Campylobacter jejuni é a espécie bacteriana mais comumente relacionada como causa de gastroenterite em humanos em vários países. Porém, o isolamento e o estudo de C. jejuni não são muito frequentes no Brasil, o que dificulta avaliar a dimensão dessa bactéria como causadora de doença em humanos e animais, bem como, determinar o impacto de sua presença em alimentos e no meio-ambiente. O objetivo desse trabalho foi avaliar a diversidade genética por cinco diferentes técnicas de tipagem molecular, o potencial patogênico pela pesquisa de 16 genes de virulência por PCR e o perfil de resistência pela concentração inibitória mínima por Etest® frente a quatro antimicrobianos e pela análise in silico de genes de resistência e pontos de mutação de linhagens de C. jejuni isoladas no Brasil. Foram estudadas 121 linhagens de C. jejuni isoladas de humanos (51), animais (35), alimentos (33) e ambiente (02) nos estados de Minas Gerais, São Paulo, Rio de Janeiro e Rio Grande do Sul, no período de 1996 a 2016. Todas as linhagens apresentaram os genes flaA, flhA, iamA, docA, ciaB, cdtA, cdtB, cdtC, racR, dnaJ, pldA, cadF, sodB e csrA. O gene wlaN foi detectado em 15 linhagens, e uma linhagem apresentou o gene virB11. Dentre as 121 linhagens estudadas, 68 linhagens foram resistentes a pelo menos um dos antimicrobianos testados. A resistência à ciprofloxacina, doxiciclina, tetraciclina e eritromicina foi observada em 43,8%, 34,7%, 34,7% e 4,9% das linhagens, respectivamente. O dendrograma de similaridade genética de Pulsed field gel electrophoresis (PFGE) agrupou as 121 linhagens estudadas em três grupos com similaridade genômica de 46,9% entre eles. Apesar da alta diversidade genômica entre as linhagens estudadas, algumas linhagens isoladas de diferentes fontes, locais e anos, apresentaram uma similaridade genotípica acima de 80% entre elas e, foram agrupadas em 21 subgrupos. Pelas sequências da SVR do gene flaA as linhagens estudadas foram agrupadas em dois grupos com linhagens isoladas de fontes clínicas e não clínicas e de humanos e animais com similaridade acima de 80,9 % entre elas e tipadas em 40 SVR-flaA alelos, sendo os alelos 57, 49 e 45 os mais frequentemente detectados. A análise do locus CRISPR por HRMA tipou as linhagens de C. jejuni em 23 diferentes variantes sendo que algumas variantes continham linhagens de origem clínica e não clínica e de humanos e animais. A árvore de SNPs gerada a partir dos dados do sequenciamento do genoma completo alocou as 116 linhagens sequenciadas em dois principais grupos. O grupo SNP-A agrupou 97 linhagens e o grupo SNP-B agrupou 19 linhagens, com linhagens de fontes clínicas e não clínicas e de humanos e animais, respectivamente. A técnica de Multilocus sequence typing (MLST) tipou as 116 linhagens de C. jejuni em 46 STs, e não foi observada a predominância de um ST. O índice de discriminação das metodologias de análise de SNPs no genoma completo, PFGE, MLST, sequenciamento das SVR do gene flaA e análise do locus CRISPR por HRMA foi 1,0, 0,982, 0,941, 0,939 e 0,874, respectivamente. Na análise in silico de genes de resistência e pontos de mutação, 95 linhagens apresentaram ao menos um gene de resistência ou ponto de mutação conhecido, sendo que a porcentagem de correlação entre os resultados de resistência fenotípicos e genotípicos foi maior que 66,7%; 94,6% e 96,8% para eritromicina, tetraciclina e ciprofloxacina, respectivamente. Conclui-se que a alta frequência da maioria dos genes de virulência pesquisados evidenciou o potencial patogênico das linhagens de C. jejuni estudadas. A resistência a antimicrobianos de primeira escolha utilizados para o tratamento da campylobacteriose encontrada nas linhagens estudadas é preocupante, podendo levar à falha terapêutica quando o tratamento é necessário. Os resultados obtidos pelas metodologias de tipagem molecular realizadas sugerem que uma possível contaminação possa ter ocorrido entre fontes clínicas e não clínicas e entre humanos e animais, ao longo de 20 anos no Brasil. Pelo índice de discriminação, foi observado que as metodologias de análise de SNPs no genoma completo e PFGE, em comparação com as outras técnicas de tipagem, foram as mais eficientes em discriminar as linhagens de C. jejuni do presente estudo. / Campylobacter jejuni is the most commonly bacterial species related as a cause of gastroenteritis in humans in several countries. However, the isolation and the study of C. jejuni have not been very frequently in Brazil, which makes it difficult to evaluate the involvement of this bacterium as a cause of diseases in humans and animals, as well as to determine the impact of its presence in food and the environment. The aim of this study was to evaluate the genetic diversity by five different molecular typing techniques, the pathogenic potential by searching for the presence of 16 virulence genes by PCR and the resistance profile by the minimum inhibitory concentration by Etest® against four antibiotics and by the in silico analyses of resistance genes and mutation points of C. jejuni strains isolated in Brazil. A total of 121 C. jejuni strains isolated from humans (51), animals (35), food (33) and the environment (02) in the States of Minas Gerais, Sao Paulo, Rio de Janeiro and Rio Grande do Sul, between 1996 to 2016 were studied. All strains presented the genes flaA, flhA, iamA, docA, ciaB, cdtA, cdtB, cdtC, racR, dnaJ, pldA, cadF, sodB and csrA. The wlaN gene was detected in 15 strains, and one strain presented the virB11 gene. Among the 121 strains studied, 68 strains were resistant to at least one of the antibiotics tested. Resistance to ciprofloxacin, doxycycline, tetracycline and erythromycin was observed in 43.8%, 34.7%, 34.7% and 4.9% of the strains, respectively. The Pulsed field gel electrophoresis (PFGE) dendrogram of genetic similarity clustered the 121 strains studied in three groups with a genomic similarity of 46.9% among them. Despite the high genomic diversity among the strains studied, some strains isolated from different sources, places and years, presented a genotypic similarity above 80% among them and were grouped into 21 subgroups. By flaA-SVR sequencing the strains studied were clustered into two groups with strains isolated from clinical and non-clinical sources and from humans and animals with a similarity above 80.9% among them and typed in 40 flaA-SVR alleles, being the alleles 57, 49 and 45 the most frequently detected. The analysis of the CRISPR locus by HRMA typed the C. jejuni strains in 23 different variants, with some variants containing strains from clinical and non-clinical origin and from humans and animals. The SNP tree generated from the whole genome sequencing data grouped the 116 strains sequenced into two major groups. SNP-A grouped 97 strains and SNP-B grouped 19 strains, with strains from clinical and non-clinical sources and from humans and animals, respectively. Multilocus sequence typing (MLST) technique typed the 116 C. jejuni strains in 46 STs, and it was not observed a predominant ST. The discrimination index of the analysis of SNPs in the whole genome, PFGE, MLST, flaA-SVR sequencing and analysis of the CRISPR locus by HRMA was 1.0, 0.982, 0.941, 0.939 and 0.874, respectively. In the in silico analyses of resistance genes and mutation points, 95 strains showed at least one resistance gene or known mutation point, and the percentage of correlation between phenotypic and genotypic resistance results was greater than 66.7%; 94.6% and 96.8% for erythromycin, tetracycline and ciprofloxacin, respectively. In conclusion, the high frequency of the majority of the virulence genes studied highlighted the pathogenic potential of the C. jejuni strains studied. Resistance to antimicrobials of first choice used for the treatment of campylobacteriosis found in the strains studied is worrying and may lead to therapeutic failure when treatment is required. The results obtained by the molecular typing methodologies performed suggest that a possible contamination may have occurred between clinical and non-clinical sources and between humans and animals over 20 years in Brazil. By the discrimination index, it was observed that the methodologies of analysis of SNPs in the whole genome and PFGE, in comparison to the other typing techniques, were the most efficients in discriminating the C. jejuni strains of the present study.

