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Antibiotic resistance is a worldwide threat to human health yet fewer new antibiotics are being approved. New antimicrobial drugs are urgently required. 3 Deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS) is a target for antimicrobial drug design. KDO8PS catalyzes the condensation of D-arabinose-5 phosphate (A5P) with phosphoenolpyruvate (PEP) to produce KDO8P. KDO8PS catalyzes the first committed step in the lipopolysaccharides (LPS) biosynthesis pathway in Gram-negative bacteria and is critical for bacterial pathogenicity/virulence. We have characterized KDO8PS from Campylobacter jejuni (cjKDO8PS), a new metal-dependent KDO8P synthase (KDO8PS). cjKDO8PS is a tetramer in solution and optimally active at pH 7.5 and 60 °C. We have kinetically established that cjKDO8PS follows a rapid equilibrium sequential ordered ter ter kinetic mechanism, where Mn2+ binds first, followed by PEP, then A5P. Pi dissociates first, before KDO8P, then Mn2+. cjKDO8PS was inhibited by KDO8P oxime, a novel slow tight-binding inhibitor. KDO8P oxime is a competitive inhibitor with respect to PEP and A5P, but uncompetitive with respect to Mn2+, with Ki = 10 ± 1 μM and an ultimate Ki* = 0.28 ± 0.10 μM. KDO8P oxime has a residence time (tR) of 5 days on the enzyme, a parameter that is highly correlated to in vivo efficacy. Crystallization conditions for the cjKDO8PS‧Mn2+‧KDO8P oxime complex have been found and can be optimized to obtain a crystal structure that shows how KDO8P oxime interacts with the active sites. / Thesis / Doctor of Science (PhD) / The relentless increase in global antibiotic resistance is, regrettably, not matched with an increase in new effective antibiotics. New antimicrobial drug discovery strategies are desperately needed. Enzymes are key targets for drug design because they catalyze the majority of biological processes. In this project we sought to study and inhibit the activity of KDO8P synthase (KDO8PS) from Campylobacter jejuni, a common cause of food poisoning. KDO8P synthase is a critical enzyme involved in the lipopolysaccharide (LPS) biosynthesis in Gram-negative bacteria. The LPS acts as a permeability barrier and is crucial for bacterial pathogenicity/virulence. We found that C. jejuni KDO8PS is potently inhibited by KDO8P oxime, a novel inhibitor of KDO8PS. This inhibitor presents a unique opportunity to study these enzymes and a platform from which antibiotics against Gram-negative bacteria can be developed.

The severity of flood events is predicted to increase as a consequence of climate change and may lead to a higher burden of waterborne infectious diseases in the United States. Contaminated floodwater transports bacterial, protozoal, and viral pathogens that typically cause moderate intestinal or respiratory disease, but can also lead to more serious disseminated infections among immunocompromised, young, and older people. Hydroclimatology and drinking water infrastructure influence the transmission of disease, but their roles are not well-understood and may vary by pathogen-type or geographic region. Specific outbreaks of waterborne disease have been attributed to major floods and cases have been positively associated with some meteorological variables, but the association between infections and flooding has not been systematically examined. In this dissertation, we examine the association between seasonal and extreme floods and parasitic and bacterial infections using multiple flood-indicator variables and exposure definitions. In Chapter 2, we use multimodel inference and generalized linear mixed models to determine the effect of seasonal meteorology on hospitalizations across the US. We found that hospitalization rates were generally higher in rural areas and in places that relied on groundwater for drinking water sources. Soil moisture, precipitation, and runoff were associated with significant increases in hospitalizations for Legionnaires' disease, Cryptosporidiosis, and Campylobacteriosis, respectively. In Chapter 3, we use 23 years of weekly case data to examine the effect of cyclonic storms on six waterborne infections in a conditional quasi-Poisson statistical model. Storm exposure was defined separately for distinct storm hazards, namely wind speed and cumulative rainfall, and effects were examined over 3 weeks post-storm. We found that exposure to storm-related rainfall was associated with immediate and lagged increases in cases. In Chapter 4, we use a nonparametric bootstrap to examine the effect of anomalous meteorological conditions, i.e. extremes unrelated to cyclonic storms, on Legionnaires' disease hospitalizations. We also assess the effect of exposure to specific cyclonic storms in a GLMM framework and compare these approaches. Extreme precipitation and months with cyclonic storms were positively associated with Legionnaires' disease hospitalizations. Determining the effect of flooding on Legionnaires' disease is particularly important as it causes severe illness and has steadily increased in incidence for 20 years. An objective of this dissertation was to develop a framework for examining flood-disease dynamics in the context of hydrometeorological and infrastructure-related factors that may influence transmission. We demonstrated that drinking water source, rurality, and geography may play an important role in these dynamics; the analyses also underscored, however, the urgent need for more extensive epidemiological surveillance and water quality data. Climate change will likely place a considerable strain on aging water infrastructure in the US. A nuanced understanding of flood-disease dynamics is central to mitigating these effects.

