Quorum-sensing regulation of phenazine production heightens Pseudomonas aeruginosa resistance to ciprofloxacin

Abstract

Quorum sensing is a type of cell-cell communication that modulates various biological activities of bacteria. Previous studies indicate that quorum sensing contributes to the evolution of bacterial resistance to antibiotics, but the underlying mechanisms are not fully understood. In this study, we grew Pseudomonas aeruginosa in the presence of sub-lethal concentrations of ciprofloxacin, resulting in a large increase in ciprofloxacin minimal inhibitory concentration. We discovered that quorum sensing-mediated phenazine biosynthesis was significantly enhanced in the resistant isolates, where the quinolone circuit was the predominant contributor to this phenomenon. We found that production of pyocyanin changed carbon flux and showed that the effect can be partially inhibited by the addition of pyruvate to cultures. This study illustrates the role of quorum sensing-mediated phenotypic resistance and suggests a strategy for its prevention.

Keywords: Pseudomonas aeruginosa; antimicrobial resistance; pyocyanin; quorum sensing.

Fig 1

P. aeruginosa evolved a high level of ciprofloxacin resistance after experimental evolution. (A) The colonies were randomly selected from lysogen broth agar and grown in shaking test tubes. The suspension was transferred to a fresh tube every 24 h with 1/2 MIC of ciprofloxacin. The MIC was measured every 4 days, and the concentration of ciprofloxacin was adjusted accordingly. The experiment proceeded for 20 days, and the same procedure without added ciprofloxacin was used as a control. (B) The MIC of all lineages. WT represents the group cultured without ciprofloxacin. eWT represents the group cultured with ciprofloxacin. (C) The MIC of WT and eWT at day 20. MIC, minimal inhibitory concentration.

Fig 2

Bacterial quorum sensing is responsible for antibiotic resistance evolution. (A) Plot of KEGG enrichment analysis of DEGs (eWT vs WT). The x-axis shows the rich factor, defined as the number of DEG enriched in the pathway/number of all genes in the background gene set. The y-axis represents the enriched pathways. The color of the bubbles represents the false discovery rate (FDR). (B) The expression of QS-related genes of eWT compared with WT measured by RNA-seq. (C) The MIC of evolved QS-deficient strains after 20 days of sub-lethal ciprofloxacin exposure. Data are mean ± SD. Different letters indicate significant differences (P <  0.05). eWT, evolved WT.

Fig 3

QS-regulated pyocyanin contributes to bacterial resistance evolution. (A) Fold change of DEGs included in the phenazine biosynthesis pathway. Significant upregulated genes (compared to WT) are noted. (B) Pyocyanin production in WT (left) and ∆phzM (right) cultures. (C) The production of pyocyanin in WT and ∆phzM before and after evolution. (D) The MIC of WT, ∆phzM, and ∆phzM complemented with pyocyanin (PYO) before and after evolution. (E) The effect of reactive oxygen species (ROS) scavengers glutathione (GSH) and vitamin C (VC) on intracellular ROS content. (F–G) MIC during a 20-day experimental evolution of PAO1 treated with GSH (F) and VC (G). (H) The expression of efflux pumps in eWT compared to that in the parent strain, analyzed using RNA-seq data. Data are mean ± SD; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. eWT, evolved WT; e∆phzM, evolved ∆phzM; ePYO, evolved ∆phzM complemented with pyocyanin; GSH, glutathione; VC, vitamin C.

Fig 4

Pyocyanin affects carbon metabolism to increase the ciprofloxacin resistance. (A) KEGG pathway analysis of DEGs in e∆phzM compared with eWT. (B) Relative expression of PA3416 (left, coding for beta chain of E1 PDH) and PA3417 (right, coding for alpha chain of E1 PDH) in eWT and e∆phzM. (C–E) The PDH activity (C), ATP production (D), and the percentage of antibiotic-resistant individuals (E) of evolved strains. (F–H) The PDH activity (F), ATP production (G), and the percentage of antibiotic-resistant individuals (H) of PAO1 treated with different concentrations of pyocyanin. The antibiotic-resistant individuals were defined as a group of bacteria that survived when exposed to a bactericidal drug concentration (5 × MIC). (I) The percentage of antibiotic-resistant individuals during the whole evolution. One-way analysis of variance (ANOVA) was used to compare means among groups. Data are mean ± SD; ^*P < 0.05, **P < 0.01. Different letters indicate significant differences (P <  0.05). eWT, evolved WT; e∆phzM, evolved ∆phzM; ePYO, evolved ∆phzM complemented with pyocyanin.

Fig 5

Pyruvate activates carbon metabolism and inhibits resistance evolution. (A–C) The PDH activity (A), ATP production (B), and percentage of antibiotic-resistant individuals (C) in WT, eWT, and ePYT. (D) The MICs of evolved WT treated with or without pyruvate in a separate experiment from the data presented in Fig. 1. eWT, evolved WT; ePYT. evolved WT treated with pyruvate. All experiments had at least three replicates. One-way analysis of variance (ANOVA) was used to compare means among groups. Data are means ± SD; ^*P < 0.05. Different letters indicate significant differences (P <  0.05).