O Brasil consolidou-se como o principal produtor e exportador mundial de carne bovina. Estudos microbiológicos, geralmente realizados com amostras de carne coletadas no comércio e não na cadeia produtiva de carne, resultam numa insuficiência de dados a respeito das características fenotípicas e genotípicas das bactérias patogênicas de relevância nos produtos destinados à exportação. Objetivando determinar a prevalência e características de Salmonella spp em carne bovina para exportação, realizou-se a coleta de amostras de superfícies de 200 bovinos adultos, provenientes de 12 fazendas, abatidos em Frigorífico Exportador em São Paulo, Brasil, no ano de 2008. Foram coletadas amostras do couro do animal, logo após a realização da sangria (Co), da carcaça do mesmo animal, após a esfola (Ca I) e da carcaça do mesmo animal, após o toalete e antes da refrigeração (Ca II). O isolamento e a identificação de Salmonella spp foram realizados de acordo com o método - ISO 6579:2002, com algumas modificações. O patógeno foi detectado em 14 amostras de couro (7,0%), 5 de carcaça I (2,5%) e 4 de carcaça II (2,0%). Verificou-se a prevalência do sorovar S. Give (52,0%), seguido de S. Abaetetuba (16,0%), S. Typhimurium (8,0%), S. Agona (4,0%) e S. Dublin (4,0%), e quatro cepas (16,0%) não tipáveis. A tipagem molecular, feita por PFGE, mostrou que as salmonelas expressaram 12 perfis genéticos distintos, sendo 10 formados por apenas uma cepa, cada. As demais cepas (15) pertenceram a dois perfis genéticos apenas, que apresentaram 91,7% de similaridade. De acordo com o teste de infecção de células Caco-2, a maioria das cepas (92,0%) apresentou Eficiência de Invasão inferior a 1,0%, indicando baixo potencial de virulência. Quanto ao perfil de resistência a antibióticos, 68,0% das cepas analisadas foram multiresistentes, apresentando 12 perfis diferentes. Animais diferentes, provenientes de uma mesma fazenda, apresentaram salmonelas de um mesmo sorovar e com o mesmo perfil genético e de resistência a drogas, comprovando a ocorrência de contaminação cruzada durante o processamento da carne bovina. A multiresistência das salmonelas isoladas e a possibilidade de disseminação desses patógenos denotam a necessidade de se adotar medidas de higiene adequadas e maior prudência no emprego de antimicrobianos, na dieta alimentar e na terapêutica veterinária. / Brazil is an important bovine meat producer and exporter. Microbiological surveys are generally run with meat samples collected at retail level and not with meat for export, explaining the lack of data on the phenotypic and genotypic characteristics of pathogens of relevance in exported food products. This study aimed to evaluate the prevalence and characteristics of Salmonella spp in bovine meat destined for export, through surface sampling of hides and carcasses of 200 animals, from 12 farms, slaughtered in 2008 in an export slaughterhouse located in São Paulo, Brazil. Sampling was done from the hides right after bleeding (Co) and from carcasses of the same animal after removal of the hide (Ca I) and after cleaning but before chilling (Ca II). Isolation and identification of Salmonella spp were done according to ISO 6579:2002, with some modifications. The pathogen was detected in 14 samples of hides (7,0%), 5 of carcasses Ca I (2,5%) and 4 of carcasses Ca II (2,0%). The most prevalent serovars were S. Give (52,0%), followed by S. Abaetetuba (16,0%), S. Typhimurium (8,0%), S. Agona (4,0%) and S. Dublin (4,0%). Four isolates (16,0%) were not typable. Molecular typing using PFGE indicated that the isolates presented 12 molecular profiles, ten of them containing one single isolate. Fifteen isolates belonged to only two distinct profiles, with 91.7% similarity. Invasion Efficiency tests, run with Caco-2 cells, indicated that most isolates (92,0%) presented low virulence potential. 68,0% of the isolates were multiresistant to antimicrobial drugs, presenting 12 different resistance profiles. Different animals, coming from the same rearing farm, harbored salmonellae belonging to same serovar and presenting the same genetic and antimicrobial resistance profiles, indicating cross contamination in the slaughterhouse during production of meat. The occurrence of salmonellae that can disseminate in the slaughterhouse and the multiresistance presented by the strains strengthen the need for adoption of proper hygiene control measures and care in the use of antibiotics in human and veterinary therapeutics.

Staphylococcus aureus is a major opportunistic pathogen causing a wide range of infections in humans and animals. Methicillin-resistant S. aureus (MRSA) has traditionally been regarded as a strictly human problem, initially confined to the healthcare settings and later a matter of concern in the general community too. All this changed in 2005 with the isolation of a specific MRSA clone, assigned to Clonal Complex (CC)398, from pigs and pig farmers in the Netherlands. These findings triggered worldwide investigation, showing the presence of this livestock-associated (LA)-MRSA clone in a variety of farm animals and in persons in contact with affected animals. Furthermore, the capacity of LA-MRSA CC398 to cause infections in humans and animals has been well documented. Recently, MRSA with a divergent mecA-homologue gene variant has been discovered in bovines and humans. Together, these emerging MRSA strains from animal sources have raised new questions as to their origin and inter-host transmission, as well as raised global concern in both veterinary and human medicine about health risks posed by MRSA prevailing in livestock.<p>In the present work, we aimed to investigate the extent and molecular epidemiology of the LA-MRSA reservoir in animal and human populations, including on livestock farms and in acute-care hospitals in Belgium. As a secondary objective, the presence of methicillin susceptible S. aureus (MSSA) CC398, from which MRSA CC398 could locally emerge by acquisition of the Staphylococal Cassette Chromosome mec (SCCmec) element, was assessed. To this end, we undertook an extensive and systematic cross-sectional survey of S.aureus and MRSA carriage among humans and animals on pig, veal calf, dairy cattle, beef cattle, broiler and horticulture farms. A questionnaire, completed by all farm residents, was used to assess occupational risk factors for human MRSA CC398 carriage. Bacterial genetic characterisation was done by spa typing, SCCmec typing and multi-locus sequence typing (MLST). Antimicrobial susceptibility profiles were determined; the presence of resistance genes and toxin genes were determined by PCR. A second set of S. aureus clinical isolates from two national surveys organised in 2005 and 2008 were characterised using the same methods.<p>Carriage of MRSA CC398 was highly prevalent in animals and humans on pig and veal calf farms and to a significantly lesser extent on beef, dairy, broiler and horticulture farms (Chapter 5.1). Persons who work with pigs or veal calves on a daily basis are at significantly higher risk for MRSA CC398 carriage compared to farm-exposed persons who work with them less regularly or never. In accordance with the results from the present work as well as those from others, it appears important to assess the impact of interventions at farm-level that aim to reduce the MRSA carriage rate in animals, as this would also reduce the risk for MRSA carriage in farmers and relatives.