Isolation of persister cells within the biofilm and relative gene expression analysis of type II toxin/antitoxin system in Pseudomonas aeruginosa isolates in exponential and stationary phases

J Glob Antimicrob Resist. 2022 Mar:28:30-37. doi: 10.1016/j.jgar.2021.11.009. Epub 2021 Dec 15.


Objectives: Chronic infections and treatment failure are concerning issues in patients with Pseudomonas aeruginosa infections. Persister cell formation in biofilm is considered a key reason for antibiotic resistance and treatment failure. In this study, expression of type II toxin/antitoxin (TA) system genes (relBE, Xre-COG5654, vapBC and Xre-GNAT) in persister cells of biofilm was evaluated in the presence of the antibiotics ciprofloxacin and colistin during exponential and stationary phases.

Methods: The impact of antibiotics on biofilm persister cell formation during exponential and stationary phases of P. aeruginosa strains was examined by colony count at different time intervals in the presence of 5-fold minimum inhibitory concentration (MIC) of ciprofloxacin and colistin. Furthermore, expression of relBE, Xre-COG5654, vapBC and Xre-GNAT genes in P. aeruginosa strains undergoing antibiotic treatment for 3.5 h during exponential and stationary phases was examined by RT-qPCR.

Results: Formation of persister cells was observed in biofilms formed by P. aeruginosa strains in the presence of 5 × MIC of ciprofloxacin and colistin compared with the control group after 3.5 h of incubation both during exponential and stationary phases. The number of biofilm persister cells was higher in stationary phase than in exponential phase. According to the findings of RT-qPCR, ciprofloxacin and colistin may induce persister cell formation by enhancing the expression of type II TA systems during stationary and exponential phases.

Conclusion: The result of this study indicate that ciprofloxacin and colistin have the potential to increase persister cells formation in biofilms by influencing the expression of type II TA systems.

Keywords: Biofilm; Exponential and stationary phases; Persister cell; Pseudomonas aeruginosa; Real-time PCR; TA system.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / therapeutic use
  • Antitoxins* / genetics
  • Antitoxins* / metabolism
  • Biofilms
  • Ciprofloxacin / pharmacology
  • Colistin / pharmacology
  • Gene Expression
  • Humans
  • Pseudomonas aeruginosa*


  • Anti-Bacterial Agents
  • Antitoxins
  • Ciprofloxacin
  • Colistin