Enterotoxigenic methicillin resistant Staphylococcus aureus contamination in salted fish from Gwadar Balochistan

Abstract Staphylococcus aureus is an important foodborne pathogen associated to food intoxication and other multiple infections in human being. Its presence in salted food is a serious issue due to its salt tolerance potential. A study was conducted to analyze the presence of enterotoxins producing drug resistance S. aureus in salted sea fish from Gwadar. Freshly persevered samples (n=50) of salted fish were subjected to analyze the presence of S. aureus using 16S rRNA and Nuc genes primers. The isolates were then evaluated for drug resistance and enterotoxins producing potential using specific primers for MecA (methicillin resistance gene), (SEA) staphylococcal enterotoxin A and (SEB) staphylococcal enterotoxin B genes. Total 13/50 (26%) of the samples were found positive for the presence of S. aureus, preliminary confirmed with biochemical profiling and finally with the help of target genes presence. The isolates were found showing 100% resistant to methicillin, which were molecularly confirmed by the presence of MecA gene present in genome. The isolates 5/13 (38%) were positive for SEA and 3/13 (23%) for SEB genes, whereas 2/13 (15%) were confirmed having both SEA and SEB genes in its genome. It was also confirmed that all the isolates were capable to form biofilm over the glass surfaces. It was concluded that the study confirmed the presence of enterotoxigenic methicillin resistance Staphylococcus aurous (MRSA) in salted fish product, that poses gross food safety concern. Preventive and control measures are necessary to handle this serious food safety concern.

Resumo Staphylococcus aureus é um importante patógeno de origem alimentar associado à intoxicação alimentar e outras infecções múltiplas em seres humanos. Sua presença em alimentos salgados é um problema sério devido ao seu potencial de tolerância ao sal. Um estudo foi realizado para analisar a presença de enterotoxinas produtoras de resistência a drogas S. aureus em peixes salgados do mar de Gwadar. Amostras recém-perseveradas (n = 50) de peixes salgados foram submetidas à análise da presença de S. aureus usando os primers dos genes 16S rRNA e Nuc. Os isolados foram então avaliados quanto à resistência a drogas e potencial de produção de enterotoxinas usando primers específicos para os genes MecA (gene de resistência à meticilina), (SEA) enterotoxina A estafilocócica e (SEB) enterotoxina B estafilocócica genes. Um total de 13/50 (26%) das amostras foi considerado positivas para a presença de S. aureus, confirmadas preliminarmente com perfis bioquímicos e finalmente com a ajuda da presença de genes-alvo. Os isolados foram encontrados com 100% de resistência à meticilina, os quais foram confirmados molecularmente pela presença do gene MecA no genoma. Os isolados 5/13 (38%) foram positivos para SEA e 3/13 (23%) para genes SEB, enquanto 2/13 (15%) foram confirmados tendo os genes SEA e SEB em seu genoma. Também foi verificado que todos os isolados foram capazes de formar biofilme sobre as superfícies de vidro. Concluiu-se que o estudo confirmou a presença de Staphylococcus aurous resistente à meticilina enterotoxigênica (MRSA) em produtos de peixe salgado, o que representa uma grande preocupação para a segurança alimentar. Medidas preventivas e de controle são necessárias para lidar com essa grave preocupação com a segurança alimentar.

Enterotoxigenic methicillin resistant Staphylococcus aureus contamination in salted fish from Gwadar Balochistan / Contaminação de Staphylococcus aureus resistente à meticilina enterotoxigênica em peixes salgados de Gwadar Balochistan

Staphylococcus aureus is an important foodborne pathogen associated to food intoxication and other multiple infections in human being. Its presence in salted food is a serious issue due to its salt tolerance potential. A study was conducted to analyze the presence of enterotoxins producing drug resistance S. aureus in salted sea fish from Gwadar. Freshly persevered samples (n=50) of salted fish were subjected to analyze the presence of S. aureus using 16S rRNA and Nuc genes primers. The isolates were then evaluated for drug resistance and enterotoxins producing potential using specific primers for MecA (methicillin resistance gene), (SEA) staphylococcal enterotoxin A and (SEB) staphylococcal enterotoxin B genes. Total 13/50 (26%) of the samples were found positive for the presence of S. aureus, preliminary confirmed with biochemical profiling and finally with the help of target genes presence. The isolates were found showing 100% resistant to methicillin, which were molecularly confirmed by the presence of MecA gene present in genome. The isolates 5/13 (38%) were positive for SEA and 3/13 (23%) for SEB genes, whereas 2/13 (15%) were confirmed having both SEA and SEB genes in its genome. It was also confirmed that all the isolates were capable to form biofilm over the glass surfaces. It was concluded that the study confirmed the presence of enterotoxigenic methicillin resistance Staphylococcus aurous (MRSA) in salted fish product, that poses gross food safety concern. Preventive and control measures are necessary to handle this serious food safety concern.

Staphylococcus aureus é um importante patógeno de origem alimentar associado à intoxicação alimentar e outras infecções múltiplas em seres humanos. Sua presença em alimentos salgados é um problema sério devido ao seu potencial de tolerância ao sal. Um estudo foi realizado para analisar a presença de enterotoxinas produtoras de resistência a drogas S. aureus em peixes salgados do mar de Gwadar. Amostras recém-perseveradas (n = 50) de peixes salgados foram submetidas à análise da presença de S. aureus usando os primers dos genes 16S rRNA e Nuc. Os isolados foram então avaliados quanto à resistência a drogas e potencial de produção de enterotoxinas usando primers específicos para os genes MecA (gene de resistência à meticilina), (SEA) enterotoxina A estafilocócica e (SEB) enterotoxina B estafilocócica genes. Um total de 13/50 (26%) das amostras foi considerado positivas para a presença de S. aureus, confirmadas preliminarmente com perfis bioquímicos e finalmente com a ajuda da presença de genes-alvo. Os isolados foram encontrados com 100% de resistência à meticilina, os quais foram confirmados molecularmente pela presença do gene MecA no genoma. Os isolados 5/13 (38%) foram positivos para SEA e 3/13 (23%) para genes SEB, enquanto 2/13 (15%) foram confirmados tendo os genes SEA e SEB em seu genoma. Também foi verificado que todos os isolados foram capazes de formar biofilme sobre as superfícies de vidro. Concluiu-se que o estudo confirmou a presença de Staphylococcus aurous resistente à meticilina enterotoxigênica (MRSA) em produtos de peixe salgado, o que representa uma grande preocupação para a segurança alimentar. Medidas preventivas e de controle são necessárias para lidar com essa grave preocupação com a segurança alimentar.

