Characterization of diarrheagenic Escherichia coli from different cattle production systems in Brazil.

Diarrheagenic E. coli (DEC) can cause severe diarrhea and is a public health concern worldwide. Cattle are an important reservoir for this group of pathogens, and once introduced into the abattoir environment, these microorganisms can contaminate consumer products. This study aimed to characterize the distribution of DEC [Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC)] from extensive and intensive cattle production systems in Brazil. Samples (n = 919) were collected from animal feces (n = 200), carcasses (n = 600), meat cuts (n = 90), employee feces (n = 9), and slaughterhouse water (n = 20). Virulence genes were detected by PCR in 10% of animal samples (94/919), with STEC (n = 81) as the higher prevalence, followed by EIEC (n = 8), and lastly EPEC (n = 5). Animals raised in an extensive system had a higher prevalence of STEC (average 48%, sd = 2.04) when compared to animals raised in an intensive system (23%, sd = 1.95) (Chi-square test, P < 0.001). From these animals, most STEC isolates only harbored stx2 (58%), and 7% were STEC LEE-positive isolates that were further identified as O157:H7. This study provides further evidence that cattle are potential sources of DEC, especially STEC, and that potentially pathogenic E. coli isolates are widely distributed in feces and carcasses during the slaughter process.

Pathogenic potential and antibiotic resistance of Yersinia enterocolitica, a foodborne pathogen limited to swine tonsils in a pork production chain from Southern Brazil.

In this study, we aimed to characterize the distribution of Yersinia enterocolitica in a pork production chain in Brazil, as well as the virulence profile and antibiotic resistance of the obtained isolates. Samples from 10 pig lots obtained from finishing farms (water, feed, and barn floors, n = 30), slaughterhouse (lairage floors, carcasses at four processing steps, tonsils, and mesenteric lymph nodes, n = 610), and processing (end cuts, processing environment, n = 160) were obtained in Paraná state, Brazil, and subjected to Y. enterocolitica detection by ISO 10,273. The obtained isolates were identified based on biochemical and molecular features (16 s rRNA, inv, bioserotyping) and subjected to PCR assays to detect virulence (ail, ystA, ystB, virF, myfA, fepA, fepD, fes, tccC, ymoA, hreP, and sat) and multidrug resistance-related genes (emrD, yfhD, and marC). Also, isolates were subjected to disk diffusion test to characterize their resistance against 17 antibiotics from 11 classes and to pulsed field gel electrophoresis (PFGE) after XbaI macro-restriction. Y. enterocolitica was detected in a single sample (tonsil), and the obtained three isolates were characterized as serotype O:3, harboring ail, ystA, virF, myfA, tccC, ymoA, hreP, emrD, yfhD, and marC, and resistant to all tested antibiotics. The three isolates presented identical macro-restriction profiles by PFGE, also identical to isolates obtained from Minas Gerais, other Brazilian state; one selected isolate was identified as biotype 4. Despite the low occurrence of Y. enterocolitica in the studied pork production, the virulence potential and the antibiotic resistance profiles of the isolates demonstrated their pathogenic potential, and the macro-restriction profiles indicate strains descending from a common subtype in the pork production chain of two Brazilian States.

Phenotypic and genotypic characterization of non-typhoidal Salmonella isolated from a Brazilian pork production chain.

Pork products are important sources of foodborne non-typhoidal Salmonella in Brazil where antibiotics are commonly used throughout the pork production process and this has the potential to selectively favor antibiotic-resistant strains. We characterized the genotypic and phenotypic diversity of S. enterica isolates (n = 41) that were isolated in Brazil. Isolates were collected from ten swine farms and one slaughterhouse. Whole-genome sequencing and in silico serotyping demonstrated that the S. enterica serovar Typhimurium was the most common serotype (n = 17), but eight additional servoars were identified. Isolates presented high similarity based on comparison of DNA sequences (minimum of 89.6%), and sequence variation grouped according to serotype. Eight multilocus sequence types were identified with ST19 being most common (n = 21). Several plasmids replicons were detected, with Col (RNAI) the most abundant (n = 30), followed by IncR (n = 22), IncI1 (n = 10) and IncA/C2 (n = 10). Minimum inhibitory concentration assays showed that the principle resistance phenotypes were for streptomycin (90.2%), tetracycline (87.8%), ampicillin (80.5%), chloramphenicol (70.7%) and ciprofloxacin (51.2%). Only two isolates were resistant to third-generation cephalosporins and no isolates were resistant to two tested carbapenems. Twenty-six unique antimicrobial-resistance genes were identified with blaTEM-1A and blaTEM-1B likely responsible for most beta-lactam resistance and floR responsible for most chloramphenicol resistance. Six strains were positive for mcr-1. At the time of collection, the sampled farms were adding ciprofloxacin to feed and this may have contributed to the high prevalence of resistance to this antibiotic. The high number of multidrug resistant Salmonella and the presence of multiple resistant genes and plasmids emphasize the diversity of Salmonella in the studied pork chain, specially from serotype Typhimurium.

