The species of Brucella were isolated and characterized almost 120 years ago and their genomes sequenced for almost 4 years. Compared to other bacterial pathogens relatively, little is known about the factors contributing to their persistence in hosts and multiplication within phagocytic cells. Also, many aspects of the interactions between Brucella and its host remain unclear. Molecular characterization of intracellular survival processes of Brucella will provide guidance for additional prevention and control measures. One of the features that distinguishes Brucella is that they do not express classic virulence factors. Thus identification of virulence factors has been elusive and some of the identified virulence genes are putative. Disruption of putative virulence genes and studying the consequent effect on attenuation in cell lines or mouse models is a widely used method. However, in most cases it is not apparent whether the mutated genes encode virulence factors or merely affect normal metabolic or biological functions. Some mutations in Brucella can be compensated by redundancy or backup mechanisms. One method for identifying putative virulence genes involved in pathogenesis is to express these genes in a nonpathogenic host and isolate recombinants with increased virulence or survival ability either in cell culture or animal model. We hypothesize that over-expression of Brucella putative virulence genes in the non-pathogenic and close phylogenic relative Ochrobactrum anthropi should enhance its survival in infection models in vivo. O. anthropi is one of the closest Brucella relatives based on DNA, rRNA, and protein analyses but it is unable to establish chronic infection and considered as opportunistic pathogen that, under certain circumstances, may produce disease in immunocompromised humans. Therefore, we established enhanced expression system in Brucella and Ochrobactrum to identify B. suis virulence genes. We created an enhanced expression system that can be used for cloning and expression of heterologous genes in Brucella and Ochrobactrum. We studied the transcriptional activity of several promoters and created some tools to enhance the expression, detection and purification of Brucella recombinant protein in Ochrobactrum. The presumable importance of alkyl hydroperoxide reductases encoded by ahpC and ahpD genes and their contribution to intracellular survival of Brucella were studied by over-expressing them. The recombinant O. anthropi expressing B. suis ahpC and ahpD genes were able to resist in vitro killing by H2O2 and or cumene hydroperoxide and survived longer in the macrophage J774 A.1 cell line. The control O. anthropi was cleared from BALB/c mice in five days while the recombinants were recovered from spleens, livers and lungs of infected mice up to eight days post-infection. We tested the contribution of B. suis cyclic glucan synthetase gene (cgs) to virulence by over-expressing it in O. anthropi. We studied the ability of the recombinant O. anthropi to resist killing in vitro and in vivo. We generated evidence that B. suis cgs when over-expressed in O. anthropi increased the amount of cyclic glucans synthesized and accumulated in the periplasmic space. This accumulation changed the virulence of the microorganism from a soil bacterium that cleared from mice in less than five days into a pathogenic organism that could survive up to 9 days and at higher doses killed the mice. In summary, several vectors have been constructed for gene expression and protein purification in Brucella and Ochrobactrum. Novel useful tools for enhancement of heterologous gene expression were created and demonstrated to work in Brucella and Ochrobactrum. Brucella putative virulence genes were studied in Ochrobactrum using the newly constructed vectors and tools. Ochrobactrum as a gain of function model for studying putative virulence genes of intracellular pathogens in general and for Brucella in particular proved to be a very useful model. / Ph. D.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-11-28T20:35:22Z No. of bitstreams: 2 Tese - Tatiane - Martins Rocha - 2013.pdf: 1983475 bytes, checksum: 7f4a4f5521b66a11dfd3ff763f5761b1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Rejected by Cláudia Bueno (claudiamoura18@gmail.com), reason: Conforme conversamos. on 2014-12-01T15:20:24Z (GMT) / Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-12-01T15:49:23Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Tatiane - Martins Rocha - 2013.pdf: 1983475 bytes, checksum: 7f4a4f5521b66a11dfd3ff763f5761b1 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-12-04T14:23:27Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Tatiane - Martins Rocha - 2013.pdf: 1983475 bytes, checksum: 7f4a4f5521b66a11dfd3ff763f5761b1 (MD5) / Made available in DSpace on 2014-12-04T14:23:27Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Tatiane - Martins Rocha - 2013.pdf: 1983475 bytes, checksum: 7f4a4f5521b66a11dfd3ff763f5761b1 (MD5) Previous issue date: 2013-12-13 / (Sem resumo em outra língua) / O presente trabalho foi desenvolvido com objetivos de detectar genes de virulência em Escherichia coli isoladas de órgãos de frangos de corte de criações industriais e alternativas, bem como identificar o perfil de resistência aos antimicrobianos utilizados em medicina humana e veterinária. Para tanto foram coletadas 496 amostras de órgãos de 32 lotes em abatedouros de diferentes regiões do Estado de Goiás com lesões de hepatite, pericardite, aerossaculite e celulite. E. coli foi identificada em 76,21% (378/496) das amostras pesquisadas com os seguintes resultados: 76,92% (80/104) em hepatite, 73,88% (99/134) em pericardite, 77,95% (99/127) em