Salmonella Não Tifóide (SNT) é um dos patógenos que mais causam gastroenterite, representando um problema de saúde pública em todo o mundo. Portanto, a presença de SNT em matrizes ambientais como águas superficiais e esgotos tem sido alvo de pesquisas em todo o mundo. Os objetivos deste estudo foram quantificar e caracterizar SNT em amostras de águas superficiais e esgotos brutos da Região Metropolitana de São Paulo (RMSP). Foram coletadas amostras, quinzenalmente, no ponto de captação de água para o abastecimento público da represa Guarapiranga e do manancial São Lourenço, assim como de esgotos brutos das cidades de Taboão da Serra e São Lourenço da Serra. A quantificação de SNT foi realizada pela técnica do Número Mais Provável (NMP). A caracterização dos isolados (identificação do sorotipo e potencial patogênico) foi realizada pela técnica de Reação em Cadeia da Polimerase (Polimerase Chain Reaction - PCR), com quatro conjuntos de iniciadores, contemplando marcadores genéticos consolidados (fliC, invA e spvC) e regiões gênicas ainda não caracterizadas. As águas superficiais apresentaram concentrações que variaram de Limite de Detecção (LD <0,06473 NMP/100 mL) a 0,67 NMP/100 mL, com frequência de 4 por cento , enquanto nos esgotos brutos as concentrações foram de LD a 54,22 NMP/100 mL, com frequência de 54 por cento . Foram isoladas sete cepas de amostras de águas superficiais, identificadas como Salmonella sp., e 499 de esgotos brutos, dentre as quais se identificaram nove sorotipos potencialmente patogênicos. Os sorotipos mais prevalentes foram Salmonella enterica subsp. enterica sor. Enteritidis e Salmonella enterica subsp. enterica sor. Typhimurium, que se apresentaram quase que exclusivamente nos meses de verão (dezembro a janeiro). Os dois últimos sorotipos apresentaram padrões atípicos de PCR, com regiões gênicas que contêm genes que expressam constituintes fimbriais e outras ainda não caracterizadas. Esta investigação evidencia a presença de Salmonella Não Tifóide potencialmente patogênica nas amostras de esgoto bruto analisadas sugerindo sua circulação na população da região estudada e corrobora com o regime sazonal de salmonelose. DeCS: Salmonella, água superficial, esgoto, sorotipo / Non-Typhoidal Salmonella (NST) is a concern pathogen which is responsible for gastroenteritis outbreaks worldwide. So its presence in environmental sources such as surface waters and sewage has been investigated around the world. The aims of this research were quantify and characterize NST in samples of surface water and raw sewage from Metropolitan Region of São Paulo (MRSP). Samples were collected fortnightly in the catchment point of supply from Guarapiranga reservoir and São Lourenço River, as well as raw sewage from the cities of Taboão da Serra and São Lourenço da Serra. Quantification of NST was performed using Most Probable Number technique (MPN). Characterization of the isolates (serotype identification and pathogenic potential) was carried out by multiplex Polimerase Chain Reaction technique (m-PCR) with four sets of primers, including genetic markers (fliC, invA and spvC) and gene regions not yet characterized. Surface water showed concentrations ranging from detection limit (DL <0.06473 MPN / 100 mL) to 0.67 MPN/100 mL with a frequency of 4 per cent . Raw wastewater concentrations were from DL to 54.22 MPN/100 mL, with a frequency of 54 per cent . Seven strains of surface water samples were isolated and identified as Salmonella sp. and 499 from raw sewage, of which nine were identified as potentially pathogenic serotypes. The most prevalent serotypes were Salmonella enterica subsp. enterica ser. Enteritidis and Salmonella enterica subsp. enterica ser. Typhimurium, which have been found almost exclusively in summer months (December-January). The two serotypes presented atypical patterns of PCR with gene regions containing genes that express fimbrial constituents and other not yet characterized. This research shows the presence of potentially pathogenic Non-Typhoidal Salmonella in raw sewage samples suggesting its circulation in the population of the study area and corroborates with the seasonal cases of salmonellosis. DeCS: Salmonella, surface water, sewage, serotype.

