Characterization of mechanisms of carbapenem resistance of clinical isolates of Enterobacter spp.

Abstract

The Enterobacter belongs to a group of bacteria called CESP (Citrobacter spp, Enterobacter spp, Serratia sp and Proteus spp), which are Gram-negative constitutive producers of chromosomal beta-lactamases such as AmpC type enzymes that during the use of antimicrobials can express an increase in production and therefore resistance to beta-lactams. So with the advent of these multi-resistant strains, the most widely used class of antibiotics for more than a decade to treat infections by these organisms were the carbapenems. However, resistance to carbapenems is starting to emerge and consequently the choice of an appropriated therapy is complicated by the fact that the majority of Enterobacter sppisresistant to many antibacterial agents or can develop resistance during treatment.These bacterial pathogens harbor a variety of antibiotic resistance mechanisms. Among them, the modification of outer membrane permeability, a porin deficiency associated with the expression of cephalosporinase activity and production of carbapenemases, is detected in clinical resistant isolates.The carbapenemases belong mostly to two molecular classes, distinguished by the conformation of its active site, the class A carbapenemases (NMC/IMI (imipenemase/ non-metallo carbapenemase-A), SME (Serratiamarcescens enzyme), KPC (Klebsiellapneumoniae carbapenemase),GES (Guiana extended-spectrum)), and metallo-²-lactamases of Ambler class B (IMP (imipenemase), VIM (Verona imipenemase), SPM (São Paulo metalo²-lactamase), GIM (German imipenemase), SIM (Seoul imipenemase) and most recently DM-1 (New Delhi).But few studies on the mechanisms of resistance to carbapenems in Enterobacterspp has been made in the world. So, the purpose of this study is characterize the mechanism of carbapenems resistance in 52 Enterobacter spp strains from three hospitals in Brazil, determining the genetic relatedness and antimicrobial susceptibility of these isolates.Therefore to characterize these resistance mechanisms the following methods will be used: antimicrobial susceptibility by microdilution broth of the following antimicrobials: Imipenem, Meropenem, Ertapenem, Polymyxin B, Amikacin, Cefepime, Tigecycline, Ciprofloxacin, Piperacillin / Tazobactam, and only for Fosfomycin the agar microdilution will be performed. Imipenem hydrolysis will be investigated by spectrophotometric. Carbapenemase-encoding genes, genes pump efflux and porins being identified by PCR assays and confirmed by amplicon sequencing. To analyze the outer membrane the SDS- PAGE method will be used. To study clonality the methodology known as PFGE will be used and the expression of pump efflux will be monitored by real-time reverse-transcription polymerase chain reaction (RT-PCR). At the same time the efflux pump will be identified in the presence and absence of efflux pump inhibitors. (AU)