Dissertations / Theses on the topic 'Coagulase-positive staphylococci'

Thesis (Ph. D.)--Illinois State University, 1993.
Title from title page screen, viewed March 6, 2006. Dissertation Committee: Brian J. Wilkinson (chair), Herman E. Brockman, H. Tak Cheung, Radheshyam K. Jayaswal, Robert L. Preston. Includes bibliographical references (leaves 162-176) and abstract. Also available in print.

The aim was to characterize 100 coagulase positive staphylococci isolates originating from pig carcasses from Swedish slaughterhouses by biotyping, antibiotic susceptibility testing, typing with pulsed field gel electrophoresis (PFGE) and real-time PCR-screening of the enterotoxin genes sea, sec, seg and sei in order to evaluate the impact on human health. The biotyping classified 56 as non host specific (NHS), 29 as human biotype, five as poultry, one as ovine, one as bovine biotype and eight were unclassified (UCF). Susceptibility testing to 16 antibiotics revealed that 49% of the isolates were resistant to penicillin, which the biotype human dominated among these isolates. The results from the PFGE showed correlation between the biotypes and the pulsotypes obtained with several groups with identical strains. The results from the 47 isolates tested for enterotoxins were that the combination of seg and sei was the most common but sea and sec were also detected. There were slaughterhouses that had certain biotypes and penicillin resistance linked to them.

Darbo tikslas: nustatyti koaguliazei teigiamų stafilokokų paplitimą tarp gyvūnų augintinių. Darbo uždaviniai: 1. išskirti koaguliazei teigiamus stafilokokus iš gyvūnų augintinių nosies ertmės; 2. išskirti koaguliazei teigiamus stafilokokus iš gyvūnų augintinių tiesiosios žarnos; 3. identifikuoti išskirtas stafilokokų padermes; 4. įvertinti įvairių veiksnių įtaką stafilokokų paplitimui; 5. nustatyti išskirtų stafilokokų atsparumą antimikrobinėms medžiagoms. Darbo apimtis – 50 puslapių. Šiame darbe yra 6 lentelės bei 14 paveikslų. Magistro darbą sudaro 4 dalys. Pirmojoje dalyje apžvelgiami literatūros šaltiniai susiję su analizuojama tema, išskiriant koaguliazei teigiamų stafilokokų virulentiškumo veiksnius, atsparumą antimikrobinėms medžiagoms, sukeliamas ligas ir šių ligų gydymą. Aptariamas Staphylococcus aureus bei Staphylococcus pseudintermedius paplitimas ir paplitimą įtakojantys veiksniai. Antrojoje dalyje nurodyti tyrimo metodai, kuriais remiantis gauti duomenys tyrimų analizei. Trečiojoje dalyje analizuojami gauti tyrimo rezultatai pagal iškeltus uždavinius. Rezultatai pateikiami atsižvelgiant į statistinių duomenų patikimumą. Ketvirtoji dalis skirta literatūros apžvalgos ir tyrimo rezultatų skirtumų ir panašumų palyginimui. Tyrimo metu iš gyvūnų augintinių nosies ertmės ir tiesiosios žarnos išskirti Staphylococcus aureus bei Staphylococcus pseudintermedius. Nustatyta, kad koaguliazei teigiamų stafilokokų paplitimas gyvūnų augintinių tarpe priklauso nuo gyvūnų... [toliau žr. visą tekstą]
The The goal of the study: to determine prevalence of coagulase positive staphylococci in companion animals. The aim of the study: 1. to isolate coagulase positive staphylococci in nasal cavity of companion animals; 2. to isolate coagulase positive staphylococci in rectum of companion animals; 3. to identificate the isolated strains of staphylococci; 4. to evaluate risk factors for prevalence of staphylococci; 5. to determine antibiotic resistance in isolated staphylococci. The master study consists of 50 pages. It includes 6 tables and 14 pictures. The master study consist of 4 major chapters. The first chapter is dedicated to review of literature that is related with analized topic. This part includes coagulase positive staphylococci virulence factors, antibiotic resistance, diseases caused by staphylococci and treatment use. Furthermore chapter contains review of the prevalence and risk factors influenced the prevalence of Staphylococcus aureus and Staphylococcus pseudintermedius. The second chapter introduce with materials and methods, that were used in the research at this master study. In the third chapter the results of the research are presented. The results are presented according to the statistical reliability. The fourth chapter is the resemblance and similarity comparision of the literature review and master study research. In this master study Staphylococcus aureus and Staphylococcus pseudintermedius were isolated from nasal cavity and rectum of companion... [to full text]

Dissertação de Mestrado Integrado em Medicina Veterinária
Methicillin-resistant coagulase-positive staphylococci (MRCPS) colonization in companion animals is an emerging and significant problem in public and animal health. During one year, nasal swabs were obtained from 40 cats and 146 dogs admitted to the Teaching Hospital of the Faculty of Veterinary Medicine - Technical University of Lisbon. MRCPS colonization was screened by plating enrichment cultures on a selective medium, Chrom MRSA ID. Bacterial species and mecA were confirmed by PCR. Clonality of the isolates was assessed by pulsed-field gel electrophoresis (PFGE). All isolates were subjected to spa and SCCmec typing. They were also tested by PCR for the lukF/lukS genes encoding Panton-Valentine leukocidin (PVL) in Staphylococcus aureus and Luk-I and Staphylococcus intermedius exfoliative toxin (SIET) in Staphylococcus pseudintermedius. Methicillin-resistant S. aureus (MRSA) was found in two cats (5 %) and one dog (0.6 %). Isolates were spa type t032, SCCmec IV and shared identical PFGE profiles. These were similar to the EMRSA-15 human clone. Strains were PVL-negative. Nine dogs carried methicillin-resistant S. pseudintermedius (MRSP) (6 %), whereas none of the cats was positive. The PFGE type A strain (n=1) showed identical characteristics as the American MRSP clone strains (CC68-MRSP-V), while PFGE cluster B grouped European MRSP isolates (CC71-MRSP-III) (n=8). All isolates were SIETnegative. The 8 European MRSP isolates were positive for the lukF/lukS genes and the American MRSP isolate was negative for both genes. Strains were multidrug-resistant, which represents a major challenge for veterinarians in terms of antibiotic therapy.
RESUMO - Frequência de colonização por staphylococci coagulase-positivo meticilinaresistente em cães e gatos internados no hospital escolar da Faculdade de Medicina Veterinária – Universidade Técnica de Lisboa, Portugal - A colonização por staphylococci coagulase-positivo meticilina-resistente (MRCPS) é um problema emergente e de grande importância em termos de saúde animal e pública. Durante um ano, zaragatoas nasais de 146 cães e 40 de gatos foram obtidas de animais internados no Hospital Escolar da Faculdade de Medicina Veterinária - Universidade Técnica de Lisboa. A colonização por MRCPSfoi pesquisada por inoculação de culturas de enriquecimento num meio selectivo, Chrom MRSA ID. As espécies de MRCPS e a amplificação do gene mecA por feita por PCR. A clonalidade dos isolados foi confirmada por PFGE. Todos os isolados foram sujeitos a tipagem spa e SCCmec. Os isolados de S. aureus meticilina-resistente (MRSA) e de S. pseudintermedius meticilina-resistente (MRSP) foram testados por PCR para a presença dos genes lukF/lukS que codificam, respectivamente, a leucocidina Panton-Valentine (PVL) e a leucocidina-I (Luk-I). Os isolados de MRSP foram ainda testados para a presença da toxina exfoliativa do S. intermedius (SIET). Nesta amostra, 0,6 % (n=1) dos cães testados e 5 % (n=2) dos gatos apresentaram MRSA. Os isolados de MRSA eram spa tipo t032, SCCmec IV e partilhavam padrões idênticos de PFGE. As estirpes eram idênticas ao clone humano EMRSA-15. Os 3 isolados eram PVL negativos. Nove cães apresentaram MRSP (6 %), enquanto nenhum dos gatos foi positivo. PFGE tipo A mostrou características idênticas ao clone americano de MRSP (CC68-MRSP-V) e PFGE tipo B agrupou os isolados europeus de MRSP (CC71-MRSP-III) (n=8). Os isolados de MRSP PFGE tipo B eram Luk-I positivos mas SIET negativos. Todas as estirpes de MRSP eram multirresistentes a várias classes de antibióticos, o que representa um desafio para os médicos veterinários em termos de estratégias de antibioterapia.

