Activity of polymyxin B combinations against genetically well-characterised Klebsiella pneumoniae producing NDM-1 and OXA-48-like carbapenemases

Background: Combination therapy can enhance the activity of available antibiotics against multidrug-resistant Gram-negative bacteria. This study assessed the effects of polymyxin B combinations against carbapenemase-producing Klebsiella pneumoniae ( K. pneumoniae). Methods: Twenty clinical K. pneumoniae strains producing NDM-1 (n = 8), OXA-48-like (n = 10), or both NDM-1 and OXA-48-like (n = 2) carbapenemases were used. Whole-genome sequencing was applied to detect resistance genes (e.g. encoding antibiotic-degrading enzymes) and sequence alterations influ-encing permeability or efflux. The activity of polymyxin B in combination with aztreonam, fosfomycin, meropenem, minocycline, or rifampicin was investigated in 24-hour time-lapse microscopy experiments. Endpoint samples were spotted on plates with and without polymyxin B at 4 x MIC to assess resistance development. Finally, associations between synergy and bacterial genetic traits were explored. Results: Synergistic and bactericidal effects were observed with polymyxin B in combination with all other antibiotics: aztreonam (11 of 20 strains), fosfomycin (16 of 20), meropenem (10 of 20), minocy-cline (18 of 20), and rifampicin (15 of 20). Synergy was found with polymyxin B in combination with fosfomycin, minocycline, or rifampicin against all nine polymyxin-resistant strains. Wildtype mgrB was associated with polymyxin B and aztreonam synergy (P = 0.0499). An absence of arr-2 and arr-3 was associated with synergy of polymyxin B and rifampicin (P = 0.0260). Emergence of populations with reduced polymyxin B susceptibility was most frequently observed with aztreonam and meropenem. Conclusion: Combinations of polymyxin B and minocycline or rifampicin were most active against the tested NDM-1 and OXA-48-like-producing K. pneumoniae. Biologically plausible genotype-phenotype as-sociations were found. Such information might accelerate the search for promising combinations and guide individualised treatment

Vancomycin MIC values (mg/L) for 13 <i>S</i>. <i>aureus</i> isolates from clinical blood cultures as determined by the Etest, macrodilution and CellDirector 3D (4 h readout).

<p>Vancomycin MIC values (mg/L) for 13 <i>S</i>. <i>aureus</i> isolates from clinical blood cultures as determined by the Etest, macrodilution and CellDirector 3D (4 h readout).</p

Antibiotic concentration ranges used in CellDirector 3D.

<p>Antibiotic concentration ranges used in CellDirector 3D.</p

MIC values in mg/L obtained with the Etest and CellDirector 3D at bacterial inocula of 10⁵ CFU/mL.

<p>MIC values in mg/L obtained with the Etest and CellDirector 3D at bacterial inocula of 10⁵ CFU/mL.</p

Vancomycin MIC values in mg/L as determined by the Etest, macrodilution and CellDirector 3D against VSSA and hVISA.

<p>Vancomycin MIC values in mg/L as determined by the Etest, macrodilution and CellDirector 3D against VSSA and hVISA.</p

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

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 (&lt;= 10(6) 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 beta-lactams, ciprofloxacin, and amikacin. Genes encoding beta-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