Heteroresistance is associated with mutations during low concentration of tigecycline therapy in multiple-resistant Klebsiella pneumoniae.

Abstract

Background: Heteroresistance can lead to treatment failure and has brought a rigorous challenge to clinical laboratories for detecting them. The aim of this study was to investigate the potential for tigecycline-susceptible Klebsiella pneumoniae (K. pneumoniae) clinical isolates to develop heteroresistance under antibiotic pressure.

Method: In this study, inducing experiment in vitro was used to acquire tigecycline heteroresistance phenotype. Population analysis profiling was used to confirm heteroresistance. Potential tigecycline heteroresistance mechanism through whole-genome sequencing and quantitative reverse-transcription PCR (qRT-PCR) were explored. Time-kill assay was used to explore the effect of tigecycline monotherapy or combination with other antibiotics.

Result: Two clinically isolated K. pneumonia strains were found to change from tigecycline susceptible to resistance during treatment of tigecycline in vivo. Experimental-evolved tigecycline heteroresistant colonies were successfully obtained by exposing to concentration of tigecycline at usual therapy of tigecycline (serum concentration of 0.1 mg/L). Heteroresistant phenotypes were stable, and the minimal inhibitory concentration sustained at resistant after 7 days serially passed in tigecycline-free medium. Frequency of heteroresistant subpopulation ranged from 7.0 × 10^-7 to 1.41 × 10^-6. Genome sequencing and analysis showed mutations of ramR, acrR and rpsJ could be responsible for the stage from tigecycline susceptible to heteroresistance and further to resistance in K. pneumoniae. Quantitative reverse-transcription PCR analysis revealed that the increased expression of tigecycline resistance genes detected in tigecycline resistant subpopulations might be associated with tigecycline heteroresistance. Time-kill assay showed the impaired efficacy of serum concentrations of 0.1 mg/L tigecycline (50 mg/q12h intravenously [i.v.]) monotherapy on tigecycline susceptible K. pneumoniae. 1 mg/L tigecycline could be effective in preventing susceptible strain but failed on heteroresistance. Combination with other antibiotics which are susceptible to target strains such as tigecycline-polymyxin B and tigecycline-amikacin can effectively inhibit the growth of resistant subpopulations.

Conclusion: The findings reveal the phenomenon where tigecycline may induce resistance in initially susceptible strains during clinical treatment, associated with several mutations of ramR, acrR and rpsJ, resulting in treatment failure. The heteroresistant strains induced by low concentrations of tigecycline in vitro provide a perspective for exploring the molecular mechanisms of tigecycline resistance in K. pneumoniae. Combination with other antibiotics like polymyxin B and amikacin would show synergistic effects in evading regrowth of resistant subpopulations.