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.

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.

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.

Comparación de tres técnicas para subtipificación molecular de Listeria monocytogenes / Comparison of three molecular subtyping techniques for Listeria monocytogenes

Abstract Listeria monocytogenes is a foodborne pathogen. The recent alert for L. monocytogenes in vegetables from Argentina warns about the importance of reinforcing its isolation, characterization and subtyping in food, clinical and environmental samples. The aim of the present study was to compare the discriminatory power of enterobacterial repetitive interpower; genic consensus polymerase chain reaction (ERIC-PCR), automated ribotyping and pulsed-field gel electrophoresis (PFGE) to subtype strains of L. monocytogenes isolated from Argentine meat and environmental samples. Simpson's Diversity Index (DI) was calculated on the basis of based on the dendrograms obtained in the by cluster analysis, showing the following discriminatory power: ApaI-PFGE (0.980), AscI-PFGE (0.966), ribotyping (0.912), ERIC-PCR (0.886). The ID values between ApaI- and AscI-PFGE and between ribotyping and ERIC-PCR were not significantly different. Of the three techniques evaluated, PFGE showed the highest discriminatory power. However, the subtyping techniques should be accompanied by effective food monitoring strategies and reliable clinical and epidemiological studies.

Resumen Listeria monocytogenes es un patógeno alimentario. La reciente alerta por la presencia de L. monocytogenes en vegetales en Argentina advierte sobre la importancia de reforzar el aislamiento, la caracterización y la subtipificación de esta bacteria en muestras clínicas de alimentos y ambientales. El objetivo del presente estudio fue comparar el poder discriminatorio de enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), la ribotipificación automatizada y la pulsed-field gel electrophoresis (PFGE) para subtipificar cepas de L. monocytogenes aisladas de carne y de muestras ambientales en Argentina. El índice de diversidad (ID) de Simpson, calculado a partir de los dendrogramas obtenidos en el análisis de agrupamiento, mostró los siguientes resultados: Apal-PFGE (0,980), AscI-PFGE (0,966), riboti-pado (0,912), ERIC-PCR (0,886). Los valores obtenidos no fueron significativamente diferentes al comparar entre Apal- y AscI-PFGE, ni entre ribotipadoy ERIC-PCR. De las técnicas evaluadas, la PFGE presentó el mayor poder discriminatorio. Sin embargo, las técnicas de subtipificación deberían acompañarse de estrategias de control de los alimentos efectivas y de estudios clínicos y epidemiológicos confiables.

Comparison of three molecular subtyping techniques for Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen. The recent alert for L. monocytogenes in vegetables from Argentina warns about the importance of reinforcing its isolation, characterization and subtyping in food, clinical and environmental samples. The aim of the present study was to compare the discriminatory power of enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), automated ribotyping and pulsed-field gel electrophoresis (PFGE) to subtype strains of L. monocytogenes isolated from Argentine meat and environmental samples. Simpson's Diversity Index (DI) was calculated on the basis of based on the dendrograms obtained in the by cluster analysis, showing the following discriminatory power: ApaI-PFGE (0.980), AscI-PFGE (0.966), ribotyping (0.912), ERIC-PCR (0.886). The ID values between ApaI- and AscI-PFGE and between ribotyping and ERIC-PCR were not significantly different. Of the three techniques evaluated, PFGE showed the highest discriminatory power. However, the subtyping techniques should be accompanied by effective food monitoring strategies and reliable clinical and epidemiological studies.

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.

Fermented whey as poultry feed additive to prevent fungal contamination.

BACKGROUND: Fungal contamination of poultry feed causes economic losses to industry and represents a potential risk to animal health. The aim of the present study was to analyze the effectiveness of whey fermented with kefir grains as additive to reduce fungal incidence, thus improving feed safety. RESULTS: Whey fermented for 24 h at 20 °C with kefir grains (100 g L(-1) ) reduced conidial germination of Aspergillus flavus, Aspergillus parasiticus, Aspergillus terreus, Aspergillus fumigatus, Penicillium crustosum, Trichoderma longibrachiatum and Rhizopus sp. Poultry feed supplemented with fermented whey (1 L kg(-1) ) was two to four times more resistant to fungal contamination than control feed depending on the fungal species. Additionally, it contained kefir microorganisms at levels of 1 × 10(8) colony-forming units (CFU) kg(-1) of lactic acid bacteria and 6 × 10(7) CFU kg(-1) of yeasts even after 30 days of storage. CONCLUSION: Fermented whey added to poultry feed acted as a biopreservative, improving its resistance to fungal contamination and increasing its shelf life.

Yeasts from kefir grains: isolation, identification, and probiotic characterization.

