Overview of heteroresistance to multiple antibiotics in clinical Klebsiella pneumoniae isolates and combination therapeutic strategies.

Objectives: To assess the prevalence of heteroresistance in 201 clinical isolates of Klebsiella pneumoniae to 16 clinically significant antibiotics. Furthermore, to investigate the interaction effects of combination antibiotic therapies for heteroresistant isolates.

Methods: Isolates were pre-screened for growth of resistant subpopulations at resistant breakpoint concentrations for each isolate/antibiotic combination. Any strain containing colony growth at the resistant breakpoint was selected as a candidate heteroresistant strain, and population analysis profiling (PAP) tested for confirmation of HR phenotype. Dual PAP and time-kill assay were conducted to assess the efficacy of antibiotic combinations in suppressing resistant subpopulations.

Results: Ninety-seven percent of isolates were shown to be heteroresistant to at least one antibiotic. Heteroresistance to at least two antibiotics was exhibited by 72.1% of strains. The prevalence of heteroresistance varied across antibiotics, with proportions ranging from 1.5% for imipenem to 85.1% for polymyxin B. The case of Kp486 was heteroresistant to amikacin, ceftazidime/avibactam, tigecycline and polymyxin B. The resistant subpopulations displayed distinct PAP curves and differences in growth and killing kinetics, indicating independent mechanisms for heteroresistance to each of the four antibiotics. Dual PAP experiments showed enhanced killing effects for combinations of antibiotics. In time-kill experiments, pairwise combinations of four drugs achieved a reduction of 3 to 6 logs within 6 h, preventing regrowth of resistant subpopulations. However, combinations with ampicillin did not enhance the activity of tigecycline, polymyxin B or ceftazidime/avibactam.

Conclusions: Heteroresistance in clinical K. pneumoniae is common and can complicate treatment outcomes. The effects of combination antibiotic therapy depend on the heteroresistance of bacteria to both drugs.