Comparison of Meat Juice Serology and Bacteriology for Surveillance of Salmonella in the Brazilian Pork Production Chain.

This study assessed an enzyme-linked immunosorbent assay (ELISA) based assay to detect Salmonella in swine as a potential tool to predict the presence of Salmonella in swine carcasses. The following samples were collected from 10 swine batches: blood (n = 100); environment (barn floor, n = 10, and lairage floor, n = 10); meat juice (n = 100, obtained after defrosting of diaphragm); tonsils (n = 100); mesenteric lymph nodes (MLNs) (n = 100); and carcasses after bleeding (n = 100), after singeing (n = 100), after evisceration (n = 100), and after final rinsing (n = 100). Blood and meat juice were subjected to ELISA to detect antibodies against Salmonella, and other samples were subjected to Salmonella detection by ISO 6579. Salmonella was detected in 3 samples from barn floors, 7 lairage floors, 45 tonsils, 43 MLNs and in 3 carcasses. Based on ELISA, Salmonella positive samples were: 86 and 46 blood serum (20% and 40% cut-offs) and 68 and 46 meat juice (20% and 40% cut-offs). Optical density readings from blood serum and meat juice presented a high and significant correlation (r = 0.93, p < 0.001), and a substantial agreement for Salmonella detection (K = 0.69, ELISA 40% cut-off). The agreement between ELISA and microbiological analysis for Salmonella detection in pig carcasses were absent or poor, with the exception of results obtained by ELISA 40% cut-off from blood serum and meat juice with MLNs (K = 0.49 and 0.50, respectively) and tonsils (K = 0.29 and 0.30, respectively). Based on the obtained results, meat juice can be considered an alternative to blood serum as a matrix for ELISA for preliminary detection of Salmonella, allowing the identification of potential sources of contamination during slaughtering.

Distribution, diversity, virulence genotypes and antibiotic resistance for Salmonella isolated from a Brazilian pork production chain.

Pigs infected with Salmonella are an important source of contamination at slaughterhouses. We characterized the distribution, virulence genotypes and antimicrobial-resistance phenotypes for Salmonella isolates that were collected from different stages of a pork production chain. Each of ten pig lots were sampled for feed (n = 10), water (n = 10), barn floor (n = 10), lairage floor (n = 10), mesenteric lymph nodes (n = 100), tonsils (n = 100), processing environment (n = 120), pork cuts (n = 40) and carcasses after bleeding (n = 100), after singeing (n = 100), after evisceration (n = 100), and after final rinsing (n = 100). Salmonella was isolated according to ISO 6579, and after confirmation the isolates were subjected to serogrouping, macro-restriction digests and pulsed-field gel electrophoresis (PFGE), detection of virulence-related genes and antimicrobial-resistance phenotyping. Salmonella was recovered from barn floors from 3 pig farms (3/10), lairage floors (7/10), carcasses after bleeding (2/100) and final washing (1/100), palatine tonsils (45/100), mesenteric lymph nodes (43/100), utensils (3/120) and cuts (4/40). The most prevalent serogroup was O: 4 (82%) followed by O:3 (7.7%); O:9 (5.1%); O:8 (2.6%) and O:7 (2.6%). Recovered strains (n = 109) were classified into 24 different pulsotypes (XbaI restriction digest), which were arranged into five different clusters. Fourteen different virulence genotypes were observed based on 15 loci, and all isolates were positive for invA, sitC, pagC and tolC. There was a high prevalence of antimicrobial resistance against streptomycin (90.5%), tetracycline (88.1%), ampicillin (81.0%), chloramphenicol (71.4%), and ciprofloxacin (50.0%). No strain was resistant to ertapenem, meropenem or kanamycin. A majority (80.9%) of isolates were considered multidrug resistant (resistant to ≥3 antibiotic classes). This study provides valuable insight about the epidemiology of Salmonella in swine production, and despite the low presence of this pathogen in carcasses and meat cuts, the majority of isolates was multidrug resistant.

Distribution, adhesion, virulence and antibiotic resistance of persistent Listeria monocytogenes in a pig slaughterhouse in Brazil.

