Rearrangement of a large novel Pseudomonas aeruginosa gene island in strains isolated from a patient developing ventilator-associated pneumonia

Abstract

Bacterial gene islands add to the genetic repertoire of opportunistic pathogens. Here, we perform comparative analyses of three Pseudomonas aeruginosa strains isolated sequentially over a 3-week period from a patient with ventilator-associated pneumonia (VAP) who received clindamycin and piperacillin-tazobactam as part of their treatment regime. While all three strains appeared to be clonal by standard pulsed-field gel electrophoresis, whole-genome sequencing revealed subtle alterations in the chromosomal organization of the last two strains; specifically, an inversion event within a novel 124-kb gene island (PAGI 12) composed of 137 open reading frames [ORFs]. Predicted ORFs in the island included metabolism and virulence genes. Overexpression of a gene island-borne putative β-lactamase gene was observed following piperacillin-tazobactam exposure and only in those strains that had undergone the inversion event, indicating altered gene regulation following genomic remodeling. Examination of a separate cohort of 76 patients with VAP for integration at this tRNA(lys) recombination site demonstrated that patients exhibiting evidence of integration at this site had significantly higher 28-day mortality. These findings provide evidence that P. aeruginosa can integrate, rapidly remodel, and express exogenous genes, which likely contributes to its fitness in a clinical setting.

FIG 1

(A) Collection of P. aeruginosa isolates during hospitalization and antibiotic treatment of patient. No abx, no antibiotics. (B) RAPD PCR profiling demonstrates that strains collected at each time point are identical. (C) Pulsed-field gel electrophoresis of SpeI-digested P. aeruginosa chromosomal DNA performed under standard conditions on each representative isolate collected at each time point demonstrates that they are clonal.

FIG 2

(A) PFGE run under nonstandard conditions to resolve various fractions of the P. aeruginosa genome reveals a repeatable shift in banding pattern (white line) between isolates 1 and 2 that persists in the latter strain. (B) Southern blot of PFGE gel using a probe designed on the PAGI-2 sequence identified by RAPD PCR analysis exhibits hybridization with the lower band in all P. aeruginosa isolates (the region of interest from the PFGE gel is enlarged for clarity).

FIG 3

Gene annotation of a novel gene island identified in clinical isolates of P. aeruginosa (strains 1 and 2). Gene function is indicated by color, and the inversion event is denoted by a hatched line.

FIG 4

qRT-PCR relative gene expression of the gene island-borne β-lactamase gene by all three clinical isolates in the absence (white bars) or presence (black bars) of piperacillin-tazobactam. P. aeruginosa C3719 acts as a negative control.

FIG 5

qRT-PCR-based relative expression of copA (A), copB (B), copC (C), and copD (D) heavy metal resistance genes in the absence (white bars) or presence (black bars) of clindamycin.