The Zymovars of Vibrio cholerae: Multilocus Enzyme Electrophoresis of Vibrio cholerae

Zymovars analysis also known as multilocus enzyme electrophoresis is applied here to investigate the genetic variation of Vibrio cholerae strains and characterise strains or group of strains of medical and epidemiological interest. Fourteen loci were analyzed in 171 strains of non-O1 non-O139, 32 classical and 61 El Tor from America, Africa, Europe and Asia. The mean genetic diversity was 0.339. It is shown that the same O antigen (both O1 and non-O1) may be present in several geneticaly diverse (different zymovars) strains. Conversely the same zymovar may contain more than one serogroup. It is confirmed that the South American epidemic strain differs from the 7th pandemic El Tor strain in locus LAP (leucyl leucyl aminopeptidase). Here it is shown that this rare allele is present in 1 V. mimicus and 4 non-O1 V. cholerae. Non toxigenic O1 strains from South India epidemic share zymovar 14A with the epidemic El Tor from the 7th pandemic, while another group have diverse zymovars. The sucrose negative epidemic strains isolated in French Guiana and Brazil have the same zymovar of the current American epidemic V. cholerae.

The neuraminidase gene is present in the non-toxigenic Vibrio cholerae Amazonia strain: a different allele in comparison to the pandemic strains

The neuraminidase gene, nanH, is present in the O1, non-toxigenic Vibrio cholerae Amazonia strain. Its location has been assigned to a 150 kb NotI DNA fragment, with the use of pulsed-field gel electrophoresis and DNA hybridization. This NotI fragment is positioned inside 630 kb SfiI and 1900 kb I-CeuI fragments of chromosome 1. Association of the pathogenicity island VPI-2, carrying nanH and other genes, with toxigenic strains has been described by other authors. The presence of nanH in a non-toxigenic strain is an exception to this rule. The Amazonia strain nanH was sequenced (Genbank accession No. AY825932) and compared to available V. cholerae sequences. The sequence is different from those of pandemic strains, with 72 nucleotide substitutions. This is the first description of an O1 strain with a different nanH allele. The most variable domain of the Amazonia NanH is the second lectin wing, comprising 13 out of 17 amino acid substitutions. Based on the presence of nanH in the same region of the genome, and similarity of the adjacent sequences to VPI-2 sequences, it is proposed that the pathogenicity island VPI-2 is present in this strain.

Occurrence and composition of class 1 and class 2 integrons in clinical and environmental O1 and non-O1/non-O139 Vibrio cholerae strains from the Brazilian Amazon

This study identified and characterised class 1 and 2 integrons in clinical and environmental Vibrio cholerae O1 and non-O1/non-O139 strains isolated from the Brazilian Amazon. The aadA2 and aadA7 gene cassettes were found in class 1 integrons in two genotypes of environmental V. cholerae non-O1/non-O139. Empty integrons were found in strains from the Brazilian cholera epidemic. A class 2 integron was detected in one strain from the V. cholerae Amazonia lineage harbouring sat1 and aadA1 genes. All isolates were resistant to aminoglycosides, indicating aadA functionality. These findings suggest that environmental bacteria act as cassette reservoirs that favour the emergence of resistant pathogens.

ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA.

Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 A pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.

Pathogenic Vibrio activate NLRP3 inflammasome via cytotoxins and TLR/nucleotide-binding oligomerization domain-mediated NF-kappa B signaling.

Vibrio vulnificus and Vibrio cholerae are Gram-negative pathogens that cause serious infectious disease in humans. The beta form of pro-IL-1 is thought to be involved in inflammatory responses and disease development during infection with these pathogens, but the mechanism of beta form of pro-IL-1 production remains poorly defined. In this study, we demonstrate that infection of mouse macrophages with two pathogenic Vibrio triggers the activation of caspase-1 via the NLRP3 inflammasome. Activation of the NLRP3 inflammasome was mediated by hemolysins and multifunctional repeat-in-toxins produced by the pathogenic bacteria. NLRP3 activation in response to V. vulnificus infection required NF-kappaB activation, which was mediated via TLR signaling. V. cholerae-induced NLRP3 activation also required NF-kappaB activation but was independent of TLR stimulation. Studies with purified V. cholerae hemolysin revealed that toxin-stimulated NLRP3 activation was induced by TLR and nucleotide-binding oligomerization domain 1/2 ligand-mediated NF-kappaB activation. Our results identify the NLRP3 inflammasome as a sensor of Vibrio infections through the action of bacterial cytotoxins and differential activation of innate signaling pathways acting upstream of NF-kappaB.