aerossaculite e 77,95% (100/131) em celulite. Nos isolados de E. coli, foi feita a pesquisa dos genes papC, tsh, iuc e iss. O gene tsh e o iuc foram os que apresentaram maiores valores, 44,4% (32/72) e 45,8% (33/72), respectivamente. Para a detecção simultânea dos genes, o maior valor (p<0,05) foi observado para tsh-iuc com 12,5% (9/72), entre tsh-iuc-iss com 11,1% (8/72) e entre os quatro genes deste estudo com 13,9% (10/72). Em criações alternativas, foram coletadas amostras de 32 propriedades onde aves não apresentavam quadros respiratórios e 32 onde apresentavam quadros respiratórios. Durante as necropsias, as alterações clínicas e anatomopatológicas foram avaliadas e fragmentos de fígado e coração, bem como conteúdo intestinal e suabes de traquéia e sacos aéreos foram coletados de cada ave. E. coli foi isolada de aves sem quadro respiratório em 5,3% (5/95), 7,4% (7/95), 4,2% (4/95), 3,2% (3/95) e 3,2% (3/95) em amostras de fígado, coração, sacos aéreos, intestino e traquéia respectivamente. Em aves com sinais respiratórios, E. coli foi identificada em 22,7% (35/154), 18,2% (28/154), 22,7% (35/154), 18,3% (29/154) e 18,3% (29/154) das amostras de fígados, coração, sacos aéreos, intestino e traquéia respectivamente. As frequências de isolamento de E. coli e de achados anatomopatológicos à necropsia foram menores (p<0,05) para criações sem quadro respiratório quando comparadas as com quadro respiratório. Para o grupo sem quadro respiratório, diferiram a detecção entre iuc, 36,4% (8/22), e iss, 9,1% (2/22). A detecção simultânea entre iuc-iss apresentou (p<0,05) os maiores resultados, 10,1% (7/69), para o grupo com quadro respiratório. Em relação à determinação do perfil de resistência e a presença do iss, para amostras de frangos de corte industrial, os maiores valores foram para doxiciclina, oxitetraciclina e enrofloxacina quando o iss esteve presente. Em isolados de frangos de corte de criações alternativas, quando o gene iss esteve presente, obteve-se maior percentual de resistência para ampicilina e ciprofloxacina. Conclui-se que os genes pap, tsh, iuc e iss estiveram presentes em isolados de E. coli, determinando maior atuação quando ocorreu associação entre pelo menos dois genes e que os níveis de resistência aos antimicrobianos foram elevados em isolados de E. coli independente do sistema de criação das aves.

E. coli avec potentiel zoonotique pourrait éclore dans les réservoirs porcins et avicoles. Cette étude consiste à examiner la présence de souches E. coli porteuses de gènes virulents associés aux STEC (E. coli producteurs de Shiga-toxines), EPEC (E. coli entéropathogène), et ExPEC (E. coli pathogène extra-intestinal) chez les porcs et volailles élevés au Vietnam. Des prélèvements d’excréments et de carcasses ont été effectués dans des fermes et abattoirs porcins et avicoles sélectionnés où les animaux ont été suivis de l’élevage à l’abattage. Un total de 13,1% des souches, toutes sources confondues, ont été catégorisées comme potentiellement contaminées par ExPEC, possédant un ou plusieurs gènes de virulence iucD, tsh, papC et cnf. Peu d’isolats d’autres pathotypes ont été observés. Tous les gènes de virulence ExPEC, à l’exception de cnf, ont été identifiés plus fréquemment dans les isolats de fèces et carcasses avicoles que dans les isolats porcins. Même constatation pour le groupe du phylogénétique D. Une multirésistance aux médicaments a été régulièrement observée chez les deux isolats ExPEC. Les isolats de fèces de volailles ont souvent été associés à une résistance à l’acide nalidixique et à la ciprofloxacine (P<0.05), de même qu’au gène blaTEM, alors que les gènes qnr et aac(6’)-Ib ont peu été rencontrés des deux côtés. Cette étude démontre que les isolats ExPEC avicoles sont potentiellement plus pathogèniques que ceux porcins et que les isolats ExPEC de carcasses porcines et avicoles peuvent provenir de leurs excréments par la contamination associée au processus d'abattage. Ainsi, la volaille, particulièrement, serait un facteur de transmission de souches ExPEC zoonotiques. / Zoonotic potential pathogenic Escherichia coli could arise from poultry and pig reservoirs. The aim of this study is to investigate the occurrence of E. coli strains carrying virulence genes associated with STEC (Shiga toxin-producing E. coli), EPEC (Enteropathogenic E. coli), and ExPEC (Extraintestinal pathogenic E. coli) in pigs and poultry on-farm and at abattoirs in Vietnam. Samples of feces and carcasses were collected at selected pig and poultry farms and abattoirs, in which animals were traced from farms to the abattoir. A total of 13.1% strains from all sources were classified as potential ExPEC, possessing one or more virulence genes iucD, tsh, papC and cnf. Few isolates of other pathotypes were observed. All ExPEC virulence genes, except cnf, were more frequently found in isolates from poultry than in isolates from pigs. A higher proportion of ExPEC isolates belonging to phylogenetic group D was observed in poultry. Multi-drug resistance was frequently observed in ExPEC isolates from both pigs and poultry. Nalidixic acid and ciprofloxacin resistance were significantly associated with poultry feces isolates (P<0.05). blaTEM gene was more frequently associated with poultry isolates, whereas qnr and aac(6’)-Ib genes were present at low prevalence in pig and poultry isolates. This study demonstrates that poultry ExPEC isolates are potentially more pathogenic than pig ExPEC isolates, and ExPEC isolates in pig and poultry carcasses may originate from pig and poultry feces, due to contamination associated to slaughtering process. Thus, meats particularly from poultry, might be a vehicle for transmission of zoonotic ExPEC strains.

