Molecular characterization of colistin resistance in carbapenem-resistant <i>Klebsiella pneumoniae</i> from a tertiary hospital in China

Colistin resistance in carbapenem-resistant <i>Klebsiella pneumoniae</i> (CRKP) poses a significant global health challenge, as colistin remains the last-resort antibiotic for treating multidrug-resistant <i>K. pneumoniae</i> infections. This study aimed to investigate the prevalence and molecular mechanisms underlying colistin resistance in CRKP (Col^r-CRKP) isolates in Henan, China, from 2021 to 2024. The minimum inhibitory concentrations of colistin for 134 <i>K</i>. <i>pneumoniae</i> isolates were determined using the broth microdilution method. Whole-genome sequencing was performed using the Illumina platform to identify carbapenemase genes and sequence types (STs). Colistin resistance mechanisms were investigated, including mutations in two-component systems (<i>pmrA</i>/<i>pmrB</i>, <i>phoP</i>/<i>phoQ</i>), inactivation of the <i>mgrB</i> gene, and the presence of plasmid-mediated <i>mcr</i> genes. Most isolates were collected from intensive care units (99/134, 73.9%), with 48.5% (59/134) of patients having no documented colistin exposure history. Notably, ST11 was the predominant sequence type among Col^r-CRKP isolates (113/134, 84.3%), all of which carried <i>bla</i>_KPC-2 as the sole carbapenemase determinant. In contrast, seven non-carbapenemase-producing isolates exhibited phenotypic resistance to carbapenems. Genomic analysis revealed inactivation or loss of the <i>mgrB</i> gene in 53.7% (72/134) of isolates, predominantly due to insertion mutations (54/72). Although 32.8% (44/134) of isolates carried mutations in two-component systems, these alterations did not exhibit pathway-specific clustering. Intriguingly, plasmid-mediated <i>mcr</i> genes were detected in only 1.5% (2/134) of cases (<i>mcr-8.2</i> and <i>mcr-1.1</i>), while 22.4% (30/134) of colistin-resistant strains lacked identifiable resistance determinants based on current detection methods. Our findings indicate that disruption of the <i>mgrB</i> gene is the primary mechanism of colistin resistance in ST11 CRKP clones. The emergence of resistance in 48.5% of patients without prior colistin exposure, combined with low <i>mcr</i> gene prevalence (1.5%) and unexplained resistance in 22.4% of isolates, suggests complex selective pressures beyond direct antimicrobial use. These findings underscore the urgent need for strengthened antimicrobial stewardship and the development of alternative therapeutic strategies to combat this high-risk pathogen.IMPORTANCEThe global rise of colistin-resistant <i>Klebsiella pneumoniae</i>, particularly in carbapenem-resistant <i>Klebsiella pneumoniae</i> (CRKP) strains, has severely restricted treatment options for multidrug-resistant infections. Our study provides the first comprehensive molecular characterization of colistin resistance in CRKP in a large tertiary hospital in central China. We identified <i>mgrB</i> disruption as the predominant resistance mechanism, while plasmid-mediated <i>mcr</i> genes were rare. Notably, nearly half of the resistant isolates occurred in patients without prior colistin exposure, suggesting alternative selective pressures driving resistance. These findings highlight the complex dynamics of colistin resistance in CRKP and underscore the need for enhanced genomic surveillance and stewardship interventions to limit further dissemination.