Determinação da diversidade bacteriana em culturas de caldo Macconkey de materiais clínicos de portadores de doença de Crohn e análise de propriedades de isolados de Escherichia coli identificados

Imbalance in intestinal bacterial composition (dysbiosis) is a hallmark of Crohn’s disease (CD), the most severe of inflammatory bowel diseases (IBD). A characteristic of IBD dysbiosis is the elevation of certain groups of bacteria such as Proteobacteria in some patients. It is believed that the augmented bacteria may contribute for the inflammatory reactions underlying the typical lesions observed in the gut of CD and ulcerative colitis patients, by stimulating the production of cytokines by local cells of the immune system. Proteobacteria, one the most prevalent bacterial phyla of the gut microbiota are Gram negative rods of which Escherichia coli (E. coli) is the most well-known member. Given the significance of Proteobacteria as pathogens or member of resident gut microbiota, this work had two purposes: 1) to investigate the occurrence of numerical variation in the population of these bacteria in CD and 2) Characterizing E. coli isolates found in this population, in search for some virulence associated properties. For the first purpose, DNA from MacConkey broth cultures of distinct clinical materials (stools and gut mucosal biopsies) of 9 CD and 8 control patients were amplified with primers for the V6 and V8 regions of the 16S ribosomal RNA gene (16S V6-V8 rDNA) and distinct gene sequences (representing particular bacterial taxa) within the amplicons were resolved by temperature gradient gel electrophoresis (TGGE), defining band profiles, which reflect the bacterial diversity. The second objective consisted in phylotyping (determining A, B1, B2 and D phylogroups), serotyping (search for O25 and O83 genes), analyzing the E. coli isolates of the cultures for adherence properties and competence to produce biofilm. Analysis of 32 cultures from the 9 CD patients and 24 cultures from 8 control subjects showed a clear case-control difference in the number of bands in the TGGE profiles of the 16S V6-V8 rDNA amplicons. Cultures from 8 of 9 CD patients had at most 2 bands, while the number of bands in the cultures from 7 of the 8 controls subjects had at least 3 bands. One of the two bands found in the cultures from CD patients had the same mobility as E. coli, put in the gelas reference for the run. PCR screening for O25 and O83 demonstrated that these serogroups were dominant among the E. coli population from both controls and CD patients (only one control subject was negative for each of these O groups). Also, most of E. coli isolates from both control and CD patients belonged to the B2 phylogroup. Partial results of bacterial adherence to Hep-2 cells revealed that all patients (controls and CD) had aggregative adherent E. coli isolates. Tests to determine the ability to produce biofilm indicated that isolates from the proximal, but not from distal region of the gut or the stools, found in controls were stronger biofilm formers than isolates from equivalent sites of CD patients. In conclusion, the data presented here reveal that intestinal population of Proteobacteria of CD patients show dysbiosis associated with the elevation of Escherichia coli and that this variation can be assessed by TGGE and confirmed by sequencing of amplicons V6 region, the technique Ion Torrent. Finally, on the basis of the bacterial typing and characterization results, was observed that samples of both the case and controls, there was a prevalence of serogroups, based on this result we can associate both study groups frequent routinely the same hospital environment and may indicate a favoring the acquisition of these bacterial types. Similar results occurred with the analysis of serogroups, where only the filogrupo D had a significant prevalence in control subjects. For biofilm production, the samples found in the proximal region of the control patients intestine produce more biofilm than DC bearing samples of the same intestinal region, indicating that the intestinal mucosa serves as a substrate may influence the formation of biofilms. Key-words: Escherichia coli; Molecular typing; Biofilms. Lista de Ilustrações Figura 1 Estrutura antigênica de E. coli.5 Figura 2 Esquema de formação de biofilme bacteriano.6 Figura 3 Aderência bacteriana em células HEp-2.7 Figura 4 Esquema para a determinação dos grupos filogenéticos de E. coli. 9 Figura 5 Eletroforese perpendicular de Enterobactérias. 16 Figura 6 TGGE de amplicons de 16S V6-V8 rDNA de algumas Enterobactérias. 21 Figura 7 TGGE de amplicons de 16S V6-V8 rDNA de diferentes materiais clínicos de paciente com DC e controle.22 Figura 8 Gráfico da proporção de sequências de gêneros bacterianos nas culturas de biópsias. 25 Figura 9 Gráfico da prevalência dos sorogrupos.26 Figura 10-Gráfico da distribuição de filogrupos EcoR por grupo de pacientes.26 Figura 11 Gráfico da formação específica de biofilme por material clínico. 27 Figura 12 Gráfico da formação específica de biofilme por E. coli do grupo controle.28