Salmonella Não Tifóide (SNT) é um dos patógenos que mais causam gastroenterite, representando um problema de saúde pública em todo o mundo. Portanto, a presença de SNT em matrizes ambientais como águas superficiais e esgotos tem sido alvo de pesquisas em todo o mundo. Os objetivos deste estudo foram quantificar e caracterizar SNT em amostras de águas superficiais e esgotos brutos da Região Metropolitana de São Paulo (RMSP). Foram coletadas amostras, quinzenalmente, no ponto de captação de água para o abastecimento público da represa Guarapiranga e do manancial São Lourenço, assim como de esgotos brutos das cidades de Taboão da Serra e São Lourenço da Serra. A quantificação de SNT foi realizada pela técnica do Número Mais Provável (NMP). A caracterização dos isolados (identificação do sorotipo e potencial patogênico) foi realizada pela técnica de Reação em Cadeia da Polimerase (Polimerase Chain Reaction - PCR), com quatro conjuntos de iniciadores, contemplando marcadores genéticos consolidados (fliC, invA e spvC) e regiões gênicas ainda não caracterizadas. As águas superficiais apresentaram concentrações que variaram de Limite de Detecção (LD <0,06473 NMP/100 mL) a 0,67 NMP/100 mL, com frequência de 4 por cento , enquanto nos esgotos brutos as concentrações foram de LD a 54,22 NMP/100 mL, com frequência de 54 por cento . Foram isoladas sete cepas de amostras de águas superficiais, identificadas como Salmonella sp., e 499 de esgotos brutos, dentre as quais se identificaram nove sorotipos potencialmente patogênicos. Os sorotipos mais prevalentes foram Salmonella enterica subsp. enterica sor. Enteritidis e Salmonella enterica subsp. enterica sor. Typhimurium, que se apresentaram quase que exclusivamente nos meses de verão (dezembro a janeiro). Os dois últimos sorotipos apresentaram padrões atípicos de PCR, com regiões gênicas que contêm genes que expressam constituintes fimbriais e outras ainda não caracterizadas. Esta investigação evidencia a presença de Salmonella Não Tifóide potencialmente patogênica nas amostras de esgoto bruto analisadas sugerindo sua circulação na população da região estudada e corrobora com o regime sazonal de salmonelose. DeCS: Salmonella, água superficial, esgoto, sorotipo / Non-Typhoidal Salmonella (NST) is a concern pathogen which is responsible for gastroenteritis outbreaks worldwide. So its presence in environmental sources such as surface waters and sewage has been investigated around the world. The aims of this research were quantify and characterize NST in samples of surface water and raw sewage from Metropolitan Region of São Paulo (MRSP). Samples were collected fortnightly in the catchment point of supply from Guarapiranga reservoir and São Lourenço River, as well as raw sewage from the cities of Taboão da Serra and São Lourenço da Serra. Quantification of NST was performed using Most Probable Number technique (MPN). Characterization of the isolates (serotype identification and pathogenic potential) was carried out by multiplex Polimerase Chain Reaction technique (m-PCR) with four sets of primers, including genetic markers (fliC, invA and spvC) and gene regions not yet characterized. Surface water showed concentrations ranging from detection limit (DL <0.06473 MPN / 100 mL) to 0.67 MPN/100 mL with a frequency of 4 per cent . Raw wastewater concentrations were from DL to 54.22 MPN/100 mL, with a frequency of 54 per cent . Seven strains of surface water samples were isolated and identified as Salmonella sp. and 499 from raw sewage, of which nine were identified as potentially pathogenic serotypes. The most prevalent serotypes were Salmonella enterica subsp. enterica ser. Enteritidis and Salmonella enterica subsp. enterica ser. Typhimurium, which have been found almost exclusively in summer months (December-January). The two serotypes presented atypical patterns of PCR with gene regions containing genes that express fimbrial constituents and other not yet characterized. This research shows the presence of potentially pathogenic Non-Typhoidal Salmonella in raw sewage samples suggesting its circulation in the population of the study area and corroborates with the seasonal cases of salmonellosis. DeCS: Salmonella, surface water, sewage, serotype.

The protein monomer of bacterial flagella, FliC, is known to stimulate human innate immunity through activation of Toll-like receptor five. Linking native Salmonella FliC with various antigens has demonstrated an increased immune response as compared to single antigen presentation. To drastically reduce production time and allow for a more cost effective recombinant vaccine adjuvant, a synthetic construct was created that enables genetic linkage of FliC to other known antigens. The construct contains the necessary components for immune system stimulation while the non-essential regions were replaced with commonly used restriction enzyme recognition sites to aid in ligation with other antigens and cloning into various expression vectors and hosts. After synthesis in the inducible expression vector pJ404, the construct was transformed into competent BL21 E. coli and expression was confirmed through SDS-PAGE, Western blot, and MALDI MS/MS. The cells were adapted to fermentation media and re-screened for expression, and upon confirmation a 20-liter fermentation was conducted. The resulting samples were analyzed for expression within the insoluble and soluble cellular fractions to further optimize fermentation conditions. Once purified, this synthetic FliC will serve as a platform technology for the standardized co-expression of the TRL5 activator with a variety of antigens in both prokaryotic and eukaryotic systems.