Public health costs of foodborne campylobacteriosis are estimated to cost more than a billion dollars in the United States annually. This pathogen has been primarily associated with chicken production and processing which is a ~$33 billion industry. To further identify practices that could reduce Campylobacter presence, concentration, and persistence in chicken prior to consumption a quantitative microbial risk assessment (QMRA) was conducted for boneless, skinless chicken breast meals prepared and consumed domestically in the contiguous United States. This QMRA model was developed with @RISK software (Palisade Corp., Ithaca, NY) and data inputs can be easily modified and updated. QMRA is a powerful analytic method that can be utilized to model the dynamics between the food pathogen, food commodity, and ingestion. It provides insight into the impacts of the process in its interaction and its surrounding environment. The baseline model determined that consumption of this product resulted in annual means of infections: 328,257, illnesses: 108,174, hospitalizations: 27,754, deaths: 37, cases of Campylobacter-associated Guillain-Barré Syndrome (GBS): 1,373, and cases of Campylobacter-associated Irritable Bowel Syndrome (IBS): 9,501. The associated annual economic burdens were ~$192 million for acute campylobacteriosis, ~$77 million for GBS, and ~$96 million for IBS. The effects of targeting and modifying the baseline model's inputs within the farm-to-fork process were studied as follows: Post grow-out (1) prevalence and (2) concentration of Campylobacter on chickens at the farm prior to transport, (3) transport crate cleaning frequency prior to loading, (4) temperature storage conditions at post processing/ pre-retail, (5) the frequency of handwashing in the preparation and handling of a chicken meal, and (6) the combination Campylobacter mitigation models using the inputs from (2) and (4). Mean yearly illnesses are estimated to decrease by approximately half if on-farm Campylobacter prevalence was lowered to 35-50% from the baseline level of 76%. An additional ~50,000 illnesses can be expected if the proportion of home preparers who do not wash their hands increases from 8.3% (baseline) to 20%. The combined effects of reducing on-farm Campylobacter concentrations and increasing the proportion of product frozen in-plant have greater impacts on reducing yearly campylobacteriosis and associated costs than either intervention alone. / Doctor of Philosophy / Campylobacter is a major foodborne bacterial pathogen that is not well known by the general public, although public health costs are estimated to cost more than a billion dollars in the United States annually. Chicken production is a ~$33 billion industry that is affected by the contamination of this pathogen. In this study, a quantitative microbial risk assessment (QMRA) was performed to study the impact of Campylobacter on chicken meals prepared in the home. Societal and economic burdens were evaluated as well. The models developed provide comprehensive simulations that describe the spread, persistence, and the concentration of Campylobacter throughout the farm to fork process in the annual consumption of boneless, skinless chicken breast meals prepared and consumed domestically in the contiguous United States. Additional simulation models were created to compare methods for reducing Campylobacter along the food chain that could lead to fewer cases of campylobacteriosis in the United States, or what could happen if there was a breakdown in a hygiene step in the preparation of the chicken meal.