Kefir-a traditional beverage whose consumption has been associated with health benefits-is a logical natural product to investigate for new probiotic strains. The aim of the present work was to isolate and identify kefir yeasts and select those with acid and bile tolerance to study their adhesion to epithelial cells and their transit through mouse gut. From 4 milky and 3 sugary kefir grains, 34 yeast strains were isolated and identified by means of classical microbiological and molecular-genetic methods (whole-cell protein pattern, internal-transcribed-spacer amplification, and analysis of restriction-fragment-length polymorphisms). We identified 4 species belonging to 3 genera-Saccharomyces cerevisiae (15 strains), Saccharomyces unisporus (6 strains), Issatchenkia occidentalis (4 strains), and Kluyveromyces marxianus (9 strains)-and selected 13 strains on the basis of resistance to low pH and bile salts. Among the strains selected, Kluyveromyces marxianus CIDCA 8154 and Saccharomyces cerevisiae CIDCA 8112 were further studied. Both strains evidenced the capacity to adhere to epithelial intestine-derived cells in vitro and to survive passage through the gastrointestinal tract of BALB/c mice. The investigation of the potential probiotic features of these kefir-yeast strains should be useful for the development of novel functional foods.

Application of culture-dependent and culture-independent methods for the identification of Lactobacillus kefiranofaciens in microbial consortia present in kefir grains.

The biological and technological characteristics of kefiran as well as its importance in grain integrity led us to analyze the microbial kefir grain consortium with focus on Lactobacillus kefiranofaciens. The presence of L. kefiranofaciens in the nine kefir grains studied was demonstrated by denaturing gradient gel electrophoresis. By culture dependent methods applying a methodology focused on the search of this species, 22 isolates with typical morphology were obtained and identified applying a combination of SDS-PAGE of whole cell proteins, (GTG)5-PCR and sequence analysis of the housekeeping gene encoding the α-subunit of bacterial phenylalanyl-tRNA synthase (pheS). This polyphasic approach allowed the reliable identification of 11 L. kefiranofaciens, 5 Lactobacillus paracasei, 4 Lactobacillus kefiri and 2 Lactobacillus parakefiri isolates. Isolated L. kefiranofaciens strains produced polysaccharide in strain-dependent concentrations and EPS produced by them also differed in the degree of polymerization. The isolation and accurate identification of L. kefiranofaciens is relevant taking into account the important role of this microorganism in the grain ecosystem as well as its potential application as starter in food fermentations.

Kefir grains as a starter for whey fermentation at different temperatures: chemical and microbiological characterisation.

We report here a comparative analysis of the growth, acidification capacity, and chemical and microbiologic composition between kefir grains after 20 subcultures in whey at 20, 30, and 37°C and the original kefir grains coming from milk along with a determination of the microbiological composition of the fermented whey as compared with that of traditional fermented milk. When fermentation was carried out repeatedly at 30 or 37°C, kefir grains changed their kefir-like appearance, exhibited reduced growth rates, had a lower diversity of yeasts and water content, and a higher protein-to-polysaccharide ratio compared with the original kefir grains. In contrast, at 20°C kefir grains could remain in whey for prolonged periods without altering their acidification capacity, growth rate, macroscopic appearance or chemical and microbiologic composition-with the only difference being a reduction in certain yeast populations after 20 subcultures in whey. At this incubation temperature, the presence of Lactobacillus kefiranofaciens, Lb. kefir, Lb. parakefir, Lactococcus lactis, Kluyveromyces marxianus, Saccharomyces unisporus, and Sac. cerevisiae was detected in kefir grains and in fermented whey by denaturing-gradient-gel electrophoresis (DGGE). In whey fermented at 20°C the number of lactic-acid bacteria (LAB) was significantly lower (P<0·05) and the number of yeast significantly higher (P<0·05) than in fermented milk. Since the DGGE profiles were similar for both products, at this temperature the microbiologic composition of fermented whey is similar to that of fermented milk. We therefore suggest a temperature of 20°C to preserve kefir grains as whey-fermentation starters.

Development of a method based on chemometric analysis of Raman spectra for the discrimination of heterofermentative lactobacilli.

In this work, a method based on Raman spectroscopy in combination with Principal Component Analysis (PCA) and Partial Least Square-Discriminant Analysis (PLS-DA) has been developed for the rapid differentiation of heterofermentative related lactobacilli. In a first approach, Lactobacillus kefir strains were discriminated from other species of heterofermentative lactobacilli: Lb. parakefir and Lb. brevis. After this first approach, PCA allowed for a clear differentiation between Lb. parakefir and Lb.brevis. For the first level of discrimination, PCA was performed on the whole spectra and also on delimited regions, defined taking into consideration the loading values. The best regions allowing a clear differentiation between Lb. kefir and non-Lb. kefir strains were found to be: the 1700-1500 cm(-1), 1500-1185 cm(-1) and 1800-400 (whole spectrum) cm(-1) Raman ranges. In order to develop a classification rule, PLS-DA was carried out on the mentioned regions. This method permitted the discrimination and classification of the strains under study in two groups: Lb. kefir and non-Lb. kefir. The model was further validated using lactobacilli strains from different culture collections or strains isolated from kefir grains previously identified using molecular methods. The second approach based on PCA was also performed on the whole spectra and on delimited regions, being the regions 1700-1500 cm(-1), 1500-1185 cm(-1) and 1185-1020 cm(-1), i.e., those allowing the clearest discrimination between Lb. parakefir and Lb. brevis. The results obtained in this work, allowed a clear discrimination within heterofermentative lactobacilli strains, proteins being the biological structures most determinant for this discrimination.