Enterotoxigenic methicillin resistant Staphylococcus aureus contamination in salted fish from Gwadar Balochistan / Contaminação de Staphylococcus aureus resistente à meticilina enterotoxigênica em peixes salgados de Gwadar Balochistan

Staphylococcus aureus is an important foodborne pathogen associated to food intoxication and other multiple infections in human being. Its presence in salted food is a serious issue due to its salt tolerance potential. A study was conducted to analyze the presence of enterotoxins producing drug resistance S. aureus in salted sea fish from Gwadar. Freshly persevered samples (n=50) of salted fish were subjected to analyze the presence of S. aureus using 16S rRNA and Nuc genes primers. The isolates were then evaluated for drug resistance and enterotoxins producing potential using specific primers for MecA (methicillin resistance gene), (SEA) staphylococcal enterotoxin A and (SEB) staphylococcal enterotoxin B genes. Total 13/50 (26%) of the samples were found positive for the presence of S. aureus, preliminary confirmed with biochemical profiling and finally with the help of target genes presence. The isolates were found showing 100% resistant to methicillin, which were molecularly confirmed by the presence of MecA gene present in genome. The isolates 5/13 (38%) were positive for SEA and 3/13 (23%) for SEB genes, whereas 2/13 (15%) were confirmed having both SEA and SEB genes in its genome. It was also confirmed that all the isolates were capable to form biofilm over the glass surfaces. It was concluded that the study confirmed the presence of enterotoxigenic methicillin resistance Staphylococcus aurous (MRSA) in salted fish product, that poses gross food safety concern. Preventive and control measures are necessary to handle this serious food safety concern.(AU)

Staphylococcus aureus é um importante patógeno de origem alimentar associado à intoxicação alimentar e outras infecções múltiplas em seres humanos. Sua presença em alimentos salgados é um problema sério devido ao seu potencial de tolerância ao sal. Um estudo foi realizado para analisar a presença de enterotoxinas produtoras de resistência a drogas S. aureus em peixes salgados do mar de Gwadar. Amostras recém-perseveradas (n = 50) de peixes salgados foram submetidas à análise da presença de S. aureus usando os primers dos genes 16S rRNA e Nuc. Os isolados foram então avaliados quanto à resistência a drogas e potencial de produção de enterotoxinas usando primers específicos para os genes MecA (gene de resistência à meticilina), (SEA) enterotoxina A estafilocócica e (SEB) enterotoxina B estafilocócica genes. Um total de 13/50 (26%) das amostras foi considerado positivas para a presença de S. aureus, confirmadas preliminarmente com perfis bioquímicos e finalmente com a ajuda da presença de genes-alvo. Os isolados foram encontrados com 100% de resistência à meticilina, os quais foram confirmados molecularmente pela presença do gene MecA no genoma. Os isolados 5/13 (38%) foram positivos para SEA e 3/13 (23%) para genes SEB, enquanto 2/13 (15%) foram confirmados tendo os genes SEA e SEB em seu genoma. Também foi verificado que todos os isolados foram capazes de formar biofilme sobre as superfícies de vidro. Concluiu-se que o estudo confirmou a presença de Staphylococcus aurous resistente à meticilina enterotoxigênica (MRSA) em produtos de peixe salgado, o que representa uma grande preocupação para a segurança alimentar. Medidas preventivas e de controle são necessárias para lidar com essa grave preocupação com a segurança alimentar.(AU)

Bacillus Cereus in Eggshell: Enterotoxigenic Profiles and Biofilm Production

A study was carried out with the objective of determining the presence of Bacillus cereus in eggshells commercialized in Mexico, the enterotoxigenic profile of the isolated strains, and the production of biofilms in different materials as well as in the eggshell. 1000 chicken eggs from four commercial brands were collected from markets and supermarkets located in the city of Chilpancingo, Mexico. Bacillus cereus was isolated from the eggshell. The molecular identification was by amplification of the gyrB gene and the enterotoxigenic profiles by the amplification of the cytK, ces, nheABC, and hblABD genes, in addition to the amplification of the tasA and sipW genes associated with the production of biofilms. In different materials and in eggshells, the production of biofilms was evaluated. The microbiological and molecular analysis of B. cereus yielded a frequency of 5.5% (55/1000), this was higher in brand III (11.6%, p=0.0001) and white eggshell (7.6%, 38/500, p≤0.001) and by marketing source, it was similar between market (5.2% / 26/500) and supermarket (5.8%, 29/500). The most common was the toxigenic profile A (23/55). Biofilm production is high in PVC in relation to other materials (p<0.0001), and the frequency of the related genes tasA and sipW was 72.7% and 40% respectively; the highest production was related to the tasA gene; in eggshell, most of the strains (54/55) were able to produce biofilm. Strains of B. cereus with toxigenic potential circulate and persist in this product, which shows the need for sanitary regulation in the country.(AU)

New findings of intestinal alkaline phosphatase: effects on intestinal and organ health of piglets challenged with ETEC F4 (K88)