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RESUMO O objetivo desse estudo foi avaliar o nível de conhecimento, atitudes e práticas dos manipuladores de alimentos em segurança alimentar; investigar a presença de estafilococos coagulase positiva resistentes à meticilina em mãos e em fossas nasais dos manipuladores; e avaliar o uso de antissépticos no controle da contaminação. O estudo desenvolveu-se em duas etapas: a primeira, através de entrevistas com o uso de um questionário auto-aplicável com 237 manipuladores e a segunda, através investigação da presença de estafilococos coagulase positiva resistentes à meticilina nas mãos e nas fossas nasais de 140 manipuladores. Este estudo está estruturado em três capítulos: o primeiro apresenta uma revisão bibliográfica com os assuntos pertinentes à pesquisa, o segundo a avaliação do nível de conhecimento, atitudes e práticas dos manipuladores em segurança alimentar e o terceiro a investigação da presença de estafilococos coagulase positiva resistentes a meticilina nas mãos e fossas nasais dos manipuladores e a avaliação in vitro para verificar a susceptibilidade das cepas isoladas aos antissépticos: álcool gel 70%, iodóforo 10% e clorexidina 2%. Os resultados demonstraram baixo nível de conhecimento para a maioria dos participantes (65,8%), apesar de 92,8% afirmarem que participaram de treinamento anterior. Em relação às atitudes e as práticas, o percentual de acertos foram de 98,3% e 73,4%, respectivamente. Foram desenvolvidos três modelos com as variáveis independentes onde se observou que a escolaridade interferiu no nível de conhecimento dos manipuladores de alimentos, mas não o treinamento. Na investigação de estafilococos coagulase positiva resistentes a meticilina nas mãos e fossas nasais, embora 100% dos manipuladores afirmassem que higienizavam suas mãos nas etapas do preparo e distribuição dos alimentos, encontrou-se 50% de amostras positivas para a presença do microrganismo, e destas 28,6% foram positivas para a presença de estafilococos coagulase positiva resistentes à meticilina (MRSA). Os isolados de MRSA apresentaram maior sensibilidade a clorexidina 2% em comparação com os outros antissépticos. Assim, pode-se concluir que o nível de conhecimento dos manipuladores em segurança alimentar é deficitário, sendo necessário reavaliar as formas de treinamento dos manipuladores de alimentos. Ainda, o uso dos antissépticos na lavagem das mãos com clorexidina 2% poderá evitar a contaminação das dietas hospitalares. Entretanto, apenas o uso de bons antissépticos não resolverá o problema se essa prática for negligenciada pelos manipuladores, ou os recursos necessários para condução da mesma não forem oferecidos pelos hospitais.
ABSTRACT The aim of this study was to evaluate the knowledge level, attitudes and practices of food handlers; investigate the presence of methicillin-resistant coagulase positive staphylococci in the hands and nares of handlers and to evaluate the use of antiseptics in the control of contamination. The study was carried out in two steps: first, through interviews with the use of a self-administered questionnaire with 237 handlers, and the second step, to investigate the presence of methicillinresistant coagulase positive staphylococci in the hands and in the nares of 140 handlers. This study is structured into three chapters: The first presents a literature review with matters relevant to the research, the second, an article on assessing the level of knowledge, attitudes and practices of food handlers and the third, an article investigating the presence of methicillin-resistant coagulase positive staphylococci (MRSA) in the hands and nares of food handlers and also the in vitro tests to check the sensitivity of these strains to antiseptics, alcohol gel 70%, iodine 10% and chlorhexidine 2%, commonly used for hand hygiene. The results showed low level of knowledge for most participants (65.8%), although 92.8% said they participated in training. Regarding the attitudes and practices, the percentage of correct answers were 98.3% and 73.4%, respectively. Three models were developed with the independent variables and were observed that education level interfered with the knowledge of food handlers. In the investigating of the presence of MRSA in the hands and nares, although 100% of the foodhandlers had informed that they sanitized their hands on the preparation and distribution of foods, we found 50% of the samples with coagulase positive staphylococci and 28.6% with MRSA. The MRSA strains were more sensitive to chlorhexidine 2% in comparison with other antiseptics. Thus, it can be concluded that the level of knowledge of food handlers in food safety is low, so it is necessary to reevaluate the ways of training of food handlers. In other side, the use of antiseptics in hand washing is essential to avoid contamination of hospital preparations. However, only the use of good antiseptics will not be sufficient if this practice is neglected by the foodhandlers, or if the hospitals do not provide the resources needed to conduct the practice.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O objetivo deste trabalho foi avaliar os riscos para a saúde pública apresentado por tilápias, produzidas em cativeiro, comercializadas em supermercados na região nordeste de São Paulo. Para tal, análises microbiológicas foram realizadas, em 40 amostras de tilápias in natura e em 50 filés de tilápias. No peixe in natura foi analisado a água de enxaguadura da pele e o trato gastrintestinal. Os números mais prováveis de coliformes totais e termotolerantes foram determinados assim como a contagem Staphylococcus coagulase positivo e a presença de Salmonella spp. de acordo com a American Public Health Association (APHA). Os resultados obtidos foram analisados utilizando valores logarítmicos e as médias comparadas pelo teste de Tukey, com nível de 5% de probabilidade. A pele foi o tecido com maior nível de contaminação em relação ao trato gastrintestinal e músculo de tilápia in natura. O número de amostras com presença de coliformes totais e termotolerantes na água de enxaguadura da pele foi, respectivamente, 72,5% e 60%, no trato gastrintestinal 25% e 12,5% e no músculo 17,5% e 5%. A carga bacteriana foi significativamente maior no filé em relação ao músculo. Em duas amostras de filé foram verificados Staphylococcus coagulase positivo, uma acima dos parâmetros microbiológicos estabelecidos pela Resolução RDC nº 12/2001 da ANVISA. Salmonella spp. não foi detectada em nenhuma amostra analisada. As amostras de peixes estavam em boas condições para consumo, com exceção de duas amostras de filé. Os filés apresentaram maior contaminação que o músculo da tilápia in natura. Indicadores de poluição fecal demonstraram que a pele foi o órgão com maior contaminação
The objective of this work was to evaluate the risks to public health of consumption of tilápia fish growed in captivity and commercialised in supermarkets in the Northwest region of São Paulo state. In order to accomplish with the objective the researcher undertook analysis in 40 samples of fresh tilápias and in 50 fillets of the fish. In the fresh fish it was also analysed the washing water of the skin and of the gastrointestinal tract. It were determined, in accordance to the American Public Health Association (APHA), the number of total and thermotolerant fecal coliforms as well as the number of coagulase-positive Staphylococcus and presence of the bacteria Salmonella spp. The results were analyzed using logarithmic values and means compared by Tukey test with 5% of probability. The skin was the tissue with higher levels of contamination comparing with the gastrointestinal tract and muscle of the fresh tilapia. The percentage of samples with total and thermotolerant fecal coliforms in the skin washing water was 72.5% and 60% respectively; 25% and 12.5% in the gastrointestinal tract and 17.5% and 5% in the muscles. Bacterial presence was significantly higher in fillets comparing to muscles. Moreover, it was noticed presence of coagulase-positive Staphylococcus in two samples of fillet; one of them showed levels above of the microbiological parameters established by the RDC resolution no. 12/2001 ANVISA. Additionally, it was not identified presence of Salmonella spp in any of the samples analyzed. Generally, the studied samples were in good condition for human consumption, except for two samples of fillets. Fillets presented higher levels of contamination comparing with muscles of the fresh tilapia. Fecal pollution indicators have proved that the skin was the organ with higher levels of contamination