E. coli avec potentiel zoonotique pourrait éclore dans les réservoirs porcins et avicoles. Cette étude consiste à examiner la présence de souches E. coli porteuses de gènes virulents associés aux STEC (E. coli producteurs de Shiga-toxines), EPEC (E. coli entéropathogène), et ExPEC (E. coli pathogène extra-intestinal) chez les porcs et volailles élevés au Vietnam. Des prélèvements d’excréments et de carcasses ont été effectués dans des fermes et abattoirs porcins et avicoles sélectionnés où les animaux ont été suivis de l’élevage à l’abattage. Un total de 13,1% des souches, toutes sources confondues, ont été catégorisées comme potentiellement contaminées par ExPEC, possédant un ou plusieurs gènes de virulence iucD, tsh, papC et cnf. Peu d’isolats d’autres pathotypes ont été observés. Tous les gènes de virulence ExPEC, à l’exception de cnf, ont été identifiés plus fréquemment dans les isolats de fèces et carcasses avicoles que dans les isolats porcins. Même constatation pour le groupe du phylogénétique D. Une multirésistance aux médicaments a été régulièrement observée chez les deux isolats ExPEC. Les isolats de fèces de volailles ont souvent été associés à une résistance à l’acide nalidixique et à la ciprofloxacine (P<0.05), de même qu’au gène blaTEM, alors que les gènes qnr et aac(6’)-Ib ont peu été rencontrés des deux côtés. Cette étude démontre que les isolats ExPEC avicoles sont potentiellement plus pathogèniques que ceux porcins et que les isolats ExPEC de carcasses porcines et avicoles peuvent provenir de leurs excréments par la contamination associée au processus d'abattage. Ainsi, la volaille, particulièrement, serait un facteur de transmission de souches ExPEC zoonotiques. / Zoonotic potential pathogenic Escherichia coli could arise from poultry and pig reservoirs. The aim of this study is to investigate the occurrence of E. coli strains carrying virulence genes associated with STEC (Shiga toxin-producing E. coli), EPEC (Enteropathogenic E. coli), and ExPEC (Extraintestinal pathogenic E. coli) in pigs and poultry on-farm and at abattoirs in Vietnam. Samples of feces and carcasses were collected at selected pig and poultry farms and abattoirs, in which animals were traced from farms to the abattoir. A total of 13.1% strains from all sources were classified as potential ExPEC, possessing one or more virulence genes iucD, tsh, papC and cnf. Few isolates of other pathotypes were observed. All ExPEC virulence genes, except cnf, were more frequently found in isolates from poultry than in isolates from pigs. A higher proportion of ExPEC isolates belonging to phylogenetic group D was observed in poultry. Multi-drug resistance was frequently observed in ExPEC isolates from both pigs and poultry. Nalidixic acid and ciprofloxacin resistance were significantly associated with poultry feces isolates (P<0.05). blaTEM gene was more frequently associated with poultry isolates, whereas qnr and aac(6’)-Ib genes were present at low prevalence in pig and poultry isolates. This study demonstrates that poultry ExPEC isolates are potentially more pathogenic than pig ExPEC isolates, and ExPEC isolates in pig and poultry carcasses may originate from pig and poultry feces, due to contamination associated to slaughtering process. Thus, meats particularly from poultry, might be a vehicle for transmission of zoonotic ExPEC strains.