The aim of this study was to assess the addition of intestinal alkaline phosphatase (IAP) to diets on the count of bacterial populations, pH of digestive organ contents, histopathological description, proinflammatory markers, hepatic glycogen reserve, and diarrhoea incidence of piglets challenged with Escherichia coli. Sixty-four crossbred piglets (7.16±0.28 kg body weight, 25-days-old) were assigned to four treatments in a completely randomised block design: negative control (NC), NC + antibiotic (ANT), NC + 15 mg IAP, or NC + 30 mg IAP kg−1 of diet, eight replications of two piglets per experimental unit. All piglets were orally challenged with 6 mL of a solution containing enterotoxigenic Escherichia coli K88 at 106 CFU mL−1 at 15 days of experimentation. The study lasted for 19 days. At the end of the experimental period, the piglets were slaughtered (six animals per treatment). Enterobacteriaceae in caecum and colon was lower in piglets on 30 mg IAP than with ANT and NC, ANT or 15 mg IAP, respectively. Enterobacteriaceae adhered to the mesenteric lymph nodes (MLN) was greater in piglets fed ANT than the other treatments. Lactic acid bacteria (LAB) count in caecum was greater in piglets fed NC and ANT. In MLN, LAB count was greater in ANT and 30 mg IAP-fed piglets compared with 15 mg IAP. Piglets in 30 mg IAP in diet showed a tendency for lowering tissue necrosis compared with NC or ANT. Piglets fed 30 mg IAP showed a reduction in diarrhoea incidence in the pre- and post-challenge compared with 15 mg IAP and all other treatments, respectively. Based on the criteria, addition of 30 mg IAP to diet inhibits Enterobacteriaceae population and suggests a potential effect in mitigating intestinal injuries, as observed in piglets in the NC for some of the parameters investigated.(AU)

Microbiological water quality monitoring of four ponds from Lagoa do Sino Farm located in São Paulo State, Brazil

This study aimed to evaluate the microbiological quality of the water of four ponds used for irrigation on the Lagoa do Sino Farm, as well as to perform the genotypic characterization of virulence factors in Escherichia coli isolates. Sampling was conducted for 11 months, between 2015 and 2016. Samples were analyzed for the presence of thermotolerant coliforms, E. coli and heterotrophs. DNA was extracted from E. coli isolates, followed by genotypic characterization by polymerase chain reaction. Agricultural activities and pesticides used in the sampling period were documented in order to assess possible relationships between agricultural activities and microbiological water quality. The absence of suitable riparian vegetation around all the ponds was observed, benefiting the entry of organic matter and contaminants in the water body. A high index of thermotolerant coliforms in some months indicated the possibility of the transmission of pathogenic microorganisms in these ponds. The values found in some months were above the regulatory limits for water potability and water intended for irrigation. The agrochemicals used in the period seem to influence the results obtained. All 17 E. coli isolates showed at least one of the virulence genes estA, stx1, stx2, and aatA, indicating enterotoxigenic, enterohaemorrhagic or enteroaggregative nature. The presence of E. coli in the waters may be associated with the presence of animals. The water samples analyzed are not suitable for irrigation of vegetables that are consumed raw and/or low lying fruits ingested without skin removal. It is essential to broaden the control of the use of chemicals, as well as the preservation of riparian vegetation to improve the quality of water used in the farm's agricultural activities.

Microbiological water quality monitoring of four ponds from Lagoa do Sino Farm located in São Paulo State, Brazil

This study aimed to evaluate the microbiological quality of the water of four ponds used for irrigation on the Lagoa do Sino Farm, as well as to perform the genotypic characterization of virulence factors in Escherichia coli isolates. Sampling was conducted for 11 months, between 2015 and 2016. Samples were analyzed for the presence of thermotolerant coliforms, E. coli and heterotrophs. DNA was extracted from E. coli isolates, followed by genotypic characterization by polymerase chain reaction. Agricultural activities and pesticides used in the sampling period were documented in order to assess possible relationships between agricultural activities and microbiological water quality. The absence of suitable riparian vegetation around all the ponds was observed, benefiting the entry of organic matter and contaminants in the water body. A high index of thermotolerant coliforms in some months indicated the possibility of the transmission of pathogenic microorganisms in these ponds. The values found in some months were above the regulatory limits for water potability and water intended for irrigation. The agrochemicals used in the period seem to influence the results obtained. All 17 E. coli isolates showed at least one of the virulence genes estA, stx1, stx2, and aatA, indicating enterotoxigenic, enterohaemorrhagic or enteroaggregative nature. The presence of E. coli in the waters may be associated with the presence of animals. The water samples analyzed are not suitable for irrigation of vegetables that are consumed raw and/or low lying fruits ingested without skin removal. It is essential to broaden the control of the use of chemicals, as well as the preservation of riparian vegetation to improve the quality of water used in the farm's agricultural activities.(AU)

Gangrenous mastitis in sheep caused by multidrug-resistant Staphylococcus haemolyticus / Mastite gangrenosa em ovinos causada por Staphylococcus haemolyticus multirresistente

Mastitis is a multifactorial disease and considered one of the most critical problems in the dairy industry worldwide. The condition is characterized by reduced milk and several abnormalities in the mammary gland. This study aimed to report an outbreak of gangrenous mastitis caused by multidrug-resistant Staphylococcus haemolyticus in a Santa Inês sheep herd. Eighteen sheep were affected, and five of them with severe clinical pictures were examined. The clinical and pathological picture were variable and characterized by apathy, anorexia, emaciation, opaque and brittle hair, apparent and congested episcleral vessels, and hyperthermia. These ewes had enlarged, firm, and painful mammary glands. Macroscopically, these lesions consisted of severe gangrenous mastitis, and microscopically, the primary lesions consisted of necrosis, thrombosis, and fibrosis of the mammary parenchyma. Milk samples from one of the five severely affected ewes were collected and cultured under aerobic or microaerophilic incubation at 37°C for 24 hours on sheep blood agar. The obtained colonies were then submitted to MALDI-TOF for speciation. The colonies were also submitted to an antimicrobial susceptibility test, genotyping of virulence factors and resistance genes were also performed. The isolates showed antimicrobial multiresistance since they were resistant to seven out of 13 tested antibiotics. The isolates were also positive for two staphylococcal enterotoxigenic genes (sec and see) and fibronectin-binding protein B (fnbB).(AU)