FundaÃÃo de Amparo à Pesquisa do Estado do CearÃ
Os moluscos bivalves sÃo alimentos de grande importÃncia nutricional sendo ususalmente consumidos nas regiÃes costeiras. Quando ingeridos in natura (crus) ou levemente cozidos, podem oferecer risco à saÃde pÃblica por serem filtradores e acumuladores de resÃduos quÃmicos e biolÃgicos, principalmente se a qualidade sanitÃria da Ãgua onde forem capturados estiverem comprometidas. A presente pesquisa tem como objetivo estudar a espÃcie Tagellus plebeius ("unha de velho") no estuÃrio do rio cearà em Fortaleza - Ce, considerando aspectos de seu ciclo reprodutivo, da sua biologia populacional e de suas condiÃÃes microbiolÃgicas. Para a anÃlise do ciclo reprodutivo os exemplares de T. plebeius foram coletados mensalmente no estuÃrio do rio cearà em Fortaleza - Ce (03, 42, 09.8 S; 35, 35, 49.0 W) no perÃodo de abril de 2006 a junho de 2007 em marÃs diurnas de sizÃgia. A cada mÃs as gÃnadas de 30 indivÃduos fram submetidas a exame histolÃgico. Para as amostragens microbiolÃgicas, as anÃlises foram feitas em dois meses da estaÃÃo seca )novembro e dezembro de 2006) e em dois meses da estaÃÃo chuvosa (marÃo e abril de 2007). Foram isoladas 22 cepas suspeitas de Salmonella spp., das quais 11 cepas foram confirmadas atravÃs do teste de sorologia com o antisoro polivalente O:H, sendo identificados 3 sorovares (S. Bredeney, S. London e S. Muenchen) e 1 (uma) cepa foi classificada atà subespÃcie: S. enterica subesp. enterica. Dentre os fatores ambientais observados neste estudo, a precipitaÃÃo pluviomÃtrica e a salinidade foram os que apresentaram as maiores amplitutes de variaÃÃo. O aumento da contaminaÃÃo por coliformes termotelorantes (45ÂC) na estaÃÃo chuvosa pode estar relacionado ao aporte de Ãgua doce no rio, como uma consequente diminuiÃÃo da salinidade associada a uma maior contaminaÃÃo bacteriologica. A correlaÃÃo significativa observada entre a salinidade e as fases "MaturaÃÃo/EliminaÃÃo Inicial" (M/E) e "Atresia Acular" (AC) mostra tambÃm a influÃncia desta variÃvel ambiental no desenvolvimento gonadal da espÃcie, prolongando ou reduzindo as fases do seu ciclo reprodutivo.
Bivalve molluscs are of great nutritional food ususalmente being consumed in the coastal regions. When ingested in nature (raw) or lightly cooked, can provide public health risk because filtrating and accumulators of chemical and biological waste, especially if the sanitary quality of water they are caught compromised. This research aims to study the species Tagellus plebeius ( "nail the old") on the estuary of the Rio Cearà in Fortaleza - Ce, considering aspects of their reproductive cycle, its population biology and their microbiological conditions. For the analysis of the reproductive cycle the specimens of T. plebeius were collected monthly in the estuary of the Rio Cearà in Fortaleza - Ce (03, 42, 09.8 S, 35, 35, 49.0 W) from April 2006 to June 2007 in the diurnal tides sizÃgia. Each month the gonads of 30 individuals subjected to histological examination FRAM. For microbiological samples, the tests were made in two months of dry season) in November and December 2006) and in two months of the rainy season (March and April 2007). We isolated 22 strains of suspected Salmonella spp., Of which 11 strains were confirmed by serology test with polyvalent antiserum O: H, and identified 3 serovars (S. Bredeney, S. London and S. Muenchen) and 1 (one ) strain was classified by subspecies: S. enterica subsp. enterica. Among the environmental factors observed in this study, rainfall and salinity were those who had the greatest amplitutes change. The increase of contamination by coliform termotelorantes (45 C) in the rainy season may be related to the intake of fresh water in the river, and a consequent decrease in salinity with increased bacterial contamination. The significant correlation observed between salinity and stages "Maturation / Initial Elimination" (M / E) and "incite atresia (CA) also shows the influence of the environmental variable in the gonadal development of the species, extending or reducing the phases of its cycle reproductive.

Dissertação de Mestrado Integrado em Medicina Veterinária
O presente trabalho foi realizado no âmbito do estágio de Mestrado Integrado em Medicina Veterinária da FMV-UTL, realizado no Posto de Inspeção Fronteiriço de Lisboa da Direção-Geral de Alimentação e Veterinária. Teve como objetivo prático verificar a ocorrência de Salmonella spp. e Listeria monocytogenes e realizar a contagem de Staphylococcus coagulase positiva e E. coli em produtos da pesca proveniente de países terceiros no período de setembro de 2010 a março de 2011. Das 30 amostras analisadas (crustáceos, moluscos bivalves e delícias do mar), todas apresentaram resultados negativos na pesquisa em 25 g, relativamente a Salmonella spp. e L. monocytogenes. No que diz respeito à contagem de Staphylococcus coagulase positivos e E. coli os resultados apresentados foram, respetivamente, inferiores a 100 ufc/g de produto e a 1 ufc/g de produto, valores considerados seguros no momento da ingestão dos alimentos, de acordo com os atuais critérios microbiológicos. Com base nos resultados obtidos conclui-se que as amostras analisadas apresentaram baixos níveis de ocorrência de Salmonella spp. e L. monocytogenes e de Staphylococcus coagulase positivos e E. coli. Ainda assim, é importante a vigilância da qualidade dos produtos da pesca, a exigência constante de implementação de boas práticas e a aplicação de sistema pró-ativos de segurança dos alimentos na cadeia dos produtos da pesca, na captura e produção em aquicultura de produtos da pesca, na comercialização, nas indústrias, bem como nos procedimentos de transporte e de receção nos postos de inspeção fronteiriços.
ABSTRACT - EVALUATION OF MICROBIOLOGIC HAZARDS IN IMPORTED FISHING PRODUCTS - BORDER INSPECTION POST OF LISBOA PORT - The present work was developed for the internship of the integrated Master’s in Veterinarian Medicine, of the FMV-UTL, and carried out at the Lisbon Border Inspection Post, of the Portuguese National Authority for Animal Health (Direção-Geral de Alimentação e Veterinária). Its objective was to verify the occurrence of Salmonella spp., Listeria monocytogenes and to perform the enumeration of coagulase positive Staphylococcus and E. coli in fish coming from third countries, from September 2010 to March 2011. All 30 analyzed samples (crustaceans, bivalve molluscs and crab sticks) presented negative results for Salmonella spp. and L. monocytogenes, in 25 g. Regarding the counting of Coagulase positive Staphylococcus and E. coli, results showed less than 100 cfu/g of product and less than 1 cfu/g of product, respectively, values which, according to the current microbiological criteria, are considered safe. On the basis of the results obtained, it is concluded that the analyzed samples presented low levels of occurrence of Salmonella spp. and L. monocytogenes, as well as Coagulase positive Staphylococcus and E. coli. Even so, it is important to monitor the quality of the fishing products, and to continue to promote the implementation of good practices, and of pro-active food safety systems in the fish chain, in the capture and production of fishing products for and in aquaculture, in the commercialization, in the industries, and also in the transportation procedures and product reception at the border inspection posts.

