A combined disc method with resazurin agar plate assay for early phenotypic screening of KPC, MBL and OXA-48 carbapenemases among Enterobacteriaceae

Abstract

Aim: To validate a combined disc method along with resazurin chromogenic agar for early screening and differentiation of Klebsiella pneumoniae carbapenemase, metallo-β-lactamase and OXA-48 carbapenemase-producing Enterobacteriaceae. Methods and Results:The combined disc test comprising of meropenem alone and with EDTA, phenylboronic acid, or both EDTA and phenylboronic acid, and temocillin alone were evaluated with the resazurin chromogenic agar plate assay against a total of 86 molecularly-confirmed Enterobacteriaceae clinical isolates (11 metallo-β-lactamases, 8Klebsiella pneumoniae carbapenemases, 11 OXA-48, 32 AmpC and 15 extended-spectrum-β-lactamase producers and 9 co-producers of extended-spectrum-β-lactamase and AmpC). The inhibition zone diameters were measured and interpreted at seven hours for the presence of carbapenemase. All carbapenemase producers were phenotypically distinguished by this assay with 100% sensitivity and specificity. Conclusions:This early phenotypic method is very simple, inexpensive, and reliable in the detection and differentiation of carbapenemase-producing Enterobacteriaceae. It could be exploited in any microbiological laboratory for diagnosis of these recalcitrant bacteria.Significance and Impact of study: This assay poses excellent performance in discrimination of Klebsiella pneumoniae carbapenemase, metallo-β-lactamase and OXA-48 carbapenemases within seven hours, which is much faster than conventional disc diffusion methods. The rapid detection could help clinicians screen patients, control infection, and provide epidemiological surveillance.

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

“…A reference strain Escherichia coli ATCC 25922 was used as a negative β-lactamase control. These bacterial isolates have previously been described in our preceding studies [11,13]. The characteristics of organisms used are presented in Table 1.…”

Performance of a new in-house medium Carba MTL-broth for the rapid detection of carbapenemase-producing Enterobacteriaceae

“…It is important to recognize that no currently described phenotypic methods are able to detect all carbapenemase types, and several are associated with very poor positive predictive values for carbapenemases detection [24]. In addition, molecularbased methods for characterization of CPE are restrictive to use due to its high cost, the requirement of skilled and experienced technicians and the inability to detect new carbapenemase-encoding genes [15]. The situation is worse in developing countries due to unavailability and/or high costs of most of these methods.…”

“…chromogenic media), mass spectrometry, biochemical-based methods (e.g. Carba NP test and Blue-Carba NP test), immunochromatography, carbapenemase inhibitor-based tests and molecular typing (PCR-based detection) [4,[10][11][12][13][14][15]. Some of these methods require specific equipments and substantial expertise and, thus, cannot be implemented in all laboratories, especially in those of developing countries [14].…”

The performance of a resazurin chromogenic agar plate with a combined disc method for rapid screening of extended‐spectrum‐β‐lactamases, AmpC β‐lactamases and co‐β‐lactamases in Enterobacteriaceae

“…In the present study, the performance of the RCA assay in rapid screening and discrimination of ESBL, AmpC, and co‐producers of ESBL and AmpC was evaluated in 86 β‐lactamase‐producing Enterobacteriaceae isolates. The organisms used in the present study are summarized in Table . The molecular types included 15 Ambler class A ESBL producers (three CTX‐M‐3, one CTX‐M‐15, one SHV‐27, one SHV‐18, one SHV, one TEM‐214, one TEM‐10, one TEM‐70, one CTX‐M‐15+SHV‐27, one SHV‐27+TEM‐53, one SHV‐27+TEM‐71, one SHV‐110+TEM‐84 and one CTX‐M+SHV+TEM), 32 Ambler class C AmpC producers (six DHA family, seven CIT family, two MOX family, 11 EBC family and six FOX family) and nine co‐producers of ESBL and AmpC (one TEM+ACT‐type, four CTX‐M+ACT‐types, one TEM+SHV+ACT‐type, one TEM+CTX‐M+ACT‐type, one SHV+ACT type and one SHV+CTX‐M‐ACT‐type).…”

“…A resazurin reduction assay, a colorimetric method, is based upon the ability of active cells to reduce blue resazurin to pink resorufin . A colorimetric (resazurin‐containing) disc susceptibility method is reportedly highly reproducible and has high sensitivity and specificity for detection and differentiation of carbapenemase‐producing Enterobacteriaceae . CPD is an attractive indicator cephalosporin for detection of ESBL production and may be used for screening according to European Committee on Antimicrobial Susceptibility Testing guidelines.…”