A mastite é uma doença multifatorial e é considerada um dos problemas mais importantes na indústria de laticínios no mundo todo. A condição é caracterizada pela redução de leite e várias anormalidades na glândula mamária. O objetivo deste estudo foi relatar um surto de mastite gangrenosa causada por Staphylococcus haemolyticus multirresistente em um rebanho ovino Santa Inês. Dezoito ovelhas foram afetadas e cinco delas com quadro clínico severo foram examinadas. O quadro clínico-patológico era variável quanto a severidade e consistia em apatia, anorexia, magreza, pelos opacos e quebradiços e vasos episclerais aparentes e ingurgitados. As ovelhas apresentavam glândulas aumentadas, firmes e dolorosas. Macroscopicamente, as principais lesões consistiam em mastite gangrenosa e microscopicamente havia necrose do parênquima glandular, trombose e fibrose. Amostras de leite de uma das cinco ovelhas severamente afetadas foram coletadas e cultivadas sob incubação aeróbica ou microaerofílica a 37°C por 24 horas em ágar sangue de ovelha. As colônias obtidas foram então submetidas ao MALDI-TOF para especiação. Além disso, as colônias foram submetidas a um teste de suscetibilidade antimicrobiana e foi realizada a genotipagem de fatores de virulência e genes de resistência. Os isolados apresentaram multirresistência antimicrobiana por serem resistentes a sete dos 13 antibióticos testados. Os isolados também foram positivos para dois genes enterotoxigênicos estafilocócicos (sec e see) e proteína B de ligação à fibronectina (fnbB).(AU)

Gangrenous mastitis in sheep caused by multidrug-resistant Staphylococcus haemolyticus / Mastite gangrenosa em ovinos causada por Staphylococcus haemolyticus multirresistente

Mastitis is a multifactorial disease and considered one of the most critical problems in the dairy industry worldwide. The condition is characterized by reduced milk and several abnormalities in the mammary gland. This study aimed to report an outbreak of gangrenous mastitis caused by multidrug-resistant Staphylococcus haemolyticus in a Santa Inês sheep herd. Eighteen sheep were affected, and five of them with severe clinical pictures were examined. The clinical and pathological picture were variable and characterized by apathy, anorexia, emaciation, opaque and brittle hair, apparent and congested episcleral vessels, and hyperthermia. These ewes had enlarged, firm, and painful mammary glands. Macroscopically, these lesions consisted of severe gangrenous mastitis, and microscopically, the primary lesions consisted of necrosis, thrombosis, and fibrosis of the mammary parenchyma. Milk samples from one of the five severely affected ewes were collected and cultured under aerobic or microaerophilic incubation at 37°C for 24 hours on sheep blood agar. The obtained colonies were then submitted to MALDI-TOF for speciation. The colonies were also submitted to an antimicrobial susceptibility test, genotyping of virulence factors and resistance genes were also performed. The isolates showed antimicrobial multiresistance since they were resistant to seven out of 13 tested antibiotics. The isolates were also positive for two staphylococcal enterotoxigenic genes (sec and see) and fibronectin-binding protein B (fnbB).(AU)

A mastite é uma doença multifatorial e é considerada um dos problemas mais importantes na indústria de laticínios no mundo todo. A condição é caracterizada pela redução de leite e várias anormalidades na glândula mamária. O objetivo deste estudo foi relatar um surto de mastite gangrenosa causada por Staphylococcus haemolyticus multirresistente em um rebanho ovino Santa Inês. Dezoito ovelhas foram afetadas e cinco delas com quadro clínico severo foram examinadas. O quadro clínico-patológico era variável quanto a severidade e consistia em apatia, anorexia, magreza, pelos opacos e quebradiços e vasos episclerais aparentes e ingurgitados. As ovelhas apresentavam glândulas aumentadas, firmes e dolorosas. Macroscopicamente, as principais lesões consistiam em mastite gangrenosa e microscopicamente havia necrose do parênquima glandular, trombose e fibrose. Amostras de leite de uma das cinco ovelhas severamente afetadas foram coletadas e cultivadas sob incubação aeróbica ou microaerofílica a 37°C por 24 horas em ágar sangue de ovelha. As colônias obtidas foram então submetidas ao MALDI-TOF para especiação. Além disso, as colônias foram submetidas a um teste de suscetibilidade antimicrobiana e foi realizada a genotipagem de fatores de virulência e genes de resistência. Os isolados apresentaram multirresistência antimicrobiana por serem resistentes a sete dos 13 antibióticos testados. Os isolados também foram positivos para dois genes enterotoxigênicos estafilocócicos (sec e see) e proteína B de ligação à fibronectina (fnbB).(AU)

Adaptação de Escherichia coli enterotoxigênica ao eugenol / Adaptation of enterotoxigenic Escherichia coli to eugenol

O objetivo do trabalho foi verificar a capacidade de adaptação de ETEC- ATCC 35401 ao antimicrobiano eugenol. Para isso, a Concentração Mínima Bactericida (CMB) de eugenol foi determinada e em seguida,as células de ETEC foram expostas a concentrações subletais de eugenol (CMB/4, CMB/8 e CMB/16). Posteriormente testadas frente a diferentes concentrações do composto (CMB/2; CMB; 1,2CMB; 1,4CMB; 1,6CMB; 1,8CMB e 2CMB), estas foram incubadas e plaqueadas em TSA (Ágar Triptona de Soja) empregando a técnica de microgotas. As células de ETEC foram classificadas como capazes de se adaptarem quando essas cresceram em placas após cultivo em presença do componente em concentração igual ou maior que a CMB do eugenol (1,0% (v/v)). As células de ETEC apresentaram a capacidade de adaptação por crescerem na CMB, após exposta a CMB/16. Os resultados demonstram a necessidade de obtenção da concentração de uso adequado de eugenol a fim de evitar a adaptação.(AU)

Adaptação de Escherichia coli enterotoxigênica ao eugenol / Adaptation of enterotoxigenic Escherichia coli to eugenol