Une exposition aux viandes comporte un risque pour la santé, et les maladies transmises par ces viandes causent un fardeau important mondialement. En Afrique centrale, le gibier est une viande communément consommée en zone urbaine. L’absence d’information sur le niveau de consommation de gibier, ainsi que sur sa contamination, limite l’évaluation des risques sanitaires associés au gibier. Une étude transversale a visé la description du niveau de consommation des viandes parmi 205 ménages de Port-Gentil (Gabon), ainsi que certains déterminants de la consommation de ces viandes. Une seconde étude transversale a quantifié la contamination musculaire de gibier vendu à Port-Gentil par Salmonella, Campylobacter et Shigella. Sur une base de trois jours, 86% des ménages ont consommé de la volaille, 84% du poisson, 44% du bœuf, 25% du porc et 24% du gibier. La consommation de gibier fut plus fréquente le dimanche et parmi les ménages à revenu élevé. Le gibier fut principalement acquis en carcasse entière sans conservation particulière, mais toujours consommé bouilli. Des trois bactéries ciblées, seule Salmonella a été isolée parmi un de 128 échantillons de gibier. Ces études fournissent des informations utiles pour mieux comprendre les facteurs de risque pour la santé associés à la consommation de viandes au Gabon. Des études sur la contamination des viandes, notamment celles des carcasses de gibier, seront nécessaires pour mieux apprécier les risques spécifiques à chaque différente bactérie pathogène. / Meat poses some risks to human health and meat-borne diseases constitute a high burden worldwide. In central Africa, bushmeat is commonly consumed in the urban setting. A lack of information on bushmeat consumption and contamination limits the evaluation of risks to human health linked to bushmeat. A cross-sectional survey was conducted among 205 households of Port-Gentil (Gabon) to quantify relative consumption levels of different meat types and to explore certain determinants of meat consumption. A separate cross-sectional study aimed to determine the prevalence of Campylobacter, Salmonella and Shigella within bushmeat sold in markets of Port-Gentil. Based on a three-day recall period, 86% of household consumed poultry compared to 84% for fish, 44% for beef, 25% for pork and 24% for bushmeat. Bushmeat consumption was more important on Sundays and within high monthly income households. Most bushmeat was acquired as whole carcasses without formal meat conservation methods, but all bushmeat was boiled prior to consumption. One Salmonella was detected among one of 128 bushmeat samples, whereas no Campylobacter or Shigella were detected. This study provides useful information to help better understand risk factors associated with the consumption of bushmeat in Gabon. Further studies on bacterial contamination of meat, including bushmeat carcasses, are required to better understand potential health risks specific to different bacterial pathogens.

Campylobacter spp., principalmente as espécies C. coli e C. jejuni, são a causa mais comum de doença bacteriana veiculada por alimentos na Europa, Estados Unidos e alguns outros locais do mundo. No Brasil, há uma escassez de estudos de C. coli, o que dificulta avaliar a dimensão do envolvimento dessa bactéria como causadora de doença nos seres humanos e em animais, bem como, determinar o impacto de sua presença em alimentos e no meio ambiente. O objetivo desse trabalho foi caracterizar molecularmente linhagens de C. coli isoladas de origens diversas no Brasil pela pesquisa da presença de genes relacionados à virulência por PCR, perfil de sensibilidade a antimicrobianos e pela análise da similaridade genotípica por métodos de tipagem molecular. Adicionalmente, o Índice de Discriminação (D) de tais metodologias foi verificado. Foram estudadas 63 linhagens de C. coli, isoladas de humanos (12), animais (21), alimentos (10) e ambiente (20), entre os anos de 1995 e 2011, nos Estados do Rio de Janeiro, São Paulo e Minas Gerais. Todas as linhagens apresentaram os genes flaA, cadF e sodB. O gene cdtB foi detectado em 20 (31,7%) linhagens, o gene flhA foi detectado em 11 (17,5%) linhagens, o gene dnaJ foi encontrado em 10 (15,9%) linhagens, o gene pldA foi detectado em sete (11,1%) linhagens, o gene iamA foi detectado em três (4,8%) linhagens, os genes cdtC e docA foram encontrados em duas (3,2%) linhagens, os genes cdtA e crsA foram encontrados em uma (1,6%) linhagem e os genes ciaB, wlaN, virB11 e racR não