Dataset Information

Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients.

ABSTRACT: The chronic airway infections with Pseudomonas aeruginosa in people with cystic fibrosis (CF) are treated with aerosolized antibiotics, oral fluoroquinolones, and/or intravenous combination therapy with aminoglycosides and β-lactam antibiotics. An international strain collection of 361 P. aeruginosa isolates from 258 CF patients seen at 30 CF clinics was examined for mutations in 17 antimicrobial susceptibility and resistance loci that had been identified as hot spots of mutation by genome sequencing of serial isolates from a single CF clinic. Combinatorial amplicon sequencing of pooled PCR products identified 1,112 sequence variants that were not present in the genomes of representative strains of the 20 most common clones of the global P. aeruginosa population. A high frequency of singular coding variants was seen in spuE, mexA, gyrA, rpoB, fusA1, mexZ, mexY, oprD, ampD, parR, parS, and envZ (amgS), reflecting the pressure upon P. aeruginosa in lungs of CF patients to generate novel protein variants. The proportion of nonneutral amino acid exchanges was high. Of the 17 loci, mexA, mexZ, and pagL were most frequently affected by independent stop mutations. Private and de novo mutations seem to play a pivotal role in the response of P. aeruginosa populations to the antimicrobial load and the individual CF host.

SUBMITTER: Greipel L

PROVIDER: S-EPMC5075079 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients.

Greipel Leonie L Fischer Sebastian S Klockgether Jens J Dorda Marie M Mielke Samira S Wiehlmann Lutz L Cramer Nina N Tümmler Burkhard B

Antimicrobial agents and chemotherapy 20161021 11

The chronic airway infections with Pseudomonas aeruginosa in people with cystic fibrosis (CF) are treated with aerosolized antibiotics, oral fluoroquinolones, and/or intravenous combination therapy with aminoglycosides and β-lactam antibiotics. An international strain collection of 361 P. aeruginosa isolates from 258 CF patients seen at 30 CF clinics was examined for mutations in 17 antimicrobial susceptibility and resistance loci that had been identified as hot spots of mutation by genome seque ...[more]

PMID: 27572404

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Project description:Clinical isolates of Pseudomonas aeruginosa from patients with cystic fibrosis (CF) are known to differ from those associated with non-CF hosts by colony morphology, drug susceptibility patterns, and genomic hypermutability. Pseudomonas aeruginosa isolates from CF patients have long been recognized for their overall reduced rate of antimicrobial susceptibility, but their intraclonal MIC heterogeneity has long been overlooked. Using two distinct cohorts of clinical strains (n = 224 from 56 CF patients, n = 130 from 68 non-CF patients) isolated in 2013, we demonstrated profound Etest MIC heterogeneity in CF P. aeruginosa isolates in comparison to non-CF P. aeruginosa isolates. On the basis of whole-genome sequencing of 19 CF P. aeruginosa isolates from 9 patients with heterogeneous MICs, the core genome phylogenetic tree confirmed the within-patient CF P. aeruginosa clonal lineage along with considerable coding sequence variability. No extrachromosomal DNA elements or previously characterized antibiotic resistance mutations could account for the wide divergence in antimicrobial MICs between P. aeruginosa coisolates, though many heterogeneous mutations in efflux and porin genes and their regulators were present. A unique OprD sequence was conserved among the majority of isolates of CF P. aeruginosa analyzed, suggesting a pseudomonal response to selective pressure that is common to the isolates. Genomic sequence data also suggested that CF pseudomonal hypermutability was not entirely due to mutations in mutL, mutS, and uvr. We conclude that the net effect of hundreds of adaptive mutations, both shared between clonally related isolate pairs and unshared, accounts for their highly heterogeneous MIC variances. We hypothesize that this heterogeneity is indicative of the pseudomonal syntrophic-like lifestyle under conditions of being "locked" inside a host focal airway environment for prolonged periods. IMPORTANCE Patients with cystic fibrosis endure "chronic focal infections" with a variety of microorganisms. One microorganism, Pseudomonas aeruginosa, adapts to the host and develops resistance to a wide range of antimicrobials. Interestingly, as the infection progresses, multiple isogenic strains of P. aeruginosa emerge and coexist within the airways of these patients. Despite a common parental origin, the multiple strains of P. aeruginosa develop vastly different susceptibility patterns to actively used antimicrobial agents-a phenomenon we define as "heterogeneous MICs." By sequencing pairs of P. aeruginosa isolates displaying heterogeneous MICs, we observed widespread isogenic gene lesions in drug transporters, DNA mismatch repair machinery, and many other structural or cellular functions. Coupled with the heterogeneous MICs, these genetic lesions demonstrated a symbiotic response to host selection and suggested evolution of a multicellular syntrophic bacterial lifestyle. Current laboratory standard interpretive criteria do not address the emergence of heterogeneous growth and susceptibilities in vitro with treatment implications.

Project description:Five hundred and thirty-four unrelated Pseudomonas aeruginosa isolates from inanimate habitats, patients with cystic fibrosis (CF) and other human infections were sequenced in 19 genes that had been identified previously as the hot spots of genomic within-host evolution in serial isolates from 12 CF lungs. Amplicon sequencing confirmed a significantly higher sequence diversity of the 19 loci in P. aeruginosa isolates from CF patients compared to those from other habitats, but this overrepresentation was mainly due to the larger share of synonymous substitutions. Correspondingly, non-synonymous substitutions were either rare (gltT, lepA, ptsP) or benign (nuoL, fleR, pelF) in some loci. Other loci, however, showed an accumulation of non-neutral coding variants. Strains from the CF habitat were often mutated at evolutionarily conserved positions in the elements of stringent response (RelA, SpoT), LPS (PagL), polyamine transport (SpuE, SpuF) and alginate biosynthesis (AlgG, AlgU). The strongest skew towards the CF lung habitat was seen for amino acid sequence variants in AlgG that clustered in the carbohydrate-binding/sugar hydrolysis domain. The master regulators of quorum sensing lasR and rhlR were frequent targets for coding variants in isolates from chronic and acute human infections. Unique variants in lasR showed strong evidence of positive selection indicated by d N/d S values of ~4. The pelA gene that encodes a multidomain enzyme involved in both the formation and dispersion of Pel biofilms carried the highest number of single-nucleotide variants among the 19 genes and was the only gene with a higher frequency of missense mutations in P. aeruginosa strains from non-CF habitats than in isolates from CF airways. PelA protein variants are widely distributed in the P. aeruginosa population. In conclusion, coding variants in a subset of the examined loci are indeed characteristic for the adaptation of P. aeruginosa to the CF airways, but for other loci the elevated mutation rate is more indicative of infections in human habitats (lasR, rhlR) or global diversifying selection (pelA).

Dataset Information

Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients.

Publications

Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients.

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