O objetivo do trabalho foi verificar a capacidade de adaptação de ETEC- ATCC 35401 ao antimicrobiano eugenol. Para isso, a Concentração Mínima Bactericida (CMB) de eugenol foi determinada e em seguida,as células de ETEC foram expostas a concentrações subletais de eugenol (CMB/4, CMB/8 e CMB/16). Posteriormente testadas frente a diferentes concentrações do composto (CMB/2; CMB; 1,2CMB; 1,4CMB; 1,6CMB; 1,8CMB e 2CMB), estas foram incubadas e plaqueadas em TSA (Ágar Triptona de Soja) empregando a técnica de microgotas. As células de ETEC foram classificadas como capazes de se adaptarem quando essas cresceram em placas após cultivo em presença do componente em concentração igual ou maior que a CMB do eugenol (1,0% (v/v)). As células de ETEC apresentaram a capacidade de adaptação por crescerem na CMB, após exposta a CMB/16. Os resultados demonstram a necessidade de obtenção da concentração de uso adequado de eugenol a fim de evitar a adaptação.

Enterotoxin-encoding genes in Staphylococcus aureus from buffalo milk

ABSTRACT: This paper investigated the occurrence of Staphylococcus aureus and the detection of enterotoxin-encoding genes of these strains in milk collected from 30 Murrah buffaloes used to produce dairy products in Brazil. A total of 68 strains of Staphylococcus aureus were found as identified by conventional laboratory tests, and thus screened for sea, seb, sec, sed, see, seg, seh and sei enterotoxin-encoding genes by polymerase chain reaction (PCR). Twelve strains containing enterotoxin-amplified genes were found, with higher expression for the sei and seh genes. These results can be attributed to animal health and inadequate cleaning of the equipment, indicating the need for better quality control in animal production and health lines. The results of this study with the presence of pathogens and their enterotoxigenic potential indicate a source of food poisoning, as well as being a pioneering study in the detection of new enterotoxins for buffalo milk.

RESUMO: Este estudo investigou a ocorrência de isolados de Staphylococcus aureus e a detecção de genes que codificam a enterotoxigenicidade dessas cepas em leite de búfala utilizado na produção de laticínios no Brasil. As amostras foram coletados em 30 búfalos da raça Murrah, identificado por testes laboratoriais convencionais, foram identificados um total de 68 cepas de S. aureus e rastreados para os genes que codificam a enterotoxina sea, seb, sec, sed, see, seg, seh and sei por reação em cadeia da polimerase (PCR). Doze cepas contendo genes da enterotoxina foram amplificadas, com maior expressão para os genes sei e seh. Esses resultados podem ser atribuídos à saúde animal e à higiene inadequada do equipamento, indicando a necessidade de melhor controle de qualidade nas linhas de produção e saúde animal. Os resultados desta pesquisa, com a presença de patógenos e seu potencial enterotoxigênico, indicam uma fonte de intoxicação alimentar, além de ser uma pesquisa pioneira na detecção de novas enterotoxinas para o leite de búfala.

Virulence genes in Escherichia coli isolates from commercialized saltwater mussels Mytella guyanensis (Lamarck, 1819) / Presença de genes de virulência em isolados de Escherichia coli provenientes de sururu Mytella guyanensis (Lamarck, 1819) comercializado

The isolation of Escherichia coli from food is a major concern. Pathogenic strains of these bacteria cause diseases which range from diarrhea to hemolytic-uremic syndrome. Therefore the virulence genes in E. coli isolates from the mussel ( Mytella guyanensis) commercialized in Cachoeira, Bahia, Brazil were investigated. Samples were purchased from four vendors: two from supermarkets and two from fair outlets. They were conditioned into isothermal boxes with reusable ice and transported to the laboratory for analysis. E. coli strains were isolated in eosin methylene blue agar, preserved in brain-heart infusion medium with 15% glycerol and stored at -20 °C, after microbiological analysis. Virulence genes in the isolated strains were identified by specific primers, with Polymerase Chain Reaction. Twenty-four isolates were obtained, with a prevalence of elt gene, typical from enterotoxigenic infection, in 75% of the isolates. The stx and bfpA genes, prevalent in enterohemorragic and enteropathogenic E. coli, respectively, were not detected. The occurrence of elt virulence-related gene in the E. coli isolates of Mytella guyanensis reveals urgent improvement in food processing, including good handling practices, adequate storage and cooking before consumption, to ensure consumers health.(AU)

O isolamento de Escherichia coli a partir de alimentos é uma grande preocupação, pois cepas patogênicas desta bactéria podem causar desde diarreia até síndrome hemolítico-urêmica. Diante do exposto, o objetivo do trabalho foi pesquisar genes de virulência em isolados de Escherichia coli provenientes do sururu Mytella guyanensis comercializado na cidade de Cachoeira, Bahia, Brasil. As amostras foram adquiridas de quatro comerciantes, sendo duas de mercados e duas em pontos de venda na feira livre da cidade de Cachoeira, acondicionadas em caixas isotérmicas com gelo reutilizável e transportadas até o laboratório para a análise. Após a análise microbiológica, as cepas de Escherichia coli foram isoladas em ágar Eosina Azul de Metileno e preservadas em caldo Brian Heart Infusion e glicerol a 15% e mantidas a - 20° C. A identificação dos genes de virulência nas cepas isoladas foi realizada utilizando primers específicos, por meio da Reação em Cadeia da Polimerase. Foram obtidos 24 isolados de Escherichia coli, destes a prevalência do gene elt , característico de Escherichia coli enterotoxigênica, foi de 75% dos isolados. Não houve a detecção dos genes stx e bfpA nos isolados, os quais são prevalentes nas cepas de Escherichia coli enterohemorrágica e Escherichia coli enteropatogênica, respectivamente. A presença do gene elt relacionado à virulência de Escherichia coli nos isolados de Mytella guyanensis revela a necessidade da melhoria no processamento, incluindo boas práticas de manipulação, armazenamento adequado e cocção previa ao consumo, visando a garantia da saúde do consumidor.(AU)