No description available.

No description available.

C. difficile is an obligate anaerobic, Gram-positive, rodshaped and spore-forming bacterium. It is a well-recognised causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis. C. difficile has emerged as an important nosocomial pathogen in recent years, associated with considerable morbidity, mortality and economic burden. Despite its importance, functional genomic studies have been lagging behind in comparison to other enteric pathogens. This is attributed to the fact that C. difficile is difficult to manipulate genetically and the lack of robust, reproducible mutagenesis systems for many years. The ideal mutation for robust functional genomic studies is a markerless, in-frame deletion of the gene of interest. All systems developed for C. difficile, up to the start of this study, involve insertional inactivation of the gene of interest. This study describes the development of a novel genetic system for C. difficile, to create precise and markerless chromosomal deletions, using the meganuclease ISceI. For validation of the system, the addBA genes in C. difficile were deleted. The AddAB enzyme complex is important in the survival of many bacteria, since it maintains genome integrity, by the repair of double-strand breaks. Deletion of addBA in C. difficile did not significantly affect growth and viability, but the mutant strains were sensitive to DNA damaging agents. In addition, it was shown that C. difficile is capable of initiating the SOS response after DNA damage and that AddAB is not necessary for the induction of this response. The genetic system was further optimised to delete type IV pili (TFP)- associated genes, particularly pilT (CD3505) and pilA (CD3507), to investigate twitching motility. TFP are important in virulence and pathogenesis of many bacteria and twitching motility is often involved. TFP in C. difficile may be expressed in vivo during infection and may be involved in biofilm formation and colonization. To study potential TFP-mediated motility, a non-flagellated C. difficile strain was first constructed by deleting the fliC gene. The pilT gene, predicted to encode a protein involved in TFP retraction, was then deleted in the ΔfliC strain. A ΔpilT strain was also generated. Preliminary experimental work using these strains did not show any evidence for twitching motility and no difference between the ΔpilT strains and the parental strains. Examination of cells from the ΔfliC strain, under various conditions, did not reveal any pili, which indicates that TFP are regulated in C. difficile and that the TFP locus might be repressed at the transcriptional level. Preliminary work to investigate an intergenic region located upstream of the TFP locus in C. difficile, that might be involved in regulation, suggested that transcription is being initiated within a 500 bp region upstream of the CD3513 gene.

Neste estudo, mostramos a possibilidade de identificação dos AgH de ETEC pela caracterização molecular do gene fliC pela análise de seu polimorfismo de restrição. Um único alelo de fliC de ETEC foi encontrado para cada antígeno flagelar, utilizando-se a endonuclease RsaI, com exceção do H21. Além de cepas móveis, isolados imóveis também puderam ser caracterizados por essa técnica molecular. A alta tipabilidade da PCR-RFLP foi comprovada por meio de sua aplicação não só a amostras de ETEC com AgH previamente desconhecidos, mas também a outras linhagens de E. coli. Observamos que após identificação do antígeno flagelar das amostras de ETEC pela PCR-RFLP, a determinação do antígeno somático pôde ser direcionada, diminuindo assim, o número de anti-soros utilizados para a pesquisa do AgO. A técnica de PCR-RFLP, em nosso estudo, mostrou uma sensibilidade de 83% e 100% de especificidade. Esta técnica foi mais rápida na identificação do AgH de E. coli (2 dias) em comparação à sorologia clássica (7 ou mais dias, dependendo da motilidade da cepa). Acreditamos que o método de soroaglutinação para determinação do AgH será substituído rapidamente pela PCR-RFLP. Contudo, a soro aglutinação não poderá ser totalmente dispensada em curto prazo. No futuro, com o perfil molecular obtido dos alelos de cepas procedentes de estudos epidemiológicos, novos padrões serão definidos para as cepas diarreiogênicas de E. coli, permitindo o abandono da sorologia para AgH. / In this study, we showed that the H antigens of ETEC can be characterised by restriction analysis of the polymorphism of the fliC gene. Only one allele of the fliC gene of ETEC was found for each flagellar antigen when restriction endonuclease RsaI was used, with the exception of H21 . Additionally, non-motile strains could also be characterised using this molecular technique. The high typeability of this technique was demonstrated by the fact that it can not only be applied to ETEC samples with previously unknown H antigens but also to all other lineages belonging to the E. coli species. Moreover, the determination of the somatic antigen was guided by the identification of the flagellar antigen of ETEC strains by PCR-RFLP, thus reducing the number of anti-AgO sera used. In this study, the PCR-RFLP technique showed a sensitivity of 83% and a specificity of 100%. This technique proved to be quicker for the identification of the E. coli AgH, taking 2 days to complete, in comparison to the 7 or more days necessary when using classic serotyping. We believe that the determination of the AgH by seroagglutination will soon be substituted by the PCR-RFLP technique. However, serotyping will still have to be used in the short run, for further studies involving PCR-RFLP must be carried out. In the future, with the determination of the molecular profiles of alleles of strains obtained in epidemiological studies, new patterns will have been described for the diarrhoeagenic strains of E. coli, thus permitting the abandonment of AgH serotyping for good.

