Detecção e caracterização de determinantes de resistência aos antibióticos ß-lactâmicos e quinolonas em bactérias Gram-negativas isoladas de amostras clínicas

Abstract

The main objective of this work was to characterize the qnr, aac(6')-Ib-cr and qepA plasmid genes, and also the blaTEM, blaSHV and blaCTX-M genes, encoding for extended spectrum β-lactamases - ESBL in a collection of Enterobacteriacecae isolated from clinical samples obtained in two hospitals in the state of Minas Gerais, Brazil. A total of 873 Enterobacteriaceae were isolated in the period of June to October 2010 of which 106 (12%) were screened for this study showing decreased susceptibility to nalidixic acid. The bacterial identification was performed by conventional biochemical tests and by the automated system MicroScan autoSCAN®-4. The antimicrobial susceptibility profile was determined by diffusion disk and the minimum inhibitory concentration was determined by broth microdilution for the isolates carrying the qnr genes e their respective transconjugantsaccording with the recommendations of the "Clinical Laboratory Standards Institute - CLSI". PCR and sequencing were performed to detect plasmid-mediated quinolone resistant (PMQR) genes as well as the analysis of mutations in chromosomal encoded genes, gyrA and parC by. The extraction of plasmids and their transconjugants containing qnr determinants was performed using the technique of Kieser. The results obtained by pulsed field gel electrophoresis were interpreted according with Tenover et al., 1995. The collection was composed as follow: Escherichia coli (n = 61, 57.6%), Enterobacter cloacae (n = 23, 21.7%), Klebsiella pneumoniae (n = 16, 15.1%), Proteus mirabilis (n= 3, 2.8%), Serratia marcescens (n = 2, 1.9%) and Proteus vulgaris (n = 1, 0.9%). Most of the enterobacteria (82%) was isolated from urinare followed by blood, swab of secretions, tracheal aspirate and catheter tip. A total of 100 (94%) isolates were resistant to nalidixic acid, 75 (71%) were resistant to ciprofloxacin and 81 (76%) showed reduced susceptibility to ceftazidime and/or cefotaxime. The qnr plasmidic determinant research resulted in the detection of qnrB and qnrS genes in 13 isolates, including E. cloacae (n=6), K. pneumoniae (n=5) andE. coli (n=2). QnrB1 was the most prevalent gene, followed by qnrS1, qnrB2 and qnrB19. In four qnr positive isolates also carried the aac(6')-Ib-cr gene, to blaSHV-like (n=6), blaCTX-M (n=11) and blaTEM gene (n=11). The qepA gene was not detected. This work indicates the dissemination of PMQR in clinical specimens in Brazil. The coexistence of ESBL and PMQR genes demonstrate the complexity of plasmids carrying resistant genesamong members of the family Enterobacteriaceae.