Indirect Tris-EDTA Disk Testing Using Imipenem and Meropenem for Detection of OXA-48 Carbapenemase Production.

Enterobacteriaceae producing class D OXA-48 and OXA-48-like carbapenemases are an increasing concern. Detection is a diagnostic challenge because (i) some isolates are carbapenem susceptible and may go unrecognized, (ii) tests that detect carbapenem hydrolysis may be insensitive due to the relatively weak hydrolytic activity of these enzymes, (iii) the enzymes are not significantly inhibited by carbapenemase inhibitors such as clavulanate, boronic acid, and EDTA, and (iv) molecular detection may yield falsely positive results due to genes encoding noncarbapenemase OXA-48-like enzymes (1). Tests utilizing commercially available Tris/EDTA disks (catalog no. 232211; BD Diagnostics, Sparks, MD) are used to detect producers of class A and B carbapenemases (2–5) but have not been evaluated for detection of OXA carbapenemases. Therefore, a study was designed to determine if Tris/EDTA-based testing could detect carbapenemase production by isolates producing OXA-48 and OXA-181. Owing to the limited availability of OXA48-producing isolates in the United States, this was a small study. The isolates were 12 Klebsiella pneumoniae strains that were previously confirmed by PCR and sequencing to produce OXA-48 (n 11) and OXA-181 (n 1). They did not produce other carbapenemases. These confirmations were the reference standard. Also tested were 12 non-carbapenemase-producing Enterobacteriaceae that produced high levels of AmpC or an extendedspectrum -lactamase (ESBL) or both. The carbapenemasepositive and -negative quality control (QC) strains used were K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae ATCC BAA 1705 and carbapenemase-negative K. pneumoniae ATCC BAA 1706. All isolates were tested by the indirect Tris/ EDTA test with imipenem and meropenem substrates. In this test, the surface of a Mueller-Hinton agar plate was lawn-inoculated with Escherichia coli ATCC 25922. Imipenem and meropenem disks were then placed on each side of the plate. Two test organisms were heavily inoculated to provide a visible inoculum on the disk surfaces of separate Tris/EDTA disks. These were then placed on the agar 1 mm from the imipenem and meropenem disks with the inoculated side contacting the agar. After overnight incubation at 35°C, an indentation of the inhibition zone near the inoculated Tris/EDTA disk indicated a positive test result (example in Fig. 1). No indentation indicated a negative test result. The tests were interpreted by individuals blinded to the -lactamase types of the isolates. All 12 OXA-producing isolates yielded positive indirect Tris/ EDTA test results. Tests with imipenem exhibited larger indentations than tests with meropenem. All carbapenemase-negative isolates yielded negative indirect Tris/EDTA test results with meropenem, but a false-positive test with imipenem occurred with an Enterobacter cloacae strain. A recent report showed that a test utilizing filter paper disks impregnated with EDTA and EDTA plus boronic acid distinguished between isolates producing OXA48-like and KPC carbapenemases (6). While not directly comparable because the EDTA disks did not contain Tris and were not commercially available, these findings indicated it may be possible to use commercially available Tris/EDTA disks to detect and distinguish between producers of OXA-48-like carbapenemases and isolates producing class A and B carbapenemases. In conclusion, the indirect Tris/EDTA test was sufficiently sensitive to detect carbapenemase production by the OXA-48 producers and the OXA-181 producer in this study. Further studies using a larger collection of producers of OXA-48 and OXA-48-like carbapenemases are now required to confirm and extend these findings.