Alicyclobacillus spp. in fruit-based products: Isolation, identification, quantitative assessment (SPME/GC-MS) of spoilage compounds and spore's resistance to thermal shocks.

Alicyclobacillus spp. is the cause of great concern for the food industry due to their spores' resistance (thermal and chemical) and the spoilage potential of some species. Despite this, not all Alicyclobacillus strains can spoil fruit juices. Thus, this study aimed to identify Alicyclobacillus spp. strains isolated from fruit-based products produced in Argentina, Brazil, and Italy by DNA sequencing. All Alicyclobacillus isolates were tested for guaiacol production by the peroxidase method. Positive strains for guaiacol production were individually inoculated at concentration of 103 CFU/mL in 10 mL of orange (pH 3.90) and apple (pH 3.50) juices adjusted to 11°Brix, following incubation at 45 °C for at least 5 days to induce the production of the following spoilage compounds: Guaiacol, 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). The techniques of micro-solid phase extraction by headspace (HS-SPME) and gas-chromatography with mass spectrometry (GC-MS) were used to identify and quantify the spoilage compounds. All GC-MS data was analyzed by principal component analysis (PCA). The effects of different thermal shock conditions on the recovery of Alicyclobacillus spores inoculated in orange and apple juice (11°Brix) were also tested. A total of 484 strains were isolated from 48 brands, and the species A. acidocaldarius and A. acidoterrestris were the most found among all samples analyzed. In some samples from Argentina, the species A. vulcanalis and A. mali were also identified. The incidence of these two main species of Alicyclobacillus in this study was mainly in products from pear (n = 108; 22.3 %), peach (n = 99; 20.5 %), apple (n = 86; 17.8 %), and tomato (n = 63; 13 %). The results indicated that from the total isolates from Argentina (n = 414), Brazil (n = 54) and Italy (n = 16) were able to produce guaiacol: 107 (25.8 %), 33 (61.1 %) and 13 (81.2 %) isolates from each country, respectively. The PCA score plot indicated that the Argentina and Brazil isolates correlate with higher production of guaiacol and 2,6-DCP/2,6-DBP, respectively. Heatmaps of cell survival after heat shock demonstrated that strains with different levels of guaiacol production present different resistances according to spoilage ability. None of the Alicyclobacillus isolates survived heat shocks at 120 °C for 3 min. This work provides insights into the incidence, spoilage potential, and thermal shock resistance of Alicyclobacillus strains isolated from fruit-based products.

Quantitative microbial risk assessment of haemolytic uremic syndrome associated with Argentinean kosher beef consumption in Israel.

The aim of this study was to perform a quantitative microbial risk assessment (QMRA) of Shiga toxin-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) linked to the consumption of Kosher beef produced in Argentina and consumed in Israel in children under 14 years. A probabilistic risk assessment model was developed to characterize STEC prevalence and contamination levels in the beef supply chain (cattle primary production, cattle transport, processing and storage in the abattoir, for export and at retail, and home preparation and consumption). The model was implemented in Microsoft Excel 2016 with the @Risk add-on package. Results of 302 surveys with data collected in Israel were as follows: 92.3% of people consumed beef, mostly at home, and 98.2% preferred levels of cooking that ensured STEC removal from the surface of beef cuts. The preferred degree of ground beef doneness was "well-done" (48.2%). Cooking preference ranged from red to "medium-well done" (51.8%). Median HUS probability from Argentinean beef cut and ground beef consumption in children under 14 years old was <10-15 and 8.57x10-10, respectively. The expected average annual number of HUS cases and deaths due to beef cut and ground beef consumption was zero. Risk of infection and HUS probability correlated with salting effect on E. coli count, processing raw beef before vegetables, ways of storage and refrigeration temperature at home, joint consumption of salad and beef cuts, degree of beef doneness and cutting board washing with detergent after each use with beef and vegetables. The STEC-HUS risk in Israel from consumption of bovine beef produced in Argentina was negligible. The current QMRA results were similar to those of previous beef cut consumption QMRA in Argentina and lower than any of the QMRA performed worldwide in other STEC-HUS linked to ground beef consumption. This study confirms the importance of QMRA to estimate and manage the risk of STEC-HUS from beef consumption. The impact variables identified in the sensitivity analysis allowed us to optimize resources and time management, to focus on accurate actions and to avoid taking measures that would not have an impact on the risk of STEC-HUS.

Prioritization of vegetable-borne biological hazards in Argentina using a multicriteria decision analysis tool.