foram detectados. Dentre as 63 linhagens estudadas, 42 foram susceptíveis a todos os antimicrobianos testados. Das 21 linhagens resistentes, 10 (15,9%) foram resistentes a tetraciclina e doxaciclina, seis (9,5%) foram resistentes a ciprofloxacina e uma (1,6 %) foi resistente a eritromicina. Somente quatro (6,3%) linhagens foram resistentes a pelo menos duas diferentes classes de antibióticos testados simultaneamente. O dendrograma de similaridade genética de Pulsed field gel electrophoresis (PFGE) agrupou as 63 linhagens estudadas em dois grupos principais denominados PFGE-A e PFGE-B com similaridade genômica de 44,9% entre eles. Entretanto, algumas linhagens isoladas de humanos, animais, ambiente e alimentos apresentaram uma alta similaridade genotípica acima de 80% entre elas e, foram agrupadas em sete subgrupos denominados PFGE-A1 a PFGE-A7. O dendrograma de similaridade genômica das sequências da SVR do gene flaA agrupou as linhagens ii estudadas em dois grupos principais designados SVR-A e SVR-B, com similaridade acima de 83,1 % entre eles. Ademais, o depósito das sequências da SVR do gene flaA no banco de dados online demonstrou que os alelos 30 e o 1647 foram os mais frequentemente encontrados e permitiu a comparação das linhagens estudadas com os alelos descritos no banco de dados. Sete alelos, dentre os 22 encontrados, não haviam sido previamente descritos. A análise do locus CRISPR por HRMA dividiu as linhagens de C. coli em quatro diferentes perfis de melting. O Multilocus sequence typing (MLST) foi utilizado para tipar 20 linhagens de C. coli e foram obtidos 18 STs diferentes dos quais apenas dois já haviam sido previamente descritos. O D das metodologias de PFGE, sequenciamento da SVR do gene flaA, análise do locus CRISPR por HRMA e MLST foi de 0,986, 0,916, 0,550 e 0,989, respectivamente. Pode- se concluir que o potencial patogênico das linhagens de C. coli não foi evidenciado o que pode estar relacionado ao fato da maioria dos estudos envolvendo patogênese terem sido realizados para a espécie C. jejuni. Algumas linhagens apresentaram-se resistentes aos antimicrobianos testados, o que é preocupante uma vez que tais linhagens podem disseminar genes de resistência a outras isoladas de diversas fontes. Os resultados gerados pelos métodos de tipagem molecular por PFGE e sequenciamento da pequena região variável (SVR) do gene flaA demonstraram uma alta similaridade genotípica entre algumas linhagens de C. coli, sugerindo que uma possível contaminação tenha ocorrido entre linhagens isoladas de fontes clínicas e não clínicas ao longo de 16 anos no Brasil. Ademais, a análise dos alelos da SVR do gene flaA nos permitiu concluir que os alelos prevalentes nas linhagens estudadas diferem daqueles encontrados nos países Europeus. Os dados obtidos por MLST sugerem que as linhagens estudadas possuem uma grande diversidade genética entre si e em comparação com as linhagens isoladas em diferentes locais do mundo. Finalmente, as técnicas de MLST e PFGE foram as mais eficientes e adequadas na genotipagem das linhagens de C. coli estudadas. / Campylobacter spp., mainly the C. coli and C. jejuni species, are the most common cause of bacterial disease conveyed by food in Europe, United States, and other places worldwide. In Brazil, there is a paucity of studies on C. coli, 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 molecularly characterize C. coli strains isolated from diverse origins in Brazil by searching for the presence of virulence-related genes by PCR, antimicrobial sensitivity profile, and analysis of the genotypic similarity by molecular typing methods. Addicionaly, the Discriminatory Index (D) of those methodologies was acessed. Sixty-three C. coli strains isolated from humans (12), animals (21), food (10), and the environment (20) between 1995 and 2011, in the States of Rio de Janeiro, São Paulo, and Minas Gerais were studied. All strains presented the flaA, cadF and sodB genes. The cdtB gene was detected in 20 (31.7%) strains; the flhA gene was detected in 11 (17.5%) strains; the dnaJ gene was detected in 10 (15.9%) strains; the pldA gene was detected in 7 (11.1%) strains ; the iamA gene was detected in three (4.8%) strains; the cdtC and docA genes were found in two (3.2%) strains; the cdtA and crsA were found in one (1.6%) strain and the ciaB, wlaN, virB11 and racR genes were not detected. Among the 63 strains studied, 42 were susceptible to all antimicrobials tested. Of the 21 resistant strains, 10 (15.9%) were