Department of Microbiology, University of Leeds and The General Infirmary, Leeds LS2 9JT, UK
Sir,
The use of second- and third-generation cephalosporins predisposes to Clostridium difficile infection (CDI), whereas aminoglycosides, fluoroquinolones and ureidopenicillins with or without ß-lactamase inhibitors, are less commonly implicated.1 Poor activity against anaerobes, and hence continued resistance by gut flora to colonization by C. difficile, is believed to explain at least in part the relative protection afforded by some antimicrobial agents. Surprisingly few studies have examined the relationship between antimicrobial activity against C. difficile and the propensity of an antibiotic to induce CDI. It has been shown that the UK epidemic C. difficile strain2 has reduced antibiotic susceptibility to both old- and new-generation fluoroquinolones,3 and so we have extended these investigations to determine whether the epidemic C. difficile clone has reduced susceptibility to other antibiotics, in vitro.
Two hundred and twenty clinical and hospital environmental isolates of C. difficile were screened by random amplified polymorphic DNA and ribosomal spacer PCR,3 selecting 23 genotypically distinct strains (three or more bands difference in DNA profile) plus 28 clonal strains that were obtained from specimens separated in time and space. The DNA clone is the UK epidemic strain (PCR ribotype 1).2 MICs of ampicillin, cefotaxime, cefuroxime, cephradine, ciprofloxacin, erythromycin, piperacillintazobactam and trimethoprim were determined by an agar incorporation method, as described previously.3 Bacteria were cultured anaerobically in Schaedler's anaerobic broth (Oxoid, Basingstoke, UK) at 37°C for 48 h, and were multipoint inoculated (104 cfu/spot, adjusted using a MacFarland standard tube) on to WilkinsChalgren agar (Oxoid) containing doubling dilutions of antibiotic (0.03512 mg/L). All media and diluents were pre-reduced in an anaerobic cabinet for at least 2 h. MICs were read after culture at 37°C anaerobically for 48 h. Bacteroides fragilis NCTC 6343 and Staphylococcus aureus NCTC 6571 were used as control strains.
MICs for genotypically distinct and clonal strains are shown in the Table. As expected, there was less variability in the MICs of all antibiotics for clonal as compared with distinct strains. There was a unimodal distribution of MICs for each antibiotic tested, with the exception of erythromycin against the genotypically distinct strains (bimodal distribution). Erythromycin MIC50s were
2 mg/L for 12 distinct strains and were
256 mg/L for the remainder. All clonal C. difficile strains had erythromycin MICs
256 mg/L. A marked difference in the susceptibility of clonal compared with distinct strains was observed only for ciprofloxacin; the mean MIC for clonal strains was 4.3-fold higher. For the other seven antibiotics tested, the population mean MICs were within one doubling dilution. Ampicillin and piperacillintazobactam were more active than the other antibiotics. Of the three cephalosporins tested, cefuroxime was the least active. All strains were resistant to trimethoprim.
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We found previously that the UK epidemic C. difficile clone was uniformly less susceptible than 26 genotypically distinct strains to six first- and second-generation fluoroquinolones.3 The present study shows that reduced fluoroquinolone susceptibility in this epidemic clone is not accompanied by other resistance traits. Also, antibiotic resistance alone does not correlate with propensity to cause CDI. For example, the mean MICs of ciprofloxacin and cefotaxime for the clonal (virulent) strains were of a similar high magnitude, and yet there is a clear difference in the associated risk of CDI with these antimicrobial agents. Likewise, mean MICs of ciprofloxacin and piperacillintazobactam for clonal strains were markedly different, despite these antimicrobial agents having similar low propensities to induce CDI.
Whilst it is possible that reduced activity against C. difficile could increase the risk of antibiotic-associated symptomatic infection, other additional factors may be important. Clindamycin-associated C. difficile diarrhoea was shown to be significantly associated with an epidemic clindamycin-resistant strain.5 However, the high prevalence of this strain may be better explained by a combination of the potent anti-anaerobic effects of clindamycin, and therefore perturbation of gut flora, coupled with other strain virulence determinants. For example, it has been shown that the UK epidemic strain sporulates more than other comparators and that this is enhanced by exposure to some, but not all disinfectants.6 This study has shown that epidemic clonal strains and distinct isolates are relatively susceptible to ampicillin. However, the epidemic strain sporulates to a greater extent in human faeces following exposure to sub-inhibitory concentrations of ampicillin, emphasizing the likely multifactorial aetiology of CDI (unpublished data). The present study therefore enhances our knowledge about the lack of correlation between antibiotic propensity to cause CDI and activity against C. difficile itself.
Notes
J Antimicrob Chemother 2001; 47: 244246
* Corresponding author. Tel: +44-113-233-5596; Fax: +44-113-233-5649; E-mail: markwi{at}pathology.leeds.ac.uk
References
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2
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Stubbs, S. L., Brazier, J. S., O'Neill, G. L. & Duerden, B. I. (1999). PCR targeted to the 16S23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes. Journal of Clinical Microbiology 37, 4613.
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Wilcox, M. H., Fawley, W., Freeman, J. & Brayson, J. (2000). In vitro activity of new generation fluoroquinolones against genotypically distinct and indistinguishable Clostridium difficile isolates. Journal of Antimicrobial Chemotherapy, 46, 5515.
4 . Barbuit, F., Decré, D., Burghoffer, B., Lesage, D., Delisle, F., Lalande, V., et al. (1999). Antimicrobial susceptibilities and serogroups of clinical strains of Clostridium difficile isolates in France in 1991 and 1997. Antimicrobial Agents and Chemotherapy 11, 260711.
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Johnson, S., Samore, M. H., Farrow, K. A., Killgore, G. E., Tenover, F. C., Lyras, D. et al. (1999). Epidemics of diarrhea caused by a clindamycin-resistant strain of Clostridium difficile in four hospitals. New England Journal of Medicine 341, 164551.
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