Formação de biofilmes em meio biótico e abiótico por Escherichia coli isoladas de mastite bovina na presença de antimicrobianos

• Ano de defesa: 2013
• Autor(a) principal: Silva, Vítor de Oliveira
• Orientador(a): Moreira, Maria Aparecida Scatamburlo
• Banca de defesa: Lage, Andrey Pereira
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Mestrado em Medicina Veterinária
• Departamento: Biotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. de
• País: BR
• Palavras-chave em Português: Leite; qRT-PCR; Microscopia eletrônica de varredura
• Palavras-chave em Inglês: Milk; qRT-PCR; Scanning electron microscopy
• Área do conhecimento CNPq: CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::MEDICINA VETERINARIA PREVENTIVA
• Link de acesso: http://locus.ufv.br/handle/123456789/5144

Escherichia coli is a microorganism of environmental origin that is highly adaptive; its ability to form biofilms under certain conditions can be critical to its resistance to antimicrobial treatments. The aim of this study was to evaluate the expression profiles of the genes luxS (autoinducer synthase-2), fliC (flagellin), csgA (large subunit of the fimbriae curli ) and fimA (largest subunit type I fimbriae), which are involved in biofilm formation, in E. coli obtained from mastitic milk in the presence of antimicrobials on abiotic and biotic environments. The genetic profile of 27 isolates was evaluated by pulsed field gel electrophoresis (PFGE) with XbaI enzyme digestion. Four isolates of E. coli, two strong biofilm producers (GBP) and two weak producers (WBP) which possessed the genes luxS, fliC, csgA and fimA, were selected by conventional PCR. The minimum inhibitory concentration (MIC) of antimicrobials was assessed for performing other tests at the concentration of ½ MIC. The biofilm production was detected in the presence of antimicrobials and visualized through scanning electron microscopy (SEM). Assay of adhesion/invasion of bacterial cells in bovine mammary alveolar cell (MAC-T) culture in the presence of antimicrobials was quantified in CFU/mL and visualization was achieved by confocal microscopy. Subsequently, the expression profiles of the genes luxS, fliC, csgA and fimA were analyzed at different times after inoculation on abiotic and biotic surfaces. Nine (I to IX) different phylogenetic groups with similarity > 90% were identified by PFGE. The results revealed the presence of the genes luxS, fimA and csgA in all 27 isolates, whereas the fliC gene was only detected in 16. The MICs ranged from 0.05 to 8 μg/mL, with the highest values obtained for gentamicin (8 μg/mL) and ampicillin (6 μg/mL). Enrofloxacin and cotrimoxazole induced biofilm formation in at least one isolated WBP, and ampicillin and gentamicin in one isolated GBP. Also, the reducing effect of enrofloxacin and cotrimoxazole was observed in at least one isolated GBP. There was no increase in the ability of adxiv hesion/invasion of the isolates of E. coli in the presence of antibiotics, but an increase in the internalization of the WBP isolate when treated with antimicrobials was observed through confocal microscopy. The four genes were differentially expressed when exposed to ½ MIC concentrations of antimicrobials with changes in the order of 1.5 to 22 times. The growth on the biotic surface favored the expression of luxS and fimA genes in relation to abiotic surfaces. The sub-inhibitory concentrations of the antimicrobials studied were able to induce the formation of biofilms by some isolates, as well as inducing invasion into host cells, which is a situation that might be occurring in vivo. This could result in a reservoir of bacteria in the udder of the animal, which could explain the increase in cases of persistent mastitis in herds.