Les objectifs de cette étude ont été de : (1) déterminer s’il existe une association entre la présence intra-utérine d'Escherichia coli dans la 1 ère semaine postpartum et le développement de la métrite postpartum, (2) déterminer s’il y a une association entre les gènes de virulence d'E. coli et la métrite postpartum, et (3) d'évaluer si les analyses bactériologiques (bactéries et gènes de virulence d'E. coli) pourraient prédire la métrite postpartum chez la vache laitière. Des écouvillons utérins ont été prélevés dans la première semaine postpartum sur 486 vaches de race Holstein et soumis au laboratoire pour détection de E. coli. Les gènes de virulence d'E. coli ont été identifiés par la technique d'hybridation des sondes radioactives. Un total de 252 vaches (52%) ont été positives à E. coli et 67 vaches positives à la métrite postpartum (13,7%). Les vaches positives à E. coli intra-utérin dès la première semaine postpartum avaient un risque 2,6 fois plus élevé de développer la métrite postpartum que les vaches sans E. coli. La plupart des E. coli possédaient un ou plusieurs gènes des E. coli d'origine extra-intestinale (ExPEC) dont fimH (89%), HlyE (87%) et iss (70%). Parmi les autres gènes ExPEC, on a retrouvé sitA (23%), fepC (20%) hra1 (20%) malX (14%) tsh (11%) et bien d'autres. Les gènes de virulence kpsMTII et hra1 ont été associés à la métrite postpartum avec un rapport de cote de 4,3 chacun. La présence d'E. coli dans l'utérus avait une valeur prédictive positive de 18% tandis que la présence des gènes kpsMTII et hra1 avait une valeur prédictive positive de 36% et 31% respectivement. La détection de certains gènes de virulence d'E. coli dans les prélèvements utérins pourrait renseigner sur le risque de développement de la métrite postpartum chez la vache laitière. Les études ultérieures pourraient tester encore plus de gènes et viser à développer des tests de dépistage simple, facilement et rapidement applicable à la ferme. / The objectives of this study were to (1) determine whether there is an association between the presence of intra-uterine Escherichia coli in the first week postpartum and the occurrence of postpartum metritis in the subsequent weeks, (2) determine whether there is an association between E. coli virulence genes and postpartum metritis, and (3) to assess whether the presence of these E. coli virulence genes could predict the occurrence of postpartum metritis in dairy cows. Uterine swabs were collected in the first week postpartum from 486 Holstein cows and submitted to the laboratory for detection of E. coli. Virulence genes of E. coli were identified using the radioactive probe hybridization method. A total of 252 cows (52%) were positive for intra-uterine E. coli and 67 cows (13.7%) were positive for postpartum metritis. Cows positive for intra-uterine E. coli in the first week postpartum had 2.6 times the odds of developing postpartum metritis compared to negative cows. Most intra-uterine E. coli possessed one or more ExPEC genes, among which FimH (89%), hlyE (87%), and iss (70%). Other ExPEC genes such as sitA (23%), fePC (20%) hra1 (20%) malX (14%) tsh (11%) and others were found with low prevalence. The presence of the virulence genes kpsMTII and hra1 was associated with 4.3 times each the odds of developing postpartum metritis compared to negative cows. The presence of E. coli in the uterus had a positive predictive value of 18%, while the presence of the genes kpsMTII and hra1 had a positive predictive value of 36% and 31% respectively. The detection of certain virulence genes of E. coli in uterine swabs could inform about the risk of developing postpartum metritis in dairy cattle. Further studies could test more virulence genes and aim at developing molecular tests that would be simple, quickly and easily applicable on farm.

Shigella spp. está entre as quatro bactérias mais isoladas de fezes diarreicas no Brasil. No mundo cerca de 164,7 milhões de casos de shigelose ocorrem anualmente, sendo a maioria em países em desenvolvimento. O gênero Shigella spp. possui quatro espécies, sendo Shigella sonnei e Shigella flexneri as espécies mais frequentemente isoladas no Brasil e no mundo. O monitoramento de linhagens resistentes de Shigella spp. é essencial, pois este garante uma terapia eficiente quando necessária. Especificamente, a maioria dos estudos realizados com linhagens de S. sonnei no país verificaram apenas a ocorrência dessa e há poucos estudos que investigaram o potencial patogênico e a diversidade genotípica dessa espécie. Os objetivos desse projeto foram analisar o potencial patogênico, o perfil de resistência a antimicrobianos e a diversidade genotípica de linhagens de S. sonnei isoladas durante três décadas no Estado de São Paulo. No total foram caracterizadas 72 linhagens de S. sonnei isoladas de humanos, entre os anos de 1983 a 2014, quanto à presença de 12 genes de virulência por PCR, perfil de suscetibilidade frente a 16 antimicrobianos por disco difusão e tipagem molecular por Pulsed-field gel electrophoresis (PFGE), Enterobacterial repetitve intergenic consensus PCR (ERIC-PCR), Multiple-locus variable-number tandem-repeat analysis (MLVA) e 20 linhagens tipadas por Multi-locus sequence typing (MLST). Todas as linhagens apresentaram os genes de virulência ipaH, iuc e sigA. O gene ipaBCD foi encontrado em 14 (19%) linhagens, os genes ial e virF em 13 (18%) linhagens e o gene sen em sete (10%) linhagens. Os genes set1A, set1B, pic, sat e sepA não foram detectados. As mais altas taxas de resistência foram frente à sulfametoxazol-trimetoprim encontrada em 42 (58,3%) linhagens e frente à tetraciclina encontrada em 30 (41,6%) linhagens. Onze (15,5%) linhagens foram resistentes à ampicilina e piperacilina. Três (4,2%) linhagens foram resistentes à cefotaxima. Três (4,2%) linhagens foram resistentes ao