Phenotypic and genotypic characterization of Acinetobacter spp. panel strains: A cornerstone to facilitate antimicrobial development

Abstract

Acinetobacterspp. have emerged as significant pathogens causing nosocomial infections. Treatment of these pathogens has become a major challenge to clinicians worldwide, due to their increasing tendency to antibiotic resistance. To address this, much revenue and technology are currently being dedicated toward developing novel drugs and antibiotic combinations to combatantimicrobialresistance. To address this issue, we have constructed apanelofAcinetobacterspp.strainsexpressing differentantimicrobialresistance determinants such as narrow spectrum β-lactamases, extended-spectrum β-lactamases, OXA-type-carbapenemase, metallo-beta-lactamase, and over-expressed AmpC β-lactamase. Bacterialstrainsexhibiting different resistance phenotypes were collected between 2008 and 2013 from Severance Hospital, Seoul.Antimicrobialsusceptibility was determined according to the CLSI guidelines using agar dilution method. Selectedstrainswere sequenced using Ion Torrent PGM system, annotated using RAST server and analyzed using Geneious pro 8.0.Genotypicdeterminants, such as acquired resistance genes, changes in the expression of efflux pumps, mutations, and porin alternations, contributing to the relevant expressed phenotype were characterized. Isolates expressing ESBL phenotype consisted ofblaPER-1gene, the overproduction of intrinsic AmpC beta-lactamase associated with ISAba1insertion, and carbapenem resistance associated with production of carbapenem-hydrolyzing Ambler class D β-lactamases, such as OXA-23, OXA-66, OXA-120, OXA-500, and metallo-β-lactamase, SIM-1. We have analyzed the relative expression of Ade efflux systems, and determined the sequences of their regulators to correlate withphenotypicresistance. Quinolone resistance-determining regions were analyzed to understand fluoroquinolone-resistance. Virulence factors responsible for pathogenesis were also identified. Due to several mutations, acquisition of multiple resistance genes and transposon insertion,phenotypicresistance decision scheme for for evaluating the resistance proved inaccurate, which highlights the urgent need for modification to this scheme. This complete illustration of mechanism contributing to specific resistance phenotypes can be used as a target for novel drugdevelopment. It can also be used as a reference strain in the clinical laboratory and for the evaluation of antibiotic efficacy for specific resistance mechanisms.