Quorum-sensing regulates biofilm formation in Vibrio scophthalmi

Background: In a previous study, we demonstrated that Vibrio scophthalmi, the most abundant Vibrio species among the marine aerobic or facultatively anaerobic bacteria inhabiting the intestinal tract of healthy cultured turbot (Scophthalmus maximus), contains at least two quorum-sensing circuits involving two types of signal molecules (a 3-hydroxy-dodecanoyl-homoserine lactone and the universal autoinducer 2 encoded by luxS). The purpose of this study was to investigate the functions regulated by these quorum sensing circuits in this vibrio by constructing mutants for the genes involved in these circuits. Results. The presence of a homologue to the Vibrio harveyi luxR gene encoding a main transcriptional regulator, whose expression is modulated by quorumsensing signal molecules in other vibrios, was detected and sequenced. The V. scophthalmi LuxR protein displayed a maximum amino acid identity of 82% with SmcR, the LuxR homologue found in Vibrio vulnificus. luxR and luxS null mutants were constructed and their phenotype analysed. Both mutants displayed reduced biofilm formation in vitro as well as differences in membrane protein expression by mass-spectrometry analysis. Additionally, a recombinant strain of V. scophthalmi carrying the lactonase AiiA from Bacillus cereus, which causes hydrolysis of acyl homoserine lactones, was included in the study. Conclusions: V. scophthalmi shares two quorum sensing circuits, including the main transcriptional regulator luxR, with some pathogenic vibrios such as V. harveyi and V. anguillarum. However, contrary to these pathogenic vibrios no virulence factors (such as protease production) were found to be quorum sensing regulated in this bacterium. Noteworthy, biofilm formation was altered in luxS and luxR mutants. In these mutants a different expression profile of membrane proteins were observed with respect to the wild type strain suggesting that quorum sensing could play a role in the regulation of the adhesion mechanisms of this bacterium.

Allelic diversity and population structure in Vibrio cholerae O139 Bengal based on nucleotide sequence analysis

Comparative analysis of gene fragments of six housekeeping loci, distributed around the two chromosomes of Vibrio cholerae, has been carried out for a collection of 29 V. cholerae O139 Bengal strains isolated from India during the first epidemic period (1992 to 1993). A toxigenic O1 ElTor strain from the seventh pandemic and an environmental non-O1/non-O139 strain were also included in this study. All loci studied were polymorphic, with a small number of polymorphic sites in the sequenced fragments. The genetic diversity determined for our O139 population is concordant with a previous multilocus enzyme electrophoresis study in which we analyzed the same V. cholerae O139 strains. In both studies we have found a higher genetic diversity than reported previously in other molecular studies. The results of the present work showed that O139 strains clustered in several lineages of the dendrogram generated from the matrix of allelic mismatches between the different genotypes, a finding which does not support the hypothesis previously reported that the O139 serogroup is a unique clone. The statistical analysis performed in the V. cholerae O139 isolates suggested a clonal population structure. Moreover, the application of the Sawyer's test and split decomposition to detect intragenic recombination in the sequenced gene fragments did not indicate the existence of recombination in our O139 population.

Vibrio spp. isolados de mamíferos marinhos capturados na região litorânea do Sudeste ao Sul do Brasil

Avaliou-se a incidência de Vibrio spp. a partir de lesões superficiais em mamíferos marinhos encalhados ou capturados em redes de pesca nas regiões litorâneas do Sudeste (Rio de Janeiro) e Sul (RS) do Brasil. Foram coletadas 198 amostras, pelas instituições de pesquisa DEENSP, GEMARS e Ceclimar, as quais foram enviadas ao Labent/IOC/FIOCruz, onde foram submetidas ao enriquecimento em Água Peptonada Alcalina (APA) adicionada de 1% e 3% de NaCl e in-cubadas a 37ºC por 18-24 horas. Em seqüência foram semeadas em meio Agar Tiossulfato Citrato Bile Sacarose (TCBS) e as colônias suspeitas submetidas à caracterização bioquímica. Foram isoladas 108 cepas bacterianas, destacando-se Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus e V. fluvialis como os principais patógenos isolados. Os resultados obtidos apontam para a necessidade de implementar atividades de vigilância e monitorização bacteriológica, particularmente de espécies selvagens, e reforçar os programas de proteção ambiental em casos de mamíferos marinhos ameaçados de extinção.

Ocorrência de Vibrio vulnificus em alguns alimentos de origem marinha

Vibrio vulnificus é uma bactéria Gram-negativa que habita águas marinhas. É patogênica para o homem e a doença está associada ao consumo de frutos do mar, com aproximadamente 60% dos casos sendo fatais em pacientes imunocomprometidos. O objetivo desta pesquisa foi estudar a ocorrência de V. vulnificus em amostras de alguns alimentos de origem marinha. As amostras de ostras, mariscos e camarões foram coletadas nos períodos de abril-agosto de 1993, maio-setembro de 1994 e fevereiro de 1995. De 55 amostras de ostras, 36 (65%) foram positivas para V. vulnificus; das 19 amostras de mariscos, 8 (42,1%) foram positivas e das 7 amostras de camarão, 1 (4,3%) foi positiva para esta bactéria. Os resultados permitiram-nos concluir que a bactéria foi recuperada durante todos os meses de análise, demonstrando que estes alimentos, principalmente quando consumidos crus, são potencialmente perigosos para os seres humanos na faixa de risco.

