Genomic Characterization of Colistin Heteroresistance in Klebsiella pneumoniae during a Nosocomial Outbreak

Abstract

dKlebsiella pneumoniae is emerging as an important nosocomial pathogen due to its rapidly increasing multidrug resistance, which has led to a renewed interest in polymyxin antibiotics, such as colistin, as antibiotics of last resort. However, heteroresistance (i.e., the presence of a subpopulation of resistant bacteria in an otherwise susceptible culture) may hamper the effectiveness of colistin treatment in patients. In a previous study, we showed that colistin resistance among extendedspectrum-beta-lactamase (ESBL)-producing K. pneumoniae isolates emerged after the introduction of selective digestive tract decontamination (SDD) in an intensive care unit (ICU). In this study, we investigated heteroresistance to colistin among ESBL-producing K. pneumoniae isolates by using population analysis profiles (PAPs). We used whole-genome sequencing (WGS) to identify the mutations that were associated with the emergence of colistin resistance in these K. pneumoniae isolates. We found five heteroresistant subpopulations, with colistin MICs ranging from 8 to 64 mg/liter, which were derived from five clonally related, colistin-susceptible clinical isolates. WGS revealed the presence of mutations in the lpxM, mgrB, phoQ, and yciM genes in colistin-resistant K. pneumoniae isolates. In two strains, mgrB was inactivated by an IS3-like or ISKpn14 insertion sequence element. Complementation in trans with the wild-type mgrB gene resulted in these strains reverting to colistin susceptibility. The MICs for colistin-susceptible strains increased 2-to 4-fold in the presence of the mutated phoQ, lpxM, and yciM alleles. In conclusion, the present study indicates that heteroresistant K. pneumoniae subpopulations may be selected for upon exposure to colistin. Mutations in mgrB and phoQ have previously been associated with colistin resistance, but we provide experimental evidence for roles of mutations in the yciM and lpxM genes in the emergence of colistin resistance in K. pneumoniae.

Challenges and opportunities for whole‐genome sequencing–based surveillance of antibiotic resistance

“…Transposases can inactivate a gene that is required for antibiotic susceptibility through insertion. This mechanism has been well characterized in K. pneumoniae , where transposon‐mediated inactivation of mgrB is a common mechanism leading to colistin resistance . Alternatively, transposon insertions can increase the expression of intrinsic resistance genes, as illustrated by transposon‐mediated upregulation of ampC in A. baumannii , causing third‐generation cephalosporin resistance, or increased expression of an efflux system in E. coli , which contributes to fluoroquinolone resistance .…”

“…It is increasingly being recognized as a crucial antibiotic of last resort, particularly in countries where carbapenem resistance is endemic . Resistance to colistin can emerge in Gram‐negative bacteria (particularly in K. pneumoniae and Acinetobacter baumannii ) owing to mutational events, particularly in genes encoding regulators of LPS modification or LPS biosynthesis proteins, which include gene amplification, gene inactivation due to the insertion of an insertion sequence (IS) element in a gene encoding a regulator, or the accumulation of point mutations . While the emergence of resistance during therapy may affect the effectiveness of colistin therapy and is an important cause of outbreaks with colistin‐resistant strains in hospitals, the mutations contributing to the resistant phenotype are fixed on the chromosome and thus cannot be transferred between bacteria.…”

“…This mechanism has been well characterized in K. pneumoniae, where transposon-mediated inactivation of mgrB is a common mechanism leading to colistin resistance. [64][65][66][67] Alternatively, transposon insertions can increase the expression of intrinsic resistance genes, as illustrated by transposon-mediated upregulation of ampC in A. baumannii, causing third-generation cephalosporin resistance, 68 or increased expression of an efflux system in E. coli, which contributes to fluoroquinolone resistance. 69 In cases where transposon-mediated resistance has been experimentally verified, these events may be predicted from genome data, using either read-based tools (ISMapper 70 ) or assembly-based analysis (ISseeker 71 ) to identify transposon insertion sites in microbial genomes.…”

“…92,98,99 Resistance to colistin can emerge in Gram-negative bacteria (particularly in K. pneumoniae and Acinetobacter baumannii) owing to mutational events, particularly in genes encoding regulators of LPS modification or LPS biosynthesis proteins, which include gene amplification, 100 gene inactivation due to the insertion of an insertion sequence (IS) element in a gene encoding a regulator, [64][65][66]101 or the accumulation of point mutations. 66,102,103 While the emergence of resistance during therapy may affect the effectiveness of colistin therapy 104,105 and is an important cause of outbreaks with colistin-resistant strains in hospitals, 106 the mutations contributing to the resistant phenotype are fixed on the chromosome and thus cannot be transferred between bacteria. The mcr-1 gene is the first known colistin-resistance determinant that was encoded on a conjugative plasmid, which may lead to a rapid spread of colistin resistance among Gram-negative bacteria.…”

Deciphering Heteroresistance to Colistin in a Klebsiella pneumoniae Isolate from Marseille, France

“…There are only 2 reports of genomic analysis of colistin-heteroresistant K. pneumoniae isolates, which were multidrug-resistant bacteria: one OXA-48 carbapenemaseproducing strain with a mutation in protein PhoP (7) and five extended-spectrum ␤-lactamase (ESBL)-expressing isolates with mutations in the phoQ, lpxM, and yciM genes, as well as two with mutations on the mgrB gene (the presence of an insertion sequence and deletion) (30). This is the first description of a colistin-heteroresistant Klebsiella pneumoniae isolate in France and the first description of a truncated MgrB protein after an insertion of a single nucleotide.…”