Abstract
BACKGROUND: This study aimed to investigate the prevalence and underlying mechanisms of colistin resistance among 557 non-duplicate CRPA isolates collected from a tertiary hospital in Jiangxi, China, between 2021 and 2024. METHODS: Antimicrobial susceptibility testing, whole-genome sequencing, bioinformatic analyses, site-directed mutagenesis, RT-qPCR and time-kill kinetics assay were performed to investigate the mechanisms of colistin resistance in CRPA. RESULTS: Among the 557 CRPA isolates, colistin minimum inhibitory concentrations (MICs) ranged from 0.125 to 32 mg/L, with MIC(50) and MIC(90) values of 1/2 mg/L. Twenty‑three isolates (23/557, 4.1%) exhibited colistin resistance (ColR‑CRPA), all showing multidrug‑resistant phenotypes. The 23 ColR‑CRPA isolates displayed extensive genetic diversity, encompassing 20 sequence types, with only two potential nosocomial transmission clusters (ST235 and ST1236) identified. Mutations in two‑component systems (pmrAB, phoPQ, parRS, and cprRS), lipid A modification genes (arnBCADTEF, eptA/eptA2/eptC), efflux regulators, and oprD were ubiquitous. Thirteen distinct amino acid substitutions were identified in PmrAB and PhoPQ, among which PmrA L71R (19/23, 82.6%) and PmrB Y345H (22/23, 95.7%) were the most prevalent. Functional characterization of all thirteen substitutions revealed that only PmrA Q109K, PmrB L107P, and PhoQ V260G significantly contributed to colistin resistance. Introducing these substitutions into the colistin‑susceptible reference strain PAO1 increased MICs by 2–4 fold, whereas reversion of PmrA Q109K or PhoQ V260G in clinical isolates reduced MICs from 32 to 4 mg/L. RT‑qPCR analysis showed that these substitutions significantly upregulated arnB and pmrA expression (P < 0.0001). Time‑kill kinetics assays further confirmed the enhanced colistin tolerance conferred by these mutations. CONCLUSIONS: Colistin resistance in CRPA remains uncommon but is mediated by complex networks of chromosomal mutations. These findings highlight the importance of continuous genomic surveillance and strict infection‑control measures to prevent the emergence and dissemination of colistin‑resistant CRPA. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12866-026-04872-5.