Vibrio parahaemolyticus produtores de urease isolados a partir de ostras (Crassostrea rizophorae) coletadas in natura em restaurantes e mexilhões (Perna perna) de banco natural

A presença de Vibrio parahaemolyticus foi avaliada em 50 amostras de moluscos bivalves marinhos compostas por 40 amostras de ostras coletadas em 15 restaurantes do Rio de Janeiro e 10 amostras de mexilhões capturados de banco natural em Ponta de Itaipú - Niterói. Foram empregadas a técnica do Número Mais Provável (NMP) para a enumeração de V. parahaemolyticus utilizando Caldo Glicosado Salgado com Teepol (GSTB) e Água Peptonada Alcalina (APA) com 3% de cloreto de sódio (NaCl). Paralelamente foi realizada técnica de enriquecimento em APA com 1 e 3% de NaCl. Decorrido o período de incubação de ambas as técnicas, foi realizado plaqueamento em ágar TCBS (Tiossulfato Citrato Bile Sacarose). Todas as cepas de V. parahaemolyticus isoladas através das duas técnicas foram testadas para o fenômeno de Kanagawa e, quanto à produção de urease. Do total de 141 cepas de V. parahaemolyticus isoladas, 62% revelaram-se urease positivas e, dentre estas, os sorotipos predominantes foram O10:K?, O11:K? e O3:K57 dentre o total de 24 sorotipos urease positivos identificados. Embora todas as cepas de V. parahaemolyticus tenham sido Kanagawa negativas, os resultados apontam elevada incidência desta espécie em ostras comercializadas em restaurantes.

Vibrio spp. isolados a partir de mexilhões (Perna perna) in natura e pré-cozidos de Estação Experimental de Cultivo, Rio de Janeiro, RJ, Brasil

A análise microbiológica dos mexilhões reflete a qualidade do habitat aquático, pois estes animais podem reter em seus organismos diversos patógenos, dentre os quais aqueles pertencentes à família Vibrionaceae. No presente estudo foi avaliada a presença de Vibrio spp. em mexilhões (in natura e pré-cozidos), comercializados na Estação Experimental de Cultivo de Mexilhões, situada em Jurujuba, Niterói, Rio de Janeiro. Foram avaliadas 86 amostras, tomando como procedimento, o enriquecimento em Água Peptonada Alcalina (APA) adicionada de 1 e 3% de NaCl, isolamento em Agar Tiossulfato Citrato Bile Sacarose (TCBS) e confirmação das colônias típicas por análise bioquímica. Dentre as 12 espécies de Vibrio identificadas destacaram-se como de maior prevalência as espécies Vibrio alginolyticus, V. cholerae não-O1, V. parahaemolyticus, V. carchariae e Vibrio vulnificus. A relevância epidemiológica destes patógenos associada a casos de gastrenterite humana após consumo de mexilhões crus ou parcialmente cozidos, reforça a importância de alertar as autoridades de Vigilância Sanitária sobre sua presença na cadeia alimentar e seus riscos para a Saúde Pública.

Ocurrence of Vibrio spp., positive coagulase staphylococci and enteric bacteria in oysters (Crassostrea gigas) harvested in the south bay of Santa Catarina island, Brazil

The aim of this study was to assess the contamination of oysters (Crassostrea gigas), harvested in six different regions of the South Bay of Santa Catarina Island, with Coliforms at 45 ºC, Escherichia coli, Vibrio spp., positive coagulase staphylococci, and Salmonella sp. over a period of one year. One hundred eighty oyster samples were collected directly from their culture sites and analyzed. Each sample consisted of a pool of 12 oysters. All of the samples analyzed showed absence of Salmonella, 18 (10%) samples showed presence of Escherichia coli, 15 (8.3%) samples were positive for V. alginolyticus, and Vibriocholerae was detected in 4 samples (2.2%). The counts of positive-coagulase staphylococci varied from <10 to 1.9 x 102 CFU.g-1, whereas the counts of Coliforms at 45 ºC and E. coli ranged from <3 to 1.5 x 102 MPN.g-1 and <3 and 4.3 x 10 MPN.g-1, respectively. Counts of V. parahaemolyticus and V. vulnificus ranged between <3 and 7 MPN.g-1, for both microorganisms. This suggests the need for monitoring these Vibrios contamination in oysters. Based on the results of the microbiological assays, the samples analyzed showed acceptable bacteriological quality, i.e., they were within the parameters established by Brazilian Legislation.

Tolerancia de Vibrio cholerae al choque ácido

Tesis (Maestría en Ciencias con Especialidad en Microbiología) UANL

Actividad de extractos de plantas en el crecimiento, la producciónde toxina y la unión de Vibrio cholerae a células

Tesis (Maestría en Ciencias con Especialidad en Microbiología) UANL

Respuesta de Vibrio cholerae a condiciones subletales de acidez y jugo biliar humano

Tesis (Doctorado en Ciencias con Especialidad en Microbiología) UANL