Controlling false-positive results obtained with the Hodge and Masuda assays for detection of class a carbapenemase in species of enterobacteriaceae by incorporating boronic Acid

Abstract

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 more-accurate 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 beta-lactamases [ESBLs], AmpC beta-lactamases, metallo-beta-lactamases [MBLs], etc.). These methods will provide the fast and useful information needed for targeting of antimicrobial therapy and appropriate infection control.

FIG. 1.

Results obtained with the modified Hodge test (MHT), the boronic acid-based MHT (BA-MHT), and the OXA-based MHT (OXA-MHT) with ETP plus OXA (1,000 μg per disk; left disks), ETP (central disks) and ETP plus APB (3,000 μg per disk; right disks) for representative isolates (K. pneumoniae M9171 [producing KPC-2], K. pneumoniae M7527 [producing VIM], Enterobacter cloacae M9251 [producing high levels of AmpC]), K. pneumoniae M9391 [producing CTX-M-2], and E. coli ATCC 35218 [producing TEM-1]). The letter “p” indicates that ETP was hydrolyzed by the streaked cells; the letter “n” indicates that ETP was not hydrolyzed by the streaked cells. Final interpretations for the MHT, the BA-MHT, and the OXA-MHT are shown below each strain.

FIG. 2.

Results obtained with the Masuda assay (MAS), the boronic acid-based MAS (BA-MAS), and the OXA-based MAS (OXA-MAS) with ETP (central disks). The left part of each photograph corresponds to the following disks, loaded with 5 μl of the crude extract: a blank disk (5 μl; left disks), an OXA disk (300 μg per disk; right disk), and an APB disk (300 μg per disk; lower disk). The right part of each photograph corresponds to the following disks, loaded with 15 μl of the crude extract: a blank disk (15 μl; left disks), an OXA disk (300 μg per disk; right disk), and an APB disk (300 μg per disk; lower disk). Representative isolates (K. pneumoniae M9171 [producing KPC-2], K. pneumoniae M11270 [producing KPC-2], Salmonella strain M5476 [producing GES-5], K. pneumoniae M7527 [producing VIM], K. pneumoniae M9391 [producing CTX-M-2], Enterobacter cloacae M9251 [producing high levels of AmpC], and E. coli ATCC 35218 [producing TEM-1]) are shown. The letter “p” indicates that ETP was hydrolyzed by the β-lactam content of the crude extract; the letter “n” indicates that ETP was not hydrolyzed by the β-lactam content of the crude extract. NC, no correspondence (the matching blank disk did not show carbapenemase activity). Final interpretations for the MAS, the BA-MAS, and the OXA-MAS are shown below each strain.