Persister cell formation in clinical isolates of Pseudomonas aeruginosa

Persister cells (PC) are non-growing and metabolically inactive cells, which lack transcription, translation, and proton motive force. PC formation was reported to occur by (ⅰ) stochastically (ⅱ) antibiotic treatment (ⅲ) nutrient deprivation and (ⅳ) other biofilm-related condition. PCs are responsible for chronic and relapse of biofilm infections as well as bacterial infections. Pseudomonas aeruginosa (Pa) is an opportunistic pathogen frequently causing chronic airway infections in patients with cystic fibrosis (CF), chronic urinary tract infection (UTI), and ventilator-associated pneumonia. For this study, Pa isolates; TP-10, ST-13, and POA1 were used. The Minimum Inhibitory Concentration (MIC) was determined and time-kill assay was carried out for PC isolation in the planktonic stage. Further, Flow Cytometry (FC) and qRT-PCR for stringent response genes (rel A, spo T and lon) and toxin-antitoxin (hig B and hig A) were done. A biphasic kill curve pattern was observed which varied across the antibiotic treatment. Starting from 12-13 log₁₀ cfu/ml mean inoculum, after ceftazidime treatment 9-10 log₁₀ cfu/ml lasted, while on gentamicin and ciprofloxacin treatment 5-6 log₁₀ cfu/ml lasted and formed biphasic pattern. The PC were analyzed through FC using Redox sensor green (RSG) and propidium iodide (PI) staining. It was observed that ceftazidime treatment has high redox activities followed by ciprofloxacin and gentamicin treatment compared to untreated isolate. Whereas ST-13 isolate had high redox activities on ceftazidime treatment followed by gentamicin and ciprofloxacin. The rel A, spo T, lon, hig B and hig A genes were upregulated on ceftazidime and gentamicin treatment compared to untreated in PAO1 and TP-10 isolates. Whereas in ST-13 isolate, genes were upregulated on gentamicin treatment. From the above study, it can be concluded that gentamicin and ceftazidime lead to PCs formation in P. aeruginosa planktonic stage.