cloranfenicol. Duas (2,8%) linhagens foram resistentes à ampicilina-sulbactam. Duas (2,8%) linhagens foram resistentes ao ácido nalidíxico. Uma (1,4%) linhagem foi resistente à amoxicilina-ácido clavulânico. Cinco (7%) linhagens foram multidroga resistentes (MDR). O dendrograma gerado pelo PFGE agrupou as 72 linhagens de S. sonnei em dois clusters designados PFGE-A e PFGE-B. O cluster PFGE-A agrupou 39 linhagens isoladas entre 1983-2014 com uma similaridade >=73,6% e mais especificamente 35 dessas linhagens apresentaram uma similaridade >=80,3%. O cluster PFGE-B agrupou 33 linhagens de S. sonnei isoladas entre 1984-2014 com uma similaridade >=74,7% e 27 dessas linhagens exibiram uma similaridade >=83,0%. Similarmente, o dendrograma gerado pelo ERIC-PCR agrupou as 72 linhagens de S. sonnei em dois clusters designados ERIC-A e ERIC-B. O cluster ERIC-A agrupou 37 linhagens isoladas entre 1983-2014 que exibiram uma similaridade >=78,8% e mais especificamente 36 dessas linhagens apresentaram uma similaridade >=82,3%. O cluster ERIC-B agrupou 34 linhagens de S. sonnei isoladas entre 1987-2014 que exibiram uma similaridade >=84,0%. Também por MLVA as linhagens foram agrupadas em dois clusters designados MLVA-A e MLVA-B. O cluster MLVA-A agrupou 31 linhagens isoladas entre 1983-2014 com uma similaridade >=40%. O cluster MLVA-B agrupou 41 linhagens isoladas entre 1983-2014 com uma similaridade >=21,6%. Todas as 20 S. sonnei foram tipadas por MLST como ST152. Conclui-se que o potencial patogênico das linhagens estudadas foi destacado pela presença de importantes genes de virulência. A alta porcentagem de resistência para alguns antimicrobianos testados, tais como, sulfametoxazol-trimetoprim e tetraciclina é preocupante e pode levar à falha terapêutica. Os resultados da tipagem molecular sugerem que existam dois subtipos prevalentes nas linhagens de S. sonnei estudadas que se diferenciaram pouco geneticamente e contaminaram humanos durante 31 anos na região metropolitana de Ribeirão Preto no Estado de São Paulo. O resultado do MLST indica que as linhagens estudadas de Shigella sonnei isoladas no Brasil descendem de um precursor comum / Shigella spp. is among the four most isolated bacteria from diarrheal faeces in Brazil. In the world about 164.7 million cases of shigellosis occur annually, mostly in developing countries. The genus Shigella spp. comprises four species, being Shigella sonnei and Shigella flexneri the most frequently isolated species in Brazil and worldwide. The monitoring of resistant strains of Shigella spp. is essential and ensures an effective therapy when necessary. Specifically, the majority of the studies with S. sonnei performed in the country verified only the occurrence of this bacterium and there are few studies that investigated the pathogenic potential and genotypic diversity of this species. The aims of this project were to analyze the pathogenic potential, antimicrobial resistance profile and genotypic diversity of S. sonnei strains isolated during three decades in the State of São Paulo. In total, 72 of S. sonnei strains isolated from humans, between the years 1983-2014, were characterized for the presence of 12 virulence genes by PCR, resistance profile against 16 antimicrobials by disk diffusion and molecular typing by Pulsed-field gel electrophoresis (PFGE), Enterobacterial repetitve intergenic consensus PCR (ERIC-PCR), Multiple-locus variable-number tandem-repeat analysis (MLVA) and 20 strains typed by Multi-locus sequence typing (MLST). All the strains contained the ipaH, iuc and sigA genes. The ipaBCD gene was detected in 14 (19%) strains, the ial and virF genes in 13 (18%) strains and the sen gene in seven (10%) strains. The set1A, set1B, pic, sepA and sat genes were not detected. The highest resistance rates were against trimethoprim-sulfamethoxazole found in 42 (58.3%) strains and against tetracycline found in 30 (41.6%) strains. Eleven (15.5%) strains were resistant to ampicillin and piperacillin. Three (4.2%) strains were resistant to cefotaxime. Three (4.2%) strains were resistant to chloramphenicol. Two (2.8%) strains were resistant to ampicillin-sulbactam. Two (2.8%) strains were resistant to nalidixic acid. One (1.4%) strain was resistant to amoxicillin-clavulanic acid. Five (7%) strains were multidrug resistant (MDR). The dendrogram generated by PFGE grouped the 72 S. sonnei strains into two clusters designated PFGE-A and PFGE-B. The PFGE-A cluster comprised, 39 S. sonnei strains isolated between 1983 and 2014 with a similarity above 73.6% and more specifically 35 of those strains exhibited a similarity >= 80.3%. The PFGE-B cluster grouped, 33 S. sonnei strains isolated between 1984 and 2014 with a similarity above 74.7%, and 27 of those strains exhibited a similarity above 83.0.Similarly, the dendrogram generated by ERIC-PCR grouped the 72 S. sonnei strains into two clusters designated ERIC-A and ERIC-B. The ERIC-A cluster comprised, 37 S. sonnei strains isolated between 1983 and 2014 that exhibited a similarity above 78.8% and specifically 36 strains of those exhibited a similarity >= 82.3%. The ERIC-B cluster grouped, 34 S. sonnei strains isolated between 1987 and 2014 that exhibited a similarity above 84.0%. Also, by MLVA strains were grouped into two clusters designated MLVA-A and MLVA-B. The MLVA-A cluster comprised 31 strains isolated between 1983 and 2014 with a similarity >=40%. The MLVA-B cluster comprised 41 strains isolated between 1983 and 2014 with a similarity >=21.6%. All the 20 S. sonnei were typed by MLST as ST152. In conclusion, the possible pathogenic potential of the strains studied was highlighted by the presence of important virulence genes. The high percentage of resistance to some of the antimicrobials tested such as trimethoprim-sulfamethoxazole and tetracycline is worrying and may lead to therapeutic failure. Molecular typing results may suggest that there are two prevalent subtypes of S. sonnei strains studied that differed little genetically and have been contaminating humans over 31 years in the metropolitan region of Ribeirão Preto in the São Paulo State in Brazil. The result of MLST indicates that the Shigella sonnei strains studied isolated in Brazil descended from a common precursor

