Comparative Evaluation of a Chromogenic Agar Medium, the Modified Hodge Test, and a Battery of Meropenem-Inhibitor Discs for Detection of Carbapenemase Activity in Enterobacteriaceae

Abstract

Three phenotypic methods (modified Hodge test, chromogenic agar, and meropenem discs combined with specific inhibitors) used for the detection of carbapenemase activity were tested on a panel of characterized Enterobacteriaceae expressing various ␤-lactamase mechanisms. Overall, the meropenem-plus-inhibitor approach was more sensitive and specific than the other methods, despite its limitation of being unable to detect class D carbapenemases.The identification of carbapenemase production in Enterobacteriaceae that results in resistance or intermediate resistance to one or more carbapenems has serious implications in hospital infection control and/or epidemiological investigations. Several approaches for the detection of these microorganisms have been described, including the modified Hodge test (MHT) (3), chromogenic agars (1,13,18), and the use of carbapenem discs with the addition of specific ␤-lactamase inhibitors (7,9,10,15,16,19,22). In this study, we compared these 3 approaches with a panel of characterized Enterobacteriaceae. The goal of this comparison was to find the most accurate method for the detection of carbapenemase activity in clinical isolates when these enzymes are suspected in laboratories after initial antimicrobial susceptibility screening.A panel of 77 genotypically characterized strains harboring different mechanisms of ␤-lactam resistance was used for this study (Table 1). Carbapenem hydrolysis was measured by spectrophotometer analysis as described previously (15). MICs were determined by the agar dilution method and Etest (bioMérieux) and interpreted using Clinical and Laboratory Standards Institute guidelines (3). The imipenem-EDTA Etest (metallo-␤-lactamase [MBL]-EDTA hereinafter) was used when necessary. The sensitivities and specificities of the inhibitor assay (7), MHT (3), and KPC chromogenic agar (Colorex KPC; Inverness Medical, Ottawa, Canada) in the detection of carbapenemase activity were compared. MHT was performed using both meropenem and ertapenem discs (Oxoid). Since our previous tests showed similar results for both meropenem and imipenem discs combined with dipicolinic acid (DPA), aminophenylboronic acid (BA), or cloxacillin (CLX) (data not shown), the discs used in this study (meropenem discs [Oxoid] The in-laboratory-prepared discs were kept at Ϫ20°C; their stability was tested once a week during 5 weeks by using the same positive controls selected from the panel studied and comparing the results to the results obtained with freshly prepared discs. Extendedspectrum ␤-lactamase (ESBL) and carbapenemase gene screening were performed when necessary (4,20).Enterobacter spp. can display a carbapenemase-resistant phenotype that is commonly due to the combination of chromosomal AmpCϩimp (8,17,21,23). In our panel, we included 26 Enterobacter spp. with different ␤-lactam resistance mechanisms (Table 1). MHT detected all serine carbapenemase producers but also the strains with AmpCϩimp, thus reducing the specificity (Table 2). All 26 Enterobacter spp. showed positive results ...

Evaluation of Carbapenemase Screening and Confirmation Tests with Enterobacteriaceae and Development of a Practical Diagnostic Algorithm

“…While chromogenic media are often used for carrier screening, the laboratory strategies for ␤-lactamase detection in routine AST consist of a screening and a confirmation step (10)(11)(12)(13)(14).…”

“…Critical diameters and MICs of ertapenem (ETP), meropenem (MEM), and imipenem (IPM) and automated microdilution expert systems have been evaluated as screening methods (14,(18)(19)(20). For carbapenemase confirmation, the modified Hodge test is recommended by CLSI and various commercial and in-house combined-disk tests (CDTs) using boronic acid derivatives and EDTA/dipicolinic acid as specific inhibitors have been described (13,(19)(20)(21)(22)(23)(24)(25). In 2014, EUCAST published new guidelines for the detection of resistance mechanisms, including carbapenemases, in which a CDT is recommended for carbapenemase confirmation (14,22,25).…”

Evaluation of the Rapidec Carba NP Test for Detection of Carbapenemases in Enterobacteriaceae

“…To confirm the production of carbapenemases, the CLSI recommends the modified Hodge test and the Carba NP I test (12). Alternatively, several commercially available and laboratory-developed combined disk tests that use boronic acid and EDTA/dipicolinic acid as specific carbapenemase inhibitors are available (11,(13)(14)(15)(16). In its 2013 guidelines for the detection of carbapenemases, EUCAST recommended combined disk tests for carbapenemase confirmation (10).…”

New Delhi metallo- -lactamase-1: local acquisition in Ontario, Canada, and challenges in detection

“…To assure that NDM-1-producing bacteria are detected for epidemiologic purposes and infection control, we recommend that laboratories adopt alternate tests, such as inhibitor-disk analysis comparing susceptibility of suspect organisms to carbapenems with and without carbapenemase in hib itors. 13,21 Alternatively, laboratories can adopt or work with reference laboratories to provide direct testing of all suspect organisms with polymerase chain reaction.…”

“…. Although a Modified Hodge Test of this isolate also gave a negative result for carbapanemases, as interpreted by both the hospital laboratory and a reference laboratory, 13 polymerase chain reaction showed that the organism possessed the gene encoding NDM-1 carbapenemase (bla NDM-1 ). Additional susceptibility testing showed that the organism possessed extensive resistance to drugs (Table 1).…”