title: Molecular mechanisms of membrane impermeability in clinical isolates of Enterobacteriaceae exposed to imipenem selective pressure.
abstract: Intrinsic mechanisms leading to carbapenem-induced membrane impermeability and multidrug resistance are poorly understood in clinical isolates of Enterobacteriaceae. In this study, molecular behaviours during the establishment of membrane impermeability in members of the Enterobacteriaceae family under imipenem selective pressure were investigated. Clinical isolates (n = 22) exhibiting susceptibility to multiple antibiotics or characterised as extended-spectrum β-lactamase (ESBL)- or AmpC-producers were submitted to progressive passages on Mueller-Hinton agar plates containing subclinical concentrations of imipenem [0.5 × the minimum inhibitory concentration (MIC)]. Changes in outer membrane permeability were evaluated by determination of antimicrobial MICs, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and gene expression analysis related to membrane permeability (i.e. omp35-like, omp36-like and acrA) and regulatory mechanisms (i.e. marA and ompR) by quantitative reverse transcription PCR. Following imipenem induction, 73% of isolates showed increased carbapenem MICs by ≥2 doubling dilutions. At an early stage of treatment, imipenem modulated the expression of porins and efflux pump genes, represented by a reduction of 78% in omp36-like and a two-fold increase in acrA expression. Transcriptional factors marA and ompR were also affected by imipenem induction, increasing mRNA expression by 14- and 4-fold, respectively. High marA expression levels were associated with higher values of acrA expression. These results suggest that imipenem is an important factor in the development of an adaptive response to carbapenems by regulating key genes involved in the control of efflux pumps and porins, which could lead to a multidrug-resistant profile in clinical isolates, contributing to possible treatment failure.
authors.affiliation: Department of Clinical Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil; Applied Molecular Biology Laboratory, Department of Basic Science, Universidad de La Frontera, Temuco, Chile. Electronic address: monica.pavez@ufrontera.cl.
authors.forename: Monica
authors.initials: M
authors.lastname: Pavez
authors.affiliation: Department of Clinical Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Camila
authors.initials: C
authors.lastname: Vieira
authors.affiliation: Laboratory of Clinical Microbiology, Hospital Beneficência Portuguesa, São Paulo, Brazil.
authors.forename: Maria Rita
authors.initials: MR
authors.lastname: de Araujo
authors.affiliation: Department of Clinical Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil; Center of Molecular Biology and Pharmacogenetics, BIOREN-CEGIN, Universidad de la Frontera, Temuco, Chile.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Cerda
authors.affiliation: Department of Clinical Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Lara Mendes
authors.initials: LM
authors.lastname: de Almeida
authors.affiliation: Department of Clinical Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Nilton
authors.initials: N
authors.lastname: Lincopan
authors.affiliation: Department of Clinical Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Elsa Masae
authors.initials: EM
authors.lastname: Mamizuka
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002462:Cell Membrane / Q000502:physiology*; D024881:Drug Resistance, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004755:Enterobacteriaceae / Q000187:drug effects*; D020869:Gene Expression Profiling; D006801:Humans; D015378:Imipenem / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D010539:Permeability; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012641:Selection, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D015378:Imipenem
keywords: Carbapenem resistance; Enterobacteriaceae; Imipenem selective pressure; Membrane impermeability