Enterotoxin-encoding genes in Staphylococcus aureus from buffalo milk / Genes codificadores de enterotoxinas em Staphylococcus aureus no leite de búfalas

This paper investigated the occurrence of Staphylococcus aureus and the detection of enterotoxin-encoding genes of these strains in milk collected from 30 Murrah buffaloes used to produce dairy products in Brazil. A total of 68 strains of Staphylococcus aureus were found as identified by conventional laboratory tests, and thus screened for sea, seb, sec, sed, see, seg, seh and sei enterotoxin-encoding genes by polymerase chain reaction (PCR). Twelve strains containing enterotoxin-amplified genes were found, with higher expression for the sei and seh genes. These results can be attributed to animal health and inadequate cleaning of the equipment, indicating the need for better quality control in animal production and health lines. The results of this study with the presence of pathogens and their enterotoxigenic potential indicate a source of food poisoning, as well as being a pioneering study in the detection of new enterotoxins for buffalo milk.(AU)

Este estudo investigou a ocorrência de isolados de Staphylococcus aureus e a detecção de genes que codificam a enterotoxigenicidade dessas cepas em leite de búfala utilizado na produção de laticínios no Brasil. As amostras foram coletados em 30 búfalos da raça Murrah, identificado por testes laboratoriais convencionais, foram identificados um total de 68 cepas de S. aureus e rastreados para os genes que codificam a enterotoxina sea, seb, sec, sed, see, seg, seh and sei por reação em cadeia da polimerase (PCR). Doze cepas contendo genes da enterotoxina foram amplificadas, com maior expressão para os genes sei e seh. Esses resultados podem ser atribuídos à saúde animal e à higiene inadequada do equipamento, indicando a necessidade de melhor controle de qualidade nas linhas de produção e saúde animal. Os resultados desta pesquisa, com a presença de patógenos e seu potencial enterotoxigênico, indicam uma fonte de intoxicação alimentar, além de ser uma pesquisa pioneira na detecção de novas enterotoxinas para o leite de búfala.(AU)

Diarrheagenic Escherichia coli in raw milk, water, and cattle feces in non-technified dairy farms / Escherichia coli diarreiogênica em leite cru, água e fezes bovinas de propriedades leiteiras não tecnificadas

This study focused on detecting diarrheagenic Escherichia coli, enteropathogenic E. coli (EPEC), Shiga-toxin-producing E. coli (STEC), enterohemorrhagic E. coli (EHEC or STEC:EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC) in raw milk, water, and cattle feces sampled from non-technified dairy farms located in the northeastern São Paulo State, Brazil. Thirty-six water samples were collected at different points, namely, water wells (8 samples), water intended for human consumption (8 samples), water from milking parlor (8 samples), and water intended for animal consumption (7 samples), headwaters (1 sample), rivers (3 samples), and reservoirs (1 sample). Three raw milk samples were taken directly from bulk tanks in each farm, totalizing 24 samples. Feces samples were collected using rectal swabs from 160 bovines (20 animals per farm). E. coli was detected in 128 feces samples (80%), 16 raw milk samples (66.67%), and 20 water samples (55.56%). STEC (26 samples, 16.25%), EPEC (10 samples, 6.25%), STEC: EPEC (5 samples, 3.13%), and STEC: ETEC (1 sample, 0.63%) were the most prevalent strains detected in samples from cattle feces. EPEC, STEC, and STEC: EPEC strains were detected in 4.17% (1 sample), 16.67% (4 samples), and 4.17% (1 sample) of raw milk samples, respectively. STEC strains were detected in water used in the milking parlor, while no EAEC strain was detected. As a conclusion, cattle feces are important contamination sources of pathogenic E. coli in non-technified dairy farms and, consequently, cross-contamination among feces, water, and/or raw milk can occur. The use of quality water and hygienic practices during milking are recommended to avoid contamination since pathogens can be transmitted to humans via raw milk or raw milk cheese ingestion.

Este estudo teve como objetivo realizar a detecção de Escherichia coli diarreiogênica (EPEC, STEC, ETEC e EAEC) em leite, água e fezes bovinas em pequenas propriedades leiteiras localizadas na Região Nordeste do Estado de São Paulo, Brasil. E. coli foi detectada em amostras obtidas de fezes (80%), leite cru (66,67%) e água (55,56%). STEC, EPEC, STEC:EPEC e STEC:ETEC foram as cepas mais prevalentes em amostras de fezes bovinas, respectivamente. Em relação ao leite cru, cepas de EPEC, STEC e STEC:EPEC foram detectadas em 4,17%, 16,67% e 4,17% das amostras, respectivamente. Ainda, detectou-se STEC na amostra de água utilizada na sala de ordenha, enquanto EAEC não foi detectada em nenhuma amostra. Conclui-se que fezes de bovinos é uma importante fonte de contaminação de E. coli patogênicas em propriedades leiteiras e podem consequentemente contaminar o leite cru e água. A importância da qualidade da água e da adoção efetiva de práticas higiênicas durante a obtenção do leite para evitar a contaminação são recomendadas devido à possibilidade de transmissão de microorganismos patogênicos a seres humanos devido a ingestão de leite cru ou queijos produzidos a partir de leite não pasteurizado.

