Molecular characterization of Aeromonas hydrophila isolates from diseased fishes in district Kasur, Punjab, Pakistan / Caracterização molecular de isolados de Aeromonas hydrophila de peixes doentes no distrito de Kasur, Punjab, Paquistão

Abstract Pakistan is an agricultural country and fisheries play a very important role in the economic development of the country. Different diseases are prevalent in Pakistani fish but information related to the causative agents is not well-known. Keeping in view the significance of bacterial pathogens as the causative agents of multiple fish diseases, the present study was conducted for identification, characterization and analysis of virulence genes of Aeromonas spp. isolated from diseased fishes. A total of fifty fish samples having multiple clinical indications were collected from different fish farms of district Kasur, Punjab Pakistan. For isolation of Aeromonas spp. samples were enriched and inoculated on Aeromonas isolation medium. Isolates were identified and characterized by different biochemical tests, Analytical Profile Index (API) 20E kit and Polymerase Chain Reaction (PCR) assays. All isolates were screened for three putative virulence genes including aerolysin (aer), haemolysin (hyl) and heat labile cytotonic enterotoxin (alt). Seven isolates of Aeromonas (A.) hydrophila were retrieved and identified based on API 20E. These isolates were further confirmed as A. hydrophila on the basis of PCR assays. Three isolates were detected positive for the presence of virulence genes (alt and hyl). Whereas aerolysin (aer) gene was not present in any of A. hydrophila isolates. The present study confirmed A. hydrophila as the causative agent of epizootic ulcerative syndrome and motile Aeromonas septicemia in fish farms of district Kasur, Punjab Pakistan. Moreover, detection of two virulence genes (alt and hyl) in A. hydrophila isolates is a threat for fish consumers of study area.

Resumo O Paquistão é um país agrícola, onde a pesca desempenha um papel muito importante para o desenvolvimento econômico. Diferentes doenças são prevalentes em peixes do Paquistão, mas as informações relacionadas aos agentes causadores não são bem conhecidas. Tendo em vista a importância dos patógenos bacterianos como agentes causadores de múltiplas doenças em peixes, o presente estudo foi conduzido para identificação, caracterização e análise de genes de virulência de isolados de Aeromonas spp. de peixes doentes. Foram coletadas 50 amostras de peixes com múltiplas indicações clínicas em diferentes fazendas do distrito de Kasur, Punjab, Paquistão. Para isolar Aeromonas spp., as amostras foram enriquecidas e inoculadas em meio de isolamento. Os isolados foram identificados e caracterizados por diferentes testes bioquímicos, kit Analytical Profile Index (API) 20E, e ensaios de reação em cadeia da polimerase (PCR). Todos os isolados foram selecionados para três genes de virulência putativos, incluindo aerolisina (aer), hemolisina (hyl) e enterotoxina citotônica termolábil (alt). Sete isolados de Aeromonas hydrophila foram recuperados e identificados com base no API 20E. Esses isolados foram posteriormente confirmados como A. hydrophila de acordo com ensaios de PCR. Três isolados indicaram a presença de genes de virulência (alt e hyl), enquanto o gene aerolisina (aer) não esteve presente em nenhum dos isolados de A. hydrophila. O presente estudo confirmou A. hydrophila como o agente causador da síndrome ulcerativa epizoótica e septicemia móvel por Aeromonas em fazendas de peixes, no distrito de Kasur, Punjab, Paquistão. Além disso, a detecção de dois genes de virulência (alt e hyl) em isolados de A. hydrophila é uma ameaça para os consumidores de peixes da área de estudo.

Virulence, biofilm formation ability and antimicrobial resistance of staphylococcus aureus isolated from cell phones of university students / Virulência, capacidade de formação de biofilme e resistência antimicrobiana de staphylococcus aureus isolado de telefones celulares de estudantes universitários

INTRODUCTION: Contamination of cell phones can contribute to the dissemination of pathogens in the community and/or hospital environment. OBJECTIVE: To characterize Staphylococcus aureus strains isolated from cell phones of university students. METHODS: Samples were collected from 100 cell phones. Detection of genes associated with virulence factors such as biofilm formation (icaA and icaD), enterotoxins production (SEA, SEB, SEC, and SED), and resistance to methicillin (mecA and mecC) was performed in S. aureus isolates by PCR. Typing mecA gene performed by multiplex PCR. Susceptibility to antimicrobials and biofilm formation rate also evaluated by using disk diffusion test and crystal violet staining. RESULTS: S. aureus was present in 40% of the total samples and about 70% of them belonged to Nursing students. Of the isolates, 85% presented resistance to penicillin and 50% were classified as moderate biofilm producers. In addition, 92.5% of isolates contained the gene icaA and 60% of the gene icaD. Approximately 25% of the isolates presented the mecA gene. Typing of the mecA gene showed the presence of staphylococcal chromosome cassette SCCmec I and c III respectively in 20% and 10% of the isolates. 70% of the samples could not be typed by the technique. Regarding the enterotoxins, the most prevalent gene was SEA (30%) followed by the SEC gene (2.5%). The presence of SED and SEB genes not observed in any of the isolates. CONCLUSION: The cleaning and periodic disinfection of cell phones can contribute to the reduction of the risk of nosocomial infection.

INTRODUÇÃO: A contaminação de celulares pode contribuir para a disseminação de patógenos na comunidade e/ou ambiente hospitalar. OBJETIVO: Caracterizar cepas de Staphylococcus aureus de telefones celulares de estudantes universitários. MÉTODOS: Foram coletadas amostras de 100 telefones celulares. Detecção de genes associados a fatores de virulência quanto a: formação de biofilme (icaA e icaD), produção de enterotoxinas (SEA, SEB, SEC e SED) e resistência à meticilina (mecA e mecC) foi realizada em isolados de S. aureus por PCR. A Tipagem do gene mecA foi realizada por PCR multiplex. A susceptibilidade a antimicrobianos e a taxa de formação de biofilme pelo teste de difusão em disco e coloração com cristal violeta. RESULTADOS: S. aureus esteve presente em 40% do total de amostras, destas, 70% pertenciam a estudantes do curso de enfermagem. Dos isolados, 85% apresentaram resistência à penicilina e 50% foram classificados com moderada formação de biofilme. Além disso, 92,5% dos isolados continham o gene icaA e 60% o gene icaD. Aproximadamente 25% dos isolados apresentaram o gene mecA. A tipagem do gene mecA mostrou a presença do cassete cromossômico estafilocócico SSCmec I e III em respectivamente 20% e 10% dos isolados. 70% das amostras não puderam ser identificadas pela técnica. Das enterotoxinas, o gene mais prevalente foi o SEA (30%), seguido pelo gene SEC (2.5%). A presença dos genes SED e SEB não foi observada nos isolados. CONCLUSÃO: A limpeza e desinfecção periódica dos telefones celulares podem contribuir para a redução do risco de infecção nosocomiais.