resistante to tetracycline and doxaciclyne, six (9.5%) showed resistance to ciprofloxacin, and one (1.6%) was resistant to erythromycin. Only four (6.3%) strains were simultaneously resistant to at least two different classes of the antibiotics tested. The dendrogram of genetic similarity of Pulsed field gel electrophoresis (PFGE) grouped the 63 strains studied into two groups namely PFGE-A and PFGE-B with a genomic similarity of 44.9% among them. However, some strains isolated from humans, animals, the environment and food presented a high genotypic similarity above 80% and were subdivided into seven groups designated as PFGE-A1 to PFGE-A7. The dendrogram of genetic similarity of the SRV-flaA gene sequences grouped the strains studied into two groups namely SVR-A and SVR-B, with similarity above 83.1% among them. Besides, the deposit of the SVR sequences of the flaA gene in the online database showed that the alleles 30 and 1647 were the iv most frequently found and allowed the comparison between the strains studied with the alleles described in the database. Seven alleles, among the 22 found have never been described before. The CRISPR locus analysis divided the C. coli strains into four different melting profiles. The Multilocus sequence typing (MLST) was used to type 20 C. coli strains and revealed 18 different STs among which just two had been previously described. The D of PFGE, SVR- flaA sequence, HRMA of CRISPR locus analysis and MLST was 0.986, 0.916, 0.550 and 0.989, respectively. In conclusion, the pathogenic potential of the C. coli strains was not highlighted, which could be related to the fact that the majority of the pathogenicity studies were performed with C. jejuni species. Some strains showed resistance to the antibiotics tested what is a concern once those strains may spread the resistance genes to other strains isolated from different sources. The results obtained by PFGE and SVR-flaA sequence showed a high genomic similarity among some C. coli strains which may suggest that a possible contamination may have occurred among clinical and non-clinical sources during 16 years in Brazil. Furthermore, the analysis of SVR- flaA alleles allowed the conclusion that the prevalent alleles in the strains studied were different from those found in European countries. The data obtained by MLST suggests that the strains studied had a high genomic diversity among them and in comparison with strains isolated from different places worldwide. Finally, the MLST and PFGE technicques were the most efficient and adequate in genotyping the C. coli strains studied.

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.

As infecções causadas por Campylobacter spp. são relatadas como causa freqüente de gastrenterites de origem alimentar em vários países do mundo. As espécies bacterianas termofílicas pertencentes ao gênero Campylobacter, principalmente Campylobacter jejuni e Campylobacter coli, têm sido isoladas de fezes de animais e estão associadas à contaminação da carne durante o processo de abate. Estas duas espécies são as mais freqüentemente envolvidas nos casos de campilobacteriose humana veiculada por alimentos. O presente estudo pretendeu avaliar a presença e a população de Campylobacter spp. no abate de bovinos e cortes refrigerados de aves e bovinos comercializados na cidade de São Paulo/SP. Um total de 198 animais foi amostrado no couro logo após a sangria, na carcaça imediatamente após a esfola e após a evisceração. As amostras foram obtidas através da técnica de swab na região do peito abrangendo uma área de 400 cm2. Foram analisados também 120 cortes refrigerados de frango e 100 cortes de carne bovina, assim distribuídos: 40 amostras de asa, 20 de coxa com sobrecoxa, 20 de coxa, 20 de coxinha da asa, 20 amostras de peito; 20 de patinho bovino (M. biceps femoris), 20 de contrafilé (M. longissimus dorsi), 20 de coxão mole (M. semi membranosus), 20 de lagarto (M. semitendinosus) e 20 de alcatra (M. glutaes medius). As amostras foram analisadas segundo os métodos ISO 10272-1 e 2 e os isolados obtidos foram confirmados como Campylobacter pela técnica de PCR. Campylobacter foi isolado em 22,7% (45/198) das amostras de couro bovino, ou seja, apenas no ponto antes da esfola, e C. jejuni foi a única espécie encontrada. Nas amostras de cortes de frango Campylobacter foi isolado em 14,2% (17/120) das amostras. A espécie prevalente em frangos foi C. coli (88%), seguido de C. jejuni (12%). Campylobacter spp. não foi isolado dos cortes