Enterotoxin-encoding genes in Staphylococcus aureus from buffalo milk / Genes codificadores de enterotoxinas em Staphylococcus aureus no leite de búfalas

This paper investigated the occurrence of Staphylococcus aureus and the detection of enterotoxin-encoding genes of these strains in milk collected from 30 Murrah buffaloes used to produce dairy products in Brazil. A total of 68 strains of Staphylococcus aureus were found as identified by conventional laboratory tests, and thus screened for sea, seb, sec, sed, see, seg, seh and sei enterotoxin-encoding genes by polymerase chain reaction (PCR). Twelve strains containing enterotoxin-amplified genes were found, with higher expression for the sei and seh genes. These results can be attributed to animal health and inadequate cleaning of the equipment, indicating the need for better quality control in animal production and health lines. The results of this study with the presence of pathogens and their enterotoxigenic potential indicate a source of food poisoning, as well as being a pioneering study in the detection of new enterotoxins for buffalo milk.(AU)

Este estudo investigou a ocorrência de isolados de Staphylococcus aureus e a detecção de genes que codificam a enterotoxigenicidade dessas cepas em leite de búfala utilizado na produção de laticínios no Brasil. As amostras foram coletados em 30 búfalos da raça Murrah, identificado por testes laboratoriais convencionais, foram identificados um total de 68 cepas de S. aureus e rastreados para os genes que codificam a enterotoxina sea, seb, sec, sed, see, seg, seh and sei por reação em cadeia da polimerase (PCR). Doze cepas contendo genes da enterotoxina foram amplificadas, com maior expressão para os genes sei e seh. Esses resultados podem ser atribuídos à saúde animal e à higiene inadequada do equipamento, indicando a necessidade de melhor controle de qualidade nas linhas de produção e saúde animal. Os resultados desta pesquisa, com a presença de patógenos e seu potencial enterotoxigênico, indicam uma fonte de intoxicação alimentar, além de ser uma pesquisa pioneira na detecção de novas enterotoxinas para o leite de búfala.(AU)

Diarrheagenic Escherichia coli in raw milk, water, and cattle feces in non-technified dairy farms / Escherichia coli diarreiogênica em leite cru, água e fezes bovinas de propriedades leiteiras não tecnificadas

This study focused on detecting diarrheagenic Escherichia coli, enteropathogenic E. coli (EPEC), Shiga-toxin-producing E. coli (STEC), enterohemorrhagic E. coli (EHEC or STEC:EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC) in raw milk, water, and cattle feces sampled from non-technified dairy farms located in the northeastern São Paulo State, Brazil. Thirty-six water samples were collected at different points, namely, water wells (8 samples), water intended for human consumption (8 samples), water from milking parlor (8 samples), and water intended for animal consumption (7 samples), headwaters (1 sample), rivers (3 samples), and reservoirs (1 sample). Three raw milk samples were taken directly from bulk tanks in each farm, totalizing 24 samples. Feces samples were collected using rectal swabs from 160 bovines (20 animals per farm). E. coli was detected in 128 feces samples (80%), 16 raw milk samples (66.67%), and 20 water samples (55.56%). STEC (26 samples, 16.25%), EPEC (10 samples, 6.25%), STEC: EPEC (5 samples, 3.13%), and STEC: ETEC (1 sample, 0.63%) were the most prevalent strains detected in samples from cattle feces. EPEC, STEC, and STEC: EPEC strains were detected in 4.17% (1 sample), 16.67% (4 samples), and 4.17% (1 sample) of raw milk samples, respectively. STEC strains were detected in water used in the milking parlor, while no EAEC strain was detected. As a conclusion, cattle feces are important contamination sources of pathogenic E. coli in non-technified dairy farms and, consequently, cross-contamination among feces, water, and/or raw milk can occur. The use of quality water and hygienic practices during milking are recommended to avoid contamination since pathogens can be transmitted to humans via raw milk or raw milk cheese ingestion.(AU)

Este estudo teve como objetivo realizar a detecção de Escherichia coli diarreiogênica (EPEC, STEC, ETEC e EAEC) em leite, água e fezes bovinas em pequenas propriedades leiteiras localizadas na Região Nordeste do Estado de São Paulo, Brasil. E. coli foi detectada em amostras obtidas de fezes (80%), leite cru (66,67%) e água (55,56%). STEC, EPEC, STEC:EPEC e STEC:ETEC foram as cepas mais prevalentes em amostras de fezes bovinas, respectivamente. Em relação ao leite cru, cepas de EPEC, STEC e STEC:EPEC foram detectadas em 4,17%, 16,67% e 4,17% das amostras, respectivamente. Ainda, detectou-se STEC na amostra de água utilizada na sala de ordenha, enquanto EAEC não foi detectada em nenhuma amostra. Conclui-se que fezes de bovinos é uma importante fonte de contaminação de E. coli patogênicas em propriedades leiteiras e podem consequentemente contaminar o leite cru e água. A importância da qualidade da água e da adoção efetiva de práticas higiênicas durante a obtenção do leite para evitar a contaminação são recomendadas devido à possibilidade de transmissão de microorganismos patogênicos a seres humanos devido a ingestão de leite cru ou queijos produzidos a partir de leite não pasteurizado.(AU)

Detection of virulence genes and antimicrobial resistance profiles of Escherichia coli isolates from raw milk and artisanal cheese in Southern Brazil / Detecção de genes de virulência e perfis de resistência antimicrobiana de Escherichia coli isoladas de leite cru e queijo artesanal no Sul do Brasil

The serrano artisanal cheese is a typical product from South region of Brazil, which is produced by skilled cheesemakers using raw milk. The contamination of this food by Escherichia coli has a great impact on public health, since it could threat the consumers health. The study evaluated the presence of virulence genes, antimicrobial susceptibility profiles and bofilm-production ability of Escherichia coli isolates obtained from raw milk and artisanal cheese produced in Southern Brazil. A total of 117 isolates of E. coli were characterized by multiplex PCR to detect the following virulence genes: eae for enteropatogenic E. coli (EPEC), lt and st for enterotoxigenic E. coli (ETEC), stx for shiga toxin-producing E. coli (STEC), stx and eae for enterohemorrhagic E. coli (EHEC), ipaH for enteroinvasive E. coli (EIEC) and aggR for enteroaggregative E. coli (EAEC). In addition, antimicrobial susceptibility profile to 22 antimicrobial agents was also performed by disk diffusion method, and we searched for extended-spectrum beta-lactamases (ESBL) and/or carbapenemase- producing isolates. Isolates that were positive for ESBL and carbapenemase were further investigated for the presence of the genes: bla(TEM), bla(SHV), bla(OXA), bla(CTX-M), for ESBL and bla(OXA-48) for carbapenemase. Further, isolates had their ability to form biofilms investigated by the red Congo agar method. Virulence genes of E. coli were identified in 21.37% of the tested isolates, which were classified as EPEC (the most prevalent pathotype) and ETEC or EAEC. Ten (8.55%) of the total studied E. coli isolates revealed a multidrug-resistant profile, since they were resistant to three or more antimicrobial classes; whereas four isolates (3.42%) were classified as ESBL-producers and showed the presence of bla(TEM) gene. None of the isolates exhibited carbapenemase activity nor did they carry carbapenemase genes. From the total of E. coli isolates, 79 (67.52%) were considered potential biofilm...(AU)

