a PHLS Anaerobe Reference Unit, University Hospital of Wales, Cardiff; b Department of Microbiology, University of Leeds and The General Infirmary, Leeds LS2 9JT, UK
Sir,
We report the first UK isolate of Clostridium difficile with reduced susceptibility to metronidazole in vitro. The isolate was recovered from the environment of a care for the elderly ward in Leeds, which was being surveyed as part of a longitudinal study of the molecular epidemiology of C. difficile.1 No similar isolate was recovered from any of the patients on this ward from whom diarrhoeal samples had been cultured for C. difficile. The origin of the isolate therefore remains obscure. It is notable, however, that the ward in question had only been converted (from a dining area for hospital staff) 3 months previously. C. difficile was not recovered from any environmental samples from this ward collected immediately prior to the ward opening, and therefore it is likely that the isolate originated from a patient. The isolate was examined at the PHLS Anaerobe Reference Unit (ARU) and gave no zone to a 5 µg metronidazole disc. By Etest, the MIC of metronidazole was 16 mg/L and it was typed as PCR ribotype 10, a non-toxigenic ribotype. Investigations for the nim genes associated with conferring resistance to metronidazole in Bacteroides species were negative.
Claims of metronidazole resistance in C. difficile have been reported rarely. Wong et al.2 recently reported that one of 100 C. difficile isolates was resistant to metronidazole (MIC 64 mg/L). Pelaez and colleagues3 reported apparent frequent resistance of C. difficile to metronidazole in Spain, particularly in isolates recovered from HIV-positive patients. Subsequently, the same group found that 9% of 469 clinically significant C. difficile isolates were resistant to metronidazole (MIC 32 mg/L).4 Resistance to metronidazole was more common in isolates from patients with recurrences of C. difficile diarrhoea (14.5% versus 7.5%, P < 0.05), as was intermediate resistance to vancomycin (MIC 816 mg/L, 6% versus 1.5%, P < 0.05).4 However, these potentially important findings were reported in abstract form only, and require independent confirmation because of the problems associated with antimicrobial susceptibility testing of anaerobes. Notably, the PHLS ARU had not previously detected metronidazole resistance in any of over 1000 C. difficile isolates tested (unpublished data), and in a study of 50 random isolates the metronidazole MIC range was 0.0941.5 mg/L. Finally, the MIC50, MIC90 and range of MICs of metronidazole for 105 equine C. difficile isolates were found to be 0.5, 16 and
0.2532 mg/L, respectively.52
The BSAC and NCCLS breakpoints for metronidazole are 8 and 16 mg/L, respectively, and therefore the isolate from Leeds may be classified as resistant or exactly on the breakpoint. Importantly, these breakpoint values do not necessarily reflect achievable faecal metronidazole concentrations. In healthy volunteers faecal metronidazole usually cannot be detected following oral administration. However, in patients with colonic disease therapeutic concentrations of metronidazole in faeces have been measured. In nine patients with C. difficile diarrhoea metronidazole concentrations were significantly higher in watery (9.3 ± 7.5 µg/g wet weight faeces, range 0.824.2) and in semi-formed stools (3.3 ± 3.6 µg/g, range 0.510.4) than in formed (1.23 ± 2.8 µg/g, range 010.2) faecal samples.6 Using these data, 96% (27/28) of samples had metronidazole concentrations below the MIC for the C. difficile isolate reported here.
What are the implications of this finding? At this stage it is not known whether similar resistance will appear in toxigenic strains with obvious consequences for treatment. Nor is it certain whether similar examples have already gone unnoticed, particularly given the decreasing use of culture for the laboratory diagnosis of C. difficile infection. Interestingly, the ARU has recently examined three isolates from Paris that displayed similar levels of metronidazole resistance (MIC range 816 mg/L) and were also found to belong to PCR ribotype 10. In a study of 370 isolates of C. difficile from the community, C. difficile PCR ribotype 10 was the most common strain detected, most often from the stools of infants. From hospital adult in-patient stools, however, they represent only 1.4% of strains received at the ARU. Of the 30 PCR ribotype 10 C. difficile isolates identified to date by the ARU, all but the four isolates mentioned here were fully susceptible to metronidazole.
Vigilance is obviously required to ascertain whether toxigenic strains will acquire this resistance and thus compromise a cornerstone for treatment of C. difficile infection.
Notes
* Corresponding author. Tel: +44-113-233-5595; Fax: +44-113-233-5649; E-mail: markwi{at}pathology.leeds.ac.uk
References
1 . Fawley, W. N. & Wilcox, M. H. (2001). Molecular epidemiology of endemic Clostridium difficile infection. Epidemiology and Infection 126, 34350.[ISI][Medline]
2 . Wong, S. S., Woo, P. C., Luk, W. K. & Yuen, K. Y. (1999). Susceptibility testing of Clostridium difficile against metronidazole and vancomycin by disk diffusion and E-test. Diagnostic Microbiology and Infectious Diseases 34, 16.[ISI][Medline]
3 . Pelaez, T., Gijon, P., Martinez, L., Catalan, P., Rivera, M. L., Bouza, E. (1997). Clostridium difficile associated diarrhea in the AIDS era. In Program and Abstracts of the Thirty-seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1997. Abstract C28. American Society for Microbiology, Washington, DC.
4 . Pelaez, T., Alcala, L., Martinez-Sanchez, L., Munoz, P., Garcia-Lechuz, J. M., Rodriguez-Creixems, M. et al. (1998). Metronidazole resistance in Clostridium difficile: a new emerging problem? In Program and Abstracts of the Thirty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract E173. American Society for Microbiology, Washington, DC.
5 . Jang, S. S., Hansen, L. M., Breher, J. E., Riley, D. A., Magdesian, K. G., Madigan, J. E. et al. (1997). Antimicrobial susceptibilities of equine isolates of Clostridium difficile and molecular characterization of metronidazole-resistant strains. Clinical Infectious Diseases 25, Suppl. 2, S2667.[ISI][Medline]
6 . Bolton, R. P. & Culshaw, M. A. (1986). Faecal metronidazole concentrations during oral and intravenous therapy for antibiotic associated colitis due to Clostridium difficile. Gut 27, 116972.[Abstract]