Time Series Genomics of Pseudomonas aeruginosa Reveals the Emergence of a Hypermutator Phenotype and Within-Host Evolution in Clinical Inpatients

Abstract

Pseudomonas aeruginosa, a common opportunistic pathogen, is one of the leading etiological agents of nosocomial infections. Many previous studies have reported the nosocomial transmission and epidemiology of P. aeruginosa infections. However, longitudinal studies regarding the dynamics of P. aeruginosa colonization and infection in health care settings are limited. We obtained longitudinal samples from aged patients with prolonged intensive care unit (ICU) stays (~4 to 19 months). P. aeruginosa was isolated from 71 samples obtained from seven patients and characterized by whole-genome sequencing. The P. aeruginosa isolates were assigned to 10 clonal complexes, and turnover of main clones was observed in sequential sputum samples from two patients. By comparing intraclonal genomic diversities, we identified two clones that had significantly higher numbers of single nucleotide polymorphisms and variations in homopolymeric sequences than the other clones, indicating a hypermutator phenotype. These hypermutator clones were associated with mutations T147I/G521S and P27L in the MutL protein, and their mutation rates were estimated to be 3.20 × 10^-5 and 6.59 × 10^-5 per year per nucleotide, respectively. We also identified 24 recurrently mutated genes that exhibited intraclonal diversity in two or more clones. Notably, one recurrent mutation, S698F in FptA, was observed in four clones. These findings suggest that convergent microevolution and adaption of P. aeruginosa occur in long-term ICU patients. IMPORTANCE Pseudomonas aeruginosa is a predominant opportunistic pathogen that causes nosocomial infections. Inappropriate empirical therapy can lead to prolonged hospital stays and increased mortality. In our study of sequential P. aeruginosa isolates from inpatients, high intrahost diversity was observed, including switching of clones and the emergence of a hypermutator phenotype. Recurrently mutated genes also suggested that convergent microevolution and adaption of P. aeruginosa occur in inpatients, and genomic diversity is associated with differences in multiple-drug-resistance profiles. Taken together, our findings highlight the importance of longitudinal surveillance of nosocomial P. aeruginosa clones.

Keywords: Pseudomonas aeruginosa; hypermutator; longitudinal study; nosocomial infections; whole-genome sequencing.

FIG 1

Timeline of sample collection from seven aged patients during prolonged ICU stays. Sequential samples are shown as triangles (P. aeruginosa cultivation positive) and dots (cultivation of non-P. aeruginosa organisms) according to their collection dates. The light-gray shadow indicates that the samples were collected when patients were in the same bed. Samples of P. aeruginosa are colored according to multilocus sequence typing (MLST). Details of clinical specimen types, diagnostics, and bacterial cultivations are presented in Table S1 in the supplemental material. F, female; M, male.

FIG 2

Phylogenetic relationships of P. aeruginosa isolates and switching of P. aeruginosa clones in two patients. (A) Maximum likelihood (ML) phylogenetic tree with 1,000 bootstrap replicates of the 71 isolates and the reference PAO1 strain. Branches are colored according to different patients. Multilocus sequence typing (MLST) and clonal complex identification (identified by group number) of the isolates were performed using goeBURST and are shown on the right correspondingly. The number of SNPs supporting each ST cluster is labeled on the branches. (B) ML phylogenetic tree with 1,000 bootstrap replicates based on the 71 isolates (red branches) and 357 publicly available P. aeruginosa strains (gray branches).

FIG 3

Timeline of switching of colonizing or infecting P. aeruginosa isolates in two patients. Isolates of different MLST types of patients P3 (A) and P5 (B) are shown with different shapes. Specimen types other than sputum are indicated.

FIG 4

Higher numbers of mutations and phylogenies of hypermutable isolates. (A) Numbers of within-lineage SNP loci in P. aeruginosa isolates of each ST. The dashed line indicates the mean number. Mutations in the MutL, MutS, and MutY proteins in strains of each ST are shown below and indicated with gray shading. The gray shading also indicates the prevalence of mutations among isolates of each ST. Stars denote that a mutation is specific for the corresponding P. aeruginosa clones. (B and C) Maximum likelihood phylogenetic relationship of ST110 (B) and ST277 (C) hypermutable isolates. Clone-specific mutations in MutL are indicated with stars at the corresponding branches. The sample collection times and numbers of SNPs of the isolates are shown next to the tips of the phylogenetic tree. SNPs were defined according to the reference genome of PAO1. (D) Antibiotic susceptibilities of ST277 isolates from patient P4. Nonhypermutator isolates are P4-S1 to P4-S4; hypermutator isolates are P4-S5 to P4-S10. Asterisks indicate a statistically significant difference (*, P < 0.05) between nonhypermutators and hypermutators as determined by Wilcoxon’s rank sum test.

FIG 5

Mutation rates and microindels of hypermutable isolates and other isolates. (A) Mutation rates of P. aeruginosa isolates of different STs. The rates of mutator ST277 isolates (with L27 in MutL) and nonmutator ST277 isolates (with P27 in MutL) are shown separately. nt, nucleotide. (B and C) Isolates with P27L or T147I/G521S mutations in MutL and nonmutators are indicated by different shapes of markers. Rates for the nonmutator isolates are shown in two categories as indicated in panel A. P values determined by a two-sided Wilcoxon rank sum test in terms of numbers (B) and percentages (C) of microindels are shown. For the box plots, boxes represent the interquartile ranges (IQRs) between the first and third quartiles. Horizontal black lines inside the boxes indicate the median values, while the lines outside the boxes represent values within 1.5 times the IQR.

FIG 6

Two large deletions harboring antibiotic resistance genes. (A and B) Multiple alignments of contigs without two large deletions were obtained with the Mauve program (58). Genetic arrangements of the two regions in the PAO1 strain are shown at the top (R package gggene [https://CRAN.R-project.org/package=gggenes]) with genes involved in antibiotic resistance and flagellar assembly (fliM, fliN, fliO, fliP, fliQ, fliR, and flhB). The amino acid sequences of PA1435 and PA1436 in the PAO1 strain presented 98.70% and 100% identities to MexM and MexN of the PAK strain, respectively. (C) Antibiotic susceptibilities of isolates from patient P1. Isolates with deletions (P1-S1, P1-S13, and P1-S14) are shown on the left. Asterisks indicate a statistically significant difference (*, P < 0.05; **, P < 0.01) between isolates with the large deletions and other isolates from patient P1 as determined by Wilcoxon’s rank sum test.