Easy synergism for colistin-resistant KPC-producing Klebsiella pneumoniae: the E-test with supplemented agar

Abstract

The renewed interest in testing synergistic interactions of antimicrobial agents is mainly related to the challenge of antimicrobial resistance. Whereas time-kill curves and checkerboard methods represent the reference tests for screening synergy, the E-test with strips in a cross formation might be an interesting alternative, but requires that minimum inhibitory concentrations (MICs) of antibiotics have been previously determined [1]. However, agreement between the different tests seems to be quite variable [1]. To be clinically useful, a synergy test should be rapid and easily interpretable.We evaluated the synergistic activity of colistin (COL) plus rifampin (RIF) against 10 clinical isolates of COL-resistant Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) comparing the checkerboard method with the E-test on Mueller-Hinton agar, alone or supplemented with RIF. The 10 tested strains resulted in KPC3 production by polymerase chain reaction and Sequence Type (ST) 512 by multilocus sequence typing (MLST) analysis (http://www.pasteur.fr/recherche/ genopole/PF8/mlst/Kpneumoniae.html). COL MICs for the checkerboard method were determined with broth microdilution in Mueller-Hinton broth alone and supplemented with RIF at the final concentration of 2, 4, 8, 16 and 32 mg/L in 96-well microtiter plates. COL MICs also were determined with the E-test method on Mueller-Hinton agar alone or supplemented with RIF at the final concentration of 2, 4, 8, 16 and 32 mg/L in Petri dishes (Fig. 1).According to the 2014 EUCAST Clinical Breakpoints, COL resistance was defined by MIC > 2 mg/L. With the checkerboard method, COL MICs for the 10 strains were 64 mg/L (5 strains) and 32 mg/L (5 strains). COL MICs decreased below the susceptibility breakpoint for 8 of 10 strains in the presence of RIF concentrations of 16 (1 strain), 8 (6 strains) and 2 mg/L (1 strain). With the E-test method, COL MICs for the 10 strains were 12 mg/L (1 strain), 8 mg/L (2 strains), 6 mg/L (4 strains) and 4 mg/L (3 strains). COL MICs for 10 of 10 strains decreased below the susceptibility breakpoint in the presence of any concentration of RIF. These results are summarized in Table 1. RIF MICs results were higher than 32 mg/L with both methods for all strains. Both methods showed similar results and were able to show that RIF decreased MICs of COL below the susceptibility breakpoint in the majority of COL-resistant KPC-Kp tested strains.The mechanism of the synergistic activity between COL and RIF might be related to the perturbation of the outer cellular membrane produced by COL that favours the penetration of RIF at intracellular concentrations able to inhibit protein synthesis [2]. The higher COL MICs values observed in our experiment with the microdilution method with respect to the E-test already have been reported and seem to be related to the adsorption of colistin to the microplate plastic wells [3,4]. Although higher COL MICs were observed with the microdilution method, the effect of adding RIF was always evident also with t...

Azithromycin possesses biofilm–inhibitory activity and potentiates non-bactericidal colistin methanesulfonate (CMS) and polymyxin B against Klebsiella pneumonia

“…More tests are needed to identify potential synergistic activity in antibacterial combinations using physiologically relevant concentrations. Antimicrobial susceptibility tests such as the Kirby-Bauer disc-diffusion assay [14,15] which can be extended to identify synergistic actions between antibiotics [16][17][18] rely on colony growth across the surface of agar and may not accurately reflect the physiological and metabolic state of liquid culture (planktonic) bacterial cells or those encased in protective biofilms and adhered to surfaces. Biofilm-formation underlies the survival and persistence of many pathogens, providing phenotypic resistance or tolerance to therapy when genetic resistance might be restricted [19,20].…”

Azithromycin possesses biofilm–inhibitory activity and potentiates non-bactericidal colistin methanesulfonate against Klebsiella pneumonia

“…However, not all antibiotic combinations make pharmaceutical sense and confusion may arise from predictions made of the efficacy of antimicrobials acting alone, and more combination tests are needed to identify potential synergistic activity in antibacterial combinations using physiologically relevant concentrations. Antimicrobial susceptibility tests such as the Kirby-Bauer disc-diffusion assay [14,15] which can be extended to identify synergistic actions between antibiotics [16][17][18] rely on colony growth across the surface of agar and may not accurately reflect the physiological and metabolic state of liquid culture (planktonic) bacterial cells or those encased in protective biofilms and adhered to surfaces. Biofilm-formation underlies the survival and persistence of many pathogens, providing phenotypic resistance or tolerance to therapy when genetic resistance might be restricted [19,20].…”