Heteroresistance to imipenem observed for clinical Clostridium difficile isolates using Etest susceptibility testing

Abstract number: P1656

Norén T., Alriksson I., Andersson J., Unemo M.

Objective: Carbapenems are frequently used in critically ill hospital patients who are at high risk of Clostridium difficile infection (CDI). The resistance rate in C. difficile, measured by minimal inhibitory concentration (MIC), is generally found low by standard agar dilution (imipenem 4–8, meropenem 1–4 and ertapenem 4–8 mg/L), thus presumably a low risk of inciting CDI. In contrast, using the Etest (Biodisk AB, Solna, Sweden), a high level of imipenem resistance (97% of 606 isolates, MIC > 32 mg/L) was observed in 1993–2008 required further evaluation.

Method: The Etest was performed on 21 toxigenic C. difficile isolates recovered consecutively during January 2008 belonging to 11 different PCR-ribotypes. Recommended IsoSensitest agar media.was used for four different concentrations of C. difficile inoculum (McFarland 0.5, 1, 2 and 4). The MIC was read at the intersection on the antimicrobial gradient strip after 48 h anaerobic incubation at 37°C. Imipenem, ertapenem and meropenem were tested. Brucella and Müeller-Hinton agar were compared and finally agar dilution was performed according to CLSI to confirm our result. Susceptible controls C. difficile (ATCC 9689), Bacteroides fragilis (ATCC 25285) and C. perfringens (ATCC13124) were included.

Results: Out of the samples investigated 14/21 had initially MICs of >32 mg/L by Etest. Double zones were read on 10 of these 14 (>32 and 2–4 mg/L). Microcolonies sub-cultured from the resistant zone gave similar results of double zones. Diluted concentrations of the inoculum (McFarland 4 to 0.5) still read±one step of MIC on the E-test strip. Müeller-Hinton or Brucella agar did not alter the MIC interpretation. Agar dilution in Brucella agar, however, lowered MICs of all but one isolate to expected levels of 1–4 mg/L. Two isolates were ertapenem resistant using the Etest while all were susceptible to meropenem according to CLSI guidelines.

Conclusion: Etest results from testing C. difficile against imipenem may show incorrect high MICs or may result the selection of a heteroresistant subpopulation within the inhibition zone of the E-strip. Accordingly caution should be made evaluating Etest results for imipenem when testing C. difficile and possibly if encountering unexpected imipenem Etest related resistance in other pathogens.

Session Details

Date: 10/04/2010
Time: 00:00-00:00
Session name: Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases
Location: Vienna, Austria, 10 - 13 April 2010
Presentation type:
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