LIMA, Ariosvana Fernandes. Staphylocuccus coagulase positiva e enterotoxinas em queijo coalho. 2005. 86 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Pós-Graduação em Tecnologia de Alimentos, centro de Ciências Agrárias, Fortaleza-CE, 2005
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It was evaluated the incidence of Staphylococcus sp. and Staphylococcus positivecoagulase in 80 samples of industrialized and artisanal “coalho” cheese commercialized in several selling points in Fortaleza-Ceará, as well the detection of staphylococcal enterotoxins in “coalho” cheese samples and in the isolated strains of Staphylococcus positive-coagulase. From the 80 samples of “coalho” cheese analysed, 44 (55%) were artisanal produced, being 33 (41,25%) samples of Ceará and 11 (13,75%) from other states; 13 (16,25%) industrialized with State Inspection Service (SIE) register and 23 (28,75%) industrialized with Federal Inspection Service (SIF) register. The incidence of Staphylococcus sp. in the analyzed samples of artisanal “coalho” cheese from Ceará, from other states and in the samples of industrialized “coalho” cheese with SIE, was of 100%; except for one unique (4,35%) industrialized “coalho” cheese sample with SIF register, which didn’t presented count of Staphylococcus sp. The incidence of samples with Staphylococcus sp. occurred in: 33 Ceará artisanal samples, varying from 6,0x104 to 8,9x107 UFC/g; in 11 samples of artisanal cheese from other states, varying from de 2,2 x 105 a 8,9 x 107 UFC/g; in 13 samples of industrialized cheese with SIE, varying from 9,0 x 104 a 4,3 x 107 UFC/g. These results showed a significative percentage (95,65%) of Staphylococcus sp. To the industrialized cheese. However, the contamination levels of Staphylococcus positive-coagulase in the analyzed “coalho” cheese were of: 23 (53,49%) artisanal Ceará cheese samples, with counts from 1,2 x 105 to 5,9 x 107 UFC/g; 7 (16,28%) artisanal samples from other states with counts from 6,9 x 105 to 2,6 x 107 UFC/g; 9 (20,93%) samples to the industrialized SIE register cheeses, with counts from 4,4 x 104 to 1,7 x 107 UFC/g; 4 (9,30%) samples to the industrialized “coalho” with SIF with counts from 4,7 x 105 to 2,7 x 106 UFC/g. From the 43 samples of “coalho” cheese contaminated with Staphylococcus positive-coagulase, the majority (21 samples) presented counts from 1 x 106 to 1 x 107 UFC/g. The high incidence of Staphylococcus positive-coagulase puts the consumer’s health in risk. For the staphylococcal enterotoxins analysis, there were obtained 43 extracts from the “coalho” cheese contaminated samples and 12 extracts of selected strain pools of Staphylococcus positive-coagulase, to be submitted to the detection of staphylococcal enterotoxins by the ELFA method in the VIDAS® system of bioMérieux. Inspite the high counts of Staphylococcus positive-coagulase, no staphylococcal enterotoxins were detected in any of the 43 samples of industrialized and artisanal “coalho” cheese. From the 12 strains pools of Staphylococcus positivecoagulase submitted to the detection of enterotoxins, only one strain pool (8,33%) was able to produce staphylococcal enterotoxins. These results indicate the need to orient the producers concerning to the raw material’s quality, suitable hygienic conditions and manipulation in the manufacturing of artisanal cheese; and a greater attention and fiscalization by the responsable organs, in order to practice the implementation of more rigid actions in the processing of this product, including Good Manufacturing Practices in the whole production chain for the guaranty of the products’ quality offered to the consumer and for the maintenance of the public health.
Avaliou-se a incidência de Staphylococcus sp. e Staphylococcus coagulase positiva em 80 amostras de queijos de coalho artesanal e industrial, comercializados em diversos pontos de venda em Fortaleza-Ceará, bem como a detecção de enterotoxinas estafilocócicas em queijos de coalho e nas cepas isoladas de Staphylococcus coagulase positiva. Das 80 amostras de queijos de coalho analisadas, 44 (55%) foram de produção artesanal, sendo 33 (41,25%) amostras do Ceará e 11 (13,75%) de outros estados; 13 (16,25%) industrializados com registro do SIE e 23 (28,75%) industrializados com registro do SIF. A incidência de Staphylococcus sp. nas amostras analisadas de queijos de coalho artesanais do Ceará, de outros estados e nas amostras de queijos de coalho industriais com registro do SIE, foi de 100%; com exceção de apenas uma (4,35%) amostra de queijo de coalho industrializado com registro do SIF que não apresentou contagem de Staphylococcus sp. A incidência de amostras com Staphylococcus sp. Ocorreu em: 33 amostras artesanais do Ceará, variando de 6,0 x 104 UFC/g a 8,9 x 107 UFC/g; em 11 amostras de queijos artesanais de outros estados, variando de 2,2 x 105 a 8,9 x 107 UFC/g; em 13 amostras de queijos industriais com SIE, variando de 2,2 x 105 a 1,9 x 107 UFC/g; em 22 amostras de queijos industriais com SIF, variando de 9,0 x 104 a 4,3 x 107 UFC/g. Esses resultados mostram uma porcentagem significativa (95,65%) de Staphylococcus sp. para os queijos industrializados. No entanto, os níveis de contaminação para Staphylococcus coagulase positiva nos queijos de coalho analisados foram de: 23 (53,49%) amostras artesanais do Ceará, com contagens entre 1,2 x 105 a 5,9 x 107 UFC/g; 7 (16,28%) amostras artesanais de outros estados com contagens entre 6,9 x 105 a 2,6 x 107 UFC/g; 9 (20,93%) amostras para queijos industriais com SIE, com contagens entre 4,4 x 104 a 1,7 x 107 UFC/g; 4 (9,30%) amostras para queijos de coalho industriais com SIF com contagens entre 4,7 x 105 a 2,7 x 106 UFC/g. Das 43 amostras de queijos de coalho contaminados com Staphylococcus coagulase positiva, a maioria (21 amostras) apresentou contagem entre 1 x 106 a 1 x 107 UFC/g. A alta incidência de Staphylococcus coagulase positiva colocam em risco a saúde do consumidor. Para as análises de enterotoxinas estafilocócicas, foram obtidos 43 extratos das amostras de queijos de coalho contaminadas e 12 extratos de “pools” de cepas selecionadas de Staphylococcus coagulase positiva, para serem submetidas à detecção de enterotoxinas estafilocócicas pelo método ELFA no sistema VIDAS® da bioMérieux. Não foram detectadas enterotoxinas estafilocócicas em nenhuma das 43 amostras de queijos de coalho artesanal e industrial, apesar das elevadas contagens de Staphylococcus coagulase positiva. Dos 12 pools de cepas de Staphylococcus coagulase positiva submetidos à detecção de enterotoxinas, somente um pool (8,33%) de cepas foi capaz de produzir enterotoxinas estafilocócicas. Esses resultados evidenciam a necessidade de orientação aos produtores com relação à qualidade da matéria-prima, condições adequadas de higiene e manipulação na fabricação de queijo artesanal; e uma maior atenção e fiscalização por parte dos órgãos responsáveis, no sentido de implementar medidas mais rígidas de controle no processamento desse produto, incluindo Boas Práticas de Fabricação em toda a cadeia de produção para a garantia da qualidade dos produtos oferecidos ao consumidor e para manutenção da saúde pública.