Listeria monocytogenes is a relevant pathogen usually associated with meat and ready-to-eat products. This study aimed to assess the distribution, adhesion, virulence and antibiotic resistance of L. monocytogenes in a pork production chain. Environment, carcass and food samples (n = 894) were obtained from different steps of a pork production chain over a 6-month period (10 samplings), including from farms and the slaughterhouse (reception, slaughtering, processing, storage and end products). L. monocytogenes was detected in samples from the reception (lairage floor, 1/10), slaughtering (drains, 2/20) and cutting room stages (conveyor belts in the final packing stage - 11/20, knife - 1/40, and cutting boards - 1/20). Positive results for conveyor belts were recorded in seven consecutive samplings. L. monocytogenes isolates (n = 87) were characterized as belonging to serogroup IVb and presented positive PCR results for inlA, inlB, inlC, inlJ, hlyA, plcA, actA and iap. Isolates were selected according to the original samples (n = 31) and subjected to Pulsed Field Gel Electrophoresis (PFGE), demonstrating their high clonal identity (98.4-100%). According to PFGE results and their original samples, isolates were selected (n = 16) and subjected to phenotypic assay to assess their adhesion potential and tested for resistance against 15 antibiotics; all tested isolates presented weak adhesion potential and were resistant to ampicillin. The present study demonstrated the persistence of L. monocytogenes in the pork processing facility, indicating the potential risk for cross-contamination with a potential virulent and resistant clone.

Occurrence of Salmonella sp. in poultry carcasses evaluated from the retail trade between 2007 and 2013 in Paraná state, Brazil / Ocorrência de Salmonella sp. em carcaças de frango avaliadas a partir do comércio varejista entre 2007 e 2013 no estado do Paraná, Brasil

Poultry meat is often involved as a vehicle for microorganisms that cause food-borne diseases. Salmonella sp. is a major pathogen involved in outbreaks around the world. Based on its importance, the objective of this study was to determine the occurrence of Salmonella sp. in frozen and chilled poultry carcasses slaughtered and marketed in the western region of Paraná state, Brazil. A total of 340 samples were collected between January 2007 and April 2013, with 66 (19.41%) carcasses positive for Salmonella sp. It can be concluded from the results obtained that the occurrence of Salmonella sp. in poultry carcasses remains high, even though it is a product marketed refrigerated or frozen, methods considered appropriate for food preservation. A lower occurrence result was expected due to the 14-year implementation of the national pathogen reduction program established by the Ministry of Agriculture, Livestock and Supply aimed at a gradual reduction in the occurrence of Salmonella sp. in these products through constant monitoring of carcasses immediately after slaughter.(AU)

A carne de aves é um alimento que frequentemente encontra-se envolvido como veículo de micro-organismos causadores de enfermidades, sendo Salmonella sp. um desses agentes comumente envolvidos nessas enfermidades em todo o mundo. Com isso, o objetivo deste estudo foi determinar a ocorrência de Salmonella sp. em carcaças de frango congeladas e refrigeradas, abatidas e comercializadas na região oeste do estado do Paraná. Um total de 340 amostras foram coletadas entre janeiro de 2007 e abril de 2013. Das amostras analisadas, 66 (19,41%) das carcaças foram positivas para Salmonella sp. De acordo com os resultados obtidos pode-se concluir que a ocorrência de Salmonella sp. em carcaças de frangos permanece alta, mesmo sendo produtos comercializados refrigerados ou congelados, métodos considerados adequados para conservação de alimentos. Um resultado de menor ocorrência era esperado devido à implementação de 14 anos do Programa Nacional de Redução de Patógenos estabelecido pelo Ministério da Agricultura, Pecuária e Abastecimento visando uma redução gradual na ocorrência de Salmonella sp. nestes produtos através de monitoramento constante de carcaças imediatamente após o abate.(AU)

Characterization of Tifton 85 bermudagrass haylage with different layers of polyethylene film and storage time.

OBJECTIVE: The objective was to characterize the fermentative and microbiological profile of Tifton 85 bermudagrass haylage with different layers of polyethylene film and storage time. METHODS: The experimental design consisted of a randomized block design with four and six wrapping layers (100 and 150 microns in total. respectively) allocated in the main plots, through repeated measures analysis (30, 60, and 90 days of storage) with four replicates. RESULTS: The storage time and number of wrapping layers did not show changes in the population of Clostridium and lactic acid bacteria. A decrease was observed in the enterobacteria population with an increase in the storage period in the two wrapping layers studied. Upon opening of the haylage at 30 days, the population of Bacillus was lower in haylages made with six layers of wrapping (3.63 log colony forming units/g). No growth of Listeria sp. or Salmonella sp. was observed during the experimental period. The fungal genera with a greater occurrence were Penicillium sp. and Fusarium sp. The following mycotoxins were not detected: ochratoxin A, fumonisins, and zearalenone. Relative to the organic butyric, propionic, and acetic acids, the haylages presented a low concentration of lactic acid; this may have prevented a drop in the pH, which was high when the silos were opened (5.4). The levels of ammoniacal nitrogen and soluble carbohydrates presented no variation among the number of wrapping layers, with an overall average of 35.55 and 38.04 g/kg. CONCLUSION: Tifton 85 bermudagrass haylage wrapped with four and six layers presented adequate fermentation and microbiological characteristics in the evaluated periods.