<p> MRSA CC398 isolates, especially those from veal calf farms, were frequently multi-resistant and thereby represent a reservoir of antimicrobial resistance determinants that could be transferred to other, more human-adapted staphylococci or other micro-organisms (Chapter 5.1). Additionally, this multi-resistance phenotype should be considered when applying empiric treatment of human staphylococcal infections in livestock-exposed persons. Only very few major “human-associated” virulence factors were detected, indicating a limited virulence capacity of LA-MRSA CC398 isolates. MRSA strains with the mecA homologue mecC, which is difficult to detect using conventional diagnostic methods, were found in beef and dairy cattle, but not in humans. <p>MSSA CC398 strains from which MRSA CC398 might locally emerge were frequently detected in humans and animals on pig, veal and broiler farms, all of which are commonly known to be affected by MRSA CC398 (Chapter 5.2). Three porcine MSSA CC398-t011 isolates harbored remnant DNA of a composite SCCmec V(5C2&5)c element, from which the mec gene complex was lacking. These findings indicate that the strains were previously involved in SCCmec recombination events. Processes similar to the one described here likely contribute to the enormous diversity of SCCmec elements observed in staphylococci. <p>Although LA-MRSA CC398 strains were frequently detected in livestock and livestock-exposed persons, they only represented a minority (~1%) of the MRSA strains from hospitalised patients. This suggests that this specific MRSA clone has not yet spread among Belgian patients without livestock contact (Chapter 5.3). However, similar to what has been seen in other countries, we observed a recent emergence of severe infections, caused by a human-adapted subclone of MSSA CC398, in hospitalised patients without livestock contact (Chapter 5.4). <p>Once more has S. aureus proven its versatility: it has optimally adapted to the selective pressure exerted by intensive animal farming by acquisition of mobile genetic elements, such as resistance determinants. Clearly MRSA is no longer a strictly human problem. Working across the human and veterinary health sectors will be essential to tackle the dissemination and pathogenic evolution of MRSA in livestock. <p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Salmonella spp. é reconhecida como uma das bactérias que mais causam doenças de origem alimentar no mundo. Dentre as diversas sorovariedades de Salmonella, a Typhimurium é uma das sorovariedades de maior ocorrência no mundo. Várias metodologias de tipagem fenotípicas e genotípicas foram desenvolvidas com o intuito de se delinear a epidemiologia e diversidade genotípica de Salmonella Typhimurium. Entretanto, a tipagem fenotípica é muitas vezes limitada por sua baixa capacidade de diferenciação de subtipos pertencentes a uma mesma sorovariedade de Salmonella, um problema minimizado pelos métodos genotípicos. No Brasil, foram realizados poucos estudos que genotiparam linhagens de S. Typhimurium. Os objetivos deste estudo foram caracterizar linhagens de S. Typhimurium isoladas de humanos, alimentos, animais e ambiente do animal no Brasil quanto ao seu potencial patogênico, perfil de resistência a antimicrobianos e diversidade genotípica. Foram estudadas 119 linhagens de S. Typhimurium, isoladas de material clínico de humanos (43), alimentos diversos (49), material clínico de suínos (22) e do ambiente de suínos (5), entre 1983 e 2013, provenientes de várias Estados do Brasil. A presença de 12 genes de virulência foi pesquisada por PCR. O perfil de resistência a 13 antimicrobianos foi realizado pelo método de discodifusão. A tipagem molecular foi realizada por Pulsed-field gel electrophoresis (PFGE), Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), Multiple-locus variablenumber tandem-repeats analysis (MLVA), Clustered regularly interspaced short palindromic repeats - Multi-virulence locus sequence typing (CRISPR-MVLST), Multilocus sequence typing (MLST) e sequenciamento do genoma completo para 92 linhagens de S. Typhimurium isoladas de humanos (43) e alimentos (46). As metodologias PFGE, ERIC-PCR e MLVA foram realizadas para 70 linhagens de S. Typhimurium isoladas de humanos (43), animais (22) e ambiente do animal (5). Todas as 119 linhagens apresentaram os genes sipA, flgK, flgL e invA. O gene sipD e o gene sopE2 foram encontrados em 118 (99,2%) linhagens. O gene fljB foi encontrado em 117 (98,3%) linhagens. O gene sopD foi presente em 114 (95,8%) linhagens, o gene sopB em 111 (93,3%) linhagens, o gene ssaR em 102 (85,7%) linhagens, o gene sifA em 86 (72,3%) linhagens e 45 (37,8%) linhagens apresentaram o gene plasmidial spvB. De um total de 119 linhagens, 64 (62,2%) linhagens foram resistentes a pelo menos um dos 13 antimicrobianos testados, sendo que 36 (30,3%) linhagens foram multi-droga resistentes (MDR). Na comparação dos isolados de humanos e alimentos, as linhagens isoladas de humanos antes de meados 1990, ficaram alocadas nos grupos PFGE-A, PFGE-B1, PFGE-B2, ERIC-A, ERIC-B, MLVA-A, MLVA-B1, MLVA-B2 e G1, G2, H para CRISPRMVLST. As linhagens isoladas de humanos após esse período ficaram alocadas nos grupos PFGE-B1, ERIC-A, MLVA-B1, MLVA-B2 e G2. As linhagens isoladas de alimentos ficaram alocadas nos grupos PFGE-A, PFGE-B1, ERIC-A, ERIC-B, MLVA-A, MLVA-B1, MLVAB2, G1 e G2. Por MLST, do total de 92 linhagens isoladas de humanos e alimentos, 77 linhagens foram tipadas como ST19. Pelo sequenciamento do genoma completo, as linhagens isoladas de alimentos e humanos ficaram alocadas no grupos I e J independente das datas de isolamento. Na comparação dos isolados de humanos e animais, as linhagens das duas origens ficaram alocadas nos grupos PFGE-D1, PFGE-D2, ERIC-C1, MLVA-C1 e MLVA-D. ii Conclui-se que a grande prevalência de genes de virulência nas linhagens de S. Typhimurium estudadas reforça o potencial das mesmas causarem doenças em humanos, bem como, os riscos de sua presença em alimentos, animais para consumo humano e ambiente. A ocorrência de S. Typhimurium multi-droga resistentes isoladas de alimentos diversos e de suínos para consumo é um alerta para o possível risco de humanos ingerirem alimentos contaminados por tais linhagens. Em conjunto os resultados de PFGE, ERIC-PCR, MLVA, CRISPR-MVLST sugerem que as linhagens de S. Typhimurium isoladas de humanos eram geneticamente mais diversificadas antes de meados de 1990, o que pode sugerir a seleção de um subtipo de S. Typhimurium mais adaptado, depois que Salmonella Enteritidis tornou-se a sorovariedade de maior ocorrência no Brasil após esse período. Com relação às linhagens isoladas de alimentos, os resultados de PFGE, ERIC-PCR, MLVA e CRISPR-MVLST sugerem que durante o período estudado houve a circulação de mais de um subtipo no país. Os resultados de MLST sugerem que tais linhagens tenham uma origem filogenética comum. Os resultados do sequenciamento do genoma completo sugerem que houve a circulação de mais de um subtipo de S. Typhimurium no país, com relação às linhagens de humanos e alimentos. Também alerta para o possível risco de linhagens MDR isoladas de alimentos contaminarem humanos e/ou disseminarem genes de resistência a antibióticos para linhagens de origem clínica e não clínica. Na comparação dos isolados de humanos e animais, os resultados de PFGE, ERICPCR e MLVA sugerem que algumas linhagens isoladas de suínos e humanos podem descender de um subtipo comum. Ademais, as linhagens MDR isoladas de suínos e do ambiente de suínos alertam para o possível risco de porcos usados para consumo contaminarem humanos, o ambiente e outros porcos. / Salmonella spp. is recognized as one of the most involved bacteria that cause food-borne diseases in the world. Among the various serovars of Salmonella, Typhimurium is one of the most frequent serovars worldwide. Several phenotypic and genotypic typing methods have been developed in order to delineate the epidemiology and genotypic diversity of Salmonella Typhimurium. However, phenotypic typing is often limited by its low capacity to differentiate subtypes belonging to the same serovar of Salmonella, a problem minimized by genotypic methods. In Brazil, few studies have been conducted that genotyped S. Typhimurium strains. The aims of this study were to characterize S. Typhimurium strains isolated from humans, food, animals and animal\'s environment in Brazil regarding its pathogenic potential, antimicrobial resistance and genotypic diversity. We studied 119 S. Typhimurium strains isolated from human clinical material (43), different foods (49), clinical material from pigs (22) and pigs environment (5), between 1983 and 2013 from various States of Brazil. The presence of 12 virulence genes was investigated by PCR. The resistance profile against 13 antimicrobial was performed by the disk diffusion method. Molecular typing was performed by Pulsed-field gel electrophoresis (PFGE), Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), Multiple-locus variable-number tandem-repeats analysis (MLVA), Clustered regularly interspaced short palindromic repeats - Multi-virulence locus sequence typing (CRISPR-MVLST), Multilocus sequence typing (MLST) and whole genome sequencing for 92 S. Typhimurium strains isolated from humans (43) and food (46). PFGE, ERIC-PCR and MLVA methods were performed for 70 S. Typhimurium strains isolated from humans (43), animals (22) and the animal\'s environment (5). All 119 strains showed the sipA, flgK, flgL and invA genes. The sipD and sopE2 genes were found in 118 (99.2%) strains. The fljB gene was