Efficacy of Antibiotic Combinations against Multidrug-Resistant Pseudomonas aeruginosa in Automated Time-Lapse Microscopy and Static Time-Kill Experiments

Abstract

Antibiotic combination therapy is used for severe infections caused by multidrug-resistant (MDR) Gram-negative bacteria, yet data regarding which combinations are most effective are lacking. This study aimed to evaluate the in vitro efficacy of polymyxin B in combination with 13 other antibiotics against four clinical strains of MDR Pseudomonas aeruginosa We evaluated the interactions of polymyxin B in combination with amikacin, aztreonam, cefepime, chloramphenicol, ciprofloxacin, fosfomycin, linezolid, meropenem, minocycline, rifampin, temocillin, thiamphenicol, or trimethoprim by automated time-lapse microscopy using predefined cutoff values indicating inhibition of growth (≤10⁶ CFU/ml) at 24 h. Promising combinations were subsequently evaluated in static time-kill experiments. All strains were intermediate or resistant to polymyxin B, antipseudomonal β-lactams, ciprofloxacin, and amikacin. Genes encoding β-lactamases (e.g., bla_PAO and bla_OXA-50) and mutations associated with permeability and efflux were detected in all strains. In the time-lapse microscopy experiments, positive interactions were found with 39 of 52 antibiotic combination/bacterial strain setups. Enhanced activity was found against all four strains with polymyxin B used in combination with aztreonam, cefepime, fosfomycin, minocycline, thiamphenicol, and trimethoprim. Time-kill experiments showed additive or synergistic activity with 27 of the 39 tested polymyxin B combinations, most frequently with aztreonam, cefepime, and meropenem. Positive interactions were frequently found with the tested combinations, against strains that harbored several resistance mechanisms to the single drugs, and with antibiotics that are normally not active against P. aeruginosa Further study is needed to explore the clinical utility of these combinations.

Keywords: Gram-negative bacteria; carbapenem resistance; combination therapy; polymyxins; synergy.

FIG 1

Results of time-kill experiments. Mean bacterial concentrations during 24-h exposure to single antibiotics and polymyxin B combinations at various drug concentrations (in mg/liter) are shown. The lower limit of detection (dotted line) was 10 CFU/ml. Abbreviations: AMK, amikacin; ATM, aztreonam; FEP, cefepime; CHL, chloramphenicol; CIP, ciprofloxacin; FOF, fosfomycin; LIN, linezolid; MEM, meropenem; MIN, minocycline; PMB, polymyxin B; RIF, rifampin; TMC, temocillin; THI, thiamphenicol; TMP, trimethoprim.

FIG 1

Results of time-kill experiments. Mean bacterial concentrations during 24-h exposure to single antibiotics and polymyxin B combinations at various drug concentrations (in mg/liter) are shown. The lower limit of detection (dotted line) was 10 CFU/ml. Abbreviations: AMK, amikacin; ATM, aztreonam; FEP, cefepime; CHL, chloramphenicol; CIP, ciprofloxacin; FOF, fosfomycin; LIN, linezolid; MEM, meropenem; MIN, minocycline; PMB, polymyxin B; RIF, rifampin; TMC, temocillin; THI, thiamphenicol; TMP, trimethoprim.