Genotypic and Phenotypic Diversity of Staphylococcus aureus Isolates from Cystic Fibrosis Patient Lung Infections and Their Interactions with Pseudomonas aeruginosa

Abstract

Staphylococcus aureus has recently overtaken Pseudomonas aeruginosa as the most commonly recognized bacterial pathogen that infects the respiratory tracts of individuals with the genetic disease cystic fibrosis (CF) in the United States. Most studies of S. aureus in CF patient lung infections have focused on a few isolates, often exclusively laboratory-adapted strains, and how they are killed by P. aeruginosa Less is known about the diversity of S. aureus CF patient lung isolates in terms of both their virulence and their interaction with P. aeruginosa To begin to address this gap, we recently sequenced 64 clinical S. aureus isolates and a reference isolate, JE2. Here, we analyzed the antibiotic resistance genotypes, sequence types, clonal complexes, spa types, agr types, and presence/absence of other known virulence factor genes of these isolates. We hypothesized that virulence phenotypes of S. aureus, namely, toxin production and the mucoid phenotype, would be lost in these isolates due to adaptation in the CF patient lung. In contrast to these expectations, we found that most isolates can lyse both rabbit and sheep blood (67.7%) and produce polysaccharide (69.2%), suggesting that these phenotypes were not lost during adaptation to the CF lung. We also identified three distinct phenotypic groups of S. aureus based on their survival in the presence of nonmucoid P. aeruginosa laboratory strain PAO1 and its mucoid derivative. Altogether, our work provides greater insight into the diversity of S. aureus isolates from CF patients, specifically the distribution of important virulence factors and their interaction with P. aeruginosa, all of which have implications in patient health.IMPORTANCEStaphylococcus aureus is now the most frequently detected recognized pathogen in the lungs of individuals who have cystic fibrosis (CF) in the United States, followed closely by Pseudomonas aeruginosa When these pathogens are found to coinfect the CF lung, patients have a significantly worse prognosis. While P. aeruginosa has been rigorously studied in the context of bacterial pathogenesis in CF, less is known about S. aureus Here, we present an in-depth study of 64 S. aureus clinical isolates from CF patients, for which we investigated genetic diversity utilizing whole-genome sequencing, virulence phenotypes, and interactions with P. aeruginosa We found that S. aureus isolated from CF lungs are phylogenetically diverse; most retain known virulence factors and vary in their interactions with P. aeruginosa (i.e., they range from being highly sensitive to P. aeruginosa to completely tolerant to it). Deepening our understanding of how S. aureus responds to its environment and other microbes in the CF lung will enable future development of effective treatments and preventative measures against these formidable infections.

Keywords: Pseudomonas aeruginosa; Staphylococcus aureus; cystic fibrosis; interspecies competition; phylogenetic analysis.

FIG 1

Core genome maximum-likelihood phylogeny of isolates used in this study. Represented is a core genome phylogeny of the 65 isolates in this study produced with IQ-Tree. The tree was built from 1,984 core genes identified by Roary with 25,651 parsimony informative sites and the GTR+F+R2 substitution model. (A) All isolates. Branches with > 90% bootstrap support are indicated with a red dot. The heatmap shows, from left to right, the sheep blood hemolysis phenotype (“+” or “–”); rabbit blood hemolysis (“+” or “–”); whether the isolate was a normal polysaccharide producer, an overproducer, or did not produce polysaccharide; the coculture group (1, 2, or 3); and whether the isolate was one of multiple taken from a patient (CFBR101, CFBR105, CFBR148, CFBR150, CFBR280, CFBR487, CFPatient5, or NA [meaning a single isolate from a patient]). (B and C) Expanded portions of the same tree focusing on CC5 and CC8 isolates, respectively.

FIG 2

Plating on Congo red agar shows three phenotypes. One representative isolate of each phenotype is shown. Isolates classified as having no polysaccharide are bright red in color and smooth or shiny in appearance. Isolates classified as having normal polysaccharide production are much darker in color than those with no polysaccharide production (many were black, as shown in this figure); these isolates are also smooth or shiny in appearance. Finally, isolates classified as overproducing polysaccharide are also darker than the no-polysaccharide isolates but have a rough or matte appearance.

FIG 3

Coculturing S. aureus CF isolates with nonmucoid and mucoid PAO1 revealed 3 interaction groups. Shown are fold changes of each S. aureus isolate after coculture with both nonmucoid (black bars) and mucoid (gray bars) PAO1. Fold change was calculated by dividing the number (per milliliter) of CFU of each S. aureus isolate grown with nonmucoid PAO1 or with mucoid PAO1 by the number of CFU of each S. aureus isolate grown alone. A fold change of <10⁻² was considered to represent significant killing, as shown by the horizontal black line, denoted a “killing line” in the text. (A) Group 1 isolates (nonmucoid kills) have black bars below 10⁻², while all gray bars are above this threshold. There were 46 isolates that fit into this interaction group, including the previously tested lab isolate JE2. (B) Group 2 isolates (both kill) have both black and gray bars below the 10⁻² threshold. Fifteen isolates are in this interaction group. (C) Group 3 isolates (neither kills) have both black and gray bars above the 10⁻² threshold. Four isolates are in this group. The averages from technical triplicates of one experiment representative of the three biological replicates performed are shown.