found in 117 (98.3%) strains. The sopD gene was present in 114 (95.8%) strains, the gene sopB in 111 (93.3%) strains, the ssaR gene in 102 (85.7%) strains, the gene sifA in 86 (72.3%) strains and 45 (37.8%) strains showed the plasmid gene spvB. From a total of 119 strains, 64 (62.2%) strains were resistant to at least one of the 13 antimicrobials tested, and 36 (30.3%) strains were multi-drug resistant (MDR). In the comparison of isolates from humans and food, the strains isolated from humans before mid-1990s were allocated in PFGE-A, PFGE-B1, PFGE-B2, ERIC-A, ERIC-B, MLVA-A, MLVA-B1, MLVA-B2 and G1, G2, H for CRISPR-MVLST. The strains isolated from humans after this period were allocated in PFGE-B1, ERIC-A, MLVA-B1, MLVA-B2 and G2 clusters. The strains isolated from food were allocated in PFGE-A, PFGE-B1, ERIC-A, ERIC-B, MLVA-A, MLVA-B1, MLVA-B2, G1 and G2 clusters. By MLST, of the total of 92 strains isolated from humans and food, 77 strains were typed as ST19. By whole genome sequencing, the strains isolated from food and humans were allocated in I and J clusters independently of its isolation date. In the comparison of isolates from humans and animals, strains of the two origins were allocated in PFGE-D1, PFGE-D2, ERIC-C1, MLVA-C1 and MLVA-D clusters. In conclusion, the high frequency of virulence genes in the S. Typhimurium strains studied reinforces their potential hazard to cause disease in humans, as well as the risk of its presence in food, animals for human consumption and the environment. The occurrence of S. Typhimurium multi-drug iv resistant isolated from various food and pigs for consumption is an alert of the possible risk for humans to ingest contaminated food with those strains. Together the results of PFGE, ERIC-PCR, MLVA e CRISPR-MVLST suggest that S. Typhimurium strains isolated from humans were genetically more diverse before mid-1990s, which might indicate the selection of a more adapted S. Typhimurium subtype after Salmonella Enteritidis became the most prevalent serovar in Brazil. Regarding the strains isolated from food, the results of PFGE, ERIC-PCR, MLVA and CRISPR-MVLST suggest that during the studied period there was circulation of more than one subtype in the country. The MLST results suggest that these strains have a common phylogenetic origin. The results of the whole genome sequencing suggest that there may be more than one subtype circulating in the country, with respect to the strains of human and food origins. Also, alerts for the possible risk of MDR strains isolated from food to contaminate humans and/or disseminate antibiotic resistance genes for strains of clinical and non-clinical origin. In the comparison of isolates from humans and animals, the results of PFGE, ERIC-PCR and MLVA suggest that some strains isolated from pigs and humans may descend from a common subtype. In addition, the MDR strains isolated from pigs and pig environment warn for the possible risk of pigs used for human consumption to contaminate humans, the environment and other pigs.

A microbiota do trato gastrointestinal dos psitacídeos é composta por bactérias Gram-positivas, entre elas as bactérias ácido láticas. Porém, há poucos estudos na literatura descrevendo a microbiota do trato gastrointestinal dessas aves. O objetivo deste estudo foi identificar e caracterizar fenotipicamente e genotipicamente, por três diferentes métodos, as bactérias ácido láticas isoladas da microbiota fecal de papagaios verdadeiros. Um total de 80 amostras bacterianas foram estudadas, sendo que 31 amostras eram provenientes da microbiota fecal de papagaios-verdadeiros de vida livre e 49 amostras eram de papagaios-verdadeiros de cativeiro. Foram realizadas provas bioquímicas convencionais, automatizadas (Vitek 2) e o sequenciamento completo do gene 16S rRNA e realizada a análise comparativa dos resultados. Das 80 amostras analisadas, em 40 (50%) destas foram identificadas as espécies, pois apresentaram concordância de identificação em pelo menos dois métodos, e as demais 40 amostras (50%) não apresentaram concordância entre os testes, não sendo possível definir a espécie. As espécies identificadas foram: Enterococcus avium (02 amostras), Enterococcus faecium (03 amostras), Enterococcus faecalis (15 amostras), Enterococcus hirae (15 amostras), Lactococcus lactis (02 amostras) e Staphylococcus warneri (02 amostras). Dentre as amostras identificadas, sete (07) amostras apresentaram concordância no resultado nas três técnicas analisadas, trinta e uma (31) amostras apresentaram concordância pelo bioquímico automatizado e pelo sequenciamento do gene 16S rRNA, e apenas duas (02) amostras apresentaram concordância pelo bioquímico convencional e pelo sequenciamento de DNA. A similaridade de utilização de substratos pelas cepas foi definida em treze (13) amostras, nas demais amostras estudadas houve uma diferença no consumo de substratos nas provas bioquímicas. O perfil de resistência a antimicrobianos evidenciou resistência em onze (11) amostras, quatro (04) a benzilpenicilina, quatro (04) a eritromicina, duas (02) a gentamicina e uma (01) a estreptomicina. A análise filogenética baseada no coeficiente de Neighbor-Join para comparar a similaridade entre as sequencias geradas pelo sequenciamento do gene 16S rRNA, mostrou uma tendência de agrupamento bacteriano de acordo com o local de onde as aves eram provenientes. Verificou-se que a identificação através do teste bioquímico convencional apresentou resultados inconsistentes quando comparados com o teste bioquímico automatizado e com o sequenciamento de DNA. Por sua vez, a técnica do sequenciamento genético demonstrou uma elevada confiabilidade nos resultados obtidos; sugerindo-se a utilização deste como teste de eleição e também como teste complementar as provas bioquímicas, para assim diminuir a probabilidade de erro de identificação bacteriana de bactérias ácido láticas em papagaios. / Gastrointestinal microbiota of pscittacines main consists largely of Gram-positive bacteria, including acid-latic bacteria. However, the literature presents very few reports on profiling the gastrointestinal microbiota of these birds. The aim of this study was to identify and to characterize phenotypicly and genotypicly acid-latic bacteria isolated from fecal microbiota of Blue-fronted Amazon Parrots. For such, three different methods were used on eighty bacterial strains. Thirty-one fecal samples were collected from free-living Blue-fronted Amazon Parrots, while fourty-nine were from captive birds. Traditional biochemical tests, automated biochemical test (Vitek 2) and complete gene sequencing of 16S rRNA were performed. Results of these three methods were compared. Forty samples (50%) had agreement between at least two of the three methods, and bacterial species identification was confirmed, while strains from the remaining 40 samples were not identified, once agreement between atleast two methods was not found. The species identified were: Enterococcus avium (02 samples), Enterococcus faecium (03 samples), Enterococcus faecalis (15 samples), Enterococcus hirae (15 samples), Lactococcus lactis (02 samples) and Staphylococcus warneri (02 samples). Agreement between the three methods was observed in seven samples. Thirty-one samples presented agreement between automated biochemical tests and sequencing of the 16S rRNA gene. Only two samples presented agreement between tradional biochemical tests and gene sequencing. Similarities of substract consumed by strains in both traditional and automated biochemical tests were observed in thirteen samples, while the other samples presented some difference in substracts utilization. Antimicrobial resistance profile showed that eleven samples were resistant to some antibiotic. Four were resistant to benzilpenicilin, four to erythromycin, two to gentamicin and one to estreptomycin. The phylogenetic test based on Neighbor-Join coefficient, which compares the similarity between 16S rRNA sequences, showed a trend for grouping of strains according to the geographical origin of each bird. However, species identification using traditional biochemical tests showed to be inconsistent when compared to automated biochemical tests and gene sequencing results. On the other hand, genetic sequencing technique results showed to be highly reliable. Thus, this method should be used both as gold standard and as complementary to the biochemical tests for acid-latic bacteria identification in Blue-fronted Amazon Parrots gastrointestinal microbiota.

