Bacteriology
Susceptibility testing of Clostridium difficile against metronidazole and vancomycin by disk diffusion and Etest

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Abstract

A prospective study on the susceptibility of Clostridium difficile to metronidazole and vancomycin using the Etest and disk diffusion test was performed over a 6-month period. One hundred strains were tested; one strain was highly resistant to metronidazole (MIC = 64 μg/mL). The zone size of inhibition by the disk diffusion test correlated with the MIC as determined by the Etest (regression coefficient = −0.043 for metronidazole and -0.044 for vancomycin, p < 0.001 for both antibiotics). However, the correlation coefficient was low for both metronidazole (r = 0.574) and vancomycin (r = 0.473); hence the zone of inhibition by disk diffusion test could not predict the MIC satisfactorily. Metronidazole is still the first-line antibiotic for the treatment of C. difficile-associated diarrhea because the incidence of metronidazole resistant strains remains very low. However, the efficacy of metronidazole in the treatment of infections attributable to isolates with high-level metronidazole resistance may be compromised because the fecal concentration of metronidazole is relatively low when compared with the MIC values of the less susceptible strains. Oral vancomycin is the drug of choice under such circumstances, as its fecal concentration is much higher than that of metronidazole.

Introduction

The value of antibiotic sensitivity testing of anaerobes has been questioned in the past owing to the predictable sensitivity to most anti-anaerobe antibiotics, lack of a simple method for testing, and little correlation between clinical treatment outcome with antibiotic susceptibility results Goldstein and Citron 1989, Wexler 1991, Zabransky 1990. Resistance of anaerobic bacteria to antibiotics has, however, been increasing even toward agents with excellent anti-anaerobe activities like metronidazole (Reysset et al. 1993), imipenem (Ueno et al. 1993), and β-lactam/β-lactamase inhibitor combinations (Goldstein et al. 1991). Antibiotic susceptibility testing of anaerobic bacteria is therefore increasingly important.

Traditionally the “gold standard” for anaerobic susceptibility testing has been the broth and agar dilution tests (National Committee for Clinical Laboratory Standards (NCCLS) 1993). They are, however, technically demanding and often too tedious for the routine clinical laboratory. The epsilometer (Etest) enables MIC to be determined in a much simpler way and studies have shown that it is a satisfactory method for MIC determination for most of the common anaerobes encountered clinically Olsson-Liljequist and Nord 1994, Rosenblatt and Gustafson 1995, Wust and Hardegger 1992. Although not generally recommended for anaerobic sensitivity testing (Wexler and Doren 1995), the agar disk diffusion test is an attractive alternative for rapidly growing anaerobes because it is much less expensive than the Etest.

Clostridium difficile is intrinsically resistant to multiple antibiotics, with metronidazole and vancomycin being two of the few antibiotics effective for the treatment of C. difficile associated diarrhea. Because there is no formal recommendation for its susceptibility testing at present, we evaluated the Etest for MIC determination and correlated this with the disk diffusion test.

Section snippets

Materials and method

Results of C. difficile culture and cytotoxin assay performed in the clinical microbiology laboratory of Queen Mary Hospital, Hong Kong, over a 6-month period from September 1996 to February 1997 were analyzed. Stool samples were cultured on cycloserine cefoxitin fructose agar plates supplemented by 5% horse blood (Oxoid, Unipath Ltd., Basingstoke, UK) and incubated in the anaerobic chamber at 37°C for 48 h before reading. Colonies that appeared grayish, opaque, and slightly rhizoid with a

Results

A total of 100 C. difficile isolates were obtained in the 6-month study period. Of the 3112 diarrheal stool samples sent for culture in the same period, 891 (28.6%) yielded a positive culture for enteric pathogens. C. difficile accounted for 11.2% (100/891) of all positive cultures. C. difficile culture was positive in 18.3% (100/546) of the stool samples with a specific request for it. There were 603 requests for C. difficile cytotoxin assay, and 57 (9.5%) of them were positive. Of the 100 C.

Discussion

C. difficile is the commonest cause of nosocomial diarrhea (Gerding et al. 1995), accounting for 15–30% of antibiotic-associated diarrhea without colitis and 90–100% of pseudomembranous colitis associated with antibiotic use (Bartlett 1992). Untreated pseudomembranous colitis carried a mortality rate of 15–30% (Chang 1985), though most other series reported an overall mortality rate of < 1% with appropriate treatment. C. difficile is also an important cause of infective diarrhea in oncology

Acknowledgements

This work was partly supported by a grant from the Committee on Research and Conference Grants.

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