RESUMO
An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.
Assuntos
Humanos , Carbapenêmicos/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Mutação , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Resistência beta-Lactâmica/genética , beta-Lactamases/metabolismo , Aminoglicosídeos/metabolismo , Anfotericina B/análogos & derivados , Anfotericina B/metabolismo , Antifúngicos/metabolismo , Brasil , Cefalosporinase/classificação , Cefalosporinase/metabolismo , Códon sem Sentido/metabolismo , Ativação Enzimática/genética , Mutação da Fase de Leitura/genética , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana Transportadoras/metabolismo , Metiltransferases/metabolismo , Nucleotidiltransferases/metabolismo , Mutação Puntual/genética , Porinas/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/isolamento & purificação , Sequências Repetitivas de Ácido Nucleico , beta-Lactamases/genéticaRESUMO
The pigmentation (pgm) locus is a large unstable area of the Yersinia pestis chromosome composed of a segment of iron acquisition (HPI) linked to a pigmentation segment. In this work we examined the mobility of HPI and the pigmentation segment in three Y. pestis isolates using successive subcultures on Congo red agar (CRA) plates. Strain P. CE 882 was shown to be highly stable while strains P. Exu 340 and P. Peru 375 dissociated into several phenotypes, PCR analysis showing evidence of changes in the pgm locus of the derived cultures. Strains P. Exu 340 and P. Peru 375 produced previously unreported cultures positive for the pesticin/yersiniabactin outer membrane receptor (psn+) but negative for the iron-regulated protein (irp2-), suggesting the occurrence of rearrangements in this chromosomal region and either a sequential loss or the loss of separated segments. These results provide evidence that besides deletion en bloc, specific rearrangements are also involved in the deletion events for that locus.