Les bactéries productrices de β-lactamase à spectre élargi (BLSE) ont été rapportées dans plusieurs pays, mais aucune information nest disponible sur les différents types de BLSE produits par les Entérobactéries au Cameroun. On distingue plus de 290 types de β-lactamases divisés en 4 classes (A, B, C, et D) et jusquaujourdhui il nexiste pas des inhibiteurs capables dinhibés toutes les classes de β-lactamase. Un total de 267 souches dEntérobactéries a été isolé entre 1995-1998 (259 souches) et 2002 (8 souches) des produits pathologiques (urines, pus et sang) des patients hospitalisés dans divers services de lHôpital Central de Yaoundé. Létude de la sensibilité in vitro de ces bactéries à 12 antibiotiques (amoxicilline, amoxicilline/acide clavulanique, pipéracilline, imipénème, céfazoline, céfoxitine, céfotaxime, ceftazidime, aztréonam, gentamicine, ofloxacine et triméthoprime/sulfaméthoxazole) par la méthode des disques a permis de noter un certains nombre de faits : limipénème, lofloxacine et la ceftazidime sont les antibiotiques les plus actifs sur les différentes Entérobactéries avec respectivement 100%, 91% et 88% de bactéries sensibles ; les différentes souches présentent un niveau de résistance élevé à lamoxicilline (90%), à la pipéracilline (79%), à la céfazoline (75%) et au triméthoprime/sulfaméthoxazole (74%). En vue de détecter les espèces bactériennes productrices de BLSE, 69 souches résistantes à au moins une céphalosporine de 3ème génération ou au monobactame ont été soumises au test de double synergie. Trente-huit (31 entre 1995-1998 ; 7 en 2002) souches ont montré un test positif. La prévalence des souches productrices de BLSE est de 12% (31/259). Ces souches présentant un phénotype BLSE sont observées chez toutes les espèces dEntérobactéries étudiées avec une prévalence de 18,8% (Klebsiella spp.), de 17,6% (Citrobacter spp.), de 14,3% (E. coli) et de 1,8% (Proteus spp.). Parmi les 38 souches potentiellement productrices de BLSE, 18 (47,4%) ont transféré le gène de résistance des oxyimino-céphalosporines aux souches E. coli HK 225, K12 et DH5α. Seize de ces gènes ont été transférés par conjugaison et deux par transformation. Le transfert de gène de BLSE a été co-transféré avec le gène de résistance à la gentamicine et/ou au triméthoprime/sulfaméthoxazole. Toutes les souches transconjugantes et transformantes ont présenté un phénotype BLSE et ont été toutes sensibles à la céfoxitine et à limipénème. Les extraits enzymatiques bruts obtenus par sonication des souches transconjugantes, transformantes et cliniques ont été capable de réduire les diamètres dinhibition autour des disques de pénicillines, céphalosporines 1ère à la 3ème génération ou monobactame en utilisant une souche dE. coli complètement sensible aux antibiotiques, mais nont pas eu deffet sur ces zones autour des disques dimipénème, de céfoxitine et damoxicilline/acide clavulanique. La détermination des points isoélectriques (pIs) des différentes souches après transfert du gène de résistance par la technique disoélectrofocalisation a montré que les souches transconjugantes et transformantes produisent des β-lactamases dont les pIs varient de 5,4 à 8,8. En effet, 12 souches produisent les BLSE de pI 8,2 ; deux souches en plus de cette BLSE produisent deux autres enzymes de pIs 5,4 et 8,5. Les souches de phénotypes CTX-M produisent soit une enzyme (pI 8,4 ; une souche) soit deux (pIs 7,3, 8,8 ; 2 souches) ou alors 3 types (pIs 5,4 ; 7,3 et 8,8 ; une souche). Les extraits dADN de 19 souches (16 transconjugantes, 2 transformantes et 1 souche clinique) soumis à la technique de PCR, PCR/NheI, en utilisant les amorces spécifiques des gènes blaSHV, blaCTX-M, blaTEM et blaOXA ont montré que les différents extraits contiennent les gènes des b-lactamases du type SHV (sulfhydryl variable), CTX-M (cefotaximase), TEM (Temoniera) et OXA (aoxacillinase). Lanalyse de la séquence des différents produits de PCR a permis de montrer que : 14 souches produisent la BLSE SHV-12, cinq la BLSE CTX-M (CTX-M-1, une souche ; CTX-M-15, 4 souches) ; quatre souches produisent en association avec les BLSE les β-lactamases TEM-1 (SHV-12, 2 souches ; CTX-M-15, 2 souches) OXA-30 (CTX-M-15, 4 souches). Lanalyse des profils de digestion des plasmides portant les gènes blaCTX-M par les enzymes de restriction, dhybridation et des profils génotypiques des souches cliniques productrice de ces enzymes par ERIC-PCR a montré que la dissémination de la BLSE CTX-M peut se faire soit par transfert de plasmide (on retrouve le même plasmide chez deux souches différentes, E. coli YC-14 et K. pneumoniae YC-17) soit par la même souche dun patient à un autre (E. coli YC-5 = E. coli YC-2). Létude de lenvironnement génétique de ce gène (blaCTX-M-15) par lanalyse des séquences a montré que CTX-M-15 est codée par deux plasmides multirésistants différents, parmi lesquels un (pYC-5b) porte lélément ISEcp1-blaCTX-M-15 flanqué par un site de réplication constituée de 5 paires de bases et inséré dans le transposon Tn2. Une forme tronquée de cet élément a été identifiée chez lautre plasmide (pYC-14). Les BLSE SHV-12, CTX-M-1 et CTX-M-15 sont décrites pour la première fois au Cameroun. Dans le but de rechercher de nouveaux inhibiteurs des β-lactamases plus actifs, nous avons étudié deux plantes médicinales, Garcinia lucida (Clusiaceae) et Bridelia micrantha (Euphorbiceae) en vue dévaluer leur activité anti- β-lactamase. Les concentrations inhibant 50 % de l'activité de l'enzyme (CI50) des extraits de G. lucida et B. micrantha sont respectivement de 0,01 mg/ml (β-lactamase P99) et de 0,019 mg/ml (β-lactamase OXA-10) indiquant ainsi une bonne inhibition des β-lactamases par les extraits de ces plantes. Le produit 4 issu de la purification par chromatographie haute performance de lextrait de G. lucida est très actif sur la β-lactamase P99 (CI50 = 0,038 mg/ml) alors que les produits 2' et 3' provenant de lextrait de B. micrantha sont actifs sur lenzyme OXA-10 (CI50 de 0,09 mg/ml et 0,11 mg/ml respectivement). La détermination de la structure des composés actifs de ces deux plantes pourrait être une voie importante dans le développement des inhibiteurs des β-lactamases. / Bacteria producing extended-spectrum β-lactamase (ESBL) have been reported in many countries, but there is no information on different type of ESBL-producing enterobacteria in Cameroon. More than 290 β-lactamases have been described and divided into four classes (A, B, C and D) and up to now, there is no good inhibitor which can inhibite all classes of β-lactamases. A total of 267 strains of Enterobacteriaceaae were isolated between 1995-1998 (259 isolates) and 2002 (eight isolates) from pathological products (urines, pus and blood) of patients at the Yaounde Central Hospital. The susceptibility of the isolates to 12 antibiotics (amoxicillin, amoxicillin/clavulanate, piperacillin, imipenem, cefazolin, cefoxitin, cefotaxime, ceftazidime, aztreonam, gentamicin, ofloxacin and trimethoprim/sulfamethoxazole) was determined using the agar diffusion disk method. Imipenem, ofloxacin and ceftazidime were the most active antibiotics against overall enterobacteria with susceptibility rates of 100%, 91% and 88% respectively. High resistance rates were observed to amoxicillin (90%), piperacillin (79%), cefazolin (75%) and trimethoprim/sulfamethoxazole (73%). Enterobacteria isolates (69) resistant to oxyimino-cephalosporin or monobactam were screened for ESBL production by the double disk (DD) synergy test. Thirty-eight (31 from 1995-1998; seven in 2002) were proved positive to the DD synergy test, suggesting the presence of ESBL. The prevalence of ESBL was 12% (31/259) and ESBL-producing strains were Klebsiella spp. (18.8%), Citrobacter spp. (17.6%), Escherichia coli (14.3%) and Proteus spp. (1.8%). Of the 38 ESBL-producing strains, 18 (47.4%) were transferred resistance to oxyimino-cephalosporins to E. coli HK 225, K12 and DH5α. Sixteen of these strains were transferred ESBL gene by conjugation and two by transformation. Resistance to gentamicin and/or trimethoprim/sulfamethoxazole was co-transferred. All transconjugant and transformant strains exhibited ESBL phenotype but remained susceptible to cefoxitin and imipenem. Crude extracts of β-lactamase-producing transconjugants, transformants and clinical strains were able to reduce the diameters of inhibition zones around disks containing penicillins, 1st to 3rd generation cephalosporins or monobactam when tested against a fully susceptible E. coli strains, but had no effect on such zones around cefoxitin-, imipenem- and amoxicillin/clavulanate disks. The determination of isoelectric points (pIs) of the various strains after transfer of resistance determinant showed that transconjugants and transformants strains produced β-lactamases with pIs 5.4 to 8.8. In fact, 12 strains produced β-lactamase with pI 8.2, 2 strains in addition to the above enzyme produced two others enzymes with pIs 5.4 and 8.5. Strains with CTX-M- phenotype produced enzyme (pI 8.4, 1 strain), two (pIs 7.3, 8.8; 2 strains) or three types pIs (5.4, 7.3, 8.8; 1 strain). PCR, PCR/NheI was performed using total or plasmid DNA from 19 strains as templates, and the primers specific to blaSHV, blaCTX-M, blaTEM and blaOXA. Direct sequencing of PCR products revealed that: 14 strains produced SHV-12 ESBL, 5 CTX-M- ESBL (CTX-M-1, one strain; CTX-M-15, 4 strains); four other strains shared non-ESBL TEM-1 (2 producing-SHV-12 strains and 2 producing-CTX-M-strains) and OXA-30 (4 producing CTX-M-15 strains). Analysis of restriction patterns of plasmid carrying blaCTX-M gene, hybridization, and genotyping patterns of CTX-M-clinical strains by ERIC-PCR showed that dissemination of blaCTX-M gene could be done by plasmid transfer (same plasmid in different strains, E. coli YC-14 and K. pneumoniae YC-17) or by strains from patient to patient (same strain, E. coli YC-2=E. coli YC-5). Sequence analysis of genetic environment of blaCTX-M-15 revealed that CTXM-15 was encoded by two different multiresistant plasmids, of which one (pYC-5b) carried an ISEcp1-blaCTX-m-15 element flanked by five bp target site duplication and inserted within a Tn2-derived sequence. A truncated form of this element in the second plasmid (pYC-14) was identified. ESBLs SHV-12, CTX-M-1, CTX-M-15 are described for the first time in Cameroon. In our effort to find new active β-lactamase inhibitors, we investigated two medicinal plants, Garcinia lucida (Clusiaceae) and Bridelia micrantha (Euphorbiaceae) for anti- β-lactamase activity. The extracts from G. lucida and B. micrantha exhibited good inhibition of β-lactamase P99 (IC50, 0.01 mg/ml) and OXA-10 (IC50, 0.02 mg/ml) respectively. Purified products from these plants by high performance liquid chromatography showed that, product 4 from G. lucida is very active on β-lactamase P99 (IC50, 0.03 mg/ml) whereas products 2 and 3 from B. micrantha are active on OXA-10 (IC50, 0.09 mg/ml; 0.11 mg/ml respectively). The structural elucidation of the active constituents of these plants will provide useful leads in the development of β-lactamase inhibitors.

Depuis quelques années et dans plusieurs pays, un nouveau type de Staphylococcus aureus résistant à la méthicilline (SARM), le séquence type (ST) 398, a été fréquemment retrouvé chez les porcs et chez les fermiers en contact avec ces porcs. Au Canada, très peu d’informations sont disponibles concernant le SARM d’origine porcine. Une première étude dans notre laboratoire a permis de récolter 107 isolats de SARM provenant de deux abattoirs porcins du Québec. Le présent travail vise à caractériser les gènes de virulence et de résistance aux antibiotiques de ces SARM, d’étudier leur formation de biofilm en relation avec la spécificité du groupe agr et de vérifier la localisation plasmidique et la transférabilité de ces gènes à des souches de SARM d’origine humaine. Plusieurs souches ont démontré différents patrons phénotypiques de résistance aux antibiotiques. Vingt-quatre souches représentatives de ces isolats ont été soumises à une caractérisation plus approfondie par une étude génotypique en utilisant une biopuce à ADN et un grand nombre de gènes de virulence a été détecté codant pour des entérotoxines staphylococcales, des leucocidines, des hémolysines, des auréolysines, des facteurs d’immunoévasion, des superantigènes, des facteurs d’adhésion et des facteurs impliqués dans la formation de biofilm. Des gènes de résistance envers les aminoglycosides, les macrolides, les lincosamides, les tétracyclines et les biocides ont été également détectés par biopuce et leur localisation plasmidique a par la suite été déterminée. La transférabilité de ces gènes de souches porcines à des souches de SARM d’origine humaine a été démontrée par conjugaison bactérienne; ainsi le transfert horizontal de certains gènes de résistance aux antibiotiques et de virulence a été observé. Ces travaux de recherche apportent une meilleure connaissance de la résistance aux antibiotiques et de la virulence des SARM d’origine porcine et de leur potentiel de contribution à l’émergence de certaines résistances et facteurs de virulence chez le SARM d’origine humaine. / In recent years and in several countries, a new type of methicillin-resistant Staphylococcus aureus (MRSA), the sequence type (ST) 398, has been frequently found in pigs and in farmers in contact with these pigs. In Canada, little information is available concerning MRSA from pigs. A previous study in our laboratory identified 107 MRSA isolates from two pig slaughterhouses in Quebec. This study was conducted to determine antimicrobial resistance and virulence genes of MRSA from abattoir pig, to study their biofilm formation in relation with agr specificity groups and to evaluate horizontal transfer of genes to a MRSA of human clinical origin. Different phenotypic patterns of antimicrobial resistance were observed in these MRSA and a representative subset of these isolates was selected for further characterization. Twenty-four porcine MRSA were characterized by a DNA microarray, the StaphyType of CLONDIAG. Our results demonstrated that the MRSA strains from the abattoirs contain several antimicrobial resistance genes responsible for macrolide and tetracycline resistance and virulence genes encoding staphylococcal enterotoxins, hemolysins, leukocidins, aureolysin, superantigens, immunoevasion, adhesion, and biofilm development. This study presents the first evidence that horizontal transfer of some of these genes can occur between MRSA of porcine and human origin. We also report for the first time biofilm formation in Livestock Associated-MRSA of porcine origin associated with agr group II. It is possible that biofilm formation favors colonization, persistence as well as zoonotic potential. This research provides a better understanding of antimicrobial resistance and virulence of MRSA from pigs and their potential contribution to the emergence of some resistance and virulence factors in MRSA of human origin.