A carne está exposta à contaminações em todas as fases do seu processamento tecnológico, particularmente nas operações em que é mais manipulada e sempre que não são tomados cuidados especiais em relação às Boas Práticas de Higiene. O patógeno mais importante em carne bovina é Salmonella spp, o principal agente causador de doenças transmitidas por alimentos no mundo. Vários estudos têm relatado a ocorrência de Salmonella spp em carne bovina in natura disponível no mercado, mas pouco se sabe sobre este patógeno em carne bovina produzida no Brasil para exportação. O presente estudo objetivou verificar a prevalência, o perfil de susceptibilidade antimicrobiana, a presença de genes de virulência e o perfil de macrorrestrição por PFGE em cepas de Salmonella spp isoladas em diferentes pontos da cadeia de produção. A pesquisa foi realizada em um abatedouro de grande porte que produz carne bovina para exportação. Amostras de superfície foram coletadas de 200 animais, em três pontos do processo do abate: no couro (CO), na carcaça após a esfola (CA1) e na carcaça após a lavagem, antes da refrigeração (CA2), com um total de 600 amostras. A metodologia utilizada para detecção de Salmonella spp foi a preconizada pela ISO 6579:2002, confirmando-se os resultados de identificação pela técnica de PCR e sorotipagem completa. O patógeno foi encontrado no CO de 31 animais (15,5%), na CA1 de 7 animais (3,5%) e na CA2 de 6 animais (3%). Houve a prevalência do sorovar S. Infantis (54,5%), seguido de S. Enteritidis (13,6%). Pesquisou-se a presença dos genes de virulência invA, sitC, spaN, sifA e msgA por PCR nos isolados de Salmonella spp de cada ponto amostrado positivo, em um total de 44 isolados. Observou-se que 59,1% dos isolados apresentaram todos os genes pesquisados e que os demais apresentaram pelo menos dois dos cinco genes de virulência estudados. Os mesmos isolados foram analisados quanto à susceptibilidade aos antimicrobianos, empregando-se as fitas M.I.C Evaluator (Oxoid) com os antimicrobianos ampicilina, cefotaxima, ciprofloxacina, gentamicina, imipenem e tetraciclina. Dos 44 isolados estudados, todos foram sensíveis a cefotaxima, ciprofloxacina, gentamicina e imipenem, enquanto que 5 (11,4%) foram resistentes à ampicilina e à tetraciclina simultaneamente. O perfil de macrorrestrição, feito por FPGE, mostrou que os isolados expressaram 26 perfis genéticos distintos, sendo que maioria dos perfis (92,3%) foi constituída por apenas um ou dois isolados. Os resultados indicam que Salmonella spp está presente no abatedouro estudado. A ocorrência de isolados pertencentes ao mesmo sorovar e ao mesmo perfil de macrorrestrição no couro de animais e também nas carcaças CA1 e CA2 indica possível contaminação cruzada durante o processo de abate, reforçando a necessidade da adoção de Boas Práticas de Higiene para evitar a disseminação do patógeno na cadeia de produção da carne bovina. / Beef is exposed to contamination at all stages of the production chain, particularly in operations where it is more manipulated and when Good Hygiene Practices are not properly followed. The most relevant pathogen in bovine meat is Salmonella spp, the major causative agent of foodborne diseases in the world. Several studies have shown that Salmonella spp can occur in raw bovine meat at retail level, but little is known about this pathogen in meat produced for export. The present study aimed to determinate the prevalence, antimicrobial susceptibility test, the presence of virulence genes and PFGE macrorestriction profiles of Salmonella spp isolates obtained at different points of the production chain. The survey was conducted in a large slaughterhouse that produces bovine meat for export. Surface samples were collected from 200 animals, at three points of the slaughtering process: hide right (CO), carcass after removal of the hide (CA1) and carcass after cleaning but before chilling (CA2), with a total of 600 samples. The methodology used for detection of Salmonella spp was that recommended by ISO 6579:2002, and results were confirmed by PCR and complete serotyping. The pathogen was found in CO of 31 animals (15.5%), in CA1 of 7 animals (3.5%) and CA2 of 6 animals (3%). The prevalent serovar was S. Infantis (54.5%), followed by S. Enteritidis (13.6%). The presence of virulence genes invA, sitC, spaN, sifA and msgA was investigated by PCR in 44 isolates. All these genes were detected in 59.1% of the isolates, and the others had at least two of these virulence genes. The same isolates were tested for antimicrobial susceptibility using the MIC Evaluator strips (Oxoid) with the antimicrobials ampicillin, cefotaxime, ciprofloxacin, gentamicin, imipenem and tetracycline. All 44 isolates were sensitive to cefotaxime, ciprofloxacin, gentamicin and imipenem, whereas five (11.4%) were resistant to ampicillin and tetracycline simultaneously. The macrorestriction profiles, determined by PFGE, the 44 isolates expressed 26 different genetic profiles, and most profiles (92.3%) contained only one or two isolates. The results indicate that Salmonella spp is present in the studied abattoir. The occurrence of isolates belonging to the same serovar and presenting the same macrorestriction profile in the hides and in the carcasses indicates possible cross contamination during the slaughtering process, strengthening the need of adoption of Good Hygiene Practices to avoid dissemination of the pathogen in beef the production chain.

