The phenotypic evolution of Pseudomonas aeruginosa populations changes in the presence of subinhibitory concentrations of ciprofloxacin

Abstract

Ciprofloxacin is a widely used antibiotic, in the class of quinolones, for treatment of Pseudomonas aeruginosa infections. The immediate response of P. aeruginosa to subinhibitory concentrations of ciprofloxacin has been investigated previously. However, the long-term phenotypic adaptation, which identifies the fitted phenotypes that have been selected during evolution with subinhibitory concentrations of ciprofloxacin, has not been studied. We chose an experimental evolution approach to investigate how exposure to subinhibitory concentrations of ciprofloxacin changes the evolution of P. aeruginosa populations compared to unexposed populations. Three replicate populations of P. aeruginosa PAO1 and its hypermutable mutant DmutS were cultured aerobically for approximately 940 generations by daily passages in LB medium with and without subinhibitory concentration of ciprofloxacin and aliquots of the bacterial populations were regularly sampled and kept at 280 8C for further investigations. We investigate here phenotypic changes between the ancestor (50 colonies) and evolved populations (120 colonies/strain). Decreased protease activity and swimming motility, higher levels of quorum-sensing signal molecules and occurrence of mutator subpopulations were observed in the ciprofloxacin-exposed populations compared to the ancestor and control populations. Transcriptomic analysis showed downregulation of the type III secretion system in evolved populations compared to the ancestor population and upregulation of denitrification genes in ciprofloxacin-evolved populations. In conclusion, the presence of antibiotics at subinhibitory concentration in the environment affects bacterial evolution and further studies are needed to obtain insight into the dynamics of the phenotypes and the mechanisms involved.

Evolution of Antibiotic Resistance in Biofilm and Planktonic Pseudomonas aeruginosa Populations Exposed to Subinhibitory Levels of Ciprofloxacin

“…In P. aeruginosa, swimming motility is driven by a single unipolar flagellum which in PAO1 is dependent on 41 genes encoding structural/ assembly and regulatory components of the flagellar organelle (40). In accordance, phenotypic characterization of P. aeruginosa populations during planktonic experimental evolution showed a decrease in the swimming motility of the populations (41), and this was also confirmed in the present study (data not shown). This suggests that during evolution under conditions of good oxygenation and sufficient nutrition, swimming motility is probably an energy-expensive property that is easily lost.…”

“…Intriguingly, mutations in the quorum-sensing system were not present in the CIP-evolved biofilm populations, indicating a selection for the maintenance of quorum-sensing-regulated factors under evolution at subinhibitory concentrations of CIP. In accordance, we have previously observed increased levels of quorum-sensing molecules in planktonic evolved populations in the presence of 0.05 mg/liter CIP compared to the controls (41). One explanation for these observations could be the protection conferred by QS-regulated catalases and superoxide-dismutases against antibiotic-related oxidative stress (44), as we have shown increased production of reactive oxygen species in P. aeruginosa biofilms exposed to ciprofloxacin (45).…”

“…Mutations in the regulatory gene of the N-acylhomoserine lactone quorum-sensing system (lasR) were identified in a high percentage of reads (more than 90% of reads) in two different lineages and correlated with a reduced production of proteases in isolates from these two lineages (Table S6). A loss of virulence traits in in vitro PAO1 evolution has been reported previously in planktonic cultures (41), showing that the occurrence of this phenotype in evolutionary studies is independent of in vivo factors, e.g., immune selection. Intriguingly, mutations in the quorum-sensing system were not present in the CIP-evolved biofilm populations, indicating a selection for the maintenance of quorum-sensing-regulated factors under evolution at subinhibitory concentrations of CIP.…”

Evolutionary diversification of Pseudomonas aeruginosa in an artificial sputum model

“…Although we have shown previously that phages can alter the mode and rate of adaptive evolution [26], comparable sets of phenotypes eventually occurred both in the presence and absence of phages. It is, however, worth noting that similar phenotypes can be driven by sub-inhibitory concentrations of antibiotics in non-biofilm systems [48]. In CF, it has been proposed that the maintenance of phenotypically diverse P. aeruginosa populations may be a consequence of the spatial heterogeneity of the lung, leading to regional isolation of separately evolving communities [15].…”

Quality Attributes and In Vitro Bioequivalence of Different Brands of Amoxicillin Trihydrate Tablets

“…The frequent prescription of broad-spectrum antibiotics rather than prescribing more precise antibiotics and the inadequate dose and duration of treatment have been particularly embroiled. The subinhibitory concentrations resulting from the later effect can modulate the bacterial virulence [19,20]. A source of subinhibitory levels of antibiotic can be from the often neglected factor that generic drugs may not be in fact therapeutically equivalent to the branded antibiotic.…”