Quorum sensing inhibition selects for virulence and cooperation in Pseudomonas aeruginosa

Abstract

With the rising development of bacterial resistance the search for new medical treatments beyond conventional antimicrobials has become a key aim of public health research. Possible innovative strategies include the inhibition of bacterial virulence. However, consideration must be given to the evolutionary and environmental consequences of such new interventions. Virulence and cooperative social behaviour of the bacterium Pseudomonas aeruginosa rely on the quorum-sensing (QS) controlled production of extracellular products (public goods). Hence QS is an attractive target for anti-virulence interventions. During colonization, non-cooperating (and hence less virulent) P. aeruginosa QS-mutants, benefiting from public goods provided by wild type isolates, naturally increase in frequency providing a relative protection from invasive infection. We hypothesized that inhibition of QS-mediated gene expression removes this growth advantage and selection of less virulent QS-mutants, and maintains the predominance of more virulent QS-wild type bacteria. We addressed this possibility in a placebo-controlled trial investigating the anti-QS properties of azithromycin, a macrolide antibiotic devoid of bactericidal activity on P. aeruginosa, but interfering with QS, in intubated patients colonized by P. aeruginosa. In the absence of azithromycin, non-cooperating (and hence less virulent) lasR (QS)-mutants increased in frequency over time. Azithromycin significantly reduced QS-gene expression measured directly in tracheal aspirates. Concomitantly the advantage of lasR-mutants was lost and virulent wild-type isolates predominated during azithromycin treatment. We confirmed these results in vitro with fitness and invasion experiments. Azithromycin reduced growth rate of the wild-type, but not of the lasR-mutant. Furthermore, the lasR-mutant efficiently invaded wild-type populations in the absence, but not in the presence of azithromycin. These in vivo and in vitro results demonstrate that anti-virulence interventions based on QS-blockade diminish natural selection towards reduced virulence and therefore may increase the prevalence of more virulent genotypes in the Hospital environment. More generally, the impact of intervention on the evolution of virulence of pathogenic bacteria should be assessed.

Trial registration: ClinicalTrials.gov NCT00610623.

Figure 1. Patient enrollment and follow-up.

Figure 2. In patient QS-inhibition in azithromycin-treated patients.

In patient QS-gene expression was determined as described. Tracheal aspirates from both day −1 and day x with bacterial RNA of adequate quality were available for twelve placebo and eleven azithromycin patients. Expression of QS-circuit gene lasI, QS-target gene rhlA and QS-independent gene trpD measured in tracheal aspirates is shown as the relative value (%) of the last accessible day (Dx) compared to day −1 (set as 100%). A horizontal line indicates the median expression levels. P values were calculated using Mann-Whitney tests.

Figure 3. Evolution of lasR mutants and elastase production in azithromycin-treated and untreated patients.

Change in the proportion of lasR mutants (a) and mean elastase production (b) through time. Solid lines and closed symbols indicate azithromycin-treated patients, and dashed lines and open symbols indicate placebo group. Note that data is presented to day 11 of colonization, despite some samples being collected up to 20 days, because of very small sample sizes (six isolates) by day 12 in the azithromycin-treated group. However, qualitatively identical results were obtained when the whole data set was analysed. The change in the proportion of lasR mutants and elastase through time was analysed using logistic regression, corrected for under-dispersion, and General linear Modelling, respectively in GenStat 10.

Figure 4. lasR mutant growth rates and invasion of wild type populations in the presence and absence of azithromycin.

In vitro densities of wildtype (black) and lasR mutant (grey) after 72 hours growth in M9 salts BSA medium (a), and selection coefficients of lasR mutant relative to wildtype (b) as a function of azithromycin (AZM). Bars show means (± SEM) of six replicates. All differences (wildtype versus lasR) in the presence of azithromycin are statistically significant (p<0.05).