# Molecular Characterization of Colistin-Resistant Clinical Acinetobacter baumannii from Northern Greece: Phenotypic Colistin Susceptibility and lpx/pmrCAB Mutational Profiles

## TL;DR

This study identifies genetic mutations linked to colistin resistance in Acinetobacter baumannii isolates from Greece, highlighting the need for surveillance and infection control.

## Contribution

The study reports novel pmrCAB mutations and clonal profiles associated with colistin resistance in A. baumannii from Northern Greece.

## Key findings

- All isolates were colistin-resistant with 95% classified as pan-drug-resistant.
- PmrB A226V and PmrC mutations were prevalent in the pmrCAB operon.
- MLST revealed a dominance of ST2 (IC2) among the isolates.

## Abstract

Background: Acinetobacter baumannii (A. baumannii) is a formidable nosocomial pathogen and is classified by the World Health Organization (WHO) as a critical-priority pathogen, owing to its rapid evolution into extensively drug-resistant (XDR) and pan-drug-resistant (PDR) strains. Colistin remains one of the last-resort therapeutic options, although resistance rates are increasing in endemic regions such as Greece. In this study, we investigated the molecular basis of colistin resistance and characterized the clonal backgrounds of clinical XDR/PDR A. baumannii isolates collected between January and June 2022 from two tertiary-care hospitals in Thessaloniki, Northern Greece. Methods: We analyzed forty non-duplicate XDR/PDR clinical isolates. Antimicrobial susceptibility was determined using the VITEK 2 system, broth microdilution, and gradient diffusion methods. The lipid A biosynthesis genes (lpxA, lpxC, lpxD) and the pmrCAB operon were amplified by PCR and sequenced for all isolates. A representative subset of strains (n = 10/40) underwent multilocus sequence typing (MLST) according to the Pasteur MLST scheme. Results: All isolates proved colistin-resistant (MIC ≥ 4 µg/mL), and 95% were classified as PDR. Sequence analysis revealed multiple nonsynonymous mutations in the pmrCAB operon, with the PmrB A226V substitution predominating and extensive amino-acid changes observed in PmrC. In contrast, lpx genes exhibited limited protein-level variation, limited to lineage-associated polymorphisms (LpxC N287D, LpxD E117K). A novel six-nucleotide insertion in pmrB was identified in one isolate. MLST demonstrated a predominance of ST2 (International Clone 2), with single representatives of ST115 (IC2) and ST1 (IC1). Conclusions: In this cohort from Northern Greece, chromosomal mutations in the pmrCAB operon, within a predominantly ST2/IC2 background, were strongly associated with colistin resistance. These findings underscore the urgent need for continued molecular surveillance and targeted infection-control measures to limit further spread of PDR A. baumannii.