Multidrug resistance and ESBL-producing Salmonella spp. isolated from broiler processing plants

Abstract The aim of this study was to investigate the occurrence of multidrug-resistant, extended spectrum beta-lactamase (ESBL) producing Salmonella spp. isolated from conveyor belts of broiler cutting rooms in Brazilian broiler processing plants. Ninety-eight strains of Salmonella spp. were analyzed. Multidrug resistance was determined by the disk diffusion test and the susceptibility of the isolated bacteria was evaluated against 18 antimicrobials from seven different classes. The double disk diffusion test was used to evaluate ESBL production. Of the 98 strains tested, 84 were multidrug resistant. The highest rates of resistance were against nalidixic acid (95%), tetracycline (91%), and the beta-lactams: ampicillin and cefachlor (45%), followed by streptomycin and gentamicin with 19% and 15% of strain resistance, respectively. By contrast, 97% of the strains were sensitive to chloramphenicol. 45% of the strains were positive for the presence of ESBL activity. In this study, high rates of multidrug resistance and ESBL production were observed in Salmonella spp.

Multidrug resistance and ESBL-producing Salmonella spp. isolated from broiler processing plants.

The aim of this study was to investigate the occurrence of multidrug-resistant, extended spectrum beta-lactamase (ESBL) producing Salmonella spp. isolated from conveyor belts of broiler cutting rooms in Brazilian broiler processing plants. Ninety-eight strains of Salmonella spp. were analyzed. Multidrug resistance was determined by the disk diffusion test and the susceptibility of the isolated bacteria was evaluated against 18 antimicrobials from seven different classes. The double disk diffusion test was used to evaluate ESBL production. Of the 98 strains tested, 84 were multidrug resistant. The highest rates of resistance were against nalidixic acid (95%), tetracycline (91%), and the beta-lactams: ampicillin and cefachlor (45%), followed by streptomycin and gentamicin with 19% and 15% of strain resistance, respectively. By contrast, 97% of the strains were sensitive to chloramphenicol. 45% of the strains were positive for the presence of ESBL activity. In this study, high rates of multidrug resistance and ESBL production were observed in Salmonella spp.

Condições de comercialização de produtos de origem animal em feiras livres de municípios da região oeste do Paraná

O objetivo deste trabalho foi constatar a situação higienicossanitária de barracas de feiras livres localizadas em cinco municípios da região oeste do Paraná e a qualidade microbiológica e físico-química dos produtos nelas comercializados. Entre fevereiro e maio de 2013, foram realizadas avaliações, por meio do uso de checklist em 52 barracas onde foram adquiridas 71 amostras sendo estas submetidas à análise microbiológica e/ou físico-química. Foi constatado que em 47,9% das barracas o armazenamento dos alimentos era realizado de forma inadequada, que 86% dos produtos de origem animal estavam acondicionados em embalagens impróprias e que em 65% das mesmas, a condição higiênica dos manipuladores foi classificada como "Regular". Foram encontradas não conformidades em 36,6% das amostras submetidas às análises microbiológicas e/ou físico-químicas, sendo detectada a presença de Salmonella sp. em uma amostra de linguiça colonial. Sendo assim, é necessário que haja um controle sanitário na produção e comércio dos produtos nestas feiras por parte dos órgãos públicos responsáveis, garantindo assim, a segurança alimentar do consumidor.

The objective of this study was to determine the hygiene conditions of street market stands that sell products of animal origin in five cities in western Parana, Brazil, as well as the microbiological and physical-chemical quality of the products that are sold there. Between February and May, 2013, a total of 52 street market stands were analyzed based on a checklist. 71 samples were collected in these stands and submitted to microbiological and/or physical-chemical analyses. It was observed that in 47.9% of the stands, food products were inadequately stored; 86% of the products of animal origin were kept in improper containers; and in 65% of the stands, handler hygiene was classified as regular. 36.6% samples did not comply with microbiological and/or physical-chemical standards, with one sample of colonial sausage showing the presence of Salmonella sp. Therefore, public organisms control of hygiene in the production and commercialization of products in street markets is highly necessary to ensure food safety.