Chromosomal Mechanisms of Colistin Resistance in Clinical Isolates of Carbapenem‐Resistant <em>Klebsiella pneumoniae</em> from a Tunisian Tertiary‐Care Hospital

Background/Objectives: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major nosocomial pathogen. Although newer agents have reduced colistin use in high-income countries, this polymyxin remains important in many low- and middle-income settings. Colistin resistance in K. pneumoniae is mainly driven by chromosomal changes in the MgrB–PhoPQ pathway or plasmid-mediated mcr genes. This study aimed to investigate chromosomally mediated colistin resistance in CRKP clinical isolates from a Tunisian ter-tiary hospital. Methods: Between 2010 and 2015, 317 non-duplicate CRKP isolates were collected at Charles Nicolle Hospital, Tunis. Colistin MICs were determined by broth microdilution. Phenotypic tests and PCR characterized carbapenemases, extended-spectrum β-lactamases, AmpC, plasmid-mediated quinolone resistance, mcr and virulence genes. Porins (OmpK35/OmpK36) and the mgrB, phoP and phoQ loci were analyzed by SDS-PAGE and sequencing. Clonal relatedness was assessed by ERIC-PCR and multi-locus sequence typing. Results: Five isolates (1.6%) were colistin-resistant. All were multidrug-resistant, pro-duced OXA-48 and two also carried NDM-1. The isolates belonged to five distinct sequence types, including high-risk clones (ST11, ST101, ST147). No mcr genes were detected. Four isolates carried disruptive mutations in mgrB, and the remaining strain harbored inactivating mutations in both phoP and phoQ with an intact mgrB. Truncating alterations in PhoP/PhoQ and frequent loss or truncation of OmpK35/OmpK36 were observed. Conclusions: In Tunisian CRKP, colistin resistance was mediated by chromosomal alterations, primarily disruption of the MgrB–PhoPQ pathway, in the absence of mcr genes. These mechanisms in both high-risk and emerging sequence types underscore the adaptability of CRKP and the need for surveillance where colistin remains an important therapeutic option.