Avaliação dos mecanismos de virulência e do fitness bacteriano de isolados clínicos de Klebsiella pneumoniae produtores de KPC-2

• Ano de defesa: 2016
• Autor(a) principal: Visconde, Marina Francisco [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Klebsiella pneumoniae; Enterobacteria; β-lactamase; KPC; Bacterial resistance; Virulence; Pathogenicity; Animal model; Caenorhabditis elegans; Galleria mellonella; Bacterial fitness; Enterobactérias; Resistência bacteriana; Virulência; Patogenicidade; Modelo animal; Fitness bacteriano
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=3775676; https://repositorio.unifesp.br/handle/11600/47144

Objective: The aim of this study was to evaluate and compare the virulence and bacterial fitness of KPC-2-producing-K. pneumoniae isolates. Methods: A total of 30 K. pneumoniae clinical isolates distributed in three distinct groups, KPC-2-producing, ES?L-producing and community-acquired, were evaluated in this study. The genetic similarity among the isolates was determined by PFGE and MLST techniques. The antimicrobial susceptibility profile was determined by agar dilution and broth microdilution methodologies. The research of resistance and virulence genes were performed by PCR followed by DNA sequencing. OMPs characterization was evaluated by PCR, sequencing and SDS-PAGE techniques. For the in vitro evaluation of virulence factors, phenotypic tests were performed, such as the presence of hypermucoviscosity, biofilm formation, heat resistance, resistance to human serum, hemolysin production and phagocytosis by macrophages. The infection models using C. elegans and G. mellonella were performed for the evaluation of the in vivo virulence factors. The bacterial fitness was evaluated by measuring the growth rate of the isolates. Results: The genetic relationships evaluation showed the presence of 26 distinct PFGE patterns (A-Z) and 24 different STs distributed among 30 clinical isolates. High resistance rates to the antimicrobials agents tested were observed among KPC and ES?L groups. The resistance genes blaKPC, blaCTX-M, blaTEM, blaSHV, and blaOKP were found in KPC and ES?L groups, while only the blaSHV and blaLEN genes were found in the community group. All isolates showed alterations in OmpK36. The PCR revealed the presence of virulence genes clpK, rmpA, mrkD, fimH, kpn, ycfm, kfu, Aero1, Aero2, iutA, entB, ybtS, fyuA and traT in all groups of this study. The hypermucoviscosity phenotype was found in only three isolates. All isolates were resistant to the factors present in human serum and able to form biofilms. All clpk carrying isolates survived the thermal shock, excepted isolated A23177. None of the isolates was able to produce hemolysin. Significant susceptibility differences in phagocytosis were observed in the KPC-2 and ES?L groups. In C. elegans model, the KPC-2, ES?L and community groups showed an LT50 of 9.65, 10.2 and 10.4 days, respectively. In G. mellonella model, there was no significant difference among the three groups evaluated, as observed in the bacterial fitness assay. Conclusion: This study demonstrated that the multi-drug resistant K. pneumoniae clinical isolates producing KPC-2 and ES?L were as virulent as those isolates recovered from the community, without significant loss in bacterial fitness, which contributed with their persistance in the hospital environment.