Real-time monitoring of Pseudomonas aeruginosa biofilm growth dynamics and persister cells eradication

Biofilm formation and appearance of persister cells within biofilms are highly responsible for conventional treatment failure and emerging antibiotic resistance. Using impedance-based measurements we studied the biofilm growth dynamics of 13 P. aeruginosa strains using different media and growth conditions and compared the methodology with the classical crystal violet biofilm quantification assay. Additionally, we investigated the effect of seven conventional antibiotics on biofilm formation as well as the ability of piperacillin-tazobactam, ceftazidime, ciprofloxacin and tobramycin to disrupt mature (24h and 48h) biofilms. Real-time experiments showed that the exposure of established P. aeruginosa biofilms to certain concentrations of ciprofloxacin, ceftazidime and tobramycin induced persister cell fraction emergence. Thus, the synergistic combination of mannitol and ciprofloxacin was evaluated on clinical 48h P. aeruginosa biofilms. Our results conclude that mannitol is capable both to prevent persister cell formation and to efficiently wake up dormant persisters when administered at 3,200 and 320 mg/L. These results were confirmed using viable colony counting. Additionally, whole genome sequencing of the wildtype and persister cells identified different SNPs and genomic duplications in multiple genes. However, these mutations were variant-specific and not uniquely associated with persisters, suggesting that the persistent phenotype mainly depends on transcriptional changes.As the tested ciprofloxacin-mannitol combination appeared to fully eradicate mature biofilms, we conclude that impedance-based biofilm diagnostics, which also permits antibiotic susceptibility testing and the identification of persister cells, is of great potential for the clinical practice and could aid in establishing clinical breakpoints for biofilm-related infections.