Orientador: Luiz Augusto do Amaral
Banca: Naiá Carla Marchi de Rezende Lago
Banca: Oswaldo Durival Rossi Junior
Resumo: O objetivo deste trabalho foi avaliar os riscos para a saúde pública apresentado por tilápias, produzidas em cativeiro, comercializadas em supermercados na região nordeste de São Paulo. Para tal, análises microbiológicas foram realizadas, em 40 amostras de tilápias in natura e em 50 filés de tilápias. No peixe in natura foi analisado a água de enxaguadura da pele e o trato gastrintestinal. Os números mais prováveis de coliformes totais e termotolerantes foram determinados assim como a contagem Staphylococcus coagulase positivo e a presença de Salmonella spp. de acordo com a American Public Health Association (APHA). Os resultados obtidos foram analisados utilizando valores logarítmicos e as médias comparadas pelo teste de Tukey, com nível de 5% de probabilidade. A pele foi o tecido com maior nível de contaminação em relação ao trato gastrintestinal e músculo de tilápia in natura. O número de amostras com presença de coliformes totais e termotolerantes na água de enxaguadura da pele foi, respectivamente, 72,5% e 60%, no trato gastrintestinal 25% e 12,5% e no músculo 17,5% e 5%. A carga bacteriana foi significativamente maior no filé em relação ao músculo. Em duas amostras de filé foram verificados Staphylococcus coagulase positivo, uma acima dos parâmetros microbiológicos estabelecidos pela Resolução RDC nº 12/2001 da ANVISA. Salmonella spp. não foi detectada em nenhuma amostra analisada. As amostras de peixes estavam em boas condições para consumo, com exceção de duas amostras de filé. Os filés apresentaram maior contaminação que o músculo da tilápia in natura. Indicadores de poluição fecal demonstraram que a pele foi o órgão com maior contaminação
Abstract: The objective of this work was to evaluate the risks to public health of consumption of tilápia fish growed in captivity and commercialised in supermarkets in the Northwest region of São Paulo state. In order to accomplish with the objective the researcher undertook analysis in 40 samples of fresh tilápias and in 50 fillets of the fish. In the fresh fish it was also analysed the washing water of the skin and of the gastrointestinal tract. It were determined, in accordance to the American Public Health Association (APHA), the number of total and thermotolerant fecal coliforms as well as the number of coagulase-positive Staphylococcus and presence of the bacteria Salmonella spp. The results were analyzed using logarithmic values and means compared by Tukey test with 5% of probability. The skin was the tissue with higher levels of contamination comparing with the gastrointestinal tract and muscle of the fresh tilapia. The percentage of samples with total and thermotolerant fecal coliforms in the skin washing water was 72.5% and 60% respectively; 25% and 12.5% in the gastrointestinal tract and 17.5% and 5% in the muscles. Bacterial presence was significantly higher in fillets comparing to muscles. Moreover, it was noticed presence of coagulase-positive Staphylococcus in two samples of fillet; one of them showed levels above of the microbiological parameters established by the RDC resolution no. 12/2001 ANVISA. Additionally, it was not identified presence of Salmonella spp in any of the samples analyzed. Generally, the studied samples were in good condition for human consumption, except for two samples of fillets. Fillets presented higher levels of contamination comparing with muscles of the fresh tilapia. Fecal pollution indicators have proved that the skin was the organ with higher levels of contamination
Mestre

Les toxi-infections alimentaires collectives staphylococciques représentent ces dernières années en France la seconde cause de toxi-infections alimentaires bactériennes. Ces dernières sont dues à l'ingestion de toxines préformées dans les aliments : les entérotoxines. Actuellement, une vingtaine d'entérotoxines (SEA àSElV) ont été décrites dans la littérature ; parmi celles-ci, toutes possédent une activité superantigéniques mais seules quelques unes possèdent une activité émétique prouvée et présentent donc un risque sanitaire pour le consommateur. Deux problèmes majeurs se posent pour confirmer le diagnostic des épisodes toxiques à staphylocoques. D'une part, les staphylocoques sont sensibles aux traitements thermiques appliqués aux aliments alors que les entérotoxines ne le sont pas, et d'autre part les méthodes de détection des entérotoxines staphylococciques actuellement disponibles ne couvrent pas l'ensemble des entérotoxines décrites à ce jour. Ainsi, et afin de proposer une alternative innovante aux tests immuno-enzymatiques utilisés pour la recherche des entérotoxines staphylococciques dans les matrices alimentaires, nous avons mis en place une démarche intégrée « du gène à la protéine » utilisant des outils de biologie moléculaire, d'immunochimie et de physisco-chimie des protéines pour caractériser, comprendre et élucider les toxi-infections alimentaires collectives à staphylocoques.