Genomic characterization, antimicrobial resistance profiles, enterotoxin, and biofilm production of methicillin-resistant Staphylococcus aureus isolated from clinical and animal products origins in Eastern Turkey / Caracterização genômica perfis de resistência antimicrobiana, produção de enterotoxinas e biofilme Staphylococcus aureus resistente à meticilina isolado de origem clínica e de produtos de origem animal no leste da Turquia

Staphylococcus aureus is an opportunistic and ubiquitous pathogen found in the skin, nares, and mucosal membranes of mammals. Increasing resistance to antimicrobials including methicillin has become an important public concern. One hundred and eight (108) S. aureus strains isolated from a total of 572 clinical and animal products samples, were investigated for their biofilm capability, methicillin resistance, enterotoxin genes, and genetic diversity. Although only one strain isolated from raw retail was found as a strong biofilm producer, the percentage of antimicrobial resistance pattern was relatively higher. 17.59% of S. aureus strains tested in this study were resistant to cefoxitin and identified as methicillin-resistant S. aureus (MRSA) isolates. mecA and mecC harboring S. aureus strains were detected at a rate of 2.79% and 0.93%, respectively. In addition, staphylococcal enterotoxin genes including Sea, Seb, Sec, and Sed genes were found to be 18.5%, 32.4%, 6.5% and 3.7%, respectively. The phylogenetic relationship among the isolates showed relationship between joint calf and cow milk isolates. Multi locus sequence typing (MLST) revealed three different sequence types (STs) including ST84, ST829, and ST6238. These findings highlight the development and spread of MRSA strains with zoonotic potential in animals and the food chain throughout the world.

Staphylococcus aureus é um patógeno dúctil e ubíquo encontrado na pele, narinas e membranas mucosas de mamíferos. O aumento da resistência aos antimicrobianos, incluindo a meticilina, tornou-se uma importante preocupação pública. Cento e oito (108) cepas de S. aureus isoladas de um total de 572 amostras clínicas e de produtos animais foram investigadas por sua capacidade de biofilme, resistência à meticilina, genes de enterotoxinas e diversidade genética. Embora apenas uma cepa isolada do cru tenha sido encontrada como forte produtora de biofilme, a porcentagem do padrão de resistência antimicrobiana foi relativamente maior. Parte das cepas (17,59%) de S. aureus testadas neste estudo eram resistentes à cefoxitina e identificadas como isolados de MRSA. mecA e mecC abrigando cepas de S. aureus foram detectados a uma taxa de 2,79% e 0,93%, respectivamente. Além disso, verificou-se que os genes da enterotoxina estafilocócica, incluindo os genes Sea, Seb, Sec e Sed, eram 18,5%, 32,4%, 6,5% e 3,7%, respectivamente. A relação filogenética entre os isolados mostrou relação entre os isolados de bezerro e leite de vaca. A tipagem de sequência multiloco (MLST) revelou três tipos de sequência diferentes (STs), incluindo ST84, ST829 e ST6238. Essas descobertas destacam o desenvolvimento e a disseminação de cepas de MRSA com potencial zoonótico em animais e na cadeia alimentar em todo o mundo.

¿La detección de toxinas de Clostridioides difficile es necesaria cuando se detecta la enzima glutamato deshidrogenasa? / Is Clostridioides difficile toxins detection necessary when the glutamate dehydrogenase enzyme is detected?

Resumen Introducción: Clostridioides difficile causa diarrea y colitis pseudomembranosa. Su diagnóstico se realiza con la detección de glutamato-deshidrogenasa (GDH) o las toxinas A y B y se confirma con pruebas de amplificación de ácidos nucleicos. Objetivo: Definir si la determinación de GDH es redundante a la de las toxinas. Métodos: Estudio observacional retrospectivo de muestras fecales de pacientes con sospecha de infección por Clostridioides difficile. Las toxinas y GDH se determinaron mediante inmunocromatografía. Se realizó una simulación bayesiana con los cocientes de probabilidad; se consideró significativo un valor de p < 0.05. Resultados: Se analizaron 329 resultados de GDH y toxinas A y B. Se encontró una prevalencia de infección de Clostridioides difficile de 18.2 %. La sensibilidad y especificidad de la prueba de GDH fue de 0.90 y 0.89, respectivamente. El cociente de probabilidad positivo fue de 8.9 y el negativo, de 0.11. Conclusiones: Un resultado negativo de GDH disminuye considerablemente la probabilidad de infección, pero no la descarta. La detección de toxinas de Clostridioides difficile puede ser necesaria en instituciones donde la amplificación de ácidos nucleicos no es económica o accesible.

Abstract Introduction: Clostridioides difficile causes diarrhea and pseudomembranous colitis. Its diagnosis is made with glutamate dehydrogenase (GDH) or toxins A and B detection and is confirmed with nucleic acid amplification tests. Objective: To define if GDH determination is redundant to that of toxins. Methods: Retrospective, observational study in diarrheal stools of patients with suspected Clostridioides difficile infection. Toxins and GDH were determined by immunochromatography. Bayesian simulation was performed with likelihood ratios; a p-value < 0.05 was regarded as significant. Results: 329 GDH and toxin A and B results were analyzed. Clostridioides difficile infection prevalence was 18.2 %. Sensitivity and specificity of the GDH test were 0.90 and 0.89, respectively. Positive likelihood ratio was 8.9, and negative was 0.11. Conclusions: A negative GDH result considerably reduces the probability of infection but does not rule it out. Clostridioides difficile toxins detection may be necessary in institutions where nucleic acid amplification is not affordable or accessible.

