TY - JOUR

T1 - Time-kill assay

T2 - An efficacy of synergy between carbapenems and clodronic acid

AU - Afinogenova, A. G.

AU - Voroshilova, T. M.

AU - Afinogenov, G. E.

AU - Maday, D. Y.

AU - Spiridonova, A. A.

PY - 2018/1/1

Y1 - 2018/1/1

N2 - Currently, a search for augmenting antibiotics activity is still crucial due to elevated frequency of detecting carbapenem-resistant Gran-positive bacterial isolates. To resolve this, it might be reasonable to combine carbapenems metal-β-lactamase (MβL) inhibitors. Unfortunately, no MβL inhibitors approved for treatment of carbapenem-resistant infections are currently available. Pathogenic bacteria may survive antibiotic attack, exert tolerance and persistence accompanied with the ongoing infectious process. In connection with this, determining dependence between antimicrobial-related bactericidal effect and exposure time on microbes at 4, 8, 12 and 24 hours after the onset, a so called time-kill assay, is necessary. A synergy between both agents was noted upon reduced microbial population by ≥ 3 log 10 . A checkerboard array followed by seeding the microplate well contents onto a dense nutrient medium at various time points were used to assess a synergistic efficacy of carbapenems applied together with clodronic acid against MβL-producing VIM-genotype P. aeruginosa 532/14 clinical isolate obtained from patients with infectious complications (minimal inhibitory concentrations [MIC] for imipenem or meropenem were 512 μg/ml), microbial burden 10 6 CFU/ml. Optical density was measured at two wavelengths (490 and 630 nm) in ELx800 reader, within 4–24 hour exposure time to determine time of logarithmic growth phase emerging in test culture. It is noteworthy that magnitude of optical density is a difference between two bichromatic measurements resulting in remarkably reduced inaccuracy due to scratches or fingerprints left on the plate. It was found that clodronic acid exhibited a synergic bactericidal effect with carbapenems against a clinically resistant MβL-producing VIM-genotype P. aeruginosa 532/14 strain. Upon that, imipenem-related antimicrobial activity was evident as early as 8 hours after the onset decreasing cell growth down to 1.4 log 10 compared to control, whereas 12 hours later it resulted in total inhibition of test strain by decreasing growth of the test strain by 6 log 10 . Meropenem in combination with clodronic acid showed a more pronounced activity: complete absence of P. aeruginosa 532/14 growth by 8 hours of incubation, growth suppression by 3.2 log 10 , which reached 6 log 10 12–24 hours after the onset. Time-kill assay allows to identify efficient combinations of carbapenems and MβL inhibitors, which is of great importance for increasing therapeutic efficacy of patients with severe purulent-septic complications.

AB - Currently, a search for augmenting antibiotics activity is still crucial due to elevated frequency of detecting carbapenem-resistant Gran-positive bacterial isolates. To resolve this, it might be reasonable to combine carbapenems metal-β-lactamase (MβL) inhibitors. Unfortunately, no MβL inhibitors approved for treatment of carbapenem-resistant infections are currently available. Pathogenic bacteria may survive antibiotic attack, exert tolerance and persistence accompanied with the ongoing infectious process. In connection with this, determining dependence between antimicrobial-related bactericidal effect and exposure time on microbes at 4, 8, 12 and 24 hours after the onset, a so called time-kill assay, is necessary. A synergy between both agents was noted upon reduced microbial population by ≥ 3 log 10 . A checkerboard array followed by seeding the microplate well contents onto a dense nutrient medium at various time points were used to assess a synergistic efficacy of carbapenems applied together with clodronic acid against MβL-producing VIM-genotype P. aeruginosa 532/14 clinical isolate obtained from patients with infectious complications (minimal inhibitory concentrations [MIC] for imipenem or meropenem were 512 μg/ml), microbial burden 10 6 CFU/ml. Optical density was measured at two wavelengths (490 and 630 nm) in ELx800 reader, within 4–24 hour exposure time to determine time of logarithmic growth phase emerging in test culture. It is noteworthy that magnitude of optical density is a difference between two bichromatic measurements resulting in remarkably reduced inaccuracy due to scratches or fingerprints left on the plate. It was found that clodronic acid exhibited a synergic bactericidal effect with carbapenems against a clinically resistant MβL-producing VIM-genotype P. aeruginosa 532/14 strain. Upon that, imipenem-related antimicrobial activity was evident as early as 8 hours after the onset decreasing cell growth down to 1.4 log 10 compared to control, whereas 12 hours later it resulted in total inhibition of test strain by decreasing growth of the test strain by 6 log 10 . Meropenem in combination with clodronic acid showed a more pronounced activity: complete absence of P. aeruginosa 532/14 growth by 8 hours of incubation, growth suppression by 3.2 log 10 , which reached 6 log 10 12–24 hours after the onset. Time-kill assay allows to identify efficient combinations of carbapenems and MβL inhibitors, which is of great importance for increasing therapeutic efficacy of patients with severe purulent-septic complications.

KW - Carbapenem-resistant Gram-negative bacteria

KW - Carbapenems

KW - Clodronic acid

KW - Metal-β-lactamase inhibitor

KW - Time-kill assay

UR - http://www.scopus.com/inward/record.url?scp=85062022883&partnerID=8YFLogxK

U2 - 10.15789/2220-7619-2018-4-497-502

DO - 10.15789/2220-7619-2018-4-497-502

M3 - Article

AN - SCOPUS:85062022883

VL - 8

SP - 497

EP - 502

JO - Russian Journal of Infection and Immunity

JF - Russian Journal of Infection and Immunity

SN - 2220-7619

IS - 4

ER -