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Heteroresistance to colistin in Klebsiella pneumoniae associated with alterations in the PhoPQ regulatory system.

ABSTRACT: A multidrug-resistant Klebsiella pneumoniae isolate exhibiting heteroresistance to colistin was investigated. The colistin-resistant subpopulation harbored a single amino acid change (Asp191Tyr) in protein PhoP, which is part of the PhoPQ two-component system that activates pmrHFIJKLM expression responsible for l-aminoarabinose synthesis and polymyxin resistance. Complementation assays with a wild-type phoP gene restored full susceptibility to colistin. Then, analysis of the colistin-susceptible subpopulation showed a partial deletion (25 bp) in the phoP gene compared to that in the colistin-resistant subpopulation. That deletion disrupted the reading frame of phoP, leading to a longer and inactive protein (255 versus 223 amino acids long). This is the first report showing the involvement of mutation(s) in PhoP in colistin resistance. Furthermore, this is the first study to decipher the mechanisms leading to colistin heteroresistance in K. pneumoniae.

SUBMITTER: Jayol A

PROVIDER: S-EPMC4394806 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Heteroresistance to colistin in Klebsiella pneumoniae associated with alterations in the PhoPQ regulatory system.

Jayol Aurélie A Nordmann Patrice P Brink Adrian A Poirel Laurent L

Antimicrobial agents and chemotherapy 20150302 5

A multidrug-resistant Klebsiella pneumoniae isolate exhibiting heteroresistance to colistin was investigated. The colistin-resistant subpopulation harbored a single amino acid change (Asp191Tyr) in protein PhoP, which is part of the PhoPQ two-component system that activates pmrHFIJKLM expression responsible for l-aminoarabinose synthesis and polymyxin resistance. Complementation assays with a wild-type phoP gene restored full susceptibility to colistin. Then, analysis of the colistin-susceptible ...[more]

PMID: 25733503

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Project description:Klebsiella 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 extended-spectrum-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.

Project description:Colistin has been considered a last-line option for the treatment of infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). Heterogeneous resistance to colistin leads to unexplained clinical colistin treatment failure for CRKP. Our study aimed to investigate the extent of colistin heteroresistance among CRKP strains in China. A total of 455 colistin-susceptible strains, collected from six tertiary care hospitals in China, were characterized. The overall rate of colistin heteroresistance was 6.2%, as determined by the population analysis profiles (PAPs). Genomic analysis revealed that 60.7% of the colistin-heteroresistant isolates belonged to the epidemic sequence type 11 (ST11) clone. Single-nucleotide polymorphisms (SNPs) suggested that 6 ST5216 strains shared the same origin. Each of the subpopulations had a ≥8-fold decrease in colistin MIC in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), which indicated that heteroresistance could be suppressed by an efflux pump inhibitor. In addition, our results suggested that the PhoPQ pathway plays an important role in the mechanisms of heteroresistance. IMPORTANCE The problem of CRKP has raised alarms concerning global health. Our study enriches the epidemiological study of colistin heteroresistance among CRKP strains in China, where the prevalence of this phenomenon was previously unknown. Importantly, colistin-heteroresistant strains may cause the failure of clinical treatment with colistin, even if the clinical laboratory reports that the strains are sensitive. The commonly used broth microdilution method is unable to detect this special phenomenon. Additionally, our results indicate that efflux pumps play a major role in colistin heteroresistance, and inhibitors can effectively reverse it. Our study is the first to provide a detailed analysis of the prevalence of colistin heteroresistance in China, as well as an analysis of the genetic mechanisms of this phenomenon.

Project description:Heteroresistance (HR) to colistin is especially concerning in settings where multi-drug-resistant (MDR) K. pneumoniae are prevalent and empiric use of colistin might lead to treatment failures. This study aimed to assess the frequency of occurrence of colistin HR (CHR) among (MDR) K. pneumoniae (n = 676) isolated from patients hospitalized in 13 intensive care units (ICUs) in six European countries in a clinical trial assessing the impact of decolonization strategies. All isolates were whole-genome-sequenced and studied for in vitro colistin susceptibility. The majority were colistin-susceptible (CS) (n = 597, MIC ≤ 2 µg/mL), and 79 were fully colistin-resistant (CR) (MIC > 2 µg/mL). A total of 288 CS isolates were randomly selected for population analysis profiling (PAP) to assess CHR prevalence. CHR was detected in 108/288 CS K. pneumoniae. No significant association was found between the occurrence of CHR and country, MIC-value, K-antigen type, and O-antigen type. Overall, 92% (617/671) of the K. pneumoniae were MDR with high prevalence among CS (91%, 539/592) and CR (98.7%, 78/79) isolates. In contrast, the proportion of carbapenemase-producing K. pneumoniae (CP-Kpn) was higher among CR (72.2%, 57/79) than CS isolates (29.3%, 174/594). The proportions of MDR and CP-Kpn were similar among CHR (MDR: 85%, 91/107; CP-Kpn: 29.9%, 32/107) and selected CS isolates (MDR: 84.7%, 244/288; CP-Kpn: 28.1%, 80/285). WGS analysis of PAP isolates showed diverse insertion elements in mgrB or even among technical replicates underscoring the stochasticity of the CHR phenotype. CHR isolates showed high sequence type (ST) diversity (Simpson's diversity index, SDI: 0.97, in 52 of the 85 STs tested). CR (SDI: 0.85) isolates were highly associated with specific STs (ST101, ST147, ST258/ST512, p ≤ 0.003). The widespread nature of CHR among MDR K. pneumoniae in our study urge the development of rapid HR detection methods to inform on the need for combination regimens.

Dataset Information

Heteroresistance to colistin in Klebsiella pneumoniae associated with alterations in the PhoPQ regulatory system.

Publications

Heteroresistance to colistin in Klebsiella pneumoniae associated with alterations in the PhoPQ regulatory system.

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