Detection of virulence factors in coagulase-negative Staphylococcus spp. strains isolated from Emmental cheese / Detecção de fatores de virulência em cepas de Staphylococcus spp. coagulase negativos isoladas de queijo Emmental

Food prepared with products derived from animals are involved in most cases of staphylococcal poisoning; therefore, the research of Staphylococcus spp. in Emmental cheese is more applicable. The objective of this study was to identify coagulase-negative Staphylococcus spp. (CNS) in cheese using biochemical and molecular techniques to detect the presence of nine genes responsible for the production of enterotoxins. From 180 samples analyzed, 204 CNS strains were obtained and identified as being 46 (22.6%) S. saprophyticus strains, 27 (13.2%) S. hominis spp. hominis strains, 22 (10.8%) S. sciuri strains, 21 (10.3%) S. xylosus strains, 19 (9.3%) S. epidermidis strains, 19 (9.3%) S. haemolyticus strains, 17 (8.3%) S. lentus strains, 17 (8.3%) S. warneri strains, 11 (5.4%) S. equorum strains and 5 (2.5%) S. cohnni . Using the PCRm protocol, 14 (6.9%) strains with the presence of the genes on the enterotoxin E (SEE)11 (78.6%), J (SEJ) 1 (7%), C (SEC) 1 (7%) and I (SEI) 1 (7%) were detected. Based on the results, the type of package is not interfered of growth and isolated that Staphylococcus spp. in cheese. It was observed that bacteria capacity to produce coagulase cannot be understood as an indicative of enterotoxigenicity; therefore, the CNS should be considered as a target of importance in the epidemiology of staphylococcal intoxications. It can be concluded that CNS need to be included in bacterial foodborne disease research, since the genes responsible for the production of toxins were detected and none of the studied samples presented Staphylococcus spp. counting above the limits allowed by legislation.(AU)

Os alimentos preparados com produtos de origem animal são os mais envolvidos em casos de intoxicação alimentar estafilocócica; portanto a pesquisa do Staphylococcus spp. em queijos tipo Emmental é relevante. O objetivo foi isolar e identificar espécies de Staphylococcus coagulase negativas (CNS)de queijo Emmental acondicionado em vários tipos de embalagem, por meio de técnicas bacteriológicas e bioquímicas e detectar, por PCR, a presença de nove genes responsáveis pela produção de enterotoxinas. Das 180 amostras, foram isoladas 204 cepas de CNS, que foram identificadas por provas bioquímicas como: 46 (22,6%) S. saprophyticus, 27 (13,2%) S. hominis spp. hominis, 22 (10,8%) S. sciuri, 21 (10,3%) S. xylosus, 19 (9,3%) S. epidermidis , 19 (9,3%) S. haemolyticus , 17 (8,3%) S. lentus , 17 (8,3%) S. warneri , 11(5,4%) S. equorum e 5 (2,5%) S. cohnii . Na PCR multiplex, em 14 (6,9%) isolados foi detectada a presença dos genes para enterotoxina E (SEE), em 11 (78,6%) J (SEJ), em 1 (7%) C (SEC) e em 1 (7%) I (SEI). Com base nos resultados, o tipo de embalagem não interferiu na multiplicação dos Staphylococcus spp. isolados dos queijos. Neste estudo, verificou-se que a capacidade para a produção de coagulase pela bactéria não pode ser concebida como indicativa de enterotoxigenicidade, portanto devem-se considerar os CNS como objeto de importância na epidemiologia das intoxicações estafilocócicas, fazendo-se necessária a atenção com relação à pesquisa dos CNS nos alimentos, uma vez que foram detectados genes responsáveis pela produção de toxinas, e nenhuma das amostras apresentou contagem para Staphylococcus spp. acima do limite permitido pela legislação.(AU)

Clostridium difficile toxins or infection induce upregulation of adenosine receptors and IL-6 with early pro-inflammatory and late anti-inflammatory pattern

Clostridium difficile causes intestinal inflammation, which increases adenosine. We compared the expression of adenosine receptors (AR) subtypes A1, A2A, A2B, and A3 in HCT-8, IEC-6 cells, and isolated intestinal epithelial cells, challenged or not with Clostridium difficile toxin A and B (TcdA and TcdB) or infection (CDI). In HCT-8, TcdB induced an early A2BR expression at 6 h and a late A2AR expression at 6 and 24 h. In addition, both TcdA and TcdB increased IL-6 expression at all time-points (peak at 6 h) and PSB603, an A2BR antagonist, decreased IL-6 expression and production. In isolated cecum epithelial cells, TcdA induced an early expression of A2BR at 2s and 6 h, followed by a late expression of A2AR at 6 and 24 h and of A1R at 24 h. In CDI, A2AR and A2BR expressions were increased at day 3, but not at day 7. ARs play a role in regulating inflammation during CDI by inducing an early pro-inflammatory and a late anti-inflammatory response. The timing of interventions with AR antagonist or agonists may be of relevance in treatment of CDI.

Characterization of Staphylococcus species isolated in production stages of brazilian colonial cheese

Colonial cheese is a culturally and economically important product from the south of Brazil. As most of its production is artisanal, the technology employed is mostly knowledge passed down from one generation to the next according to family tradition and may be produced with raw or pasteurized milk. It is noted for its spicy flavour and variable composition and is often classified as a medium to high-moisture cheese. This intrinsic feature increases the risk of microbial spoilage and food poisoning. One of the main bio-indicators of contamination in colonial cheese is coagulase positive Staphylococcus. The purpose of this study was the phenotypic identification of Staphylococcus species isolated from the products and surfaces in the main production stages of colonial cheese. Staphylococcus sp. isolates from the food and the production environment were obtained from two colonial cheese-production agro-industries in Rio Grande do Sul. Samples of fresh milk, curd, ripening and final colonial cheese were collected. In addition, surface sampling was performed on the coagulation tanks, production tables, molds, cheese ripening shelves and on the hands of the handlers. Staphylococcus sp. isolates in the cheese and the production environments tested in this study were identified by phenotypic techniques through biochemical and MALDI-TOF MS analyses. These isolates were subjected to gene expression analysis for enterotoxins A, B, C, D, and E. All isolates (72) were identified as Staphylococcus sp., and 43% of the total isolates tested were coagulase positive. Staphylococcus aureus was the predominant species in the raw milk and production tanks. Regarding coagulase negative staphylococci isolates, S. warneri and S. sciuri were most abundant. The sea and seb genes were detected in 4% of the Staphylococcus isolates. The results indicate eleven different species of Staphylococcus present in the colonial cheese production environments studied. The predominant presence of S. aureus in the different samples of milk, curd, ripened cheese, ready-to-eat cheese and hands of the handlers indicates that there are issues with the selection of milk-producing animals, pasteurization process and/or hygiene control of handlers. The sea and seb genes were detected in samples of raw milk and colonial cheese. No enterotoxin genes were detected in coagulase negative staphylococci.

Brote de intoxicación alimentaria en un jardín de infantes de la provincia de Buenos Aires / Food poisoning outbreak in a kindergarten in the Province of Buenos Aires, Argentina

Resumen El 27 de noviembre de 2008 ocurrió un brote de intoxicación alimentaria asociado al consumo de salpicón de ave en un jardín de infantes de Hurlingham, provincia de Buenos Aires. Treinta y siete niños y 10 adultos presentaron síntomas gastrointestinales. Cinco niños fueron internados con signos de deshidratación, y uno de ellos requirió cuidados intensivos. Se aisló Staphylococcus aureus subsp. aureus del alimento involucrado, de 4/5 muestras de materia fecal de pacientes y de 3/5 manipuladores (nariz del manipulador 1, manos de manipuladores 2 y 3). Las cepas aisladas portaban los genes que codifican las enterotoxinas SEA y SED. Por electroforesis de campo pulsado con la enzima SmaI, los patrones de macrorrestricción presentaron 100% de similitud. La investigación oportuna del brote permitió identificar al agente causal de la intoxicación, determinar las fallas en la elaboración del alimento e implementar las medidas correctivas correspondientes.

