Plasmid pHXY0908 confers ciprofloxacin heteroresistance to Salmonella enterica serovar typhimurium ATCC 14028 by regulating efflux pump gene expression

Salmonella enterica Serovar Typhimurium (S. Typhimurium) is a major global foodborne pathogen. In our previous study, the S. Typhimurium ATCC 14028 strain harboring plasmid pHXY0908 survived at fourfold its ciprofloxacin MIC (MIC0908 = 0.125 µg/mL), but the mechanism remained unclear. Heteroresistance, where minority subpopulations show elevated resistance, is among the key contributors to chronic, recurrent, and high-fatality infections. This study aimed to elucidate the molecular mechanisms by which plasmid pHXY0908 promotes ciprofloxacin heteroresistance in S. Typhimurium ATCC 14028. Ciprofloxacin heteroresistance in ATCC 14,028-pHXY0908 was confirmed by population analysis profiling, antimicrobial susceptibility testing, and stability assessment. Whole-genome sequencing (WGS) and transcriptome (RNA-seq) analyses were performed to identify genomic variations and global transcriptional changes linked to the development of heteroresistance. WGS revealed minimal DNA mutations in subclones with increased MIC, with no single nucleotide variants detected in known ciprofloxacin resistance genes. Notably, plasmid pHXY0908 acquisition altered chromosomal interaction domains (CIDs), potentially affecting gene regulation. Weighted gene co-expression network analysis (WGCNA) of transcriptomic profiles identified a “brown” module comprising 168 eigengenes that were significantly correlated with MIC values. Within this module, the efflux pump genes acrA, acrB, and tolC exhibited high gene significance and module membership scores. Additional candidate genes potentially contributing to ciprofloxacin heteroresistance were identified, including sodB, yhiH, yfaV, STM14_2687, and pHXY0908-99. Notably, STM14_2687 (a LysR-type transcriptional regulator), negatively regulated acrAB-tolC; its overexpression suppressed acrA and enhancing susceptibility to ciprofloxacin. Furthermore, functional validation demonstrated that plasmid pHXY0908 could confer heteroresistance to ciprofloxacin in other Salmonella enterica strains, Escherichia coli, and Klebsiella pneumoniae, suggesting its broad-host-range potential in mediating heteroresistance. Our study reveals that transcriptional regulation drives ciprofloxacin heteroresistance in S. Typhimurium ATCC 14028 carrying plasmid pHXY0908. We show that regulation of efflux pumps (e.g., acrAB-tolC), superoxide dismutase (sodB), DNA protection systems, and regulatory networks contributes to heteroresistance development. Plasmid pHXY0908 acquisition was found to confer ciprofloxacin heteroresistance across species, suggesting its potential for horizontal transfer and increased transmission risk. Notably, STM14_2687 was identified and validated as a transcription factor that represses acrAB-tolC expression and may serve as a potential therapeutic target.