Reduction of Shiga toxin-producing Escherichia coli in a beef abattoir.

The aim of this work was to reinforce actions tending to reduce Shiga toxin-producing Escherichia coli (STEC) in beef products from an Argentinean commercial abattoir implementing Hazard Analysis and Critical Control Point (HACCP) practices. An environmental stx map was built with 421 environmental samples from the slaughter, quartering, cool chamber and deboning sectors (February-May 2013). For stx determination, 125 carcass and 572 anatomical cut samples were used. Based on the environmental stx mapping results, improvement actions were designed and implemented (June and July 2013). After implementing improvement actions, 160 carcass and 477 anatomical cut samples were collected to identify stx and verify the impact of improvement actions (August-December 2013). Our results showed stx-positivity in pre-operational (10.1%) and operational (15.5%) environmental samples and in carcass and beef cut samples before (4.8 and 10.1%; p = 0.144) and after (1.2 and 4.8%; p = 0.0448) implementing improvement actions, respectively. Although improvement actions reduced stx in beef cuts, it is difficult to implement and sustain a system based on stx zero-tolerance only by reinforcing Good Manufacturing Practices, Sanitation Standard Operating Procedures and HACCP practices. The application of combined intervention strategies to reduce STEC in carcasses and beef cuts should be therefore considered.

Development and In-House Validation of a Real-Time Polymerase Chain Reaction for the Detection of Listeria monocytogenes in Meat.

Listeriosis is a foodborne disease caused by Listeria monocytogenes. The aims of this work were to develop and validate an in-house real-time polymerase chain reaction (RT-PCR) for the detection of L. monocytogenes, and to determine its prevalence in raw ground beef samples from 53 butcheries that also sell ready-to-eat foods. One set of primers and one hydrolysis probe were designed for hly gene detection and then challenged with pure strains. The detection was successful for all L. monocytogenes strains analyzed and negative for all non-L. monocytogenes strains (detection limit, 10 colony forming unit [CFU]/mL). Inclusivity, exclusivity, and analytical accuracy were 100%. L. monocytogenes was detected in 41.5% of raw ground beef samples from the 53 butcheries analyzed. This RT-PCR may be a valuable method for rapid detection of L. monocytogenes in meat.

Isolation and characterization of non-O157 Shiga toxin-producing Escherichia coli from beef carcasses, cuts and trimmings of abattoirs in Argentina.

Several foods contaminated with Shiga toxin-producing Escherichia coli (STEC) are associated with human diseases. Some countries have established microbiological criteria for non-O157 STEC, thus, the absence of serogroups O26, O45, O103, O104, O111, O121, and O145 in sprouts from the European Union or ground beef and beef trimmings from the United States is mandatory. While in Argentina screening for O26, O103, O111, O145 and O121 in ground beef, ready-to-eat food, sausages and vegetables is mandatory, other countries have zero-tolerance for all STEC in chilled beef. The aim of this study was to provide data on the prevalence of non-O157 STEC isolated from beef processed in eight Argentinean cattle slaughterhouses producing beef for export and local markets, and to know the non-O157 STEC profiles through strain characterization and genotypic analysis. Samples (n = 15,965) from 3,205 beef carcasses, 9,570 cuts and 3,190 trimmings collected between March and September 2014 were processed in pools of five samples each. Pools of samples (n = 3,193) from 641 carcasses, 1,914 cuts and 638 trimming were analyzed for non-O157 STEC isolation according to ISO/CEN 13136:2012. Of these, 37 pools of carcasses (5.8%), 111 pools of cuts (5.8%) and 45 pools of trimmings (7.0%) were positive for non-O157 STEC. STEC strains (n = 200) were isolated from 193 pools of samples. The most prevalent serotypes were O174:H21, O185:H7, O8:H19, O178:H19 and O130:H11, and the most prevalent genotypes were stx2c(vh-b) and stx2a/saa/ehxA. O103:H21 strain was eae-positive and one O178:H19 strain was aggR/aaiC-positive. The prevalence of non-O157 STEC in beef carcasses reported here was low. None of the non-O157 STEC strains isolated corresponded to the non-O157 STEC serotypes and virulence profiles isolated from human cases in Argentina in the same study period. The application of microbiological criteria for each foodstuff should be determined by risk analysis in order to have a stringent monitoring system. Likewise, zero-tolerance intervention measures should be applied in beef, together with GMP and HACCP. Further, collaborative efforts for risk assessment, management and communication are extremely important to improve the safety of foodstuffs.

Comprehensive Evaluation and Implementation of Improvement Actions in Butcher Shops.