Abstract On November 27, 2008, a foodborne disease outbreak associated with the consumption of chicken salad occurred in a kindergarten in the District of Hurlingham, Province of Buenos Aires. Thirty-seven children and 10 adults with gastrointestinal symptoms were affected. Five children were hospitalized with signs of dehydration, one of them requiring intensive care. Staphylococcus aureus subsp. aureus was isolated from the mentioned food in 4 out of 5 stool specimens from the patients, and in 3 out of 5 food handlers (nose of food handler #1, hands of food handlers #2 and 3). The isolates carried the genes coding for enterotoxins SEA and SED. The macrorestriction patterns showed 100% similarity by pulsed-field gel electrophoresis using the SmaI enzyme. A timely outbreak investigation allowed us to identify the causative agent of the food poisoning as well as the failures in food processing and to implement corrective measures.

Comparación de tres métodos de recuento de Staphylococcusaureus en queso fresco de expendio en los mercados populares de la ciudad de La Paz-Bolivia / Comparison of three methods of counting Staphylococcus aureus in fresh cheese for sale in the central markets of the city of La Paz-Bolivia

Staphylococcus aureus puede contaminar una gran gama de alimentos, constituyéndose el queso fresco en un buen medio diferencial y selectivo para el desarrollo de este microorganismo. La intoxicación estafilocóccica trasmitida por alimentos, resulta de la ingesta de enterotoxina termoestable preformada en el alimento que fue generada por una cepa toxigénica de Staphylococcus aureus llegando a constituirse en un riesgo para la salud de los consumidores. El presente estudio tiene el objetivo de comparar tres métodos: i) ISO 6888-1:2003 ii) NB 32004:2004 y iii) Placas secas rehidratables (Placas Petrifilm) para el recuento de Staphylococcus aureus en queso fresco de expendio en los mercados populares de la ciudad de La Paz. La comparación entre el método de cultivo convencional empleando medio Agar Baird Parker y el método alternativo por placa seca rehidratable fue hecha por medio del muestreo al azar de 30 muestras de queso fresco, el análisis fue hecho por los tres métodos métodos, donde por el método de placa seca rehidratable 10 muestras se encontraban dentro de norma y 20 se encontraban fuera de norma; por medio del método de cultivo convencional 22 muestras se encontraban en norma y 8 fuera de norma. Concluyendo que el método de placa seca rehidratable es más sensible y fácil de aplicar en comparación con el método convencional según norma Bolivia y norma ISO.

Staphylococcus aureus can contaminate a wide range of foods, constituting fresh cheese in a good differential and selective medium for the development of this microorganism. Staphylococcal foodborne poisoning results from the intake of thermostable enterotoxin preformed in the food that was generated by a toxigenic strain of Staphylococcus aureus, becoming a risk to the health of consumers. The present study has the objective of comparing three methods: i) ISO 6888-1: 2003 ii) NB 32004: 2004 and iii) Dry rehydratable plates (Petrifilm plates) for the recount of Staphylococcus aureus in fresh cheese for sale in popular markets from the city of La Paz. The comparison between the conventional culture method using Baird Parker Agar medium and the alternative method by rehydratable dry plate was made by random sampling of 30 samples of fresh cheese, the analysis was done by the three methods methods, where by the method dry rehydratable plate 10 samples were within norm and 20 were out of norm; By means of the conventional cultivation method, 22 samples were in standard and 8 out of norm. Concluding that the rehydratable dry plate method is more sensitive and easier to apply compared to the conventional method according to the Bolivia standard and ISO standard.

Aislamiento de Bacillus cereus en restaurantes escolares de Colombia / Isolation of Bacillus cereus in school restaurants in Colombia

Resumen Introducción. Bacillus cereus es reconocido como un agente patógeno causante de intoxicaciones alimentarias. Se trata de una bacteria de metabolismo aerobio facultativo capaz de formar esporas, lo que le permite sobrevivir a la pasteurización y el calentamiento e, incluso, a la irradiación con los rayos gamma usados para reducir los agentes patógenos de los alimentos. Objetivo. Estudiar la presencia de B. cereus y su toxina diarreica en el arroz y en alimentos a base de cereales, harinas o féculas listos para el consumo en restaurantes escolares de algunos departamentos de Colombia. Materiales y métodos. Se hizo un estudio descriptivo y transversal con alimentos listos para el consumo distribuidos en restaurantes escolares de los departamentos que más notifican enfermedades transmitidas por alimentos al sistema de vigilancia, así como en los de menor notificación. Resultados. Se recolectaron 479 muestras en ocho departamentos, 74 municipios y 363 restaurantes escolares; el 63 % correspondió a muestras de arroz y el 37 % a alimentos como coladas. El 9 % de las muestras analizadas fueron positivas para B. cereus y, en el 91 % de estas, se detectó la toxina diarreica. Conclusiones. En todos los departamentos estudiados se encontró B. cereus. El manejo de materias primas y el inadecuado tratamiento térmico de los alimentos fueron los factores directamente relacionados con las enfermedades transmitidas por alimentos. Es importante reforzar la vigilancia e incentivar la investigación y la notificación de los brotes de enfermedades transmitidas por alimentos para mejorar la calidad de la información, llevar a cabo acciones de comunicación, prevención y coordinación intersectorial, y con los manipuladores, con el fin de adoptar las medidas necesarias que garanticen la inocuidad de los alimentos, así como la eliminación de los factores de riesgo de estas enfermedades.

Abstract Introduction: Bacillus cereus is recognized as a pathogen that causes food poisoning. It is a facultative aerobic metabolism bacterium capable of forming spores, which allows it to survive pasteurization and heating even by the gamma irradiation used to reduce pathogens in food. Objective: To study the presence of Bacillus cereus and its diarrheal toxin in rice and ready-to-eat cereals, flours, and starches in school restaurants in Colombia. Materials and methods: We conducted a descriptive cross-sectional study of ready-to-eat foods distributed in school restaurants in the departments with the most and the least notification of foodborne diseases to the surveillance system. Results: A total of 479 samples were collected from eight departments, 74 municipalities, and 363 school restaurants, 63% of which were rice samples and 37%, starchy food samples; 9% of them tested positive for Bacillus cereus. In 91% of the samples that tested positive, the bacterium was isolated with the presence of the diarrheal toxin. Conclusions: In all the departments with B. cereus in the samples, the factors directly related to food-borne diseases were the handling of raw materials and the poor thermal treatment of food. Strengthening surveillance by stimulating research and reporting on outbreaks of foodborne diseases is important to improve the quality of information, to develop communication, prevention and intersectional coordination and manipulation measures, as well as to take the necessary actions to guarantee the safety of food and to eliminate the risk factors that may contribute to this problem.

