Estudo epidemiológico molecular da resistência aos carbapenêmicos em Pseudomonas aeruginosa isoladas de sangue: produção de β-lactamases, perda de porina OprD e hiperexpressão de bombas de efluxo

Abstract

Introduction: The spread of Pseudomonas aeruginosa variants associated with the emergence of carbapenem resistance genotypes, is of great concern worldwide. Objectives: Molecular epidemiological study of carbapenem resistance in isolates of P. aeruginosa from blood. Material and Methods: We conducted a prospective observational study through active surveillance in the HC-UFU Microbiology Laboratory for detection of patients with P. aeruginosa bacteremia, from May/2009 to December/2012. The mechanisms of resistance to carbapenems were studied based on phenotypic methods, including Synergy Test with Double Disk and Hodge Test, and genotypic to detection of carnapenemase genes, including MBL genes (blaIMP, blaVIM, blaSIM, blaGIM e blaSPM) and OXA genes (blaOXA-51, blaOXA-23, blaOXA-40 e blaOXA-58) by multiplex PCR and gene blaKPC by end point PCR. The qRT-PCR technique was conducted to determine the intrinsic resistance mechanisms: overproduction of AmpC, overexpression of efflux pumps (MexABOprM, MexXY, MexCDOprJ, MexEFOprN) and loss of OprD porin. The PFGE was used to molecular typing of isolates presenting MBL genes. Results: 157 patients admitted to the HC-UFU with P. aeruginosa bacteremia were included. A total of 162 episodes/isolated bacteremia were observed, with the adult ICU presenting the highest rates of isolates resistant to carbapenems. A high consumption of ceftriaxone, cefepime and meropenem was observed, with an increasing trend until the end of the study period, and no positive correlation with carbapenems resistant isolates, however we observed an outbreak of these isolates in December 2010, predominantly in adult ICU. The mortality rate was high (43.3%) and the multivariate analysis showed that the predictors independently associated with death were patients with cancer and with inappropriate antimicrobial therapy, particularly from cefepime use. Risk factors associated with antimicrobial resistance were: hospital stay ≥ 30 days, ICU admission, prior use of antibiotics, and presence of invasive procedures, including tracheostomy, hemodialysis and urinary catheter. The mortality rates were higher among patients with resistant isolates. The inappropriate antibiotic therapy was a strong marker of worse prognostic. The MBL production was conducted in 56 isolated, with positivity of 21.4% by the phenotypic test, of which 75.0% presented amplicon consistent with MBL genes, being 66.7% of blaSPM-1 type and 33.3% of blaVIM. Of the 47 non-MBL carbapenems resistant isolates, none had another type of carbapenemase, and 14 were included in the study of resistance mechanisms from qRT-PCR, with rates of AmpC overproduction and OprD porin loss of 71,4%, for both. The rates of MexABOprM and MexXY overexpression were 57,1% and 64.3%, respectively. The evaluation of the clonal relationship among isolates with MBL genes, showed high similarity of those isolates presenting blaSPM, which was not observed for those containing the blaVIM gene. Conclusions: Our results showed that inappropriate therapy is a significant worse prognosis factor among patients with bacteremia resistant P. aeruginosa. In the absence of effective carbapenemase, P. aeruginosa isolates presented association of other resistance mechanisms. Furthermore, this study showed a cross spread of blaSPM clone and the first report of blaVIM at HC-UFU.