Selective Sweeps and Parallel Pathoadaptation Drive Pseudomonas aeruginosa Evolution in the Cystic Fibrosis Lung

Abstract

Pulmonary infections caused by Pseudomonas aeruginosa are a recalcitrant problem in cystic fibrosis (CF) patients. While the clinical implications and long-term evolutionary patterns of these infections are well studied, we know little about the short-term population dynamics that enable this pathogen to persist despite aggressive antimicrobial therapy. Here, we describe a short-term population genomic analysis of 233 P. aeruginosa isolates collected from 12 sputum specimens obtained over a 1-year period from a single patient. Whole-genome sequencing and antimicrobial susceptibility profiling identified the expansion of two clonal lineages. The first lineage originated from the coalescence of the entire sample less than 3 years before the end of the study and gave rise to a high-diversity ancestral population. The second expansion occurred 2 years later and gave rise to a derived population with a strong signal of positive selection. These events show characteristics consistent with recurrent selective sweeps. While we cannot identify the specific mutations responsible for the origins of the clonal lineages, we find that the majority of mutations occur in loci previously associated with virulence and resistance. Additionally, approximately one-third of all mutations occur in loci that are mutated multiple times, highlighting the importance of parallel pathoadaptation. One such locus is the gene encoding penicillin-binding protein 3, which received three independent mutations. Our functional analysis of these alleles shows that they provide differential fitness benefits dependent on the antibiotic under selection. These data reveal that bacterial populations can undergo extensive and dramatic changes that are not revealed by lower-resolution analyses.

Importance: Pseudomonas aeruginosa is a bacterial opportunistic pathogen responsible for significant morbidity and mortality in cystic fibrosis (CF) patients. Once it has colonized the lung in CF, it is highly resilient and rarely eradicated. This study presents a deep sampling examination of the fine-scale evolutionary dynamics of P. aeruginosa in the lungs of a chronically infected CF patient. We show that diversity of P. aeruginosa is driven by recurrent clonal emergence and expansion within this patient and identify potential adaptive variants associated with these events. This high-resolution sequencing strategy thus reveals important intraspecies dynamics that explain a clinically important phenomenon not evident at a lower-resolution analysis of community structure.

FIG 1

Antibiotic treatment history and the relative abundance of the two clades over time. (A) Black bars indicate antibiotic administration, and hashed bars indicate intermittent exposure in that time block. The method of antibiotic administration is shown as intravenous (iv), inhaled (inh), or oral (po). Sputum samples were collected at the time points indicated by green lines, which extend to panels B and C. (B) Relative abundance at the genus level is shown as a percentage. (C) Relative abundance of clade A (blue shades) and clade B (red shades) genotypes over time. Genotypes are defined as whole-genome sequences differing by one or more SNPs segregating in at least two isolates. The same color and shading are used to identify specific genotypes across specimens.

FIG 2

Maximum likelihood and network-based (neighbor-net) phylogenetic analyses. The phylogenetic structure of 233 P. aeruginosa isolates was characterized based on genome-wide single nucleotide polymorphisms (SNPs) using the maximum likelihood (A) and neighbor-net (B) algorithms. The structure of the resulting tree revealed two populations, clades A (blue) and B (red). The 152 isolates in clade B produce a star phylogeny consistent with a recent expansion of a clonal population, while the 81 isolates in clade A show longer branches with more phylogenetic structure. (A) The scale bar shows genetic distance using the maximum composite distance and all segregating SNPs. The strain names shown on the tree are a composite of the specimen number from which the clone was isolated, an arbitrary clone letter, and the clade designation. (B) Individual strain names at the tips of each branch have been replaced with pie charts indicating the distribution of dates during which the strains were sampled (indicated by the circular legend). The scale bar indicates genetic distances.

FIG 3

Frequency spectrum of SNPs in clades A and B. (A) The frequencies of the number of SNP differences between isolates within clade A and B reveal different distributions. While in clade A, most pairwise SNP differences occurred evenly, in clade A, pairwise SNP differences lower than 5 are overrepresented. (B) Distribution of pairwise SNP differences within clades A and B shows different profiles (P <<< 0.0001, t test).

FIG 4

dN/dS ratio calculation over time. Shown is the nonsynonymous over synonymous substitution rate (dN/dS) for multispecimen SNPs calculated for each sample time and partitioned based on clade membership. Clade B arose between sampling specimens 1 and 2; therefore, there are no data for clade B.

FIG 5

Distribution of pbpB alleles in clade A (A) and clade (B) over the 12 sampling time points. Clades A and B share an ancestral major allele. A minor allele is observed specifically in clade A, and two others segregate only in clade B.

FIG 6

Relationship between pbpB alleles and MICs to selected antipseudomonal antibiotics. Pseudomonas aeruginosa isolates were grouped by pbpB genotype based on the presence of major and minor alleles, as well as clade membership in clade A (ancestral) and clade B (sweep) populations. Antibiotic MICs were assayed for aztreonam (A), cefsulodin (B), ceftazidime (C), and piperacillin (D). The presence of the minor alleles was associated with significantly increased resistance to aztreonam and cefsulodin in both clades but to ceftazidime and piperacillin only for the clade A minor allele. Statistical significance was determined by Mann-Whitney U test and is indicated with asterisks. (**, P ≤ 0.01; ***, P ≤ 0.001).