Vegetables, especially those eaten raw, have been implicated in several foodborne disease outbreaks. Since multiple vegetable matrices and hazards are involved, risk managers have to prioritize those with the greatest impact on public health to design control strategies. In this study, a scientific-based risk ranking of foodborne pathogens transmitted by leafy green vegetables in Argentina was performed. The prioritization process included hazard identification, evaluation criteria identification and definition, criteria weighting, expert survey design and selection and call for experts, hazard score calculation, hazard ranking and variation coefficient, and result analysis. Regression tree analysis determined four risk clusters: high (Cryptosporidum spp., Toxoplasma gondii, Norovirus), moderate (Giardia spp., Listeria spp., Shigella sonnei), low (Shiga toxin-producing Escherichia coli, Ascaris spp., Entamoeba histolytica, Salmonella spp., Rotavirus, Enterovirus) and very low (Campylobacter jejuni, hepatitis A virus and Yersinia pseudotuberculosis). Diseases caused by Norovirus, Cryptosporidium spp. and T. gondii do not require mandatory notification. Neither viruses nor parasites are included as microbiological criteria for foodstuff. The lack of outbreak studies did not allow to accurately identify vegetables as a source of Norovirus disease. Information on listeriosis cases or outbreaks due to vegetable consumption was not available. Shigella spp. was the main responsible for bacterial diarrhea, but it has not been epidemiologically associated with vegetable consumption. The quality of the available information for all hazards studied was very low and low. The implementation of good practice guidelines throughout the entire vegetable production chain could prevent the presence of the identified hazards. The current study allowed the identification of vacancy areas and could help reinforce the need for performing epidemiological studies on foodborne diseases potentially associated with vegetable consumption in Argentina.

Molecular subtyping of Salmonella spp. strains in provincial abattoirs with no hazard analysis critical control point from Buenos Aires, Argentina / Subtipificación molecular de Salmonella spp. en frigoríficos provinciales sin análisisde peligros y puntos críticos de control ubicados en Buenos Aires, Argentina

Abstract We subtyped 32 Salmonella enterica strains isolated from carcasses (n = 10), theenvironment (n = 14), head meat (n = 1) and viscera washing and chilling water (n = 7) in provin-cial abattoirs with no Hazard Analysis Critical Control Point (HACCP) system from Buenos Aires,Argentina, before and after implementing improvement actions. Pulsed-field gel electrophore-sis (PFGE) was carried out using the XbaI restriction enzyme. Strains belonged to six serovars,from which 10 restriction patterns were obtained (five unique patterns and five clusters). Wefound different clones of S. enterica serovars in the same abattoir by XbaI-PFGE. In addition topromoting good hygiene practices, the implementation of an HACCP plan is necessary to meetthe zero-tolerance criteria for Salmonella on beef.

Resumen Subtipificamos en total 32 cepas de Salmonella enterica aisladas de carcasas(n = 10), medio ambiente (n = 14), carne de cabeza (n = 1) y agua de lavado y enfriamientode vísceras (n = 7) en frigoríficos provinciales de Buenos Aires (Argentina) sin análisis de peli-gros y puntos críticos de control (hazard analysis critical control point [HACCP]); la toma demuestras se efectuó antes y después de implementar acciones de mejora. Se llevó a cabo elec-troforesis en gel de campo pulsado (PFGE) utilizando la enzima de restricción XbaI. Las cepaspertenecían a 6 serovares y presentaron 10 patrones de restricción (5 patrones únicos y 5 clus-ters). Demostramos la presencia de diferentes serovares de S. enterica en un mismo frigorífico.

Molecular subtyping of Salmonella spp. strains in provincial abattoirs with no hazard analysis critical control point from Buenos Aires, Argentina.

We subtyped 32 Salmonella enterica strains isolated from carcasses (n=10), the environment (n=14), head meat (n=1) and viscera washing and chilling water (n=7) in provincial abattoirs with no Hazard Analysis Critical Control Point (HACCP) system from Buenos Aires, Argentina, before and after implementing improvement actions. Pulsed-field gel electrophoresis (PFGE) was carried out using the XbaI restriction enzyme. Strains belonged to six serovars, from which 10 restriction patterns were obtained (five unique patterns and five clusters). We found different clones of S. enterica serovars in the same abattoir by XbaI-PFGE. In addition to promoting good hygiene practices, the implementation of an HACCP plan is necessary to meet the zero-tolerance criteria for Salmonella on beef.

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.

Analysis of scenarios to reduce the probability of acquiring hemolytic uremic syndrome associated with beef consumption.

