Phenotypic and Genotypic Characterization of Acinetobacter spp. Panel Strains: A Cornerstone to Facilitate Antimicrobial Development

Abstract

Acinetobacter spp. 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 combat antimicrobial resistance. To address this issue, we have constructed a panel of Acinetobacter spp. strains expressing different antimicrobial resistance determinants such as narrow spectrum β-lactamases, extended-spectrum β-lactamases, OXA-type-carbapenemase, metallo-beta-lactamase, and over-expressed AmpC β-lactamase. Bacterial strains exhibiting different resistance phenotypes were collected between 2008 and 2013 from Severance Hospital, Seoul. Antimicrobial susceptibility was determined according to the CLSI guidelines using agar dilution method. Selected strains were sequenced using Ion Torrent PGM system, annotated using RAST server and analyzed using Geneious pro 8.0. Genotypic determinants, 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 of bla PER−1 gene, the overproduction of intrinsic AmpC beta-lactamase associated with IS Aba1 insertion, 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 with phenotypic resistance. 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, phenotypic resistance 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 drug development. It can also be used as a reference strain in the clinical laboratory and for the evaluation of antibiotic efficacy for specific resistance mechanisms.

A Diverse Panel of Clinical Acinetobacter baumannii for Research and Development

“…This narrow strain set does not represent the true clinical diversity of circulating A. baumannii and does not capture emerging epidemic clones. In 2019, a second Acinetobacter panel was also created by D'Souza and colleagues, but it contains just 8 A. baumannii strains, 3 Acinetobacter pittii strains, and 1 strain of Acinetobacter nosocomialis collected from a single hospital (46). Although the isolates had diverse antimicrobial resistance profiles, none of the eight A. baumannii strains belongs to a successful epidemic clone.…”

Acinetobacter baumannii Antibiotic Resistance Mechanisms

“…Quinolone resistance-determining regions (QRDRs) refer mainly to alteration of target sites in gyrase (Ser83Leu, Gly81Asp, and Ser81Leu mutations preventing quinolones from binding its alpha-subunit) and topoisomerase IV (mutations Ser80Leu, Glu84Lys, and Gly78Cys, and Ser84Leu in its subunit C). Although a single point mutation in DNA gyrase is usually not enough for resistance to fluoroquinolones in A. baumannii (maybe only against levofloxacin; single parC mutations link with ciprofloxacin resistance), concurrent mutations within QRDR regions of the gyrA and parC genes are linked with significantly higher level of quinolone resistance [ 130 , 131 ]. Alterations in gyrB and parE genes are of minor significance [ 126 ].…”

In Vitro Activity of a Novel Siderophore-Cephalosporin, GT-1 and Serine-Type β-Lactamase Inhibitor, GT-055, against Escherichia coli, Klebsiella pneumoniae and Acinetobacter spp. Panel Strains

“…Acinetobacter spp. and K. pneumoniae isolates were selected from our previously characterised panel strains [21,22]. Three K. pneumoniae strains (YMC2016/01/R859, YMC2016/02/N207 and YMC2016/04/N62) exhibiting carbapenem resistance were also added.…”