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O setor hoteleiro vem se expandindo mundialmente a cada ano e dentre os serviços oferecidos, a gastronomia é um dos mais requisitados, através da qual busca-se a satisfação do cliente, oferecendo produtos seguros e de qualidade. Paralelamente ao incremento do turismo, a incidência de doenças de origem alimentar está crescendo em todo mundo. Acredita-se que o consumo de alimentos contaminados seja a principal causa de doenças em turistas. Neste contexto o objetivo deste estudo foi avaliar a aplicação das boas práticas em restaurantes hoteleiros da cidade de Pelotas/RS e Rio Grande/RS e relacioná-las com a qualidade microbiológica dos alimentos oferecidos, além de identificar fontes de contaminação. O setor de alimentos e bebidas (A&B) de quatro hotéis foi avaliado quanto às condições higiênicossanitárias em duas visitas, com intervalo mínimo de 30 dias, através de aplicação de check list fundamentado na norma da RDC 216/2004, e de análises microbiológicas de amostras coletadas no local. Foram realizadas coletas de amostras de superfícies de utensílios, equipamentos e mãos de manipuladores para contagem de Staphylococcus coagulase positiva e de coliformes termotolerantes. Isolados obtidos das culturas dessas contagens foram comparados através de perfis de genéticos obtidos por rep-PCR. Considerando as não conformidades observadas no check list, na primeira visita apenas um hotel foi classificado como ótimo, dois foram classificados como bons e um recebeu a classificação ruim. Entretanto, na segunda visita o hotel classificado como ruim passou para bom, devido à realização de melhorias no restaurante. Alguns resultados das análises microbiológicas apresentaram limite acima do permitido para a contagem dos micro-organismos analisados, identificando falha na aplicação das boas práticas. Através da técnica de rep-PCR, foi identificada uma mesma cepa em amostras de queijo e mãos, manteiga e tábua de corte, queijo e tábua de corte e salada de frutas e mãos, evidenciando contaminação cruzada devido falhas no processo de higiene e manipulação dos alimentos.
The hotel industry has been expanding every year and among the services offered, the cuisine is one of the most requested, which seeks customer satisfaction by offering safe and quality products. Parallel to the tourism development, the incidence of foodborne diseases is growing worldwide. It is believed that the consumption of contaminated food is the major cause of disease in tourists. This study goal was to evaluate the application of best practices in hotel restaurants in the cities of Pelotas/RS and Rio Grande/RS and relate it to the microbiological quality of the food offered, besides to identify sources of contamination. The sector of food and beverages (F&B) of four hotels was evaluated in two visits with an interval of 30 days regarding hygiene-sanitary conditions, by application of check list and microbiological analyzes of samples collected on site. Surface, utensils, equipment and hands handlers samples have been collected for counting coagulase positive Staphylococcus and thermotolerant coliform. Isolates obtained from these counting cultures were compared by bands profiles obtained by rep-PCR. According to the non-conformity observed in the check list on the first visit, only one hotel was rated as great, two were classified as good and one received a bad rating. However, on the second visit, the one considered bad got a good rate due to improvements in its unit. Some microbiological analysis showed a limit above the allowed for the count of analyzed microorganisms, identifying failure in the hygiene-sanitary process. By rep-PCR, was identified the same strain in samples of cheese and hands, butter and cutting board, cutting board and cheese and fruit salad and hands, showing cross-contamination due to flaws in the process of care and handling of food.

Orientador: Luiz Augusto do Amaral
Banca: Laudicéia Giacometti Lopes
Banca: Maria da Glória Buzinaro
Resumo: O objetivo deste trabalho foi determinar o número de coliformes totais, termotolerantes, Staphylococcus coagulase positivo e a presença de bactéria do gênero Salmonella no músculo, no tecido superficial, no trato gastrintestinal de peixes e na água de cultivo de pesque-pagues situados na microbacia do Córrego Rico, região nordeste do estado de São Paulo. Não foi detectado Staphylococcus coagulase positivo em nenhuma amostra de água e peixe. Na contagem de Staphylococcus sp. a água de enxaguadura da pele apresentou números que variam de 1,0 x 102 a 3,3 x 105 UFC.mL-1; no músculo de 1,0 X 102 a 6,7 x 103 UFC.g-1; no trato gastrintestinal de 1,0 x 103 a 2,9 x 105 UFC.g-\ e na água de cultivo de < 20 a 3,3 x 102 UFC.mL-1. O número mais provável (NMP) de coliformes totais na pele variou de 1,5 x 103 a > 1,1 x 104 NMP.100mL-1; no músculo variou de 2,0 x 10 a> 1,1 x 103 NMP.g-1; no trato gastrintestinal variou de 2,6 x 103 a> 1,1 x 104 NMP.g-1; e na água de cultivo variou de 4,2 x 104 a> 2,4 x 105 NMP.100mL-1. Para coliformes termotolerantes a água de enxaguadura da pele variou de < 3,0 x 10 a 1,4 x 103 NMP.IOOmL-1; no músculo variou de < 3 a 6 NMP.g-1; no trato gastrintestinal variou de 1,2 X 103 a 5,1 x 103 NMP.g-1; e na água de cultivo variou de 3,8 x 102 a 2,0 x 104 NMP.100mL-1. Não houve diferença estatística (P>0,05) entre as populações de microrganismos pesquisados na água, pele e trato gastrintestinal o que reflete a relação direta entre a presença dos microrganismos na água e nesses dois locais analisados. No que se refere à musculatura verifica-se que existe diferença (P<0,05) entre a população de microrganismos na água e na musculatura, sendo na musculatura sempre menor. Foi isolada Salmonella sp. em uma amostra de músculo e em duas amostras de trato gastrintestinal. O pescado pode ser veículo de contaminação cruzada, tendo como... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The aim of this work was to determine the number of total coliforms, thermotolerant, Staphylococcus coagulase positive and the presence of bacteria of the genus Salmonella in the muscle, in the surface tissue, in the gastrointestinal tract of fish and pond water of fee- fishing located in Cón-ego Rico microwatershed, northeast region of São Paulo state. Staphylococcus coagulase positive was not detected in any sample of pond water and fish. In the Staphylococcus sp. counts the surface tis sue ranged from 1,0 x 102 to 3,3 X 105 UFC.mL-1; in the musc1e from 1,0 x 102 to 6,7 x 103 UFC.g-1; in the gastrointestinal tract of fish from 1,0 x 103 to 2,9 X 105 UFC.g-1; and in pond water from < 20 to 3,3 X 102 UFC.mL¬1. The most probable number (NMP) of total coliforms in the surface tissue ranged from 1,5 x 103 to > 1,1 X 104 NMP.100mL-1; in the muscle from 2,0 x 10 to > 1,1 X 103 NMP.g-1; in the gastrointestinal tract offish from de 2,6 x 103 to> 1,1 X 104 NMP.g-1; and in pond water from 4,2 x 104 to > 2,4 X 105 NMP.1 00mL-1 . In thermotolerant coliforms the surface tissue ranged from < 3,0 x 10 to 1,4 X 103 NMP.100mL-1; in the musc1e from < 3 to 6 NMP.g-1; in the gastrointestinal tract of fish from de 1,2 x 103 to 5,1 X 103 NMP.g-1; and in pond water from 3,8 x 102 to 2,0 X 104 NMP.100mL-1. There was no statistical difference (P>0,05) among the studied populations of microorganisms in water, skin and gastrointestinal tract that reflects the relationship between the presence of microorganisms in water and in these two tissues analyzed. Conceming the musc1e there is difference (P<0,05) among the population of microorganisms in water and muscle, in which muscles were always smaller. Salmonella sp. was isolated in a sample of muscle and in two samples of gastrointestinal tract. The fish ean be a vehic1e of cross contamination, and the gastrointestinal tract and skin as a source of microorganisms... (Complete abstract click electronic access below)
Mestre

