nitrocefin and Enterobacteriaceae-Infections

In this study, a rapid and simple chromogenic method for screening of carbapenemase-producing Enterobacteriaceae (CPE), namely the Nitro-Carba test (NCT), was developed.. The NCT was validated using a total of 31 carbapenemase-producing isolates [9 Klebsiella pneumoniae carbapenemase (KPC), 11 metallo-β-lactamase (MBL) and 11 OXA-48] and 56 non-carbapenemase-producing isolates. The assay relies on the hydrolysis of nitrocefin by carbapenemases in the presence of carbapenem antibiotics. Carbapenemases were extracted with lysis buffer prior to addition to wells with and without imipenem (IPM), meropenem (MEM) and ertapenem (ETP). Following addition of nitrocefin, a change in colour from yellow to red, indicating carbapenemase production, was observed within 20min. The susceptibility profiles of each bacterial isolate were also investigated.. The NCT detected all 31 CPE within a timeframe of only 10s to 12min. All carbapenemase-producers hydrolysed nitrocefin in all wells. No colour change in wells with carbapenems was observed in non-carbapenemase-producers. The sensitivity for all three carbapenems was 100%, whilst the specificity of IPM, MEM and ETP was 64.3%, 91.1% and 100%, respectively. IPM, MEM and ETP had minimum inhibitory concentrations (MICs) against all carbapenemase-producing strains ranging from 0.5μg/mL to ≥256μg/mL, 0.25μg/mL to ≥256μg/mL and 1μg/mL to ≥256μg/mL, respectively. OXA-48-producing isolates showed lower MICs compared with MBL- and KPC-producing isolates.. This assay is a promising method for detecting CPE rapidly. The NCT is a simple and reliable method capable of detecting CPE even in carbapenem-susceptible strains.

Topics: Bacterial Proteins; Bacteriological Techniques; beta-Lactamases; Carbapenem-Resistant Enterobacteriaceae; Carbapenems; Cephalosporins; Enterobacteriaceae Infections; Humans; Hydrolysis; Microbial Sensitivity Tests; Reproducibility of Results

Reliable, simple and rapid methods for laboratory detection of carbapenemases are important for an appropriate antibiotic administration. A nitrocefin disc containing ertapenem for rapid screening of carbapenemase production among Enterobacteriaceae is developed in the present study. A total of 87 molecularly-confirmed Enterobacteriaceae including 31 carbapenemase producers and 56 non-carbapenemase producers, were tested with nitrocefin discs supplemented with and without ertapenem (20 μg/disc). Nitrocefin discs with ertapenem successfully discriminated all 31 carbapenemase and all non-carbapenemase producers within 30 minutes. The sensitivity and specificity of the method were 100%. The minimum inhibitory concentrations (MICs) of ertapenem against all carbapenemase-producing isolates ranged from 1 to ≥256 μg/mL. This simple test could help to minimize the treatment failure and control the dissemination of infections caused by carbapenemase-associated resistant bacteria. It is a promising approach that could be performed routinely in any laboratory.

Topics: Anti-Bacterial Agents; Bacterial Proteins; beta-Lactamases; beta-Lactams; Carbapenem-Resistant Enterobacteriaceae; Carbapenems; Cephalosporins; Disk Diffusion Antimicrobial Tests; Enterobacteriaceae; Enterobacteriaceae Infections; Ertapenem; Humans; Microbial Sensitivity Tests; Sensitivity and Specificity

We developed a novel chromogenic method, Penta-well test, which enables the rapid detection and classification of β-lactamases in clinical Enterobacteriaceae isolates. This test is based on a combination of nitrocefin and 3 β-lactamase inhibitors specific to classes A, B, and/or C, with nitrocefin hydrolysis by β-lactamases being assessed by optical density measurements at 490 nm. When the cutoff value for each β-lactamase class was determined (0.09, 0.4, and 0.55 for class A, class B, and class C β-lactamase producers, respectively), the sensitivity and specificity of classification were 93.5% and 68.8% for class A, 93.8% and 100% for class B, and 86.7% and 100% for class C, respectively. Moreover, this method allowed accurate β-lactamase classification in 20 of 23 (87.0%) isolates producing plural class of β-lactamases. Thus, the Penta-well test can provide information that would be useful in the accurate detection and classification of β-lactamases produced by causative bacteria.

Topics: Bacteriological Techniques; beta-Lactamases; Cephalosporins; Chromogenic Compounds; Enterobacteriaceae; Enterobacteriaceae Infections; Humans; Hydrolysis; Sensitivity and Specificity; Time Factors