Glutamato deshidrogenasa: Su valor diagnóstico en la diarrea por Clostridioides difficile / Glutamate dehydrogenase: Its diagnostic value in Clostridioides difficile diarrhea

Clostridioides difficile es el principal agente causal de diarreas asociadas al cuidado de la salud. Esta bacteria produce toxinas y una enzima que se encuentra muy conservada en la especie: la glutamato deshidrogenasa (GDH). El diagnóstico rápido y el tratamiento efectivo permiten la pronta mejoría del paciente, con el consecuente control de la diseminación del microorganismo. Sin embargo, aún no se cuenta con un método diagnóstico óptimo y se propone la realización de diversas pruebas, cuyos resultados se interpretan en el contexto de ciertos algoritmos. En este trabajo se evaluó el desempeño de la GDH como prueba de tamizaje en el diagnóstico de la diarrea por c. difficile. Se estudiaron 615 muestras de materia fecal. Se determinó la presencia de GDH y de toxinas mediante el equipo diagnóstico de enzimoinmunoensayo de membrana C. DIFF QUIK-CHEK COMPLETE® (TECHLAB) y se realizaron cultivos para la búsqueda de C. difficile. Se calcularon los valores de sensibilidad, especificidad, VPP y VPN con un nivel de significación p < 0,05. Se detectó GDH en 266 muestras (43,25%), con una sensibilidad del 100% y una especificidad del 87,10%, IC95: 84,58-91,42. Se hallaron toxinas en 218 muestras (35,45%) y C. difficile desarrolló en 235 cultivos (38,21%). De 48 muestras GDH positivas y sin producción de toxina/s, 17 fueron positivas al cultivo de C. difficile, con 15 aislamientos toxigénicos y 2 no toxigénicos. No hubo desarrollo de C. difficile en las 31 muestras restantes. Ninguna muestra GDH negativa dio resultado positivo de toxina/s ni desarrollo en el cultivo, por lo cual el VPN de la GDH fue del 100%, mientras que el VPP fue del 81,9%. Concluimos que la determinación de GDH representa un screening adecuado para descartar casos de diarrea por C. difficile, por lo tanto de valor en los algoritmos diagnósticos de las diarreas infecciosas.

Clostridioides difficile is the main etiological agent of diarrhea associated with health care, it produces toxins and glutamate dehydrogenase (GDH), an enzyme that is highly conserved in this species. Rapid diagnosis and effective treatment produce prompt improvement of the patient and subsequent control of the microorganism spread. There are several techniques whose results are interpreted in the context of algorithms. However, the optimal diagnostic method is yet unknown. The performance of GDH as a screening test for the diagnosis of C. difficile diarrhea was assessed. Six hundred and fifteen stool samples were studied. The presence of GDH and toxins presence was determined by TECHLAB® C. DIFF QUIK-CHEK COMPLETE and the samples were cultured for the search of C. difficile. The values of sensitivity, specificity, PPV and NPV were calculated with a p value of 0.05 or less. GDH was detected in 266 samples (43.25%), with a sensitivity of 100% and specificity of 87.10%, IC95: 84.58-91.42; toxin/s were detected in 218 (35.45%) and C. difficile developed in 235 cultures (38.21%). From 48 samples with positive GDH and negative toxin/s, 15 toxigenic and 2 non-toxigenic isolates were obtained, the remaining 31 samples were negative for C. difficile. All GDH-negative samples were negative for toxins or culture, therefore, GDH NPV was 100%, while PPV was 81.9%. We conclude that GDH is a suitable screening test for the diagnostic algorithm of C. difficile diarrhea.

Detección y expresión de superantígenos y de resistencia antimicrobiana en aislamientos obtenidos de mujeres portadoras de Staphylococcus aureus que cuidan y alimentan niños / Detection and expression of superantigens and resistance in isolates from women carriers of Staphylococcus aureus who take care of and feed children

Resumen Introducción. Staphylococcus aureus coloniza mucosas y piel, y causa graves infecciones en el hombre y los animales. Es importante establecer el estatus de portadoras de cepas enterotoxigénicas de este microorganismo en manipuladoras de alimentos, con el fin de prevenir intoxicaciones alimentarias. Objetivo. Establecer las correlaciones entre los genes de enterotoxinas clásicas, el gen tsst-1, la producción de toxinas en cultivo y la resistencia antimicrobiana en aislamientos de S. aureus provenientes de manipuladoras de alimentos que cuidan niños en sus comunidades. Materiales y métodos. Se cultivaron muestras de las fosas nasales y las yemas de los dedos de las manos, y se identificó S. aureus empleando las pruebas de rutina y métodos automatizados. La extracción de ADN se hizo mediante el método de bromuro de cetil-trimetil-amonio (Cetyl-Trimethyl- Ammonium Bromide, CTAB) modificado. Para la detección de superantígenos se emplearon pruebas de reacción en cadena de la polimerasa (PCR) simple y múltiple, y para la de toxinas, estuches comerciales. Resultados. Se encontró que el 22,0 % de los aislamientos correspondía a portadoras de S. aureus: 17,0 % en los aislamientos de fosas nasales; 5,0 % en los de las manos y 6,7 % simultáneamente en los dos sitios. La prevalencia de superantígenos fue de 73,7 %. El genotipo más frecuente fue el sea-tsst-1, con 10,0 %. La resistencia a un solo antibiótico fue de 74,7 % y, a cuatro antibióticos, de 3,2 %; de los aislamientos, el 93,7 % correspondía a cepas productoras de betalactamasas. La detección de genes clásicos y de tsst-1 mediante PCR fue de 48,4 % y la de toxinas en el sobrenadante, de 42,1 %, con una correlación de 95,7 %. Las mayores correlaciones se establecieron entre las toxinas TSST-1 (22/22) y SEA (17/18). La correlación del gen tsst-1 con la proteína y la resistencia fue de 100 %. Todos los aislamientos con el genotipo sea-tsst-1 t fueron resistentes y productores de las toxinas. Conclusión. La tasa de aislamientos de S. aureus toxigénicos y resistentes obtenidos de mujeres que cuidan y preparan alimentos para niños fue de más de 70 %, lo que demostró su gran virulencia y la consecuente necesidad de aplicar estrictamente las normas higiénicas y sanitarias vigentes para evitar el riesgo de intoxicación alimentaria.

