Heteroresistance to Amikacin in Carbapenem-Resistant Klebsiella pneumoniae Strains.

Feiyang Zhang,Guangxi Wang,Jiawei Bai,Qin Li,Xiangjin Yan,Manlin Ding,Baoli Zhu,Yingshun Zhou

doi:10.3389/fmicb.2021.682239

Abstract

Heteroresistance can lead to treatment failure and is difficult to detect by the methods currently employed by clinical laboratories. The aim of this study was to investigate the prevalence of the amikacin-heteroresistant Klebsiella pneumoniae strains and explore potential amikacin heteroresistance mechanism through whole-genome sequencing (WGS) and quantitative reverse-transcription PCR (qRT-PCR). In this study, 13 isolates (8.39%) were considered as amikacin-heteroresistant K. pneumoniae strains among a total of 155 K. pneumoniae strains. The majority of the heterogeneous phenotypes (11/13, 84.61%) was unstable and the minimal inhibitory concentrations (MICs) fully or partially reverted back to the level of susceptibility of the parental isolate. The frequency of heteroresistant subpopulation ranged from 2.94×10−7 to 5.59×10−6. Whole-genome sequencing and single-nucleotide variants (SNVs) analysis showed that there were different nucleotide and resultant amino acid alterations among an amikacin-heteroresistant strain S38 and the resistant subpopulation S38L in several genes. Quantitative reverse-transcription PCR analysis revealed that the increased expression of aminoglycoside resistance genes detected in amikacin-heteroresistant K. pneumoniae strains might be associated with amikacin heteroresistance. The findings raise concerns for the emergence of amikacin-heteroresistant K. pneumoniae strains and the use of amikacin as therapy for the treatment of multidrug-resistant K. pneumoniae strains.

Highlights

Heteroresistance refers to a phenomenon where there are different subpopulations of seemingly isogenic bacteria which exhibit a range of susceptibilities to antibiotics (El-Halfawy and Valvano, 2015)
The minimal inhibitory concentrations (MICs) and the proportion of resistant subpopulation were decreased to different extent in the strains whose heterogeneous phenotypes were unstable after they were sub-cultured for 7 days without amikacin
Quantitative RT-PCR analysis revealed that amikacin-resistant subpopulation S30L and F35L had increased expression of aac (6′)-Ib compared to the amikacin-resistant parental strains S30 and F35 (18.22- and 11.4-fold increased expression, respectively), which was statistically significant (p < 0.05)

Summary

Introduction

Heteroresistance refers to a phenomenon where there are different subpopulations of seemingly isogenic bacteria which exhibit a range of susceptibilities to antibiotics (El-Halfawy and Valvano, 2015). In a previous study at our center about the susceptibility of K. pneumoniae clinical isolates to different antibiotics using the disk diffusion method, the appearance of distinct colonies growing inside the clear zone of inhibition in amikacin disk was found. This phenomenon has attracted our attention and was defined as heteroresistance by relevant articles. The major concern about this form was that more resistant subpopulation may pass a switch of heteroresistance to homogeneous high-level resistance and could pose a serious threat to antibiotic treatment. Biofilm formation, growth rate analysis, and quantitative reverse-transcription PCR (qRTPCR) were performed to explore the potential amikacin heteroresistance mechanism in the current study

Objectives

Methods

Results

Conclusion