The objective of this study was to develop a quantitative microbial risk assessment (QMRA) model to evaluate potential risk mitigation strategies to reduce the probability of acquiring hemolytic uremic syndrome (HUS) associated with beef consumption in Argentina. Five scenarios were simulated to evaluate the effect of interventions on the probability of acquiring HUS from Shiga toxin-producing Escherichia coli (STEC)-contaminated ground beef and commercial hamburger consumption. These control strategies were chosen based on previous results of the sensitivity analysis of a baseline QMRA model. The application of improvement actions in abattoirs not applying Hazard Analysis and Critical Control Points (HACCP) for STEC would result 7.6 times lower in the probability that consumers acquired HUS from ground beef consumption, while the implementation of improvements in butcher shops would lead to a smaller reduction. In abattoirs applying HACCP for STEC, the risk of acquiring HUS from commercial hamburger consumption was significantly reduced. Treatment with 2% lactic acid, hot water and irradiation reduced 4.5, 3.5 and 93.1 times the risk of HUS, respectively. The most efficient interventions, in terms of case reduction, being those that are applied in the initial stages of the meat chain.

Quantitative risk assessment of haemolytic uremic syndrome associated with beef consumption in Argentina.

We developed a quantitative microbiological risk assessment (QMRA) of haemolytic uremic syndrome (HUS) associated with Shiga toxin-producing Escherichia coli (STEC)-contaminated beef (intact beef cuts, ground beef and commercial hamburgers) in children under 15 years of age from Argentina. The QMRA was used to characterize STEC prevalence and concentration levels in each product through the Argentinean beef supply chain, including cattle primary production, cattle transport, processing and storage in the abattoir, retail and home preparation, and consumption. Median HUS probability from beef cut, ground beef and commercial hamburger consumption was <10-15, 5.4x10-8 and 3.5x10-8, respectively. The expected average annual number of HUS cases was 0, 28 and 4, respectively. Risk of infection and HUS probability were sensitive to the type of abattoir, the application or not of Hazard Analysis and Critical Control Points (HACCP) for STEC (HACCP-STEC), stx prevalence in carcasses and trimmings, storage conditions from the abattoir to retailers and home, the joint consumption of salads and beef products, and cooking preference. The QMRA results showed that the probability of HUS was higher if beef cuts (1.7x) and ground beef (1.2x) were from carcasses provided by abattoirs not applying HACCP-STEC. Thus, the use of a single sanitary standard that included the application of HACCP-STEC in all Argentinean abattoirs would greatly reduce HUS incidence. The average number of annual HUS cases estimated by the QMRA (n = 32) would explain about 10.0% of cases in children under 15 years per year in Argentina. Since other routes of contamination can be involved, including those not related to food, further research on the beef production chain, other food chains, person-to-person transmission and outbreak studies should be conducted to reduce the impact of HUS on the child population of Argentina.

Characterization and molecular subtyping of Shiga toxin-producing Escherichia coli strains in provincial abattoirs from the Province of Buenos Aires, Argentina, during 2016-2018.

We characterized Shiga toxin-producing Escherichia coli (STEC) O157 (n = 20) and non-O157 (n = 68) isolated from carcasses (n = 54), the environment (n = 20), head meat (n = 3) and viscera washing and chilling water (n = 11) in provincial abattoirs before and after implementing improvement actions. The strains were tested for eae, saa, ehxA and fliCH7 genes. Variants stx1 and stx2 were also determined. Pulsed-field gel electrophoresis (PFGE) was carried out with restriction enzymes XbaI and BlnI. All twenty O157 STEC strains [H7; H21; HNM] carried genes rfbO157 and ehxA; 90.0 % were positive for eae and 15.0 % were negative for fliCH7 and positive for saa. Results of PFGE showed 17 XbaI patterns, of which 14 were unique and three formed clusters. From the 68 non-O157 STEC strains, 66.2 %, 55.9 % and 2.9 % were positive for ehxA, saa and eae genes, respectively. Fifty-three XbaI patterns were obtained (49 unique and four forming clusters). Cross-contamination between products and between the environment and products was confirmed in all abattoirs. While the proposed improvements reduced the risk of contamination, Good Hygiene Practices and Good Manufacturing Practices should be implemented in provincial abattoirs, stressing the importance of having a uniform national food safety standard.

