Differentiation in quinolone resistance by virulence genotype in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a leading pathogen that has become increasingly resistant to the fluoroquinolone antibiotics due to widespread prescribing. Adverse outcomes have been shown for patients infected with fluoroquinolone-resistant strains. The type III secretion system (TTSS) is a major virulence determinant during acute infections through the injection of effector toxins into host cells. Most strains exhibit a unique TTSS virulence genotype defined by the presence of either exoS or exoU gene encoding two of the effector toxins, ExoS and ExoU, respectively. Specific TTSS effector genotype has been shown previously to differentially impact virulence in pneumonia. In this study, we examined the relationship between TTSS effector genotype and fluoroquinolone resistance mechanisms in a collection of 270 respiratory isolates. We found that a higher proportion of exoU+ strains were fluoroquinolone-resistant compared to exoS+ strains (63% vs 49%, p = 0.03) despite its lower overall prevalence (38% exoU+ vs 56% exoS+). Results from sequencing the quinolone resistance determining regions (QRDRs) of the 4 target genes (gyrA, gyrB, parC, parE) indicated that strains containing the exoU gene were more likely to acquire ≥ 2 mutations than exoS+ strains at MICs ≤ 8 µg/ml (13% vs none) and twice as likely to have mutations in both gyrA and parC than exoS+ strains (48% vs 24% p = 0.0439). Our findings indicate that P. aeruginosa strains differentially develop resistance-conferring mutations that correlate with TTSS effector genotype and the more virulent exoU+ subpopulation. Differences in mutational processes by virulence genotype that were observed suggest co-evolution of resistance and virulence traits favoring a more virulent genotype in the quinolone-rich clinical environment.

Figure 1. Frequency of fluoroquinolone-resistance among exoU vs. exoS containing isolates.

Fluoroquinolone (FQ) resistance is defined as levofloxacin MIC ≥4 ug/ml. FQ-susceptible population is predominated by exoS+ isolates while FQ-resistant population is predominated by exoU+ isolates. *p = 0.03.

Figure 2. Cumulative Frequency of Isolates with ≥2 target site mutations at each given MIC comparing exoU- vs exoS-containing isolates.

The proportion of isolates that has acquired at least two target site mutations is greater with exoU+ than exoS+ isolates and increases in a linear fashion as MIC increases, with exoU+ isolates starting at lower MICs. At a levofloxacin MIC ≤8 ug/ml, 25% of exoU+ isolates compared to 0% of exoS+ isolates have 2 or more TSMs whereas for isolates with MICs ranging from 16–32 ug/ml, 36% exoU+ isolates compared to 22% of exoS+ isolates, and for isolates with MICs ranging from 64–128 ug/ml, 52% exoU+ isolates compared to 39% of exoS+ isolates have 2 or more TSMs.