bovinos. A população de Campylobacter spp. foi < 13 UFC/cm2 em carcaças bovinas, < 2 UFC/g em amostras de frango e < 10 UFC/cm2 em cortes bovinos. A susceptibilidade de 120 isolados de frango e couro bovino foi determinada frente a 8 agentes antimicrobianos usando o método de disco-difusão. A resistência às quinolonas (ác. nalidíxico e ciprofloxacina) foi frequentemente observada nas cepas de C. jejuni (72,2%) e C. coli (50,8%) isoladas dos frangos. Entre os isolados de C. jejuni obtidos do couro bovino maior taxa de resistência foi observada para estreptomicina (32%), seguida da eritromicina (16%) e do ácido nalidíxico (14%). / Campylobacter spp. infections are reported as a frequent cause of foodborne gastroenteritis in many countries. The thermophilic bacterial species belonging to the genus Campylobacter, particularly Campylobacter jejuni and Campylobacter coli have been isolated from feces of animals and are associated with the contamination of meat during the slaughtering process. These two species are the most frequently involved in cases of human campylobacteriosis conveyed by food. The aim of the present study was to evaluate the presence and population of Campylobacter spp. during cattle slaughter and in refrigerated chicken and beef cuts commercialized in the city of Sao Paulo/SP. A total of 198 animals were sampled in the hide after bleeding, the carcass immediately after skinning and after evisceration. Samples were obtained by swab technique in the chest area encompassing an area of 400 cm2. We also analyzed 120 refrigerated chicken cuts and 100 beef cuts. The samples were analyzed according to ISO 10272-1 and 2 methods and the isolates were confirmed as Campylobacter by PCR technique. Campylobacter was isolated only in the hide samples (45/198), and C. jejuni was the only species found. Campylobacter was isolated in 14.2% (17/120) of chicken samples. The most prevalent species in chickens was C. coli (88%), followed by C. jejuni (12%). Campylobacter spp. was not isolated from beef cuts. The counts of Campylobacter spp. was < 13 CFU/cm2 in bovine carcasses, < 2 CFU/g in chicken samples and < 10 CFU/cm2 in beef cuts. The susceptibility to 8 antimicrobial agents of 120 isolates of chicken and bovine hide was determined using the disk-diffusion method. The resistance to quinolones (ciprofloxacin and nalidixic acid) was frequently observed in strains of C. jejuni (72.2%) and C. coli (50.8%) isolated from chickens. Among strains of C. jejuni obtained from bovine hide highest resistance rate was observed to streptomycin (32%), followed by erythromycin (16%) and nalidixic acid (14%).

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-07-29T11:13:47Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PesquisaCampylobactersppGranjas.pdf: 649227 bytes, checksum: c39eeff01123d4d81e68a9da4c4b01ec (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-08-28T14:41:49Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PesquisaCampylobactersppGranjas.pdf: 649227 bytes, checksum: c39eeff01123d4d81e68a9da4c4b01ec (MD5) / Made available in DSpace on 2014-08-28T14:41:49Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PesquisaCampylobactersppGranjas.pdf: 649227 bytes, checksum: c39eeff01123d4d81e68a9da4c4b01ec (MD5) Previous issue date: 2007 / As infecções de origem alimentar no homem, causadas por Campylobacter spp., resultam em grandes perdas econômicas e estão relacionadas à produção e o abate de frangos, etapas importantes na disseminação dessas bactérias. Baseando-se na importância do Campylobacter spp. na saúde pública e tendo em vista os dados constantes na literatura de que as aves comercializadas estão constantemente contaminadas com esse agente, sentiu-se a necessidade de realizar um estudo envolvendo a criação e o abate de frangos na região amazônica para que medidas profiláticas e de controle possam ser adotadas. O trabalho teve como objetivo estudar a ocorrência de Campylobacter spp. em granjas e abatedouro avícolas na mesorregião metropolitana de Belém – PA; isolar e identificar as espécies de Campylobacter spp. e identificar as fontes de contaminação nas granjas e os pontos críticos no abate. Foi coletado um total de 120 amostras em três granjas avícolas: 30 amostras de “swab” cloacal, 30 amostras de cama de frango, 30 amostras de ração e 30 amostras de água dos bebedouros. No abatedouro, foram colhidas 126 amostras: 36 amostras de água em 12 pontos diferentes da linha de abate e mais 30 amostras de pele do