Aspectos biológicos da interação bactéria-droga-hospedeiro em modelo de Infecção por Bacteroides fragilis e tratamento com metronidazol em concentração subinibitória

Abstract

Bacteroides fragilis, a member of the resident intestinal microbiota is the most important pathogen associated to endogenous infections. Meanwhile, metronidazole persists as the main drug available for treating these diseases. As other resident microorganisms, these bacteria are exposed to ecological disturbances, often resulting from subinhibitory concentrations of antimicrobial agents that can trigger changes in microbial biology and which may affect potential aggressor. Considering that the studies of drug-bacteria interactions usually uses in vitro assays, questions about the true extension of the phenomenon in a real situation of microbial infection and exposure to antimicrobials in subinhibitory concentrations remains. Thus, for a better understanding of drug-bacteria-host relationship in real situation of infection, an in vivo model of interaction has been proposed. Wistar rats were subjected to laparotomy to insert a perforated table tenis ball into the peritoneal cavity, followed by percutaneous challenge with B. fragilis ATCC 43859. The animals were divided into three experimental groups: negative control (NC) (n = 18) animals without bacterial inoculation using the vehicle only; positive control (PC) (n = 30) animals infected with the parental bacterial strain; experimental group (EG) (n = 24) animals infected with the parental bacterial strain, treated with subinhibitory concentration of metronidazole (1µg/mL percutaneous) for a period of eight days in 48 hour intervals. To evaluate the impact of metronidazole in the bacteria-host relationship the experimental infections were evaluated two times during the infectious process; first, 8 days after the treatment with MTZ and 8 days after the end of the treatment (16 days). The dynamics of the microbial colonization into the tissue cage was evaluated by conventional PCR, real time PCR and Gram stain. In the host, histological and transcriptional analysis of cytokine expression was performed. Morpho-physiological, biochemical and molecular characteristics of bacteria selected in vivo and in vitro were evaluated by Gram stain, kit API20, experimental biofilm formation, tolerance to oxidative stress (H2O2), and genotyping by AP-PCR. The results show that in vivo infection by Bacteroides fragilis remained as monoinfection throughout the experimental period with bacterial viability even after the treatment with sub inhibitory concentration of metronidazole. Histological analysis of the tissue formed into the tissue cage showed exacerbated inflammatory infiltrate after 8 days of treatment in both infected groups. However, after 16 days the inflammatory infiltrate decreased in the EG group. Increased cytokine expression was observed when infected groups were compared, mainly related to interleukin 10. Although changes in the bacterial biochemical profile were not observed, aberrant morphology, increased biofilm formation and decreased sensitivity to oxidative stress were observed in the in vitro selected strains. Genetic polymorphism was not detected by AP-PCR. Our results suggest that low concentrations of metronidazole interfere in the bacteria-host relationship, which could have implications in the infection process even without showing obvious alterations in the bacterial cells as demonstrated in vitro. Therefore, the complexity of drug-bacteria-host interactions in real infection situations and its consequences in microbial biology and pathogenesis is clear.