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Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Neste estudo, foram analisadas 167 cepas [82 B. thuringiensis (Bt), 70 B. cereus (Bc) e 15 B. mycoides], isoladas no Brasil e provenientes de diversas origens. Foram avaliados alguns aspectos fenotípicos, tais como, a capacidade de motilidade, atividade hemolítica, produção de lecitinase e hidrólise do amido, bem como, genótipos relacionados ao potencial toxigênico e de virulência. A maioria das estirpes produziu enzimas extracelulares, tais como, amilase e lecitinases, o que ressalta o potencial deteriorante dessas espécies. A motilidade e a atividade hemolítica apresentaram proporções semelhantes nas cepas analisadas. A maioria das estirpes (155,93%) foi \03B2-hemolítica. A expressão dos genes toxigênicos e de virulência foram investigados através da PCR. De acordo com a ocorrência dos genes das enterotoxinas e da toxina emética, as cepas selecionadas foram divididas em 7 perfis toxigênicos. O perfil predominante, o perfil I, incluiu 119 cepas (71%) que foram positivas para todos os genes das enterotoxinas. As enterotoxinas HBL e NHE foram detectadas em 143 (86%) e 164 (98%) das cepas, respectivamente. Todas as cepas possuem genótipo positivo para entFM. O gene da cytK-2 estava presente em 134 cepas (80%). Todas as cepas foram negativas para os genes cytK-1 e ces. Na distribuição dos fatores de virulência, o perfil predominante, o perfil I, (20%, 34/167) incluiu cepas positivas para todos os determinantes hemolíticos, sendo divididos em 24 perfis. Entre os genes de virulência a hlyIII foi o mais prevalente (130/78%). O gene piplc foi encontrado em 112 cepas (67%) e o gene pcplc em 118 cepas (71%). Já o gene sph foi detectado em 76 estirpes (45%)
O gene menos comum, a hlyII foi detectada em 69 das estirpes (41%). Para ambos os perfis, a espécie Bt apresentou alta prevalência desses genótipos. Somente o genótipo mesofílico (gene cspF) foi detectado nas cepas selecionadas. Além das 28 cepas que amplificaram todos os fatores de virulência (20 Bt e 8 Bc), 12 cepas (3 Bt e 9 Bc) foram selecionadas randomicamente entre as 167 estirpes, as quais foram submetidas a caracterização genotípica por rep-PCR e MLST. Através da análise do rep-PCR, observa-se que a distribuição das sequências rep-PCR em Bt, tende a ser menos variáveis, havendo uma propensão para o agrupamento dos perfis eletroforéticos específicos de cepas de Bt, não sendo a mesma orientação apresentada pelas cepas de Bc, que demonstraram perfis altamente polimórficos. O MLST permitiu caracterizar e observar as relações filogenéticas entre as estirpes selecionadas agrupando-as em 20 Sts, com 17 dos Sts apresentando perfis alélicos novos (ST156 a ST170). A análise por MLST demonstrou que as cepas de distintas fontes compartilham origens clonais comuns surgindo de diferentes grupos filogenéticos. A expressiva distribuição dos determinantes de virulência observada entre as cepas estudadas atesta o dinamismo e o alto potencial toxigênico e deteriorante, fatos que provavelmente contribuem para entender a epidemiologia e a biodiversidade das espécies que compõem o B. cereus l.s. / In this study, 167 isolates [82 B. thuringiensis (Bt), 70 B. cereus (Bc) and 15 B. mycoides], isolated in Brazil from diverse sources were analyzed. Some phenotypic aspects such as motility, hemolytic activity, lecithinase production and hydrolysis of starch, as well as toxigenic and virulence genotypes were evaluated. Most isolates produced extracellular enzymes, such as amylase and lecithinases, which emphasizes the spoilage potential of these species. Motility and hemolytic activity were found in similar proportions among the isolates analyzed. The majority of the isolates (155, 93%) were -hemolytic. Detection of toxigenic and virulence factors were investigated by PCR. According to the occurrence of genes encoding enterotoxins and the emetic toxin, the isolates were divided into 7 toxigenic patterns. The predominant toxigenic pattern was type I (119, 71%) which included isolates positive for all toxin genes but ces. The nonhemolytic enterotoxin (NHE) was found in 164 isolates (98%) positive for the three genes (nheA, nheB, nheC). All isolates were positive for entFM. Enterotoxic HBL complex (hblA, hblC and hblD) was found in 143 (86%) isolates. The cytK-2 gene was present in 134 (80%) isolates. All isolates were negative for cytK-1 gene. For the additional virulence factors analyzed, 24 patterns were observed and the predominant pattern was type I (20%, 34/167) which included isolates positive for all hemolytic genes. Among the additional virulence genes studied hlyIII was the most prevalent and was found in 130 (78%) isolates. piplc was found in 112 (67%) isolates and pcplc in 118 (71%) isolates. sph was detected in 76 (45%) isolates. Far less common, hlyII was detected in 69 (41%) of the isolates. For both, toxigenic and virulence patterns, Bt lineages showed high prevalence of genotypes including the highest numbers of genes. Only the mesophilic genotype (gene cspF) was detected in the isolates. The isolates that amplified all virulence determinants (20 Bt and 8 Bc) and 12 isolates (3 Bt and 9 Bc) randomly selected were genotyped by rep-PCR and MLST. By rep-PCR analysis, it was observed that the distribution of rep-PCR sequences in Bt, tend to be less variable, there is a tendency for clustering of specific electrophoretic profiles of Bt isolates, which was far less common for Bc isolates and demonstrated highly polymorphic profiles. MLST allowed observing the phylogenetic relationships among the 40 isolates selected, grouping them into 20 STs, with 17 of STs assigned new allelic pattern (ST156 to ST170). The MLST analysis demonstrated that the isolates from distinct sources share common clonal origin located in different phylogenetic groups. The significant distribution of virulence determinants observed among isolates studied attests to the dynamism and the high toxigenic and spoilage potential among the isolates, facts that probably contribute to understand the epidemiology and the biodiversity of species that comprise the B. cereus l.s.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.arca.fiocruz.br:icict/14234 |
| Date | January 2015 |
| Creators | Chaves, Jeane Quintanilha |
| Contributors | Zahner, Viviane, Fracalanzza, Sergio Eduardo Longo, Andrade, João Ramos Costa, Vivoni, Adriana Marcos, Gomes, Clara de Fátima, Rabinovitch, Leon |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da FIOCRUZ, instname:Fundação Oswaldo Cruz, instacron:FIOCRUZ |
| Rights | info:eu-repo/semantics/openAccess |
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