Prevalence of Campylobacter jejuni and Campylobacter coli from broilers at conventional and kosher abattoirs and retail stores / Prevalencia de Campylobacter jejuni y Campylobacter coli en carcasas de pollo procedentes de frigoríficos, faena tradicional y kosher, y de locales de expendio

Abstract We studied and compared the prevalence of Campylobacter jejuni and Campylobacter coli in chicken carcasses from conventional and kosher broiler abattoirs and retail stores. The prevalence of thermotolerant Campylobacter-positive carcasses was 94.0 (kosher) and 32.0% (conventional) (p< 0.0001), while the prevalence of samples contaminated with C. jejuni, C. coli and simultaneously with both species was 36.0, 2.0 and 56.0% (kosher) and 26.0, 4.0 and 2.0% (conventional) (p< 0.0001), respectively. Samples of chicken carcasses (n = 25) and food contact surfaces (tables, n = 25; knives, n=25) from 25 retails were collected and risk quantification was performed. Retails were categorized as high-risk (n = 11), moderate-risk (n = 11) and low-risk (n = 3). Nineteen (76.0%) carcasses, 20 (80.0%) tables and 18 (72.0%) knives were Campylobacter-positive. Retails and abattoirs proved to be sources of carcass contaminaron with Campylobacter spp. Carcasses from kosher abattoirs were mostly contaminated with Campylobacter spp., whereas C. coli was the most prevalent species isolated from carcasses in retail stores.

Resumen El objetivo del estudio fue determinar y comparar la prevalencia de Campylobacter jejuni y Campylobacter coli en carcasas de pollo obtenidas en frigoríficos por faena convencional y kosher, y en locales de expendio. La prevalencia de Campylobacter spp. termotolerante fue del 94,0 (kosher) y del 32,0% (convencional) (p< 0,0001). La prevalencia de muestras contaminadas con C. jejuni, C. coli y con ambas especies fue del 36,0, del 2,0 y del 56,0% (Kosher) y del 26,0, del 4,0 y del 2,0% (convencional) (p< 0,0001), respectivamente. Se tomaron muestras de carcasas (n = 25) y superficies (tablas, n = 25; cuchilla, n = 25) en 25 locales. Los locales fueron categorizados como de riesgo alto (n = 11), moderado (n = 11) y bajo (n = 3). Diecinueve (76,0%) carcasas, 20 (80,0%) tablas y 18 (72,0%) cuchillas fueron positivas para Campylobacter spp. Frigoríficos y locales fueron fuente de contaminación de carcasas con Campylobacter spp. La prevalencia de Campylobacter spp. fue mayor en carcasas kosher. Campylobacter coli fue la especie más prevalente en carcasas de locales.

Comprehensive evaluation and implementation of improvement actions in bovine abattoirs to reduce pathogens exposure.

The slaughter process plays an important role in animal welfare, meat quality, safety and public health through the meat production chain. In this study, we performed a three-stage evaluation: I) comprehensive evaluation, II) implementation of improvement actions and III) verification of the success of the actions implemented in three abattoirs from Argentina during 2016-2018. Risk was estimated using two checklists, quantified on a 1-100 scale and classified as high (1-40), moderate (41-70) and low (71-100). In stages I and III, Salmonella spp., E. coli O157:H7 and non-O157 STEC were detected and isolated in samples from carcasses (n = 252), the environment (n = 252); head meat (n = 21) and viscera washing and chilling water (n = 105). Carcass samples were analyzed for mesophilic aerobic organisms, coliforms and E. coli enumeration. Of 201 water samples taken, 42.0-75.6 % were non-potable quality. After the implementation of improvement actions in stage II (building, processes, systems for water purification and training), the estimation of risk of contamination was reduced from high to moderate in all three abattoirs, the count of indicator microorganisms decreased in two abattoirs, and the presence of pathogens significantly decreased. Salmonella spp. was not isolated from any of the samples collected in two abattoirs. Isolation of E. coli O157:H7 decreased in carcass and was not isolated from viscera washing and chilling water. Isolation of non-O157 STEC decreased in carcass but not in environmental samples. Finally, 75.0-95.0 % of water samples were of potable quality. Although this was only the first step in the process of change and improvement of abattoirs, the assessment of the situation and the proposal of solutions to correct deviations in a joint effort with the health authorities helped to implement a work model for enhancing food safety before meat reaches consumers.

Prevalence of Campylobacter jejuni and Campylobacter coli from broilers at conventional and kosher abattoirs and retail stores.

We studied and compared the prevalence of Campylobacter jejuni and Campylobacter coli in chicken carcasses from conventional and kosher broiler abattoirs and retail stores. The prevalence of thermotolerant Campylobacter-positive carcasses was 94.0 (kosher) and 32.0% (conventional) (p<0.0001), while the prevalence of samples contaminated with C. jejuni, C. coli and simultaneously with both species was 36.0, 2.0 and 56.0% (kosher) and 26.0, 4.0 and 2.0% (conventional) (p<0.0001), respectively. Samples of chicken carcasses (n=25) and food contact surfaces (tables, n=25; knives, n=25) from 25 retails were collected and risk quantification was performed. Retails were categorized as high-risk (n=11), moderate-risk (n=11) and low-risk (n=3). Nineteen (76.0%) carcasses, 20 (80.0%) tables and 18 (72.0%) knives were Campylobacter-positive. Retails and abattoirs proved to be sources of carcass contamination with Campylobacter spp. Carcasses from kosher abattoirs were mostly contaminated with Campylobacter spp., whereas C. coli was the most prevalent species isolated from carcasses in retail stores.