O queijo artesanal serrano é um produto típico da região sul do Brasil e se caracteriza por ser produzido a partir de leite cru. A contaminação desse alimento por Escherichia coli assume grande relevância para a saúde pública, pois oferece risco a saúde dos consumidores. Esse estudo avaliou a presença de genes de virulência, os perfis de susceptibilidade antimicrobiana e a capacidade de produção de biofilme de isolados de E. coli obtidos a partir de leite cru e queijo artesanal produzido no Sul do Brasil. Um total de 117 isolados de E. coli foram caracterizados por multiplex PCR para detecção dos seguintes genes de virulência: eae para E. coli enteropatogênica (EPEC), lt e st para E. coli enterotoxigênica (ETEC), stx para E. coli produtora da toxina shiga (STEC), stx e eae para E. coli enterohemorrágica (EHEC), ipaH para E. coli enteroinvasiva (EIEC) e aggR para E. coli enteroagregativa (EAEC). Adicionalmente, o perfil de susceptibilidade antimicrobiana a 22 agentes antimicrobianos foi determinado pelo método de disco difusão e a busca de isolados produtores de beta-lactamases de espectro estendido (ESBL) e carbapenemase foram realizadas. Os isolados positivos para ESBL e carbapenemase foram investigados quanto a presença dos genes: bla(TEM), bla(SHV), bla(OXA), bla(CTX-M) para ESBL e bla(OXA-48) para carbapenemase. Além disso, a potencial capacidade dos isolados de E. coli em produzir biofilme foi determinada pela técnica do ágar vermelho Congo. Os genes de virulência de E. coli foram identificados em 21,37% dos isolados testados, que foram classificados como EPEC (patótipo mais prevalente), ETEC ou EAEC. Dez (8,55%) isolados de E. coli apresentaram perfil de multirresistência, pois foram resistentes a três ou mais classes de antimicrobianos; enquanto que quatro isolados (3,42%) foram classificados como produtores de ESBL, sendo identificado o gene blaTEM. Nenhum isolado foi classificado como produtor de carbapenemase. Do total de...(AU)

Virulence factors and antimicrobial resistance profile of Escherichia coli isolated from nursery piglets and drinking water

Background: One of the most frequent health problems in the swine industry is the post-weaning diarrhea in nursery pigs, which leads to significant losses due to weight loss, dehydration, cost of medication and mortality. Escherichia coli (E. coli) is one of the main bacterial agents of the post-weaning diarrhea. To investigate the possibility of enterotoxigenic E. coli (ETEC) transmission through drinking water to nursery piglets, the objective of this study was to isolate, characterize by virulence factors, and compare the antimicrobial resistance profiles of E. coli from drinking water samples in nurseries and from rectal swabs of their piglets presenting post-weaning colibacillosis.Materials, Methods & Results: Fifteen rectal swabs from diarrheic piglets in their first three weeks after weaning and one water sample were collected from each of ten nurseries located in Rio Grande do Sul State, south of Brazil. After enrichment with a commercial broth medium, water samples were cultured in blood agar, as well as the rectal swab samples, and the characteristic colonies were identified by standard biochemical analysis. Following isolation and identification of E. coli, the colonies from water samples and their corresponding piglets’ samples were characterized by multiplex PCR in order to determine specific ETEC fimbria and toxin genes. Finally, all E. coli isolates were submitted to antimicrobial susceptibility testing. Virulence factors and antimicrobial sensitivity could then be compared between water and piglets’ samples. The difference in the antimicrobial resistance frequency for each of the sample groups were compared using the multi comparison test. E. coli was isolated in four out of the ten water samples, although none of the water samples presented ETEC virulence factors.[...]

Virulence factors and antimicrobial resistance profile of Escherichia coli isolated from nursery piglets and drinking water

Background: One of the most frequent health problems in the swine industry is the post-weaning diarrhea in nursery pigs, which leads to significant losses due to weight loss, dehydration, cost of medication and mortality. Escherichia coli (E. coli) is one of the main bacterial agents of the post-weaning diarrhea. To investigate the possibility of enterotoxigenic E. coli (ETEC) transmission through drinking water to nursery piglets, the objective of this study was to isolate, characterize by virulence factors, and compare the antimicrobial resistance profiles of E. coli from drinking water samples in nurseries and from rectal swabs of their piglets presenting post-weaning colibacillosis.Materials, Methods & Results: Fifteen rectal swabs from diarrheic piglets in their first three weeks after weaning and one water sample were collected from each of ten nurseries located in Rio Grande do Sul State, south of Brazil. After enrichment with a commercial broth medium, water samples were cultured in blood agar, as well as the rectal swab samples, and the characteristic colonies were identified by standard biochemical analysis. Following isolation and identification of E. coli, the colonies from water samples and their corresponding piglets samples were characterized by multiplex PCR in order to determine specific ETEC fimbria and toxin genes. Finally, all E. coli isolates were submitted to antimicrobial susceptibility testing. Virulence factors and antimicrobial sensitivity could then be compared between water and piglets samples. The difference in the antimicrobial resistance frequency for each of the sample groups were compared using the multi comparison test. E. coli was isolated in four out of the ten water samples, although none of the water samples presented ETEC virulence factors.[...](AU)