Salmonella é o agente etiológico mais comumente envolvido em casos e surtos de doenças diarréicas de origem alimentar no Brasil e outros países. A preocupação com este patógeno é, ainda, maior quando se verifica o surgimento e disseminação de cepas multi-resistentes e potencialmente mais patogênicas. O presente estudo teve como objetivo caracterizar 237 cepas Salmonella spp. distribuídas entre 50 sorovares diferentes, isoladas de alimentos associados e não associados à toxinfecções alimentares, quanto ao perfil de susceptibilidade antimicrobiana e presença dos genes de virulência spvC, invA, sefA e pefA. O gene invA foi detectado em todas as cepas de Salmonella. Com relação aos demais genes estudados, spvC e pefA foram encontrados em 48,1% e 44,3% das cepas, respectivamente. O gene sefA foi detectado em 31,6% das cepas, estando presente somente entre as cepas de S. Enteritidis. Ainda com relação à presença dos genes de virulência, as cepas de S. Enteritidis foram classificadas em três perfis, com predominância (90,7%) do perfil constituído pelos quatro genes de virulência. Quanto ao perfil de susceptibilidade antimicrobiana, 46,8% do total de cepas avaliadas foram sensíveis a todos os agentes antimicrobianos, 51,9% resistentes à pelo menos uma droga e 1,3% das cepas apresentaram apenas resistência intermediária. Multi-resistência foi observada em 10,5% das cepas. As maiores taxas de resistência foram observadas para estreptomicina (35,9%), ácido nalídixico (16,9%), tetraciclina (5,9%) e gentamicina (4,6%). Não foram detectadas cepas resistentes à cefoxitina, cefalotina, cefotaxima, amicacina, ciprofloxaxina e imipenem. Os resultados do presente estudo mostram a ampla distribuição dos genes de virulência e ocorrência de resistência antimicrobiana tanto nas cepas associadas a surtos como naquelas não envolvidas em toxinfecções alimentares, sendo os produtos de origem avícola fontes importantes de Salmonella com estas características. / Salmonella is the most common causative agent of cases and outbreaks of foodborne diarrhoeal diseases in Brazil and other countries. The concern with this pathogen is even greater considering the emergence and spread of multi-resistant and potentially more pathogenic strains. In this study, the antimicrobial susceptibility profile and the presence of virulence genes spvC, invA, sefA and pefA were examined in 237 Salmonella strains belonging to 50 serovars, isolated from foods associated and nonassociated to foodborne disease outbreaks. The gene invA was detected in all Salmonella strains. The genes spvC and pefA were found in 48.1% and 44.3% of strains, respectively. The sefA gene was found in 31.6% of strains and detected only in S. Enteritidis strains. According to the presence of virulence genes, S. Enteritidis strains were grouped in into three profiles, being the one consisting of four virulence genes the most common profile (90.7%). Among strains, 46.8% were sensitive to all antibiotics, 51.9% resistant to at least one drug and 1.3% of the strains presented intermediate resistance. Multi-resistance was seen in 10.5% of the strains. The highest rates of resistance were observed for streptomycin (35.9%), nalidixic acid (16.9%), tetracycline (5.9%) and gentamicin (4.6%). No resistance was observed to cefoxitin, cephalothin, cefotaxime, amikacin, ciprofloxaxina and imipenem. The results of this study show the wide distribution of virulence genes and occurrence of antimicrobial resistance in strains both associated and non-associated to foodborne disease outbreaks, being poultry products the major sources of Salmonella with these characteristics.

