Analysis Critical Control Point interventions yielded only 1 isolate each of generic E. coli and Campylobacter (1/1653) and no Salmonella (0/1653). Catch basin water specimens were positive for generic E. coli in both the spring (62%, 13/21) and the fall (52%, 11/21). Other bacteria were detected only in the spring water specimens, including E. coli O157 (29%, 6/21), Salmonella (5%, 1/21), and Campylobacter (52%, 11/21). Generic E. coli was frequently isolated from soil specimens (30%, 27/88), but E. coli O157 was not found in soil samples obtained in the spring and was only occasionally detected in the fall samples (9%, 3/32). Salmonella were occasionally found in the soil specimens collected in the spring (3%, 2/56), but not in the fall season (0/32). Campylobacter jejuni was frequent in cattle manure (66%, 1070/1623), but rare in carcass and environmental samples. E. coli O157 and Salmonella were rarely detected in cattle or the environment. Generic E. coli and Salmonella were rarely detected on carcasses.

Effect of low temperature on stability of theta-type plasmids in Carnobacterium maltaromaticum.

The heterologous production of useful peptides such as bacteriocins by lactic acid bacteria (LAB) has been studied for use in the biopreservation of foods. Recombinant plasmids can suffer drawbacks such as segregational instability affecting the production of these peptides in certain environments such as absence of selective pressure or low temperature. The link between growth temperature characteristics of parental strains and stability of theta-type plasmids at a low temperature was investigated. The growth of four parental strains at 4 degrees C and stability of five derivative theta-type plasmids transformed into Carnobacterium maltaromaticum UAL26 at 25 and 4 degrees C were determined. Two plasmids (pCD11 and pCaT) derived from psychrotrophic LAB and plasmid, pHW800, from Enterococcus faecium 226 with unknown growth temperature characteristics, had excellent stability when strains were grown at 4 degrees C. Plasmids (pTRKH2 and pUCB820) derived from LAB that did not grow at refrigeration temperatures were not stable at 4 degrees C. When a DNA fragment from pCD11 containing 22-bp repeats, a putative replication initiation site, and the gene for the RepA protein was inserted into pTRKH2, the resulting derivative plasmid was 100% stable at 4 degrees C.

Presence of non-O157 Shiga toxin-producing Escherichia coli in feces from feedlot cattle in Alberta and absence on corresponding beef carcasses.

The study objectives were to determine the prevalence and serotypes of non-O157 Shiga toxin-producing Escherichia coli (STEC) in pens of feedlot cattle and on corresponding beef carcasses. We collected 25 fecal samples from 84 pens in 21 Alberta feedlots and 40 carcass swabs from each preslaughter pen for analysis by culture and polymerase chain reaction (PCR). Non-O157 STEC were recovered from feces from 12 (14%) of the 84 pens and 12 (57%) of the 21 feedlots by examination of 1 E. coli isolate positive for 4-methylumbelliferyl-beta-beta-glucuronide per sample. Twelve non-O157 serotypes were detected, but 7 of the 15 STEC isolates lacked the accessory virulence genes eae and hlyA. Although 115 (7%) of the carcass broths were PCR-positive, no STEC isolates were recovered from the 1650 carcasses sampled. Our data indicate that multiple non-O157 STEC serotypes may be present in cattle feces, yet are unlikely to be recovered from the corresponding beef carcasses when 20 colonies per sample from PCR-positive broth cultures are analyzed.

Evaluation of detection methods for screening meat and poultry products for the presence of foodborne pathogens.