The mozzarella cheese is a product of high consumption, but very susceptible to microbiological contamination. Therefore, this study aimed to evaluate the microbiological and physical-chemistry of sliced mozzarella cheese in bulk and vacuum-packed, sold in supermarkets in the Northwest region of Rio Grande do Sul State. We evaluated five marks (50 samples) of vacuum-packed mozzarella cheese (A, B, C, D and E) and a mark (25 samples) sold in bulk (F). Microbiological tests were performed according to Normative Instruction 62, August 26th, 2003: fecal coliform counts (45ºC), Salmonella sp. and coagulase positive Staphylococcus in two different periods during the validity period (period I) and after the expiration of the samples (period II). Were also conducted analysis of pH, fat and moisture, according to the Normative Instruction 68, December 12th, 2006. The physicochemical parameters classified the samples as fat cheese and medium humidity and all attended the legal parameters established by the Brazilian legislation. The microbiological results showed that all samples were within the standards for fecal coliform and coagulase positive Staphylococcus in both periods of analysis. However, in period I detected the presence of Salmonella sp. in one of the marks (16.67 %), being improper for human consumption.
O queijo Mussarela é um produto de elevado consumo, porém muito susceptível à contaminação microbiológica. Portanto, este trabalho teve como objetivo avaliar a qualidade microbiológica e físico-química do queijo Mussarela fatiado à granel e embalado à vácuo, comercializado em supermercados da região Noroeste do estado do Rio Grande do Sul. Foram avaliadas 5 marcas de queijo Mussarela embalado à vácuo (A, B, C, D e E), totalizando 50 amostras, e uma marca comercializada à granel (F), totalizando 25 amostras. As análises microbiológicas realizadas foram: contagem de coliformes termotolerantes (45°C), Salmonella sp. e Staphylococcus coagulase positiva, em dois diferentes períodos, durante o prazo de validade (período I) e após o vencimento das amostras (período II). Também foram realizadas as análises de pH, teor de gordura e umidade. Os parâmetros físico-químicos classificaram as amostras como sendo queijos gordos e de média umidade e atenderam os padrões exigidos pela legislação brasileira. Os resultados microbiológicos mostraram que todas as amostras apresentaram-se dentro dos padrões para coliformes termotolerantes e Staphylococcus coagulase positiva, nos dois períodos de análise, porém, no período I, foi detectada a presença de Salmonella sp. em uma das marcas (16,67 %), encontrando-se imprópria para o consumo humano.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Universidade Estadual Paulista (UNESP)
O objetivo deste trabalho foi determinar o número de coliformes totais, termotolerantes, Staphylococcus coagulase positivo e a presença de bactéria do gênero Salmonella no músculo, no tecido superficial, no trato gastrintestinal de peixes e na água de cultivo de pesque-pagues situados na microbacia do Córrego Rico, região nordeste do estado de São Paulo. Não foi detectado Staphylococcus coagulase positivo em nenhuma amostra de água e peixe. Na contagem de Staphylococcus sp. a água de enxaguadura da pele apresentou números que variam de 1,0 x 102 a 3,3 x 105 UFC.mL-1; no músculo de 1,0 X 102 a 6,7 x 103 UFC.g-1; no trato gastrintestinal de 1,0 x 103 a 2,9 x 105 UFC.g-\ e na água de cultivo de < 20 a 3,3 x 102 UFC.mL-1. O número mais provável (NMP) de coliformes totais na pele variou de 1,5 x 103 a > 1,1 x 104 NMP.100mL-1; no músculo variou de 2,0 x 10 a> 1,1 x 103 NMP.g-1; no trato gastrintestinal variou de 2,6 x 103 a> 1,1 x 104 NMP.g-1; e na água de cultivo variou de 4,2 x 104 a> 2,4 x 105 NMP.100mL-1. Para coliformes termotolerantes a água de enxaguadura da pele variou de < 3,0 x 10 a 1,4 x 103 NMP.IOOmL-1; no músculo variou de < 3 a 6 NMP.g-1; no trato gastrintestinal variou de 1,2 X 103 a 5,1 x 103 NMP.g-1; e na água de cultivo variou de 3,8 x 102 a 2,0 x 104 NMP.100mL-1. Não houve diferença estatística (P>0,05) entre as populações de microrganismos pesquisados na água, pele e trato gastrintestinal o que reflete a relação direta entre a presença dos microrganismos na água e nesses dois locais analisados. No que se refere à musculatura verifica-se que existe diferença (P<0,05) entre a população de microrganismos na água e na musculatura, sendo na musculatura sempre menor. Foi isolada Salmonella sp. em uma amostra de músculo e em duas amostras de trato gastrintestinal. O pescado pode ser veículo de contaminação cruzada, tendo como...
The aim of this work was to determine the number of total coliforms, thermotolerant, Staphylococcus coagulase positive and the presence of bacteria of the genus Salmonella in the muscle, in the surface tissue, in the gastrointestinal tract of fish and pond water of fee- fishing located in Cón-ego Rico microwatershed, northeast region of São Paulo state. Staphylococcus coagulase positive was not detected in any sample of pond water and fish. In the Staphylococcus sp. counts the surface tis sue ranged from 1,0 x 102 to 3,3 X 105 UFC.mL-1; in the musc1e from 1,0 x 102 to 6,7 x 103 UFC.g-1; in the gastrointestinal tract of fish from 1,0 x 103 to 2,9 X 105 UFC.g-1; and in pond water from < 20 to 3,3 X 102 UFC.mL¬1. The most probable number (NMP) of total coliforms in the surface tissue ranged from 1,5 x 103 to > 1,1 X 104 NMP.100mL-1; in the muscle from 2,0 x 10 to > 1,1 X 103 NMP.g-1; in the gastrointestinal tract offish from de 2,6 x 103 to> 1,1 X 104 NMP.g-1; and in pond water from 4,2 x 104 to > 2,4 X 105 NMP.1 00mL-1 . In thermotolerant coliforms the surface tissue ranged from < 3,0 x 10 to 1,4 X 103 NMP.100mL-1; in the musc1e from < 3 to 6 NMP.g-1; in the gastrointestinal tract of fish from de 1,2 x 103 to 5,1 X 103 NMP.g-1; and in pond water from 3,8 x 102 to 2,0 X 104 NMP.100mL-1. There was no statistical difference (P>0,05) among the studied populations of microorganisms in water, skin and gastrointestinal tract that reflects the relationship between the presence of microorganisms in water and in these two tissues analyzed. Conceming the musc1e there is difference (P<0,05) among the population of microorganisms in water and muscle, in which muscles were always smaller. Salmonella sp. was isolated in a sample of muscle and in two samples of gastrointestinal tract. The fish ean be a vehic1e of cross contamination, and the gastrointestinal tract and skin as a source of microorganisms... (Complete abstract click electronic access below)