Salmonella é o agente etiológico mais comumente envolvido em casos e surtos de doenças diarréicas de origem alimentar no Brasil e outros países. A preocupação com este patógeno é, ainda, maior quando se verifica o surgimento e disseminação de cepas multi-resistentes e potencialmente mais patogênicas. O presente estudo teve como objetivo caracterizar 237 cepas Salmonella spp. distribuídas entre 50 sorovares diferentes, isoladas de alimentos associados e não associados à toxinfecções alimentares, quanto ao perfil de susceptibilidade antimicrobiana e presença dos genes de virulência spvC, invA, sefA e pefA. O gene invA foi detectado em todas as cepas de Salmonella. Com relação aos demais genes estudados, spvC e pefA foram encontrados em 48,1% e 44,3% das cepas, respectivamente. O gene sefA foi detectado em 31,6% das cepas, estando presente somente entre as cepas de S. Enteritidis. Ainda com relação à presença dos genes de virulência, as cepas de S. Enteritidis foram classificadas em três perfis, com predominância (90,7%) do perfil constituído pelos quatro genes de virulência. Quanto ao perfil de susceptibilidade antimicrobiana, 46,8% do total de cepas avaliadas foram sensíveis a todos os agentes antimicrobianos, 51,9% resistentes à pelo menos uma droga e 1,3% das cepas apresentaram apenas resistência intermediária. Multi-resistência foi observada em 10,5% das cepas. As maiores taxas de resistência foram observadas para estreptomicina (35,9%), ácido nalídixico (16,9%), tetraciclina (5,9%) e gentamicina (4,6%). Não foram detectadas cepas resistentes à cefoxitina, cefalotina, cefotaxima, amicacina, ciprofloxaxina e imipenem. Os resultados do presente estudo mostram a ampla distribuição dos genes de virulência e ocorrência de resistência antimicrobiana tanto nas cepas associadas a surtos como naquelas não envolvidas em toxinfecções alimentares, sendo os produtos de origem avícola fontes importantes de Salmonella com estas características. / Salmonella is the most common causative agent of cases and outbreaks of foodborne diarrhoeal diseases in Brazil and other countries. The concern with this pathogen is even greater considering the emergence and spread of multi-resistant and potentially more pathogenic strains. In this study, the antimicrobial susceptibility profile and the presence of virulence genes spvC, invA, sefA and pefA were examined in 237 Salmonella strains belonging to 50 serovars, isolated from foods associated and nonassociated to foodborne disease outbreaks. The gene invA was detected in all Salmonella strains. The genes spvC and pefA were found in 48.1% and 44.3% of strains, respectively. The sefA gene was found in 31.6% of strains and detected only in S. Enteritidis strains. According to the presence of virulence genes, S. Enteritidis strains were grouped in into three profiles, being the one consisting of four virulence genes the most common profile (90.7%). Among strains, 46.8% were sensitive to all antibiotics, 51.9% resistant to at least one drug and 1.3% of the strains presented intermediate resistance. Multi-resistance was seen in 10.5% of the strains. The highest rates of resistance were observed for streptomycin (35.9%), nalidixic acid (16.9%), tetracycline (5.9%) and gentamicin (4.6%). No resistance was observed to cefoxitin, cephalothin, cefotaxime, amikacin, ciprofloxaxina and imipenem. The results of this study show the wide distribution of virulence genes and occurrence of antimicrobial resistance in strains both associated and non-associated to foodborne disease outbreaks, being poultry products the major sources of Salmonella with these characteristics.

Silicon-based electric DNA chip arrays were utilized to fast identify pathogenic microorganisms with respect to the capacity to produce toxins involved in foodborne poisoning and infections. Bacteria of the B. cereus and the enterohemorrhagic E. coli (EHEC) groups contain different set-ups of various virulence factors that are encoded by the corresponding genes. The purpose of this work was to develop a fast and simple method for determination of the presence of these virulence genes in a colony from primary enrichment cultures. A target gene is detected through hybridization to a surface-immobilized specific capture probe and biotin-labeled detection probe. Following binding of an enzyme conjugate to this sandwich hybrid complex, a current signal is generated by electronic redox recycling of the enzymatic product paminophenol (pAP). Two versions of the assay were developed. In the first version the capture probes were immobilized on magnetic beads, which carried out all reactions until the pAP generation, while the final electric signal was created by transferring pAP to a single-electrode chip surface. In the second version a silicon chip array with 16 parallel sensing electrode positions each of them functionalized by capture probes, carried out all assay steps on the chip surface. This instrument can realize automatic and multiplexed gene detection. The kinetics of bacterial cell disruption and impact of DNA fragmentation by ultrasound were determined. The experimental data suggested that the increased signal after first minutes of ultrasonication were due to the accumulation of released DNA amount, while the further signal increase resulted from the improved hybridization with the shortened target DNA strands. Studies on probe localization on the 16-electrode chip assays indicated that the probe-targeting site, which was located at the 5’-end of strands, gave rise to the highest signal level due to the efficient targetprobes hybridization and the following enzyme binding. When these functionalized chip arrays were exposed to the cell homogenates, the sensing electrodes were fouled by cellular proteins and therefore led to dramatically decreased redox-recycling current. To circumvent this, samples were treated by DNA extraction after the 1st sonication and then DNA fragmentation by a 2nd time sonication. The DNA extract removed most of the interfering components from bacterial cell. This sample treatment was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the chip arrays functionalized by eight DNA probes. The signal patterns of eight virulence genes from chip assays agreed well with PCR control analyses for both strains. By simply adding the SDS detergent to cell homogenates, chip surface blocking effect can be significantly reduced even without DNA extraction treatment. After optimization of some critical factors, the 16-electrode DNA chips with the improved sensing performance can directly detect multiple virulence genes from a single E. coli colony in 25 min after the introduction of supernatant of ultrasonicated cell lysate. / QC 20100824