21st European Congress of Clinical Microbiology and Infectious Diseases

A simple phenotypic algorithm for direct and specific detection of KPC and MBL carbapenemase-producing Enterobacteriaceae in rectal screening

Abstract number: P683

Zarkotou O., Kristo I., Poulou A., Chrysos G., Themeli-Digalaki K., Pournaras S., Tsakris A.

Objectives: Carbapenemase-producing Enterobacteriaceae are rapidly spreading. Early detection of carriers by faecal screening is crucial for their restriction; it is performed by various non-specific tests using carbapenem discs or selective agar plates supplemented with carbapenems. We evaluated a simple phenotypic algorithm for specific detection of MBL and KPC producers directly from rectal swabs.

Methods: Rectal swabs obtained from 55 hospitalized patients during September-December 2010 were suspended in 1ml saline and subsequently cultured onto two MacConkey agar plates (MC). A MC was streaked for colony isolation and two ertapenem (ERT) discs were added at the end of first and second quadrant; an inhibition zone of 27 mm was applied for detection of carbapenemase producer. A second MC was streaked for confluent growth and four meropenem (MER) discs were placed containing: i) MER alone, ii) MER plus 20 micro-L of 20 mg/ml phenylboronic acid (PBA, KPC inhibitor), iii) MER plus 10 micro-L of 0.1M EDTA (MBL inhibitor), and iv) MER plus both PBA and EDTA. Inhibition zone around MER disc alone of <23 mm was considered positive result for carbapenemase production. A difference of 5 mm in the inhibition zone between discs containing MER without and with inhibitors (PBA, EDTA or both) was considered a positive result for detection of KPC, MBL or both carbapenemases, respectively. Rectal suspensions were further tested for carbapenemase genes by PCR. Enterobacterial colonies grown at the edge of the inhibition halo around MER plus inhibitors and ERT discs were identified by phenotypic tests and PCR.

Results: 21 of the 55 screened patient-samples were PCR-positive for carbapenemase genes (9 KPC, 3 VIM, 9 KPC and VIM). By ERT disc test, 20 of the 21 PCR-positive samples (sensitivity 95%) were carbapenemase-positive. By the combined MER disc test, 19/21 PCR-positive samples (sensitivity 90%) were positive for carbapenemases, while all 34 PCR-negative samples were negative (specificity 100%). In all 19 cases the combined MER disc tests correctly differentiated KPC producers from those producing MBL or both enzymes.

Conclusion: This simple combined disc algorithm enables direct and specific detection of carbapenemase production from the first day of rectal screening without further testing, supporting the timely and costly implementation of infection control measures. It could be effectively used in hospitals with high rates of carbapenemase producers.