Rapid and molecular technologies such as enzyme-linked immunosorbent assay (ELISA), PCR, and lateral flow immunoprecipitation can reduce the time and labor involved in screening food products for the presence of pathogens. These technologies were compared with conventional culture methodology for the detection of Salmonella, Campylobacter, Listeria, and Escherichia coli O157:H7 inoculated in raw and processed meat and poultry products. Recommended protocols were modified so that the same enrichment broths used in the culture methods were also used in the ELISA, PCR, and lateral flow immunoprecipitation assays. The percent agreement between the rapid technologies and culture methods ranged from 80 to 100% depending on the pathogen detected and the method used. ELISA, PCR, and lateral flow immunoprecipitation all performed well, with no statistical difference, compared with the culture method for the detection of E. coli O157:H7. ELISA performed better for the detection of Salmonella, with sensitivity and specificity rates of 100%. PCR performed better for the detection of Campylobacter jejuni, with 100% agreement to the culture method. PCR was highly sensitive for the detection of all the foodborne pathogens tested except Listeria monocytogenes. Although the lateral flow immunoprecipitation tests were statistically different from the culture methods for Salmonella and Listeria because of false-positive results, the tests did not produce any false negatives, indicating that this method would be suitable for screening meat and poultry products for these pathogens.

Bacteriology and storage life of moisture-enhanced pork.

This study was undertaken to determine the impact of the moisture enhancement process on the bacterial contamination and storage life of vacuum-packaged pork loins. Bone-in and boneless pork loins injected with brine (sodium chloride, sodium phosphate, lemon juice) were obtained from a commercial processing facility and stored for 5 weeks in vacuum packaging at 2 and 5 degrees C. At weekly intervals, samples were excised to determine numbers of spoilage bacteria and pathogens. The loins were subjectively evaluated by a sensory panel to quantify appearance and odor acceptability. Moisture-enhanced loins were initially contaminated with a population of psychrotrophic bacteria that was approximately 2 log units higher than that for noninjected boneless loins. This difference was largely due to contamination by larger numbers of pseudomonads in the brine-injected loins. There were no significant differences in the initial numbers of lactic acid bacteria, Enterobacteriaceae, or Brochothrix thermosphacta. Similar trends in spoilage bacterial populations were observed for moisture-enhanced loins with bones, but Enterobacteriaceae counts were also found to be approximately 1 log unit higher for the injected product. Brine-injected loins generally had larger bacterial numbers at each storage time, but there were no consistent injection treatment effects on bacterial growth. Brine injection did not affect color or odor deterioration, and the storage life for vacuum-packaged loins was the same as that for noninjected controls. The incidence of Listeria monocytogenes was 21% for control loins and 27% for moisture-enhanced loins. Although the brine injection process resulted in an increase in bacterial contamination, there was no evidence that this contamination would affect the storage life of vacuum-packaged loins, and further research is necessary to determine the significance of the increased incidence of L. monocytogenes.

Antimicrobial susceptibility of hazard analysis critical control point Escherichia coli isolates from federally inspected beef processing plants in Alberta, Saskatchewan, and Ontario.

A survey to estimate the prevalence of antimicrobial resistance in Escherichia coli was conducted in 7 Canadian federally inspected processing plants during 2001. Escherichia coli isolates were recovered during routine hazard analysis critical control point sampling from beef carcasses and trim and subsequently tested for their antimicrobial susceptibility by using susceptibility panels. Of the 2653 isolates analyzed, 68% were sensitive to all 18 antimicrobials tested. For 14 of the 18 antimicrobials evaluated, the percentage of resistant isolates was < or = 1. Twenty-five percent of the isolates were resistant to tetracycline, 9% to sulfamethoxazole, 7% to streptomycin, and 3% to ampicillin. Multiple resistance was found in 12% of the isolates, with 7% showing resistance to 2 antimicrobials, 2% to 3 antimicrobials, 2% to 4 antimicrobials, and 1% to 5 or more antimicrobials. Forty-five different antimicrobial resistance patterns were observed. The reasons for the development of the antimicrobial resistance were not investigated in this study. This study was useful as a pilot to help to develop a national antimicrobial resistance surveillance program in Canada. This study indicates that laboratory standardization is possible for consistent results across the country and that the indicator organism, E. coli, is fairly easy to obtain for surveillance but Salmonella are not, due to their low prevalence in beef.

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.