Genetic characterization indicates that a specific subpopulation of Pseudomonas aeruginosa is associated with keratitis infections

Abstract

Pseudomonas aeruginosa is a common opportunistic bacterial pathogen that causes a variety of infections in humans. Populations of P. aeruginosa are dominated by common clones that can be isolated from diverse clinical and environmental sources. To determine whether specific clones are associated with corneal infection, we used a portable genotyping microarray system to analyze a set of 63 P. aeruginosa isolates from patients with corneal ulcers (keratitis). We then used population analysis to compare the keratitis isolates to a wider collection of P. aeruginosa from various nonocular sources. We identified various markers in a subpopulation of P. aeruginosa associated with keratitis that were in strong disequilibrium with the wider P. aeruginosa population, including oriC, exoU, katN, unmodified flagellin, and the carriage of common genomic islands. The genome sequencing of a keratitis isolate (39016; representing the dominant serotype O11), which was associated with a prolonged clinical healing time, revealed several genomic islands and prophages within the accessory genome. The PCR amplification screening of all 63 keratitis isolates, however, provided little evidence for the shared carriage of specific prophages or genomic islands between serotypes. P. aeruginosa twitching motility, due to type IV pili, is implicated in corneal virulence. We demonstrated that 46% of the O11 keratitis isolates, including 39016, carry a distinctive pilA, encoding the pilin of type IV pili. Thus, the keratitis isolates were associated with specific characteristics, indicating that a subpopulation of P. aeruginosa is adapted to cause corneal infection.

Fig. 1.

Positions of keratitis isolates in the P. aeruginosa population. (A) Position of all analyzed keratitis isolates of this study in an eBurst analysis containing all published, SNP-typed P. aeruginosa strains. Each dot represents a clone based on the AT profile. Two dots connected by a single line differ in only one locus (single-locus variants [SLV]). Most keratitis isolates represent a closely related subgroup. (B) Two-dimensional projection of the distance matrix of SNP-typed P. aeruginosa strains. The plot was performed with the correlations between values in the distance matrix. Hence, this is a plot of the correlations of the genetic distances of the strains to each other, making it easy to see that most keratitis isolates are very closely related. Distance values were normalized and mean centered. The first two components, represented by the axes in the figure, contributed 90% of the variance of the SNP patterns. As for the eBurst analysis, most keratitis isolates fall within a closely related subgroup. Completely unrelated mathematical models were used for A and B, but both plots indicated that the keratitis isolates represent a specific, closely related subpopulation within the global P. aeruginosa population structure and may have arisen from a common ancestor. The locations of keratitis isolates are indicated in red, with the number of keratitis isolates at each location (if more than one) also shown. For example, 2× indicates two isolates at one location. The positions of clone D (blue circle) and the reference strains PAO1 (large black circle) and PA14 (red circle with black circumference, indicating that this clone type also was present among the keratitis isolates) are shown. The same keratitis strains clustered using both methods.

Fig. 2.

Dendrogram based on PilA amino acid sequences. The figure shows a phylogenetic tree comparing 28 database PilA sequences, including those of the commonly used strains PAO1 (P04739) and PA14 (AAL12242), to the PilA sequence of strain 39016, indicating the considerable diversity in P. aeruginosa PilA sequences.