Genetic Alterations Associated with Polymyxin B Resistance in Nosocomial KPC-2-Producing Klebsiella pneumoniae from Brazil

Abstract

The rapid increased multidrug resistance in Klebsiella pneumoniae has led to a renewed interest in polymyxin antibiotics, such as colistin, as antibiotics of last resort, not least in low/middle income countries. We conducted a genomic survey of clinical polymyxin-resistant K. pneumoniae to investigate the genetic alterations in isolates harboring bla_KPC-2. Whole-genome sequencing was performed using an Illumina NextSeq 500 paired-end reads. Mutations and insertion sequence detection were analyzed to seven isolates recovered from clinical specimens of patients hospitalized in Brazil, focusing on key genes associated with polymyxin resistance. Furthermore, the levels of mRNA expression of genes associated with resistance to polymyxin B and other antimicrobials were evaluated by quantitative real-time PCR. Eighty-five percent of the isolates were assigned to clonal complex 258, with a minimum inhibitory concentration range of 4 to >256 mg/L for polymyxin B. It was possible to observe the presence of one important insertion element, ISKpn13, in a strain recovered from the blood that have bla_KPC-2. Deleterious mutations reported in PmrB (R256G), YciM (N212T), and AcrB (T598A) were common, and mobile colistin resistance (mcr) genes were absent in all the isolates. RT-qPCR analysis revealed an overexpression of the pmrC (1.160-fold), pmrD (2.258-fold), and kpnE (1.530-fold) genes in the polymyxin B-resistant isolates compared with the expression of the polymyxin B-susceptible K. pneumoniae isolate. Overall, these results demonstrate the diversity of genetic variations in polymyxin-resistant populations derived from the different clonal strains, but the same sequence types, and suggest that there are still unknown mechanisms of polymyxin resistance in K. pneumoniae.