Neste estudo, mostramos a possibilidade de identificação dos AgH de ETEC pela caracterização molecular do gene fliC pela análise de seu polimorfismo de restrição. Um único alelo de fliC de ETEC foi encontrado para cada antígeno flagelar, utilizando-se a endonuclease RsaI, com exceção do H21. Além de cepas móveis, isolados imóveis também puderam ser caracterizados por essa técnica molecular. A alta tipabilidade da PCR-RFLP foi comprovada por meio de sua aplicação não só a amostras de ETEC com AgH previamente desconhecidos, mas também a outras linhagens de E. coli. Observamos que após identificação do antígeno flagelar das amostras de ETEC pela PCR-RFLP, a determinação do antígeno somático pôde ser direcionada, diminuindo assim, o número de anti-soros utilizados para a pesquisa do AgO. A técnica de PCR-RFLP, em nosso estudo, mostrou uma sensibilidade de 83% e 100% de especificidade. Esta técnica foi mais rápida na identificação do AgH de E. coli (2 dias) em comparação à sorologia clássica (7 ou mais dias, dependendo da motilidade da cepa). Acreditamos que o método de soroaglutinação para determinação do AgH será substituído rapidamente pela PCR-RFLP. Contudo, a soro aglutinação não poderá ser totalmente dispensada em curto prazo. No futuro, com o perfil molecular obtido dos alelos de cepas procedentes de estudos epidemiológicos, novos padrões serão definidos para as cepas diarreiogênicas de E. coli, permitindo o abandono da sorologia para AgH. / In this study, we showed that the H antigens of ETEC can be characterised by restriction analysis of the polymorphism of the fliC gene. Only one allele of the fliC gene of ETEC was found for each flagellar antigen when restriction endonuclease RsaI was used, with the exception of H21 . Additionally, non-motile strains could also be characterised using this molecular technique. The high typeability of this technique was demonstrated by the fact that it can not only be applied to ETEC samples with previously unknown H antigens but also to all other lineages belonging to the E. coli species. Moreover, the determination of the somatic antigen was guided by the identification of the flagellar antigen of ETEC strains by PCR-RFLP, thus reducing the number of anti-AgO sera used. In this study, the PCR-RFLP technique showed a sensitivity of 83% and a specificity of 100%. This technique proved to be quicker for the identification of the E. coli AgH, taking 2 days to complete, in comparison to the 7 or more days necessary when using classic serotyping. We believe that the determination of the AgH by seroagglutination will soon be substituted by the PCR-RFLP technique. However, serotyping will still have to be used in the short run, for further studies involving PCR-RFLP must be carried out. In the future, with the determination of the molecular profiles of alleles of strains obtained in epidemiological studies, new patterns will have been described for the diarrhoeagenic strains of E. coli, thus permitting the abandonment of AgH serotyping for good.

