The detection of class A serine-carbapenemases among species of Enterobacteriaceae remains a challenging issue. Methods of identification for routine use in clinical microbiology laboratories have not been standardized to date. We developed a novel screening methodology suitable for countries with high basal levels of carbapenem resistance due to non-carbapenemase-mediated mechanisms and standardized several simple confirmatory methods that allow the recognition of bacteria producing class A carbapenemases, including KPC, Sme, IMI, NMC-A, and GES, by using boronic acid (BA) derivatives. A total of 28 genetically unrelated Enterobacteriaceae strains producing several class A carbapenemases were tested. Thirty-eight genetically unrelated negative controls were included. The isolates were tested against imipenem (IPM), meropenem (MEM), and ertapenem (ETP) by MIC and disk diffusion assays in order to select appropriate tools to screen for suspected carbapenemase production. It was possible to differentiate class A carbapenemase-producing bacteria from non-carbapenemase-producing bacteria by using solely the routine IPM susceptibility tests. The modified Hodge test was evaluated and found to be highly sensitive, although false-positive results were documented. Novel BA-based methods (a double-disk synergy test and combined-disk and MIC tests) using IPM, MEM, and ETP, in combination with 3-aminophenylboronic acid as an inhibitor, were designed as confirmatory tools. On the basis of the performance of these methods, a sensitive flow chart for suspicion and confirmation of class A carbapenemase production in species of Enterobacteriaceae was designed. By using this methodology, isolates producing KPC, GES, Sme, IMI, and NMC-A carbapenemases were successfully distinguished from those producing other classes of -lactamases (extended-spectrum -lactamases, AmpCs, and metallo--lactamases, etc). These methods will rapidly provide useful information needed for targeting antimicrobial therapy and appropriate infection control.
The modified Hodge method (MHT) has been recommended by the CLSI for confirmation of suspected class A carbapenemase production in species of Enterobacteriaceae. This test and the Masuda method (MAS) have advantages over traditional phenotypic methods in that they directly analyze carbapenemase activity. In order to identify the potential interferences of these tests, we designed a panel composed of diverse bacterial genera with distinct carbapenem susceptibility patterns (42 carbapenemase producers and 48 nonproducers). About 25% of results among carbapenemase nonproducers, mainly strains harboring CTX-M and AmpC hyperproducers, were observed to be false positive. Subsequently, we developed an optimized approach for moreaccurate detection of suspicious isolates of carbapenemase by addition of boronic acid (BA) derivatives (reversible inhibitor of class A carbapenemases and AmpC cephalosporinases) and oxacillin (inhibitor of AmpCs enzymes). The use of the modified BA-and oxacillin-based MHT and MAS resulted in high sensitivity (>90%) and specificity (100%) for class A carbapenemase detection. By use of these methodologies, isolates producing KPCs and GES, Sme, IMI, and NMC-A carbapenemases were successfully distinguished from those producing other classes of ß-lactamases (extended-spectrum -lactamases [ESBLs], AmpC -lactamases, metallo--lactamases [MBLs], etc.). These methods will provide the fast and useful information needed for targeting of antimicrobial therapy and appropriate infection control.Class A carbapenemases (Klebsiella pneumoniae carbapenemases [KPCs], Sme, NMC-A, IMI, and some allelic variants of GES/IBC) have become more prevalent within the Enterobacteriaceae family (23,25). Early recognition of carbapenemase producers has become mandatory, as clinical failure associated with these enzymes has been described (8). Therefore, different strategies are needed for their detection in any attempt aimed for their control and eradication. An accurate identification of class A carbapenemases will therefore rely on the availability of specific, sensitive, and simple assays able to differentiate carbapenemase producers from nonproducers. The modified Hodge test (MHT) (5) and similar tests, such as the Masuda assay (MAS) (15), directly analyze the carbapenemase activity in unbroken cells and enzyme crude extracts, respectively. These tests preformed better than routine phenotypic methods for detection of carbapenemase-producing bacteria, especially when combined mechanisms were present. Additionally, a practical and fast approach that efficiently disrupts bacterial cells has become available to the routine clinical microbiological laboratory, making the MAS even easier to perform (12). Thus, given these features, these methods started to gain more adherents among microbiologists. Moreover, the CLSI has issued recommendations for phenotypic screening of carbapenemase producers among species of Enterobacteriaceae and included the MHT as a confirmatory assay (5). Thus, MICs of ertapenem (ETP), meropenem (MEM...
We evaluated the ability of the combination disk test (CDT) and the Modified Hodge Test (MHT) to discriminate between various carbapenemase-producing Pseudomonas aeruginosa isolates (KPC, n = 36; metallo-β-lactamase (MBL), n = 38) and carbapenemase non-producers (n = 75). For the CDT, the optimal inhibitor concentrations and cut-off values were: 600 μg of 3-aminophenylboronic acid (APB) per disk (an increment of ≥4 mm), 1000 μg of dipicolinic acid (DPA) per disk (an increment of ≥5 mm) and 3000 μg of cloxacillin per disk (an increment of ≥3 mm). APB had excellent sensitivity (97%) and specificity (97%) for the detection of KPC enzymes. DPA detected MBL enzymes with a sensitivity and specificity of 97% and 81%, respectively. The MHT resulted in a low sensitivity (78%) and specificity (57%). The CDT could be very useful in daily practice to provide fast and reliable detection of KPC and MBL carbapenemases among P. aeruginosa isolates.
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