Alejandra Vera-Leiva1, Sergio Carrasco-Anabalón1, Celia A Lima1, Nicolás Villagra2, Mariana Domínguez1, Helia Bello-Toledo1, Gerardo González-Rocha3. 1. Universidad de Concepción, Facultad de Ciencias Biológicas, Departamento de Microbiología, Laboratorio de Investigación en Agentes Antibacterianos, Concepción, Chile. 2. Universidad Andres Bello, Facultad de Medicina, Laboratorio de Patogénesis Molecular y Antimicrobianos, Santiago, Chile. 3. Universidad de Concepción, Facultad de Ciencias Biológicas, Departamento de Microbiología, Laboratorio de Investigación en Agentes Antibacterianos, Concepción, Chile. Electronic address: ggonzal@udec.cl.
Abstract
OBJECTIVES: KPC-producing strains present a wide range of carbapenem minimum inhibitory concentrations (MICs). This variation may be due to differential expression of blaKPC and porin genes, efflux pump activity and the production of extended-spectrum β-lactamases and/or AmpC β-lactamases. The aim of this study was to determine the role of efflux pumps inhibited by phenylalanine-arginine β-naphthylamide (PAβN) in resistance to carbapenems in Chilean clinical isolates of blaKPC-harbouring Klebsiella pneumoniae. METHODS: MICs were determined by the agar dilution method for imipenem, meropenem, ertapenem and ciprofloxacin in the presence and absence of PAβN (25mg/L) in 17 carbapenem-resistant KPC-producing K. pneumoniae strains. Outer protein membrane (OMP) profiles were determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Expression levels of the ompK35 and ompK36 genes were also determined by real-time quantitative reverse transcription PCR (qRT-PCR). RESULTS: No contribution of PAβN-inhibited efflux pumps to carbapenem resistance was found, unlike ciprofloxacin resistance. However, a ≥4-fold increase in the MIC of at least one carbapenem was observed in 13 isolates in the presence of PAβN. Additionally, decreased gene expression of ompK35 and ompK36 in the presence of PAβN was detected, however no obvious differences in porin band intensity were observed by SDS-PAGE. CONCLUSIONS: The presence of PAβN resulted in an increase in carbapenem MICs unrelated to efflux pump inhibition, and a decrease in the expression of ompK35 and ompK36 genes without an obvious difference in OMP profiles observed by SDS-PAGE. Therefore, additional factors are responsible for the increase in carbapenem MIC in the presence of PAβN.
OBJECTIVES: KPC-producing strains present a wide range of carbapenem minimum inhibitory concentrations (MICs). This variation may be due to differential expression of blaKPC and porin genes, efflux pump activity and the production of extended-spectrum β-lactamases and/or AmpC β-lactamases. The aim of this study was to determine the role of efflux pumps inhibited by phenylalanine-arginine β-naphthylamide (PAβN) in resistance to carbapenems in Chilean clinical isolates of blaKPC-harbouring Klebsiella pneumoniae. METHODS: MICs were determined by the agar dilution method for imipenem, meropenem, ertapenem and ciprofloxacin in the presence and absence of PAβN (25mg/L) in 17 carbapenem-resistant KPC-producing K. pneumoniae strains. Outer protein membrane (OMP) profiles were determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Expression levels of the ompK35 and ompK36 genes were also determined by real-time quantitative reverse transcription PCR (qRT-PCR). RESULTS: No contribution of PAβN-inhibited efflux pumps to carbapenem resistance was found, unlike ciprofloxacin resistance. However, a ≥4-fold increase in the MIC of at least one carbapenem was observed in 13 isolates in the presence of PAβN. Additionally, decreased gene expression of ompK35 and ompK36 in the presence of PAβN was detected, however no obvious differences in porin band intensity were observed by SDS-PAGE. CONCLUSIONS: The presence of PAβN resulted in an increase in carbapenem MICs unrelated to efflux pump inhibition, and a decrease in the expression of ompK35 and ompK36 genes without an obvious difference in OMP profiles observed by SDS-PAGE. Therefore, additional factors are responsible for the increase in carbapenem MIC in the presence of PAβN.
Authors: Maria Spencer-Sandino; Roberto Riquelme-Neira; William C Shropshire; An Q Dinh; Gerardo González-Rocha; Paulina González-Muñoz; Alejandra Vera-Leiva; Rafael Araos; Blake Hanson; Cesar A Arias; José M Munita Journal: JAC Antimicrob Resist Date: 2021-06-21