Phenotypic heterogeneity of Pseudomonas aeruginosa populations in a cystic fibrosis patient

Abstract

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the lower airways of patients with cystic fibrosis. Throughout the course of infection this organism undergoes adaptations that contribute to its long-term persistence in the airways. While P. aeruginosa diversity has been documented, it is less clear to what extent within-patient diversity contributes to the overall population structure as most studies have been limited to the analysis of only a few isolates per patient per time point. To examine P. aeruginosa population structure in more detail we collected multiple isolates from individual sputum samples of a patient chronically colonized with P. aeruginosa. This strain collection, comprised of 169 clonal isolates and representing three pulmonary exacerbations as well as clinically stable periods, was assayed for a wide selection of phenotypes. These phenotypes included colony morphology, motility, quorum sensing, protease activity, auxotrophy, siderophore levels, antibiotic resistance, and growth profiles. Each phenotype displayed significant variation even within isolates of the same colony morphotype from the same sample. Isolates demonstrated a large degree of individuality across phenotypes, despite being part of a single clonal lineage, suggesting that the P. aeruginosa population in the cystic fibrosis airways is being significantly under-sampled.

Figure 1. Levels of P. aeruginosa during three pulmonary exacerbation periods over the course of one year.

A, Total levels, expressed as colony forming units (CFU) per mL of sputum are shown in green and the numbers of mucoid and non-mucoid isolates are indicated in red and blue, respectively. B, The CFU per ml for each of the 14 colony types are shown.

Figure 2. Genetic diversity present in collected P. aeruginosa isolates from a single patient.

The Dice coefficient from pulse field gel electrophoresis patterns of each isolate was used to construct a UPGMA tree (unrooted). Isolates from this study were no more than 20% different from each other but were more than 40% different from PA01. Isolates with identical PFGE patterns are grouped into a single node where the size of that node represents the number of isolates. Colouring represents the phenotypic group that the isolate falls into and the colours match the colouring in Figure 5. Two PES strains were included in the analysis and are coloured in yellow. The cluster highlighted by purple is a cluster where the phenotypic and genetic groups are similar.

Figure 3. Phenotypic diversity present in collected P. aeruginosa isolates from a single patient.

The data points are coloured according to whether the isolate was classified as mucoid or not. Grey lines indicate the phenotype value measured for the common lab strain, PA01.

Figure 4. Distribution of antibiotic resistance among the analyzed isolates.

Shown are zones of clearing from disc diffusion assays for five commonly used antibiotics in CF clinics and the resistance cutoffs for each antibiotic are given by the dashed lines. Resistance profiles for each colony morphotype are shown individually and coloured according to whether that colony type was initially identified as mucoid or not. Cutoff values for resistant, intermediate and sensitive are indicated by the colour of the background; red, yellow and blue, respectively. Cutoff values for trimethoprim/sulfamethoxazole are not published for P. aeruginosa by the CLSI.

Figure 5. Hierarchical cluster analysis of the phenotype profiles.

Green indicates a value above the mean and red indicates a value below the mean. The three colours on the left side indicate the three main cluster groups. Purple bars on the right side indicate the isolates that group closely in both the phenotypic and genetic analysis. Missing values were removed prior to analysis.

Figure 6. Phenotypic diversity of P. aeruginosa isolates from a single sample.

Phenotype profiles were obtained from a total of 88 isolates and were comprised of two different colony morphologies, which are shown as orange and green on the plot.