Abstract Introduction: Staphylococcus aureus colonizes mucous membranes and skin causing severe infections in humans and animals. It is important to determine carrier status of enterotoxigenic strains of this microorganism in food handlers to prevent food poisoning. Objective: To establish the correlations among classic enterotoxigenic genes, tsst-1 gene, the production of toxins in cultures and antimicrobial resistance in S. aureus isolates from women who handle the food, feed and take care of children in their communities. Materials and methods: Nasal swab and finger samples were cultured and S. aureus was identified using routine methods and automated systems. DNA extraction was done by the CTAB modified method, and superantigen detection by simple and multiplex PCR, while toxins were detected using commercial kits. Results: We found that 22.0% of subjects were S. aureus carriers: 17.0% corresponded to nose samples, 5.0% to hands and 6.7% to both nose and hands. The prevalence of superantigens was 73.7%. The most frequent genotype was sea-tsst-1 with 10%. Resistance to one antibiotic was 74.7%, and to four antibiotics, 3.2%; 93.7% of the isolates were betalactamase-positive. Classical genes and tsst-1 gene were detected by PCR in 48.4% of samples and toxins in supernatant were detected in 42.1% of them with 95.7% of correlation.The highest correlations were established for TSST-1 and SEA with 100% and 94.4%, respectively. The correlation of tsst-1 gene with toxin production and resistance was 100%. All isolates with genotype sea-tsst-1 were toxin-positive and resistant. Conclusion: The rate of toxigenic and resistant S. aureus isolates from women in charge of feeding and taking care of children was higher than 70%, which demonstrates its high virulence. This requires the strict application of hygienic and sanitary regulations in order to avoid the risk of food poisoning.

Detection of toxigenic Clostridioides --#1;Clostridium--#3; difficile: Usefulness of two commercially available enzyme immunoassays and a PCR assay on stool samples and stool isolates / Detección de Clostridioides --#1;Clostridium--#3; difficile toxigénico: utilidad de dos métodos de enzimoinmunoensayo comerciales y una PCR en las muestras de materia fecal y en los respectivos aislamientos

The best laboratory diagnostic approach to detect Clostridioides --#1;Clostridium--#3; difficile infection (CDI) is a subject of ongoing debate. With the aim of evaluating four laboratory diagnostic methods, 250 unformed stools from patients with suspected CDI submitted to nine medical center laboratories from November 2010 to December 2011, were studied using: (1) an immunochromatographic rapid assay test that combines the qualitative determination of glutamate dehydrogenase (GDH) plus toxins A and B (QAB), the CDIFF QUIK CHEK COMPLETE assay; (2) an enzyme immunoassay for qualitative determination of toxins A and B, the RIDASCREENTC. difficile Toxin A/B assay (RAB); (3) a PCR for the toxin B gene assay (PCR); and (4) the toxigenic culture (TC).C. difficile isolates from direct toxin negative stools by QAB, RAB and PCR were evaluated for toxigenicity by the same direct tests, in order to assess the contribution of the TC (QAB-TC, RAB-TC, PCR-TC). A combination of the cell culture cytotoxicity neutralization assay (CCCNA) in stools, and the same assay on isolates from direct negative samples (CCCNA-TC) was considered the reference method (CCCNA/CCCNA-TC). Of the 250 stools tested, 107 (42.8%) were positive by CCCNA/CCCNA-TC. The GDH and PCR/PCR-TC assays were the most sensitive, 91.59% and 87.62%, respectively. The QAB, RAB, QAB/QAB-TC and RAB/RAB-TC had the highest specificities, ca. 95%. A negative GDH result would rule out CDI, however, its low positive likelihood ratio (PLR) of 3.97 indicates that a positive result should always be complemented with the detection of toxins. If the RAB, QAB, and PCR assays do not detect toxins from direct feces, the toxigenic culture should be performed. In view of our results, the most accurate and reliable methods to be applied in a clinical microbiology laboratory were the QAB/QAB-TC, and RAB/RAB-TC, with PLRs >10 and negative likelihood ratios <0.30.

El mejor procedimiento para realizar el diagnóstico de laboratorio de la infección causada por Clostridioides --#1;Clostridium--#3; difficile (ICD) es aún objeto de debate. Con el fin de evaluar cuatro métodos diagnósticos de laboratorio, se estudiaron 250 muestras de heces diarreicas provenientes de pacientes con sospecha de ICD remitidas a los laboratorios de nueve centros médicos entre noviembre de 2010 y diciembre de 2011. Dichas muestras se analizaron mediante los siguientes métodos:1) un ensayo rápido inmunocromatográfico que combina la detección cualitativa de la glutamato deshidrogenasa (GDH) y de las toxinas Ay B (QAB), CDIFF QUIK CHEK COMPLETE;2) un enzimoinmunoanálisis para la determinación cualitativa de las toxinas A/B, RIDASCREENTC. difficile Toxin A/B (RAB);3) un método molecular basado en PCR para la detección del gen que codifica la toxina B (PCR) y 4) el cultivo toxigénico (TC). Como método de referencia se utilizó la combinación del ensayo de citotoxicidad sobre cultivo de células con la neutralización de toxina mediante anticuerpo específico en los filtrados de las heces (CCCNA) y el mismo método en sobrenadantes de aislamientos de C. difficile (CCCNA-TC). La toxigenicidad de las cepas aisladas de muestras directas negativas con QAB, RAB y PCR se evaluó con los mismos métodos, con el propósito de detectar la contribución del TC (QAB-TC, RAB-TC, PCR-TC). De las 250 muestras estudiadas, 107 (42,8%) fueron positivas por CCCNA/CCCNA-TC. Los métodos GDH y PCR/PCR-TC fueron los más sensibles: 91,59 y 87,62%, respectivamente. Los métodos QAB, RAB, QAB/QAB-TC y RAB/RAB-TC mostraron las mayores especificidades, del 95%, aproximadamente. Un resultado negativo para GDH excluiría la ICD, pero su baja razón de verosimilitud positiva (PLR), que fue 3,97, indica que un resultado positivo debe complementarse con la detección de toxinas. Cuando no se detectan toxinas directas por RAB, QAB ni PCR, debería realizarse el TC. De acuerdo con nuestros resultados, los métodos más precisos y confiables para ser aplicados en un laboratorio de microbiología clínica son QAB/QAB-TC y RAB/RAB-TC, con una PLR> 10 y una razón de verosimilitud negativa < 0,30.