conjunto peito/ pescoço, 30 amostras de fígado e 30 amostras de moela. As amostras foram colhidas entre os meses de janeiro e maio de 2007 para o isolamento e identificação das espécies de Campylobacter spp. As amostras foram processadas na Seção de Bacteriologia e Micologia do Instituto Evandro Chagas – IEC da Secretaria de Vigilância em Saúde (SVS), Ministério da Saúde, Ananindeua – PA. Campylobacter spp. foi isolado em 33,3% (40/120) das amostras das granjas. Não houve diferença significativa (p>0,05) entre os percentuais de isolamentos positivos entre as três granjas pesquisadas. Ao analisar as freqüências dos isolados de Campylobacter spp. para cada tipo de amostra das granjas, observou-se que 96,6% (29/30) das amostras de “swab” cloacal, 33,3% (10/30) das amostras de cama e 3,3% (1/30) das amostras de água foram positivas para Campylobacter spp. Não foi isolada a bactéria nas amostras de ração. Campylobacter jejuni foi identificado bioquimicamente em 82,5% (33/40) das cepas isoladas nas granjas. No abatedouro, todas as cepas isoladas foram identificadas como C. jejuni., sendo isolado a bactéria em 8,73% (11/126) das amostras provenientes da linha de abate. Ao analisar as freqüências dos isolados de C. jejuni para cada tipo de amostra do abatedouro, observou-se que 27,8% (10/36) das amostras de água foram positivas para C. jejuni, seguido pela moela com 3,3% (1/30) das amostras positivas. Não foi isolado Campylobacter spp. nas amostras de fígado e de pele do conjunto peito/ pescoço. Houve diferença significativa (p<0,0001) entre os isolamentos positivos, negativos e os tipos de amostras processadas nas granjas e no abatedouro. As principais fontes de contaminação nas granjas foram o “swab” cloacal, a cama e, em menor escala, a água. Os principais pontos críticos observados no abatedouro foram a água, seguido pela moela. C. jejuni foi identificado em elevado percentual entre as cepas isoladas nas granjas e em todas as cepas do abatedouro. / The human infections of food origin caused by Campylobacter spp. result in high economic losses and these infections are correlated to poultry flock and slaughter, important steps in Campylobacter spp. dissemination. Based on the importance of this microorganism in public health and in the literature data that show the high poultry contamination by Campylobacter spp., we realize a study involving the flock and the poultry slaughter in Amazon region for adoption of prophylactic and control measures. The objective of this work was to investigate the occurrence of Campylobacter spp. in poultry flock and slaughterhouse in Amazon region and to isolate and to identify the Campylobacter species and to identify the sources of infection in the flock and the critical points in the slaughterhouse. We collected 120 samples in three flocks: 30 cloacal swab samples, 30 poultry litter samples, 30 feed samples and 30 water samples. In the slaughterhouse, 126 samples were collected: 36 water samples from 12 different points in the abattoir, 30 neck/ chest skin samples, 30 liver samples and 30 gizzard samples. The samples were collected from January to May 2007. The samples were processed in the Bacteriology and Micology Laboratory in Evandro Chagas Institute – Health Surveilance Office – Brazil’s Health Ministery. Campylobacter spp. was isolated in 33,3% (40/120) flock samples. There was no significant difference (p>0,05) between positive isolates in the three flocks. Campylobacter spp. was isolated in 96,6% (29/30) cloacal swab samples, 33,33% (10/30) poultry litter samples and 3,3% (1/30) water samples. There were no positive samples in the feed. Campylobacter jejuni was identified by biochemistry reactions in 82,5% (33/40) of isolates from flocks. In the slaughterhouse, all isolates were identified as C. jejuni. This microorganism was isolated in 8,73% (11/126) of slaughterhouse samples. C. jejuni was isolated in 27,8% (10/36) water samples and in 3,3% (1/30) gizzard samples. Campylobacter spp. was not isolated in liver samples neather in neck/ chest skin samples. There was significant difference (p<0,0001) between positive, negative isolates and among all kinds of samples collected in the flocks and at the slaughterhouse. The infections sources identified in the flocks were the cloacal swab, the poultry litter and the water. In the slaughterhouse, the critical points identified were the water and the gizzard. C. jejuni was identified in high levels in the flocks and in all isolates from the slaughterhouse.