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Staphylococcus aureus, Staphylococcus hyicus e Staphylococcus intermedius are species of genus Staphylococcus capable of producing enterotoxins, as well as the enzymes termonuclease and coagulase, and are called Staphylococcus Coagulase Positive (SCP). They show very similar biochemical and morphological characteristics, this fact, makes their differentiation and they identification in food by phenotypic techniques very difficult. In this way, the aim of this work was the development of a Multiplex PCR (mPCR), targetting the nuc gene (encoding for thermonuclease) as target, for the identification of S. aureus, S. intermedius, and S. hyicus. Initially, the mPCR was standartizeted using reference strains of S. aureus, S. warneri, S. lugdunensis and Listeria monocytogenes and isolates of S. hyicus and S. intermedius, affer words it was tested with 16 isolates of the three species of SCP. The primers annealing to each of the three species, the conditions for mPCR, the suitability of the Internal Amplification Control (IAC) for the genus Staphylococcus, and the sequences obtained through the amplification by PCR were evaluated. After this, the results were compared with those achieved by biochemical tests for differentiation of SCP. Primers NUC1-NUC2 (for sequences of the S. aureus nuc gene), NUC5-NUC6 (for sequences of the S. intermedius nuc gene), NUC7-NUC8 (for sequences of the S. hyicus nuc gene) and, as IAC, the primers 16S1 16S2 (for sequences of the 16S of rRNA gene) were used. The mPCR proposed enable the identification of S. aureus, S. hyicus e S. intermedius, and the IAC was suitable for the genus. The sequences obtained in PCR showed a high degree of similarity with the sequences of the thermonuclease gene deposited in GenBank. The mPCR showed greater discriminatory power that the biochemical tests for identification the vii S. aureus, S. hyicus e S. intermedius, as well as reduced the time required for the identification of ECP (reduction of 24h) or identification of specie (reduction of 72h).
Staphylococcus aureus, Staphylococcus hyicus e Staphylococcus intermedius são espécies do gênero Staphylococcus com capacidade de produzir enterotoxinas, bem como as enzimas termonuclease e coagulase, sendo denominadas de Estafilococos Coagulase Positiva (ECP). Apresentam características morfológicas e bioquímicas extremamente semelhantes, o que torna difícil sua diferenciação assim como sua identificação em alimentos através de técnicas fenotípicas de análise microbiológica. Dessa forma, este trabalho teve como objetivo o desenvolvimento de um Multiplex PCR (mPCR), tendo-se como alvo o gene nuc (codifica para a termonuclease), para a identificação de S. aureus, S. intermedius e S. hyicus. Inicialmente, o mPCR foi padronizado utilizando-se cepas padrão de S. aureus, S. warneri, S. lugdunensis e Listeria monocytogenes e isolados de S. hyicus e S. intermedius, e, posteriormente, foi testado em 16 isolados das três espécies de ECP. Foram avaliadas a sensibilidade dos primers para cada uma das três espécies, as condições da mPCR, a sensibilidade do Controle Interno de Amplificação (IAC) para o gênero Estafilococos, e as seqüências obtidas através da amplificação por PCR. Posteriormente, os resultados foram comparados aos obtidos com testes bioquímicos de diferenciação de ECP. Foram utilizados os primers NUC1-NUC2 (para seqüências do gene nuc de S. aureus), NUC5-NUC6 (para seqüências do gene nuc de S. intermedius), NUC7-NUC8 (para seqüências do gene nuc de S. hyicus) e, para amplificação do IAC, os primers 16S1 16S2 (para seqüências do gene 16S do rRNA). O mPCR proposto possibilitou a identificação das espécies S. aureus, S. hyicus e S. intermedius, bem como o IAC utilizado apresentou sensibilidade para o gênero. As seqüências obtidas na PCR apresentaram elevado v grau de similaridade com as seqüências do gene da termonuclease depositadas no GenBank. A mPCR apresentou maior poder discriminatório que os testes bioquímicos para identificação de S. aureus, S. hyicus e S. intermedius, bem como reduziu significativamente o tempo necessário para a identificação de ECP (redução de 24h) ou de identificação em nível de espécie (redução de 72h).

Antimicrobials are not only considered as beneficial agents to human and animal health, but are also potentially harmful, both in terms of promoting antimicrobial resistance (AMR), and the potentially negative effects they may have on the gut microbiota. Focusing on these two major detrimental effects, this thesis aims to determine the frequency of AMR in companion animal pathogens isolated from Australia, as well as identify risk factors associated with infection my multidrug-resistant strains; and to describe the gut microbial community changes and other adverse effects in rabbits in response to administration of pradofloxacin, a new veterinary antimicrobial belonging to the critically important fluoroquinolone class. In the first nation-wide survey of AMR in companion animal pathogens, resistance to antimicrobials from more than ten different classes was described for a total 883 clinical Escherichia coli isolated from dogs (n=514), cats (n=341) and horses (n=28). Resistance to critically important antimicrobials not registered for use in animals (imipenem) was not detected in all clinical E. coli isolates. Resistance to amikacin, another critically important antimicrobial not registered for use in Australian animals, was low in dog and cat isolates (<2%) but moderately high in horse isolates (10.7%), reflecting its increased use in this species. Resistance to other third line antimicrobials such as third generation cephalosporins (3GC) and fluoroquinolones (FQN), was generally lower in cat isolates (3.8%-5%) compared with dog isolates (9.1%-10.9%) and horse isolates (25%-35.7%). A higher proportion of resistance was observed among the first and second line antimicrobials, ranging from 25%-100% for ampicillin and amoxicillin-clavulanic acid, mainly due to the new interpretation by veterinary-specific clinical breakpoints for dogs and cats which are much lower than human clinical breakpoints. The frequency of multidrug-resistant (MDR) E. coli was 18.1%, 11.7% and 42.9% in dog, cat and horse isolates respectively, which was positively associated with the chronicity of infection and prior antimicrobial treatment, in particular for urinary tract infections from dogs. Furthermore, among coagulase-positive staphylococci (CoPS) isolates from companion animals the frequency of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and methicillin-resistant Staphylococcus aureus (MRSA) in the isolate collections was 11.8% (74/629) and 12.8% (15/117), respectively. Resistance to FQN in S. pseudintermedius ranged from 8.1%-8.8% and was highly associated with MRSP isolates from dogs (OR 287; 95%CI 91.2–1144.8). MRSA isolates were also co-resistant to FQN (OR 5.4, 95%CI 0.6–252.1), with a frequency ranging from 8.5%-11.8% of total S. aureus from dogs (n=47) and cats (n=14). By contrast MRSA isolates from horses, were most likely to be co- resistant to amikacin and rifampicin (OR 6.5, 95%CI 0.7–315.2) with a frequency of 9.4% of total S. aureus from horses (n=53). A risk factor analysis showed that MRSP isolates from dogs were significantly more likely in surgical site infections (SSI) and skin and soft tissue infections (SSTI), particularly if the animals had received prior antimicrobial treatment. Compared with other countries where similar studies have been undertaken, rates of AMR in E. coli, were generally lower. However, due to the abrupt emergence and spread of MRSP infections in Australia, rates of AMR in S. aureus and S. pseudintermedius were moderately higher. Faecal and gut microbiota profiling using V3-V4 16S rRNA gene Illumina MiSeq DNA sequence analysis confirmed that the veterinary fluoroquinolone pradofloxacin (which has a much broader spectrum of activity compared to earlier generations) had quite different effects on the microbial community in the rabbit stomach and caecum; compared to hard faeces, and soft faeces. Microbial richness and diversity decreased significantly in hard faeces at the end of the 3- day treatment but not in the lumen of the stomach and caecum. Pseudomonas spp. was depleted significantly in hard faeces while Anaeroplasma significantly diminished in the stomach and caecum. The abundance of several bacterial taxa from the Ruminococcaceae and Lachnospiraceae was significantly overrepresented in all samples. However, overproliferation of bacteria causing enteritis such as Clostridium spp. and coliforms was not detected in the two gut compartments nor hard and soft faeces. Further, we hypothesized that stomach microbiota would be less diverse compared with caecum microbiota but our data show that the stomach harboured higher levels of microbial diversity compared with the caecum, regardless of pradofloxacin treatment, most likely due to coprophagy. Although the short-term use of pradofloxacin appears safe in rabbits as we observed no clinically adverse effects, these results confirm that pradofloxacin did result in significant disturbance of the faecal microbial community and changes of metabolic functional diversity. In conclusion, the first nation-wide AMR survey provides an important snapshot of the current situation of AMR in companion animal pathogens in Australia. Further, this thesis underlined the public health (AMR) and individual health-related (dysbiosis) consequences which should increase the awareness of prudent use of antibiotics especially in animals and broadly in human medicine.
Thesis (Ph.D.) -- University of Adelaide, School of Animal & Veterinary Sciences, 2018