Prevalence of polymyxin heteroresistance in Klebsiella pneumoniae under sustained antibiotic selection during experimental evolution.

The swift increase in resistance exhibited by Klebsiella pneumoniae, primarily a nosocomial pathogen, against all frontline antibiotics has significantly restricted the available therapeutic options. Additionally, the rising resistance to polymyxins, frequently considered the last-resort antibiotic, has further exacerbated the situation. The molecular mechanisms responsible for developing high levels of polymyxin resistance are well understood. However, the evolutionary trajectory under antibiotic selection that mimics the clinical setting is not fully elucidated. This study aimed to investigate the development of stable polymyxin resistance in K. pneumoniae using adaptive laboratory evolution (ALE). The K. pneumoniae (ATCC 13883) strain was subjected to ALE with stepwise increasing concentrations of polymyxin-B (PB) and colistin (Col) for 17 days (106 generations). Phenotypic characterisation indicated that the MIC of the polymyxins among the evolved strains increased by 64- to 128-fold. The evolved strains produced smaller hypo-mucoid colonies on LB agar than their ancestral strain and produced significantly more biofilm. However, the phenotypic distinction in the LB broth among the ancestral and the evolved strains was not apparent. Cross-resistance between Col and PB and increased resistance to trimethoprim among the evolved strains was observed. Interestingly, the evolved strains developed collateral sensitivity to ampicillin, kanamycin, and gentamycin. Furthermore, the evolved strains showed polymyxin heteroresistance (HR), where a subpopulation of isogenic bacteria exhibits differential antibiotic susceptibility, often responsible for antibiotic treatment failure. HR was detected using the Kirby-Bauer disk diffusion assay and the E-test and further confirmed by population analysis profiling (PAP) assay. Polymyxin resistance and HR phenotypes remained stable for 100 generations even without antibiotic selection. However, nucleotide sequencing of the targeted TCS genes (phoP, phoQ, pmrA, pmrB, mgrB, and pmrD) revealed no mutations in the evolved strains in comparison to the parental strain, suggesting other genetic mechanisms are likely responsible for the observed phenotypes, warranting further investigation.