Foodborne pathogens can cause acute and chronic diseases and produce a wide range of symptoms. Since the consumption of ground beef is a risk factor for infections with some bacterial pathogens, we performed a comprehensive evaluation of butcher shops, implemented improvement actions for both butcher shops and consumers, and verified the impact of those actions implemented. A comprehensive evaluation was made and risk was quantified on a 1-100 scale as high-risk (1-40), moderate-risk (41-70) or low-risk (71-100). A total of 172 raw ground beef and 672 environmental samples were collected from 86 butcher shops during the evaluation (2010-2011) and verification (2013) stages of the study. Ground beef samples were analyzed for mesophilic aerobic organisms, Escherichia coli and coagulase-positive Staphylococcus aureus enumeration. Salmonella spp., E. coli O157:H7, non-O157 Shiga toxin-producing E. coli (STEC), and Listeria monocytogenes were detected and isolated from all samples. Risk quantification resulted in 43 (50.0%) high-risk, 34 (39.5%) moderate-risk, and nine (10.5%) low-risk butcher shops. Training sessions for 498 handlers and 4,506 consumers were held. Re-evaluation by risk quantification and microbiological analyses resulted in 19 (22.1%) high-risk, 42 (48.8%) moderate-risk and 25 (29.1%) low-risk butcher shops. The count of indicator microorganisms decreased with respect to the 2010-2011 period. After the implementation of improvement actions, the presence of L. monocytogenes, E. coli O157:H7 and stx genes in ground beef decreased. Salmonella spp. was isolated from 10 (11.6%) ground beef samples, without detecting statistically significant differences between both study periods (evaluation and verification). The percentage of pathogens in environmental samples was reduced in the verification period (Salmonella spp., 1.5%; L. monocytogenes, 10.7%; E. coli O157:H7, 0.6%; non-O157 STEC, 6.8%). Risk quantification was useful to identify those relevant facts in butcher shops. The reduction of contamination in ground beef and the environment was possible after training handlers based on the problems identified in their own butcher shops. Our results confirm the feasibility of implementing a comprehensive risk management program in butcher shops, and the importance of information campaigns targeting consumers. Further collaborative efforts would be necessary to improve foodstuffs safety at retail level and at home.

Isolation of Shiga Toxin-Producing Escherichia coli from Ground Beef Using Multiple Combinations of Enrichment Broths and Selective Agars.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens, and beef cattle are recognized as the principal reservoir. The aims of this study were (1) to identify the most sensitive combination of selective enrichment broths and agars for STEC isolation in artificially inoculated ground beef samples, and (2) to evaluate the most efficient combination(s) of methods for naturally contaminated ground beef samples. A total of 192 ground beef samples were artificially inoculated with STEC and non-stx bacterial strains. A combination of four enrichment broths and three agars were evaluated for sensitivity, specificity, and positive predictive value for STEC isolation from experimentally inoculated samples. Enrichments with either modified tryptic soy broth (mTSB) containing 8 mg/L novobiocin (mTSB-8) or modified Escherichia coli (mEC) broth followed by isolation in MacConkey agar were the most sensitive combinations for STEC isolation of artificially inoculated samples. Independently, both enrichments media followed by isolation in MacConkey were used to evaluate ground beef samples from 43 retail stores, yielding 65.1% and 58.1% stx-positive samples by RT-PCR, respectively. No difference was observed in the isolate proportions between these two methods (8/25 [32%] and 8/28 [28.6%]). Identical serotypes and stx genotypes were observed in STEC strains isolated from the same samples by either method. In this study, no single enrichment protocol was sufficient to detect all STEC in artificially inoculated samples and had considerable variation in detection ability with naturally contaminated samples. Moreover, none of the single or combinations of multiple isolation agars used were capable of identifying all STEC serogroups in either artificially inoculated or naturally occurring STEC-contaminated ground beef. Therefore, it may be prudent to conclude that there is no single method or combination of isolation methods capable of identifying all STEC serogroups.

Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat.

Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n=103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stx-positive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1×10(2) CFU mL(-1) limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P<0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation.

Shiga toxin-producing Escherichia coli in beef retail markets from Argentina.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause mild or serious diseases and can lead to people death. This study reports the prevalence and characteristics of STEC O157 and non-O157 in commercial ground beef and environmental samples, including meat table, knife, meat mincing machine, and manipulator hands (n = 450) obtained from 90 retail markets over a nine-month period. The STEC isolates were serotyped and virulence genes as stx (Shiga toxin), rfb(O157)] (O157 lipopolysaccharide), fliC(H7) (H7 flagellin), eae (intimin), ehxA (enterohemolysin) and saa (STEC autoagglutinating adhesin), were determined. STEC O157 were identified in 23 (25.5%) beef samples and 16 (4.4%) environmental samples, while STEC non-O157 were present in 47 (52.2%) and 182 (50.5%), respectively. Among 54 strains isolated, 17 were STEC O157:H7 and 37 were STEC non-O157. The prevalent genotype for O157 was stx(2)/eae/ehxA/fliC(H7) (83.4%), and for STEC non-O157 the most frequent ones were stx(1)/stx(2)/saa/ehxA (29.7%); stx(2) (29.7%); and stx(2)/saa/ehxA (27%). None of the STEC non-O157 strains were eae-positive. Besides O157:H7, other 20 different serotypes were identified, being O8:H19, O178:H19, and O174:H28 the prevalent. Strains belonging to the same serotype could be isolated from different sources of the same retail market. Also, the same serotype could be detected in different stores. In conclusion, screening techniques are increasingly sensitive, but the isolation of STEC non-O157 is still a challenge. Moreover, with the results obtained from the present work, although more studies are needed, cross-contamination between meat and the environment could be suspected.