Genes codificadores para enterotoxinas de aislamientos de estafilococos coagulasa negativos y coagulasa positivos a partir de muestras de mastitis bovina / Coding genes for enterotoxins of isolated coagulase-negative and coagulase-positive staphylococci from bovine mastitis samples / Genes codificadores de enterotoxinas em isolados de estafilococos coagulase positivo em amostras com mastite bovina

Introducción. La mastitis bovina es la inflamación de glándulas mamarias y tejidos secretores. El género Staphylococcus es el agente causal más importante, por su capacidad de producir diferentes factores de virulencia. Las enterotoxinas estafilocócicas son un grupo importante de toxinas que permiten al microorganismo invadir células y tejido huésped, son diseminadas por medio de productos alimenticios y son responsables de graves intoxicaciones alimentarias en el mundo. Objetivo. Determinar la presencia de genes codificadores para las enterotoxinas estafilocócicas (Staphylococcal Enterotoxins, SE) SEA, SEB, SEC, SED y SEE, en cepas de Staphylococcus spp. asociadas con mastitis bovina. Materiales y métodos. Se trata de un estudio cuantitativo, descriptivo y transversal. Se identificaron especies por medio de la amplificación de la región r16S. Los genes SEA, SEE, SEC, SED y SEE se detectaron mediante la amplificación por PCR convencional, usando iniciadores específicos para cada gen, y se evidenciaron los amplicones con electroforesis. Resultados. Hubo predominio del grupo Staphylococcus coagulasa positivo (65,2 %) sobre el grupo coagulasa negativo (37,5 %). Staphylococcus aureus fue la cepa más frecuente (88,5 %). El gen SEA (1,7 %) se detectó en S. sciuri, el gen SEB (3,6 %), en S. pasteuri y S. warneri, y el gen SEC (3,6 %), en S. sciuri y S. saprophyticus SEC. No se detectaron los genes SED y SEE en ninguna de las cepas evaluadas Conclusiones. Los resultados apoyan que la mastitis bovina también es causada por Staphylococcus coagulasa negativo, lo cual indica la posibilidad de que este grupo adquiera atributos genéticos, como enterotoxinas y factores de virulencia, por transferencia horizontal.

Introduction: Bovine mastitis is an inflammation of mammary glands and secretory tissues. Staphylococcus gender is the main causal agent, due to its capacity to produce different virulence factors. Staphylococcal enterotoxins are a main group of toxins which permit the microorganism to spread in cells and guest tissue, which are disseminated through food products, being responsible of serious food poisoning cases around the world. Objective: To determine the presence of coding genes for staphylococcal enterotoxins (SE); SEA, SEB, SEC, SED and SEE, in Staphylococcus spp. strains related to bovine mastitis. Materials and methods: Quantitative study, descriptive and cross-sectional. It was made an identification of species through. They were identified 57 Staphylococcus spp. strains at specie level by amplification of r16S region. The detection of SEA, SEE, SEC, SED, y SEE genes was made through conventional PCR, using specific primers for each gene, they were evinced amplicons through electrophoresis. Results: It was evinced predominance of coagulase-positive Staphylococcus group (65.2%), being Staphylococcus aureus the strain with the highest presence (88.5%), whereas coagulase-negative Staphylococcus group was 37.5%. SEA gene was detected in S. sciuri (1.7%); SEB in S. pasteuri and S. warneri (3.6%); SEC was identified in S. sciuri and S. saprophyticus (3.6%); they were not detected SED y SEE genes in any of the researched strains. Conclusions: The results support that the development of bovine mastitis is also caused by coagulase-negative Staphylococcus, indicating the possibility that this group acquires genetic attributes as enterotoxins and virulence factors by horizontal gene transfer.

Introdução. A mastite bovina é a inflamação das glândulas mamárias e dos tecidos secretórios. O gênero Staphylococcus é o agente causador mais importante, devido à sua capacidade de produzir diferentes fatores de virulência. As enterotoxinas estafilocócicas são um importante grupo de toxinas que permitem que o microorganismo invada as células e tecidos do hospede, são disseminadas através de produtos alimentícios e são responsáveis por intoxicações alimentares graves no mundo. Objetivo. Determinar a presença de genes que codificam enterotoxinas estafilocócicas (Staphylococcal Enterotoxins, SE) SEA, SEB, SEC, SED e SEE, em cepas de Staphylococcus spp. associada à mastite bovina. Materiais e métodos. Estudo quantitativo, descritivo e transversal. As espécies foram identificadas por amplificação da região r16S. Os genes SEA, SEE, SEC e SED foram detectados por amplificação PCR convencional utilizando primers específicos para cada gene, e os amplicons foram evidenciados com eletroforese. Resultados. Houve predomínio do grupo Staphylococcus coagulase positivo (65,2%) sobre o grupo negativo da coagulase (37,5%). Staphylococcus aureus foi a cepa mais frequente (88,5%). O gene SEA (1.7%) foi detectado em S. sciuri, o gene SEB (3.6%) em S. pasteuri e S. warneri, SEC (3.6%) em S. sciuri e SEC em S. saprophyticus. Os genes SED e SEE não foram detectados em nenhuma das cepas avaliadas. Conclusões. Os resultados confirmam que a mastite bovina também é causada por Staphylococcus coagulase-negativo, o que indica a possibilidade de que este grupo adquira atributos genéticos, como enterotoxinas e fatores de virulência, por transferência horizontal.

Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin

BACKGROUND The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant. OBJECTIVES We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead. METHODS BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response. FINDINGS Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide. MAIN CONCLUSIONS The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.

Portación de Staphylococcus aureus enterotoxigénico tipo A, en frotis nasofaríngeos en manipuladores de alimentos / Carriage of Staphylococcus aureus among food service workers

Background Staphylococcus aureus produces 11 serotypes of endotoxins that may cause food poisoning. Aim To determine the prevalence of type A enterotoxigenic Staphylococcus aureus carriage among food service workers in Chillan, Chile. Material and Methods Pharyngeal swabs were obtained from 100 food service workers and were cultured in Agar plates. After identifying the presence of Staphylococcus aureus, DNA was extracted to identify type A toxin by conventional PCR. Results Thirty eight percent of samples were colonized with Staphylococcus aureus. Among these, 26% were toxin A producers. Conclusions Half of the sampled workers carried Staphylococcus aureus and a quarter of these produced type A enterotoxin.

