Fig. 5: Diagram showing the combination of ciprofloxacin-tobramycin to drive different ceftazidime-avibactam and ceftazidime resistant clinical strains of P. aeruginosa to extinction.

A Short-term evolution of population 1 from 5 different clinical isolates (MAD02-001, CLE03-004, CLM02-004, BAL02-001, and MAD04-002) previously evolved on ceftazidime-avibactam (CZA), represented as bacterial cells with different colors, 4 replicate populations of each parental population, was performed during 3 days in the presence of the ciprofloxacin-tobramycin (CIP-TOB) combination. Growth of the 40 control populations was confirmed in the 2 drugs independently used, at the same concentrations used for the drugs combinations. Extinct populations at the end of the experimental evolution are represented in black, while surviving populations are represented in gray. 6 out of 20 ceftazidime-avibactam resistant populations 1 submitted to short-term ALE in the presence of the antibiotic combination became extinct. These results are statistically significant (p = 0.002) and indicate that the ciprofloxacin-tobramycin CS-based therapeutic strategy previously proposed for the treatment of concern clinical strains may be ineffective on strains resistant to the last-resort combination ceftazidime-avibactam; especially those presenting mutations in mexR. B Short-term evolution of population 1 from 5 different clinical isolates (MAD02-001, CLE03-004, CLM02-004, BAL02-001 and MAD04-002) previously evolved on ceftazidime (CAZ), represented as bacterial cells with different colors, 4 replicate populations of each parental population, was performed during 3 days in the presence of the ciprofloxacin-tobramycin (CIP-TOB) combination. In addition, population 4 from the ceftazidime resistant clinical isolate BAL02-001 was added (BAL02-001 CAZ4), since this population acquired a genetic variation in mexR, something that frequently happened on ceftazidime-avibactam (CLM02-004 CZA, BAL02-001 CZA and MAD04-002 CZA). Growth of the 48 control populations was confirmed in the 2 drugs independently used, at the same concentrations used for the drugs combinations. Extinct populations at the end of the experimental evolution are represented in black, while surviving populations are represented in gray. All the populations 1 submitted to short-term ALE in the presence of the antibiotic combination became extinct while the treatment was ineffective for all the populations from BAL02-001 CAZ4, containing a genetic variation in mexR. These results are statistically significant (p = 8.415e-14) and indicate that the ciprofloxacin-tobramycin CS-based therapeutic strategy previously proposed for the treatment of concerned clinical strains may be ineffective on strains presenting mutations in mexR.