RESUMO
In the absence of a molecule that would collectively inhibit both metallo-ß-lactamases and serine-reactive carbapenemases, containment of their genes is the main weapon currently available for confronting carbapenem resistance in hospitals. Cost-effective methodologies rapidly detecting carbapenemase-producing enterobacteria (CPE) would facilitate such measures. Herein, a low-cost CPE detection method was developed that was based on the direct colorimetry of the yellow shift caused by the accumulation of diketopiperazines-products of the acid-catalyzed imipenem oligomerization-induced by carbapenemase action on dense solutions of imipenem/cilastatin. The reactions were studied by spectrophotometry in the visible spectrum using preparations of ß-lactamases from the four molecular classes. The effects of various buffers on reaction mixtures containing the potent carbapenemases NDM-1 and NMC-A were monitored at 405 nm. Optimal conditions were used for the analysis of cell suspensions, and the assay was evaluated using 66 selected enterobacteria, including 50 CPE as well as 16 carbapenemase-negative strains overexpressing other ß-lactamases. The development of the yellow color was specific for carbapenemase-containing enzyme preparations, and the maximum intensity was achieved in acidic or unbuffered conditions in the presence of zinc. When applied on bacterial cell suspensions, the assay could detect CPE with 98% sensitivity and 100% specificity, with results being comparable to those obtained with the Carba NP technique. Direct colorimetry of carbapenemase-induced imipenem decomposition required minimum reagents while exhibiting high accuracy in detecting CPE. Therefore, it should be considered for screening purposes after further clinical evaluation. IMPORTANCE Currently, the spread of multidrug-resistant (MDR) carbapenemase-producing enterobacteria (CPE), mostly in the clinical setting, is among the most pressing public health problems worldwide. In order to effectively control CPE, use of reliable and affordable methods detecting carbapenemase genes or the respective ß-lactamases is of vital importance. Herein, we developed a novel method, based on a previously undescribed phenomenon, that can detect CPE with few reagents by direct colorimetry of bacterial suspensions and imipenem/cilastatin mixtures.
Assuntos
Enterobacteriaceae , Imipenem , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Cilastatina/farmacologia , Colorimetria , Análise Custo-Benefício , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , Suspensões , beta-Lactamases/genéticaRESUMO
VIM-producing Klebsiella pneumoniae isolates are usually associated with high MICs to carbapenems. Preclinical studies investigating the pharmacokinetic-pharmacodynamic (PK-PD) characteristics of carbapenems against these isolates are lacking. The in vitro antibacterial activity of meropenem against one WT and three VIM-producing K. pneumoniae clinical isolates (median MICs 0.031, 8, 16 and 128âmg l- 1) was studied in a dialysis-diffusion PK-PD model and verified in a thigh infection neutropenic animal model by testing selected strains and exposures. The in vitro PK-PD target associated with bactericidal activity was estimated and the target attainment for different dosing regimens was calculated with Monte Carlo analysis. The in vitro model was correlated with the in vivo data, with log10CFU/ml reduction of < 1 for the VIM-producing (MIC 16âmg l- 1) and >2 for the WT (MIC 0.031âmg l- 1) isolates, with %fâT >MIC 25 and 100%, respectively. The in vitro bactericidal activity for all isolates was associated with 40 % fâT>MIC and attained in >90% of cases with the standard 1âg q8 0.5âh infusion dosing regimen only for isolates with MICs up to 1âmg l- 1. For isolates with MICs of 2-8âmg l- 1, prolonged infusion regimens (4âh infusion q8 or 2âh infusion q4) of standard (1âg) and higher (2âg) doses or continuous infusion regimens (3-6âg) are required. For isolates with a MIC of 16âmg l- 1 the unconventional dosing regimen of 2âg as 2âh infusion q4 or 12âg continuous infusion will be required. Prolonged and continuous infusion regimens of meropenem may increase efficacy against VIM-producing K. pneumoniae isolates.