Imunogenicidade da proteína M recombinante de Streptococcus equi subsp. equi coadministrada com um adjuvante molecular / Immunogenicity of recombinant M protein of Streptococcus equi subsp. equi co administered with a molecular adjuvant

The strangles is an infectious disease that affects horses from all ages and causes important economic losses in the equine-related business. The aim of this work was to evaluate the immunogenicity of the recombinant M protein from Streptococcus equi (rSeM) co-administered with the recombinant heat-labile enterotoxin B subunit from Escherichia coli (rLTB) in mice and horses. A total of 72 female Balb-c mice were divided into eight groups and 18 horses were divided into six groups. The animals were inoculated by intramuscular (IM) or intranasal (IN) routes with different treatments of rSeM, rLTB and/or Al(OH)3. The results obtained in both species, independent of administration routes, demonstrated that rSeM + rLTB had higher levels of specific serum immunoglobulins, however, in mucosal immunity the increase was not identified. Thus, the use of rSeM as vaccine antigen and rLTB as adjuvant can be a potential tool in the control of equine strangles.(AU)

Frequência de genes codificadores de toxinas em Staphylococcus aureus isolados de leite de tanques expansão comunitários / Frequency of toxin-encoding genes in Staphylococcus aureus isolated from community milk tanks

A capacidade de produção de toxinas pelo Staphylococcus aureus no leite e produtos derivados está relacionado com surtos de intoxicação alimentar. Objetivou-se nesta pesquisa, estudar a ocorrência de genes que codificam para enterotoxinas estafilocócicas (sea, seb, sed, seg, seh e sei) e toxinas α e ß hemolítica (hla e hlb) em S. aureus isolados de 53 amostras de leite de tanques expansão comunitários no Estado de Alagoas, Brasil. Foram identificados 27 isolados (50,94%) como S. aureus pela amplificação do gene nuc. 13/27 isolados (48,1%) foram positivos para pelo menos um gene das enterotoxinas estudadas, sendo as frequências dos genes sea 33,3%, seh 18,5%, sei 11,1% e sed 7,4%; não entanto não foram identificados os genes seb e seg nestas bactérias. Para as toxinas hemolíticas, 51,9% dos isolados portavam ambos genes (hla e hlb), sendo a frequência para o gene hla de 81,5% e para o gene hlb de 51,9%. A frequência de genes das toxinas avaliadas é alta o que constitui um risco potencial para a saúde pública em especial, as enterotoxinas por serem termoestáveis e estarem asssociados com surtos de intoxicação alimentar.(AU)

The capacity of toxin production by Staphylococcus aureus in milk and dairy products is associated with food poisoning outbreaks. The objective of this research was to study the frequency of genes encoding staphylococcal enterotoxin (sea, seb, sed, seg, seh and sei) and α and ß hemolytic toxins (hla and hlb) in S. aureus isolates from 53 milk samples from community tanks in the State of Alagoas, Brazil. Twenty-seven isolates (50.94%) were identified as S. aureus by nuc gene amplification; 13/27 isolates (48.1%) were positive for at least one gene of the studied enterotoxins and the frequency of genes sea was 33.3%, seh 18.5%, sei 11.1% and sed 7.4%; the seb and sec genes have not been identified in the bacteria. For the hemolytic toxins, 51.9% of isolates harbored both genes (hla and hlb), the frequency of hla gene was 81.5% and 51.9% for the hlb gene. The evaluated toxin-encoding gene frequency is high and constitutes a potential risk for public health, especially staphylococcal enterotoxin genes; because they are heat-stable enterotoxins and have been associated with food poisoning.(AU)

Enterotoxin genes, multidrug resistance, and molecular typing of Staphylococcus spp. isolated from organic bovine milk / Genes de enterotoxinas, multirresistência a antimicrobianos e caracterização molecular de espécies de Staphylococcus spp. isoladas de leite bovino orgânico

The multidrug resistant and the emergence of methicillin-resistant staphylococci isolated from animals, food, and humans are public health concern. These microorganisms produce different toxins related to food poisoning in humans. This study aimed to characterize Staphylococcus spp. isolated from two organic milk farms in Brazil. A total of 259 milk samples were collected, from which 58 (22.4%) Staphylococcus spp. were isolated. The highest sensibility to ceftiofur and sulfamethoxazole/trimethoprim was observed in 96.6% of Staphylococcus spp., and whereas 89% were resistant to penicillin G. The mecA gene was detected in 13.8% of the isolates. SEA and SEC were the most common enterotoxins detected. PFGE revealed genetic heterogeneity from S. intermedius and S. warneri analyzed, while S. aureus presented similar profiles among isolates from the two studied herds. To the best of our knowledge, the current study describes for the first time presence of enterotoxins, mecA gene, and genetic diversity of staphylococci isolated from organic dairy farms in Brazil.(AU)

A emergência de estafilococos multirresistentes e resistentes à meticilina, isolados de animais, alimentos e humanos é uma preocupação em saúde pública. Esses micro-organismos produzem diferentes toxinas relacionadas à intoxicação alimentar em humanos. Este estudo caracterizou Staphylococcus spp. isolados em duas fazendas orgânicas no Brasil. Foram coletadas 259 amostras de leite em duas propriedades leiteiras orgânicas, nas quais 58 (22,4%) estirpes de Staphylococcus spp. foram isoladas. A maior sensibilidade dos isolados foi observada para ceftiofur e sulfametoxazol/trimetoprim em 96,6%. Em contraste, acima de 89% de resistência dos estafilicocos foi encontrada para penicilina G. O gene mecA foi identificado em 13,8% dos isolados. SEA e SEC foram as enterotoxinas mais comumente detectadas. PFGE revelou heterogeneidade genética entre S. intermedius e S. warneri, enquanto S. aureus demonstraram perfis semelhantes entre isolados dos dois rebanhos estudados. Relata-se pela primeira vez no Brasil a detecção de enterotoxinas, o gene mecA e diversidade genética em estafilococos isolados de vacas em produção orgânica.(AU)

Plesiomonas shigelloides exports a lethal cytotoxic-enterotoxin (LCE) by membrane vesicles

ABSTRACT Plesiomonas shigelloides isolated from water in Brazil was previously described as a hemorrhagic heat-labile cytotoxic-enterotoxin producer. We purified this toxin from culture supernatants using ion metallic affinity chromatography (IMAC) followed by molecular exclusion chromatography. The pure toxin presented molecular mass of 50 kDa and isoelectric point (pI) around 6.9 by 2D electrophoresis. When injected intravenously, the purified cytotoxic-enterotoxin induced also severe spasms followed by sudden death of mice. Hence, we entitled it as lethal cytotoxic-enterotoxin (LCE). The presence of membrane vesicles (MVs) on cell surfaces of P. shigelloides was observed by scan electron microscopy (SEM). From these MVs the LCE toxin was extracted and confirmed by biological and serological assays. These data suggest that P. shigelloides also exports this cytotoxic-enterotoxin by membrane vesicles, a different mechanism of delivering extra cellular virulence factors, so far not described in this bacterium.