Evaluation of intervention measures at different stages of the production chain in Argentinian exporting abattoirs.

Antimicrobial treatments could help to decrease the transmission of microorganisms to beef carcasses and abattoir environments. The aim of this study was to evaluate the effectiveness of interventions in reducing Shiga toxin genes (stx1 and stx2) presence in a commercial abattoir. Intervention measures included the application of electrolytically generated hypochlorous acid to steer pens (experiment 1), chlorinated water, electrolytically generated hypochlorous acid, and isoclor to steer pens (experiment 2), electrolytically generated hypochlorous acid to knocking pens (experiment 3), and aqueous ozone and electrolytically generated hypochlorous acid onto beef carcasses (experiment 4). Detection of stx in samples was performed with BAX® System Real-Time PCR Assay. Our results showed that treatment with pressurized electrolytically generated hypochlorous acid and isoclor were effective to reduce stx presence from hides on steer pens. Although there is no single strategy to ensure the reduction of stx presence in a commercial abattoir, the combined application of several antimicrobial interventions would be ideal.

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.

Characterization and Molecular Subtyping of Shiga Toxin-Producing Escherichia coli Strains in Butcher Shops.

Shiga toxin-producing Escherichia coli (STEC) are important emerging foodborne human pathogens. Ruminants are the main animal reservoir of STEC currently known, and meat can become contaminated at different stages of the production chain. The aim of this work was to subtype and establish the epidemiological relatedness of non-O157 STEC strains isolated from ground beef and the environment in butcher shops before (evaluation stage, 2010-2011 period) and after (verification stage, 2013) implementing improvement actions. Sixty-eight non-O157 STEC strains were tested for eae, saa, ehxA, iha, efa1, toxB, subAB, cdt-V, astA, aggR, and aaiC genes, and stx1 and stx2 variants were determined. Pulsed-field gel electrophoresis (PFGE) was carried out with XbaI and XmaJI. From the 68 strains, 92.6%, 75.0%, 58.8%, 53.5%, 10.3%, 7.3%, and 4.4% were positive for iha, ehxA, subAB, saa, cdt-V, astA, and eae, respectively. All strains were aggR/aaiC-negative. PFGE showed that 19 strains grouped in 9 clusters and 41 showed unique XbaI patterns. During the evaluation stage (2010-2011), we identified clonal strains in different samples, circulating clones in different butcher shops, and more than one different strain in the same butcher shop. The bovine origin of meat and its manufacturing process could not ensure the total absence of all non-O157 STEC serotypes in this foodstuff. Most strains isolated during the evaluation (2010-2011) and verification (2013) stages did not exhibit a genotypic profile associated with human disease. It is necessary to conduct periodic reviews of the new epidemiological information and verify that the analyses of non-O157 STEC in food are appropriate to identify strains affecting the population.

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.

High prevalence of clade 8 Escherichia coli O157:H7 isolated from retail meat and butcher shop environment.

Escherichia coli O157:H7 is an enteric pathogen associated with food safety threats and with significant morbidity and mortality worldwide. In Argentina, post-enteric hemolytic uremic syndrome (HUS) is endemic, with >70% of cases associated with E. coli O157 infection. To date the biological basis behind the severity among E. coli O157 infections is unknown. However, single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades, of which clade 8 strains are associated with severe disease cases. The aim of this study was to characterize a collection of 20 STEC O157:H7 strains isolated between 2011 and 2013 from ground beef and different environmental samples from butcher shops of Argentina. All strains harbored the eae, ehxA, fliCH7, efa, iha, and toxB genes, with stx2a/stx2c as the predominant genotype (75%). The XbaI-PFGE analysis showed that the E. coli O157 strains had high genetic diversity. Nine strains were grouped in four XbaI-PFGE clusters, whereas 11 strains showed unique XbaI-PFGE patterns. In contrast, the SNP analysis allowed us to separate the strains in two distinct lineages representing clade 8 (70%) and clade 6 (30%). Our results show the molecular characterization of E. coli O157 strains isolated from ground beef and environmental samples from Argentinean butcher shops.

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.