Quorum quenching quandary: resistance to antivirulence compounds

Abstract

Quorum sensing (QS) is the regulation of gene expression in response to the concentration of small signal molecules, and its inactivation has been suggested to have great potential to attenuate microbial virulence. It is assumed that unlike antimicrobials, inhibition of QS should cause less Darwinian selection pressure for bacterial resistance. Using the opportunistic pathogen Pseudomonas aeruginosa, we demonstrate here that bacterial resistance arises rapidly to the best-characterized compound that inhibits QS (brominated furanone C-30) due to mutations that increase the efflux of C-30. Critically, the C-30-resistant mutant mexR was more pathogenic to Caenorhabditis elegans in the presence of C-30, and the same mutation arises in bacteria responsible for chronic cystic fibrosis infections. Therefore, bacteria may evolve resistance to many new pharmaceuticals thought impervious to resistance.

Figure 1

The mexR and nalC mutations render P. aeruginosa less sensitive to QQ compound C-30 during growth in adenosine minimal medium. (a) Growth (at 37 °C) of wild-type P. aeruginosa PA14 (blue squares), transposon mutant mexR (red circles) and transposon mutant nalC (green triangles). (b) Growth of spontaneous quorum quenching quandary mutants (sQQQ) 1 (red circles) and sQQQ3 (green triangles) vs wild-type PA14 (blue squares). (c) Growth of CF clinical isolate 1253 (red circles) and CF Liverpool epidemic strain 12142 (green triangles) with enhanced mexA expression vs control strain AHP (blue squares). Open symbols indicate the presence of 50 μ C-30, whereas closed symbols indicate the absence of C-30. Data represent the mean±s.d.; n=3.

Figure 2

The mexR and nalC mutations decrease C-30 inhibition of P. aeruginosa QS phenotypes. (a) Swarming motility at 37 °C after 24 h using rhlR as a negative control. Three independent cultures were used for each strain, and two swarming plates were used for each culture. Representative plates are shown. (b) Pyocyanin production after incubation in LB medium for 9 h. Mutant phzM was used as a negative control. C-30 was used at 50 μ. Data represent the mean±s.d.; n=3. ^*P<0.01 by Student's t-test compared with the value in the presence of C-30.

Figure 3

The mexR mutation decreases C-30 inhibition of killing of C. elegans by P. aeruginosa. L4 stage hermaphrodite worms were exposed to wild-type P. aeruginosa (blue squares) and mexR (red circles) grown on PGS medium with (open symbols) or without (closed symbols) 50 μ C-30. Worms exposed to E. coli OP50 on the same medium plates were used as the negative control, which showed 100% viability. Data represent the mean±s.d.; n=4 plates, 20 worms per plate.