Development of colistin resistance via heteroresistance modeling in Klebsiella pneumoniae: A diagnostic study

Abstract

Purpose: Colistin heteroresistance mediates the failure of antibiotic treatments, is prone to lead to colistin resistance, and has been frequently reported in <i>Klebsiella pneumoniae</i>. This study investigated origins of the increase in colistin resistance following colistin exposure to colistin-heteroresistant <i>K. pneumoniae</i>.Methods: A modeled colistin-heteroresistant <i>K. pneumoniae</i> strain (i.e., mimicking colistin- heteroresistant [m-CL^HR]) was generated using a susceptible <i>K. pneumoniae</i> strain ATCC 10031 and its resistance-induced mutant. Heteroresistance patterns were investigated through population analysis profiling (PAP), competition assays, and fluctuation and stability tests. An <i>in vitro</i> time-killing assay for colistin was performed for the m-CL^HR to identify the change in susceptible and resistant populations before and after colistin exposure.Results: The generated m-CL^HR strain showed a typical heteroresistance pattern for colistin in PAP, and its competition assay did not show fitness costs for any population. The ratio of resistant cells to susceptible cells did not deviate significantly from the range of heteroresistance in the fluctuation test, while the ratios of resistant cells preserved their stability. The <i>in vitro</i> time-killing assay thus showed that the resistant cells increased upon colistin exposure; sequencing analyses confirmed that the surviving resistant cells did not originate from susceptible cells by mutation. This study successfully modeled a colistin-heteroresistant <i>K. pneumoniae</i> strain, i.e., m-CL^HR.Conclusion: In this modeled heteroresistant strain, only the colistin-resistant population survived the colistin treatment—this suggests that the development of colistin resistance from heteroresistant strain is because of the selection of a resistant population and not by the induction of resistance through mutations in susceptible populations.