Nitro-Carba test, a novel and simple chromogenic phenotypic method for rapid screening of carbapenemase-producing Enterobacteriaceae

Abstract

Highlights: The present study developed Nitro-Carba Test (NCT), a rapid and simple chromogenic method for screening of Carbapenemase-producing Enterobacteriaceae (CPE). The NCT detected all 31 CPEs within a timeframe of only 10 seconds to 12 min. The sensitivity for all three carbapenems was 100%, while specificity of IPM, MEM and ETP were 64.29%, 91.07% and 100%, respectively.  IPM, MEM and ETP against all carbapenemase-producing strains had MIC values ranging from 0.5 to ≥256 µg/mL, 0.25 to ≥256 µg/mL and 1 to ≥256 µg/mL, respectively. OXA-48-producing isolates showed lower MIC values compared with producers of MBL and KPC. 2 AbstractObjectives: The present study developed Nitro-Carba Test (NCT), a rapid and simple chromogenic method for screening of Carbapenemase-producing Enterobacteriaceae (CPE). Methods:The NCT was validated with a total of 31 carbapenemase-producing isolates (9 KPCs, 11 MBLs and 11 OXA-48s) and with 56 non-carbapenemase-producing strains. The assay relies on the hydrolysis of nitrocefin in the presence of carbapenems. The carbapenemases were extracted with lysis buffer prior to addition to wells with and without imipenem (IPM), meropenem (MEM) and ertapenem (ETP). Following the addition of nitrocefin, a change in colour from yellow to red, indicating carbapenemase production, was observed within 20 min. The susceptibility profiles of each bacterial strain were also investigated. Results:A NCT detected all 31 CPEs within a timeframe of only 10 seconds to 12 min. All carbapenemase producers hydrolyzed nitrocefin in all wells. No colour change in the wells with carbapenems was observed in non-carbapenemase producers. The sensitivity for all three carbapenems was 100%, while specificity of IPM, MEM and ETP were 64.29%, 91.07% and 100%, respectively. IPM, MEM and ETP against all carbapenemase-producing strains had MIC values ranging from 0.5 to ≥256 µg/mL, 0.25 to ≥256 µg/mL and 1 to ≥256 µg/mL, respectively. OXA-48-producing isolates showed lower MIC values compared with producers of MBL and KPC. Conclusion:This assay is a promising method detecting CPE rapidly. The NCT is a simple and reliable method, capable of detecting CPE in even carbapenem-susceptible strains.

NitroSpeed-Carba NP Test for Rapid Detection and Differentiation between Different Classes of Carbapenemases in Enterobacterales

“…A rapid, sensitive, and specific test has been developed here, not only for ultrarapid and simple detection of carbapenemase-producing Enterobacterales but also to identify the different classes of carbapenemases. This test is a derivative of the Nitro-Carba test ( 21 ) but presents here the significant advantage of identifying the carbapenemases according to the classes they belong to. The NitroSpeed-Carba NP test allows rapid identification of all types of carbapenemase producers, including isolates producing KPC, OXA-48-types, NDM, VIM, and IMP, with a turnaround time of ca.…”

Anti-carbapenamase activity of Camellia japonica essential oil against isolated carbapenem resistant klebsiella pneumoniae (MN396685)

“…The CR K. pneumoniae strain was procured from Government Head Quarters Hospital, Tiruchirappalli, Tamil Nadu, India, which cause urinary tract infections. The carbapenemase production of selected CR K. pneumoniae strain was confirmed by Nitro-Carba test, followed by previous report of Teethaisong et al, (2019) . All the chemicals, reagents, plates and antibiotic discs of this study was procured from Hi-Media Laboratories, Mumbai, India.…”

“…According to the WHO reports, carbapenem resistant GNB as the number 1 critical list including Acinetobacter baumannii , Klebseilla pneumoniae , Pseudomonas aeruginosa and Proteus mirabilis ( WHO, 2017 ). In 2019, the review of Antimicrobial resistance committee was announced that the carbapenem resistant GNB increase the number of death, it attributed to antimicrobial resistance could increase 1 million each year ( Teethaisong et al, 2019 ). Five fold increased death rate was reported recently in developing nations compared to developed nations.…”

<p>Evaluation of Resistance Mechanisms in Carbapenem-Resistant <em>Enterobacteriaceae</em></p>

“…The increase in CRE is a serious matter due to high case-fatality rates (>30%). [24][25][26] Studying the mechanisms of CRE is an essential step in planning a national public health strategy for this emerging pathogen. 27 Several studies have been studied the mechanisms of resistance to carbapenem in CRE isolates from Iran.…”