The evolutionary trajectories of P. aeruginosa in biofilm and planktonic growth modes exposed to ciprofloxacin: beyond selection of antibiotic resistance

Abstract

Ciprofloxacin (CIP) is used to treat Pseudomonas aeruginosa biofilm infections. We showed that the pathways of CIP-resistance development during exposure of biofilms and planktonic P. aeruginosa populations to subinhibitory levels of CIP depend on the mode of growth. In the present study, we analyzed CIP-resistant isolates obtained from previous evolution experiments, and we report a variety of evolved phenotypic and genotypic changes that occurred in parallel with the evolution of CIP-resistance. Cross-resistance to beta-lactam antibiotics was associated with mutations in genes involved in cell-wall recycling ( ftsZ , murG ); and could also be explained by mutations in the TCA cycle ( sdhA ) genes and in genes involved in arginine catabolism. We found that CIP-exposed isolates that lacked mutations in quorum-sensing genes and acquired mutations in type IV pili genes maintained swarming motility and lost twitching motility, respectively. Evolved CIP-resistant isolates showed high fitness cost in planktonic competition experiments, yet persisted in the biofilm under control conditions, compared with ancestor isolates and had an advantage when exposed to CIP. Their persistence in biofilm competition experiments in spite of their fitness cost in planktonic growth could be explained by their prolonged lag-phase. Interestingly, the set of mutated genes that we identified in these in vitro-evolved CIP-resistant colonies, overlap with a large number of patho-adaptive genes previously reported in P. aeruginosa isolates from cystic fibrosis (CF) patients. This suggests that the antibiotic stress is contributing to the bacterial evolution in vivo, and that adaptive laboratory evolution can be used to predict the in vivo evolutionary trajectories.

Pseudomonas aeruginosa: An Audacious Pathogen with an Adaptable Arsenal of Virulence Factors

“…Cross-resistance to β-lactam antibiotics was associated with mutations in genes involved in cell wall recycling ( ftsZ, murG ) and could also be explained by mutations in TCA cycle genes ( sdhA ) and genes involved in arginine catabolism. Interestingly, the set of identified mutated genes overlaps with a large number of pathoadaptive genes previously reported in P. aeruginosa isolates from CF patients [ 304 ]. Furthermore, pyomelanin-resistant mutants frequently coexist with other morphotypes in CF patients [ 326 ].…”

rpoS-mutation variants are selected in Pseudomonas aeruginosa biofilms under imipenem pressure

“…Previous studies showed that acquired mutations conferring beneficial traits such as antibiotic resistance will dominate the populations when exposing biofilm bacteria to high concentrations of antibiotic [ 37 ]. Therefore, the enrichment of the hyperbiofilm rpoS mutation variants could have been achieved due to their enhanced resistance to imipenem.…”

Shared and Unique Evolutionary Trajectories to Ciprofloxacin Resistance in Gram-negative Bacterial Pathogens

“…The double mutant NfxB:Leu62fs/PilW:Ala164fs expanded to ~85% of the population on Day 3 and provided a genetic background for the appearance of the three GyrA mutant variants on Day 4 (Figure 2c, Supplementary Table 2D). The loss of type IV pili has been observed previously under CIP stress in P. aeruginosa (79). While the mechanistic rationale for this class of events remains unclear, it was hypothesized that the loss of type IV pili, known receptors for filamentous phages implicated in chronic infection, contributes to resistance against superinfection and lysis under ciprofloxacin stress (80).…”