La pollution microbienne des eaux récréatives peut engendrer un risque pour la santé des populations exposées. La contamination fécale de ces eaux représente une composante importante de ce risque, notamment par la présence possible d’agents pathogènes et par l’exposition à des micro-organismes résistants aux antimicrobiens. Les sources de pollution fécale sont multiples et incluent entre autres les activités agricoles et les productions animales. Ce projet visait donc à mieux comprendre les facteurs influençant la qualité microbiologique des eaux récréatives du Québec méridional, en ciblant le rôle possible des activités agricoles, ainsi qu`à proposer et évaluer de nouvelles sources de données pouvant contribuer à l’identification de ces facteurs. Dans un premier temps, une évaluation de la présence d’Escherichia coli résistants aux antimicrobiens dans les eaux récréatives à l’étude a été effectuée. À la lumière des résultats de cette première étude, ces eaux représenteraient une source de micro-organismes résistants aux antimicrobiens pour les personnes pratiquant des activités aquatiques, mais l’impact en santé publique d’une telle exposition demeure à déterminer. Les déterminants agroenvironnementaux associés à la présence de micro-organismes résistants aux antimicrobiens ont par la suite été explorés. Les résultats de ce chapitre suggèrent que les activités agricoles, et plus spécifiquement l’épandage de fumier liquide, seraient reliées à la contamination des eaux récréatives par des bactéries résistantes aux antimicrobiens. Le chapitre suivant visait à identifier des déterminants agroenvironnementaux temps-indépendants d’importance associés à la contamination fécale des eaux à l’étude. Différentes variables, regroupées en trois classes (activités agricoles, humaines et caractéristiques géohydrologiques), ont été explorées à travers un modèle de régression logistique multivarié. Il en est ressorti que les eaux récréatives ayant des sites de productions de ruminants à proximité, et en particulier à l’intérieur d’un rayon de 2 km, possédaient un risque plus élevé de contamination fécale. Une association positive a également été notée entre le niveau de contamination fécale et le fait que les plages soient situées à l’intérieur d’une zone urbaine. Cette composante nous permet donc de conclure qu’en regard à la santé publique, les eaux récréatives pourraient être contaminées par des sources de pollution fécale tant animales qu’humaines, et que celles-ci pourraient représenter un risque pour la santé des utilisateurs. Pour terminer, un modèle de régression logistique construit à l’aide de données issues de la télédétection et mettant en association un groupe de déterminants agroenvironnementaux et la contamination fécale des eaux récréatives a été mis au point. Ce chapitre visait à évaluer l’utilité de telles données dans l’identification de ces déterminants, de même qu`à discuter des avantages et contraintes associées à leur emploi dans le contexte de la surveillance de la qualité microbiologique des eaux récréatives. À travers cette étude, des associations positives ont été mises en évidence entre le niveau de contamination fécale des eaux et la superficie des terres agricoles adjacentes, de même qu’avec la présence de surfaces imperméables. Les données issues des images d’observation de la Terre pourraient donc constituer une valeur ajoutée pour les programmes de suivi de la qualité microbiologique de ces eaux en permettant une surveillance des déterminants y étant associés. / Microbial pollution of recreational waters may constitute a health risk for exposed populations. Fecal contamination of these waters represents an important component of this risk, especially due to the possible presence of pathogens and exposure to antimicrobial resistant microorganisms. Sources of fecal pollution are many and include agricultural activities and animal production. This project aims to better understand agroenvironmental factors influencing the microbiological quality of recreational waters of Southern Quebec and also suggests and assesses new sources of data which may contribute to the identification of these factors. An evaluation of the presence of antimicrobial resistant Escherichia coli in the recreational waters studied was first performed. Given the positive results found, certain bodies of waters may represent a source of antimicrobial resistant microorganisms for people engaged in water activities. However the public health impact of such exposure is still unknown. Agroenvironmental determinants associated with the presence of antimicrobial resistant microorganisms were investigated. Results of this chapter suggest that agricultural activities, especially liquid manure spreading, may be associated with the contamination of recreational waters with antimicrobial resistant microorganisms. The next chapter assesses key time-independent agroenvironmental determinants associated with the fecal contamination of studied recreational waters. Various variables – classified as either agricultural activities, human activities or geo-hydrological characteristics – are explored through a multivariate logistic regression model. It appears that recreational waters with ruminant productions in their proximal environment, especially when they are within a radius of 2 km, may present a higher risk of fecal contamination. A positive association was also noticed between the level of fecal contamination of a beach and its presence in an urban area. From a public health perspective, recreational waters may be contaminated by animal and human sources and these sources may represent a risk for bathers’ health. Finally, association between fecal contamination of recreational waters and agroenvironment determinants was studied through a logistic regression model built from remote sensing data. This chapter assesses the utility of such data in the identification of these determinants and discusses the strengths and the limitations of their use in the context of the monitoring of the microbiological quality of recreational waters. In this study, positive associations were found between the level of fecal contamination and both the proximity of agricultural lands and the presence of impervious surfaces. Earth observation data may constitute an added value for the water quality monitoring program in allowing the surveillance of the determinants associated with this quality.

Les Escherichia coli pathogènes de la volaille (APEC) font partie des E. coli extra-intestinaux pathogènes (ExPEC) et seraient un réservoir possible de gènes de virulence et de résistance aux antimicrobiens (RAM) des ExPEC chez l’humain. L’objectif de cette étude était d’évaluer l’effet d’un prébiotique et d’un mélange d’acide organique et d’huiles essentielles encapsulés sur la prévalence des gènes de virulence des ExPEC et de RAM, ainsi que les associations entre ces gènes chez E. coli de l’intestin du poulet sain. Des échantillons de contenus caecaux de poulets de 29 jours d’âge ayant reçu un de ces ingrédients alimentaires comparativement à des témoins ont été analysés pour la présence des gènes de virulence iucD, tsh, papC et des gènes de RAM blaTEM, blaSHV, tetA, tetC, blaCMY-2, aadA1, aac3 par PCR. La prévalence d’iucD était supérieure dans le groupe témoin comparativement aux groupes «prébiotique» et «acide organique» et la prévalence de papC était affectée dans le groupe «acide organique». La prévalence d’isolats d’E.coli positifs pour blaCMY-2 était supérieure dans le groupe témoin comparée aux groupes «prébiotique» et «acide organique», tel que démontré par la technique d’hybridation de l’ADN sur HGMF (Hydrophobic Grid Membrane Filter). De plus, la prévalence des isolats d’E. coli positifs pour tetA, blaTEM, aadA1 ou tsh était affectée par les ingrédients alimentaires. Dans l’ensemble, des associations entre la présence de tsh et iucD, blaTEM et aadA1, et iucD et blaCMY-2 ont été observées. .Cette étude démontre l’utilité de certains ingrédients alimentaires pour dimunier le risque d’exposition en santé publique. / Avian Pathogenic E. coli (APEC) belong to the extra-intestinal pathogenic E. coli (ExPEC) pathotype, and may be a virulence and antimicrobial resistance (AMR) gene reservoir for ExPEC in humans. The aim of this study was to evaluate the effect of addition to the feed of a prebiotic or an organic acid on the prevalence of ExPEC-associated virulence genes and antimicrobial resistance (AMR) genes and the association between these genes in E. coli of the intestinal microflora of healthy chickens. Caecal contents from 29-day-old chickens having received one of these feed ingredients in comparison to a control group were examined for the presence of virulence genes iucD, tsh, and papC and AMR genes blaTEM, blaSHV, tetA, tetC, blaCMY-2, aadA1, and aac3 by PCR. The prevalence of iucD was significantly higher in the control group than in the prebiotic and organic acid groups and prevalence of papC was affected by the use of the organic acid. The prevalence of blaCMY-2-positive E. coli isolates was higher in the control group than the prebiotic or organic acid groups, as demonstrated by Hydrophobic–grid membrane filter (HGMF) DNA probe colony hybridization. In addition, the prevalence of E. coli isolates positive for tetA, blaTEM, aadA1 or tsh was affected by the use of these feed ingredients. Overall, associations between the presence of iucD and tsh, blaTEM and aadA1, and iucD and blaCMY-2 were observed. This study demonstrates that the use of certain feed ingredients could reduce the risk of exposure in a public health perspective.