Clostridium difficile (CD) est l’entéropathogène le plus fréquemment responsable d'infections nosocomiales intestinales post-antibiotiques. L'apparition de cas graves liés à l'émergence de souches hypervirulentes ces dernières années a contribué à accroître la morbidité et la mortalité. Les toxines TcdA et TcdB contribuent directement aux lésions intestinales associées aux infections à CD (ICD), mais d'autres facteurs bactériens sont nécessaires à l’adhésion et la colonisation intestinale. Les flagelles de CD, qui confèrent la mobilité et la chimiotaxie, pourraient jouer un rôle dans la pathogenèse en contribuant à la réponse inflammatoire de l’hôte et aux lésions de la muqueuse. En effet, en activant le récepteur TLR5, les flagelles peuvent provoquer l'activation des cascades de signalisation cellulaire des MAPK et de NF-κB conduisant à la sécrétion de cytokines pro-inflammatoires. Notre objectif était d’étudier ce rôle potentiel des flagelles de CD in vitro et in vivo. Nous avons montré que l'interaction de la flagelline de CD avec le TLR5 active principalement la voie de NF-κB, et, dans une moindre mesure, la voie des MAPK, conduisant ainsi à la régulation de l'expression de gènes pro-inflammatoires et à la synthèse de médiateurs pro-inflammatoires. De plus, en utilisant un modèle murin d’ICD, nous avons démontré un effet synergique des flagelles et des toxines dans l’induction d’une réponse inflammatoire de la muqueuse caecale. Dans ce modèle, l'absence de flagelles diminue considérablement le degré d'inflammation de la muqueuse caecale et la seule présence de toxines, sans flagelles, ne suffit pas à provoquer d’importantes lésions épithéliales. Ces résultats mettent en évidence le rôle des flagelles de CD dans l’induction d’une réponse inflammatoire intestinale en synergie avec l’action des toxines bactériennes sur l'épithélium. / Clostridium difficile (CD) is the most common enteropathogen responsible for intestinal nosocomial post-antibiotic infections. The appearance of severe cases related to the emergence of hypervirulent strains these last years has contributed to increased mortality and morbidity. The CD TcdA and TcdB toxins contribute directly to CD infection (CDI)-associated lesions of the gut, but other bacterial factors are needed for the bacteria to adhere and colonize the gut. The CD flagella, which confer motility and chemotaxis for successful intestinal colonization, could play an additional role in bacterial pathogenesis by contributing to the inflammatory response of the host and mucosal injury. Indeed, by activating the TLR5, flagella can elicit activation of the MAPK and NF-κB cascades of cell signaling, leading to the secretion of pro-inflammatory cytokines. Our objective was to study the potential role of CD flagella in vitro and in vivo. We reported that the interaction of CD flagellin-TLR5 predominantly activates the NF-κB, and, in a lesser degree, the MAPKs pathways, thus leading to up-regulation of pro-inflammatory gene expression and subsequent synthesis of pro-inflammatory mediators. Moreover, by using a mouse model of CDI, we demonstrated a synergic effect of flagella and toxins in eliciting an inflammatory mucosal response. In this model, the absence of flagella dramatically decreased the degree of mucosal inflammation in mice and the sole presence of toxins without flagella was not enough to elicit epithelial lesions. These results highlight the important role of CD flagella in eliciting mucosal lesions as long as the toxins exert their action on the epithelium.

A espécie Escherichia coli constitui um grupo de bactérias tipicamente não patogênicas e que fazem parte do trato intestinal de humanos e animais. As amostras são sorotipadas com base em seus antígenos de superfície O (somático), H (flagelar) e K (capsular). O antígeno flagelar correspondente ao filamento é formado pela polimerização da flagelina, codificada pelo gene fliC. O presente trabalho empregou a técnica de PCR-RFLP para analisar os padrões de antígenos flagelares de 112 amostras de EPEC e STEC. Quatorze amostras não amplificaram o gene fliC, 17 tiveram seu antígeno flagelar determinado apenas por PCR-RFLP e 75 amostras tiveram seus antígenos flagelares confirmados por esta técnica. Três antígenos H com padrões irregulares foram clonados e sequenciados. Após o sequenciamento, inserções e remoções de nucleotídeos foram encontradas. Até o momento, poucos estudos utilizam um número abrangente de amostras de STEC e EPEC provenientes de diferentes animais para a determinação do antígeno H empregando a técnica de PCR-RFLP do gene fliC. De acordo com os resultados encontrados neste estudo, podemos concluir que a técnica de PCR-RFLP do gene fliC é mais rápida, menos trabalhosa e mais eficiente que a metodologia de sorotipagem clássica. / The Escherichia coli species consists of a group of typically non-pathogenic bacteria present in the intestinal tract of humans and animals. Strains are serotyped according to their O (somatic), H (flagellar) and K (capsular) surface antigens, in order to distinguish these microorganisms from the non-pathogenic members of the intestinal microbiota. The flagellar antigen corresponding to the filament is formed by the polymerization of the flagellin, codified by the fliC gene. This study employed the PCR-RFLP technique to analyze flagellar antigen patterns from 112 EPEC and STEC strains. Fourteen strains have not amplified the fliC gene, 17 had their flagellar antigen determined only by the PCR-RFLP and 75 strains had their flagellar antigen confirmed by this technique. Three H antigens with irregular patterns were cloned and sequenced. After sequencing, insertions and deletions of nucleotides were discovered. So far, few studies used a significant number of STEC and EPEC strains originated from different animals to determine H antigens employing the PCR-RFLP technique of the fliC gene. According to the findings of this study, we assumed that PCR-RFLP of the fliC gene is faster, less laborious and more efficient than classic serotyping methodology.