Les entérocoques font partie de la flore normale intestinale des animaux et des humains. Plusieurs études ont démontré que les entérocoques d’origine animale pouvaient représenter un réservoir de gènes de résistance aux antibiotiques pour la communauté humaine et animale. Les espèces Enterococcus faecalis et Enterococcus faecium sont importantes en santé publique; elles sont responsables d’environ 12% de toutes les infections nosocomiales aux États-Unis. Au Canada, les cas de colonisation et/ou d’infections à entérocoques résistants à la vancomycine ont plus que triplé de 2005 à 2009. Un total de 387 isolats E. faecalis et E. faecium aviaires, et 124 isolats E. faecalis porcins ont été identifiés et analysés pour leur susceptibilité aux antibiotiques. De hauts pourcentages de résistance envers les macrolides et les tétracyclines ont été observés tant chez les isolats aviaires que porcins. Deux profils phénotypiques prédominants ont été déterminés et analysés par PCR et séquençage pour la présence de gènes de résistance aux antibiotiques. Différentes combinaisons de gènes de résistance ont été identifiées dont erm(B) et tet(M) étant les plus prévalents. Des extractions plasmidiques et des analyses par hybridation ont permis de déterminer, pour la première fois, la colocalisation des gènes erm(B) et tet(M) sur un plasmide d’environ 9 kb chez des isolats E. faecalis porcins, et des gènes erm(B) et tet(O) sur un plasmide de faible poids moléculaire d’environ 11 kb chez des isolats E. faecalis aviaires. De plus, nous avons démontré, grâce à des essais conjugatifs, que ces plasmides pouvaient être transférés. Les résultats ont révélé que les entérocoques intestinaux aviaires et porcins, lesquels peuvent contaminer la viande à l’abattoir, pouvaient représenter un réservoir de gènes de résistance envers la quinupristine-dalfopristine, la bacitracine, la tétracycline et les macrolides. Afin d’évaluer l’utilisation d’un antisérum polyclonal SA dans l’interférence de la résistance à de fortes concentrations de bacitracine (gènes bcrRAB), lors d’un transfert conjugatif répondant aux phéromones, un isolat multirésistant E. faecalis aviaire a été sélectionné. Après induction avec des phéromones produites par la souche réceptrice E. faecalis JH2-2, l’agrégation de la souche donatrice E. faecalis 543 a été observée ainsi que des fréquences de transfert élevées en bouillon lors d’une courte période de conjugaison. Le transfert conjugatif des gènes asa1, traB et bcrRAB ainsi que leur colocalisation a été démontré chez le donneur et un transconjugant T543-1 sur un plasmide de 115 kb par électrophorèse à champs pulsé (PFGE) et hybridation. Une CMI de > 2 048 µg/ml envers la bacitracine a été obtenue tant chez le donneur que le transconjuguant tandis que la souche réceptrice JH2-2 démontrait une CMI de 32 µg/ml. Le séquençage des gènes asa1, codant pour la substance agrégative, et traB, une protéine régulant négativement la réponse aux phéromones, a révélé une association de cet élément génétique avec le plasmide pJM01. De plus, cette étude présente qu’un antisérum polyclonal SA peut interférer significativement dans le transfert horizontal d’un plasmide répondant aux phéromones codant pour de la résistance à de fortes doses de bacitracine d’une souche E. faecalis aviaire multirésistante. Des isolats cliniques E. faecium d’origine humaine et canine ont été analysés et comparés. Cette étude rapporte, pour la première fois, la caractérisation d’isolats cliniques E. faecium résistants à l’ampicilline (EFRA) d’origine canine associés à CC17 (ST17) au Canada. Ces isolats étaient résistants à la ciprofloxacine et à la lincomycine. Leur résistance envers la ciprofloxacine a été confirmée par la présence de substitutions dans la séquence en acides aminés des gènes de l’ADN gyrase (gyrA/gyrB) et de la topoisomérase IV (parC/parE). Des résistances élevées envers la gentamicine, la kanamycine et la streptomycine, et de la résistance envers les macrolides et les lincosamides a également été observées. La fréquence de résistance envers la tétracycline était élevée tandis que celle envers la vancomycine n’a pas été détectée. De plus, aucune résistance n’a été observée envers le linézolide et la quinupristine-dalfopristine. Les données ont démontré une absence complète des gènes esp (protéine de surface des entérocoques) et hyl (hyaluronidase) chez les isolats canins EFRA testés tandis qu’ils possédaient tous le gène acm (adhésine de liaison au collagène d’E. faecium). Aucune activité reliée à la formation de biofilm ou la présence d’éléments CRISPR (loci de courtes répétitions palindromiques à interespaces réguliers) n’a été identifiée chez les isolats canins EFRA. Les familles de plasmide rep6 and rep11 ont significativement été associées aux isolats d’origine canine. Les profils PFGE des isolats d’origine humaine et canine n'ont révélé aucune relation (≤ 80%). Ces résultats illustrent l'importance d'une utilisation judicieuse des antibiotiques en médecine vétérinaire afin d’éviter la dissémination zoonotique des isolats EFRA canins. Nous pensons que ces résultats contribueront à une meilleure compréhension des mécanismes de résistance aux antibiotiques et de leurs éléments mobiles ainsi qu’à de nouvelles stratégies afin de réduire le transfert horizontal de la résistance aux antibiotiques et des facteurs de virulence. / Enterococci are part of normal intestinal gut flora of animals and humans. Many studies have shown that enterococci from animal origin could represent an antimicrobial resistance genes reservoir for the human community. The two species Enterococcus faecalis and Enterococcus faecium are important in public health; they are responsible for approximately 12% of all nosocomial infections in the United States. In Canada, cases of colonization and/or infections to vancomycin resistant enterococci have more than tripled from 2005 to 2009. A total of 387 poultry E. faecalis and E. faecium isolates, and 124 porcine E. faecalis isolates were identified and analyzed for their antibiotic susceptibilities. High percentages of resistance to macrolides and tetracyclines were found in both avian and porcine isolates. Two predominant phenotypic profiles were determined and analyzed by PCR and sequencing for the presence of antimicrobial resistance genes. Various combinations of antibiotic resistance genes were detected; erm(B) and tet(M) were the most common genes. For the first time, plasmid extraction and hybridization revealed colocalization of erm(B) and tet(M) on a plasmid of ~9 kb in porcine E. faecalis isolates, and of erm(B) and tet(O) on a low-molecular-weight plasmid of ~11 kb in poultry E. faecalis isolates. Furthermore, we demonstrated, through mating experiments, these plasmids could be transferred. Results indicate that the intestinal enterococci of healthy pigs and poultry, which can contaminate meat at slaughter, could be a reservoir for quinupristin-dalfopristin, bacitracin, tetracycline, and macrolide resistance genes. To assess the use of a polyclonal antiserum AS on the contact interference of a high level bacitracin resistant (bcrRAB genes) pheromone-responsive plasmid, a multiresistant E. faecalis isolate of poultry origin was selected. After induction with pheromones produced by the recipient strain E. faecalis JH2-2, clumping of the donor E. faecalis strain 543 was demonstrated as well as high transfer frequencies in short time broth mating. Conjugative transfer of asa1, traB and bcrRAB genes and their co-localization were also demonstrated in the donor strain and a transconjugant T543-1 on a plasmid band of 115 kb by PFGE and Southern blotting. A MIC to bacitracin of > 2 048 µg/ml was obtained for both strains 543 and T543-1 whereas the recipient strain JH2-2 demonstrated a MIC of 32 µg/ml. Sequencing of the asa1 gene encoding for an AS, and traB for a pheromone shutdown protein, confirmed the association of this genetic element to the pheromone-responsive plasmid related to pJM01. More significantly, this study presents the evidence that a polyclonal antiserum AS can significantly interfere with the horizontal transfer of a pheromone-responsive plasmid encoding high-level bacitracin resistance of a poultry multidrug resistant E. faecalis strain. Clinical isolates of E. faecium of human and canine origin were analyzed and compared. This report describes for the first time the characterization of canine clinical ampicillin-resistant E. faecium (AREF) isolates related to CC17 (ST17) in Canada. These isolates were resistant to ciprofloxacin and lincomycin. Resistance to ciprofloxacin was confirmed by amino acid substitutions in DNA gyrase (gyrA/gyrB) and topoisomerase IV (parC/parE) genes. High-level gentamicin, -kanamycin and -streptomycin resistances and macrolides resistance were also observed. The frequency of tetracycline resistance was high whereas vancomycin resistance was not detected. Also, no resistance was observed to linezolid and quinupristin-dalfopristin antibiotics. Data demonstrated the complete absence of enterococcal surface protein (esp) and hyaluronidase (hyl) genes among the canine AREF isolates tested while all were acm (collagen adhesin from E. faecium) positive. However, most of them were shown to harbor efaAfm gene, encoding for a cell wall adhesin. No biofilm formation or clustered regularly interspaced short palindromic repeats (CRISPR) elements were identified in these canine AREF isolates. rep6 and rep11 families of plasmids were significantly associated with isolates from dogs. The PFGE patterns of human and dog isolates were considered unrelated (≤ 80%). These findings also support the importance of prudent use of antibiotics in veterinary medicine to avoid zoonotic spread of canine AREF isolates. We are confident that our results may help to better understand the mechanisms of antibiotic resistance and mobile element carrying them as well as new strategies to reduce the horizontal transfer of antibiotic resistance and virulence traits.