Department of Microbiology, University of Leeds and The General Infirmary, Leeds LS2 9JT, UK
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Abstract |
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Introduction |
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New generation fluoroquinolones have improved activity against both Gram-positive bacteria and anaerobes.8 It is therefore uncertain whether these antimicrobial agents will share the low propensity of progenitor antibiotics such as ciprofloxacin to cause C. difficile infection. There are few published data available to determine the comparative activity of new generation fluoroquinolones against C. difficile.9,10 Furthermore, there is evidence that there is much clonal spread of C. difficile strains, and for example, 55% of C. difficile strains submitted to the Public Health Laboratory Service Anaerobe Reference Unit by UK hospitals are indistinguishable genotypically.11 In Leeds General Infirmary this epidemic strain causes c.80% of symptomatic C. difficile infections in elderly patients (unpublished data). Despite these observations, in vitro antibiotic activity studies are performed commonly on C. difficile isolates that have not been fingerprinted to ensure that varied strains are selected for study. This situation is analogous to the selection of MRSA isolates for study without regard for the fact that very limited numbers of DNA clones are responsible for the great majority of infections.12
We therefore aimed to compare the activities of ciprofloxacin and levofloxacin with those of the newer fluoroquinolones grepafloxacin, moxifloxacin, sparfloxacin and trovafloxacin against both genotypically distinct and indistinguishable C. difficile isolates.
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Materials and methods |
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Results |
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Discussion |
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Other workers have not addressed the issue of clonal versus distinct strains when selecting study isolates, and this may reflect the fact that potentially epidemic C. difficile strains have only recently been recognized. Interestingly, in three studies that have examined the activity of fluoroquinolones against C. difficile, MIC90s were found to be lower than those observed in the present study for clonal strains.9,10,16 Fluoroquinolone MICs for genotypically distinct C. difficile strains observed in the present study using conventional inocula preparation are similar to those reported by Nord (ciprofloxacin)16 and Woodcock et al.10 (ciprofloxacin and moxifloxacin). Edlund et al.9 reported an MIC90 of moxifloxacin for 50 C. difficile isolates (of uncertain relationship) of 2 mg/L. While the corresponding figure in the present study was 16 mg/L, the geometric mean MIC90 was only 1.3 mg/L, indicating that the MIC was elevated by a small number of relatively resistant strains.
We opted to use two different methods to prepare inocula for MIC determinations in an attempt to overcome the often poor growth that was obtained. For 17 of 54 strains (31%) we were unable to read MICs (i.e. no growth on the antibiotic-free control plate) following inocula preparation using MuellerHinton broth. Hence, we used instead Schaedler's anaerobic broth to prepare inocula for MIC determinations. Schaedler's broth supports the growth of some anaerobes better than standard media such as MuellerHinton broth and may be used as an alternative medium for MIC studies.17 Using this medium we were able to read MICs for 93% of strains examined. We adjusted inocula prepared in each of the media to 104 cfu/spot, using a MacFarland standard tube. Such an approach may mean that the relative proportions of spores and vegetative cells in inocula may differ, but, for practicality reasons, this is the best option available. Geometric mean MICs were uniformly higher (2.5- to 5.4-fold) using Schaedler's broth as opposed to MuellerHinton broth. This difference probably reflects the improved growth of strains prepared in Schaedler's broth. The issue of how to determine MICs for anaerobes is clearly contentious,13,18 and for example, end-point determination can be difficult during reading, particularly when hazy growth occurs at several different concentrations.
We have shown previously that the endemic C. difficile strain (PCR ribotype 1) sporulates significantly more than other strains when cultured in Schaedler's broth, although no significant differences were observed when strains were cultured in a human faecal emulsion.19 The results of the present study indicate a further potential virulence determinant for this C. difficile strain, namely reduced antibiotic (fluoroquinolone) susceptibility compared with other strains found in the nosocomial setting. This may increase the likelihood of selection of C. difficile PCR ribotype 1 when antibiotic pressure exists. We are currently investigating whether the epidemic C. difficile clone examined here is more resistant to other antibiotics compared with other strains.
The broad-spectrum activity of new fluoroquinolones offers the possibility of single agent oral therapy for the empirical treatment of sepsis where Gram-positive and/or Gram-negative pathogens are suspected. Enhanced pneumococcal activity in particular will open the possibility of single agent treatment of respiratory tract infection.8,10 However, new agents should not increase the susceptibility of elderly patients to C. difficile infection if they are to have a useful role in this age group. Early generation fluoroquinolones, such as ciprofloxacin, have rarely been implicated as causing C. difficile diarrhoea.20 In the few case reports that have been published it is notable that there are often other explanations for gut flora disturbance, such as salmonella gastroenteritis.21,22 Interestingly, ciprofloxacin has occasionally been used successfully to treat C. difficile infection.23 The likelihood of an antibiotic inducing C. difficile infection will be determined in part by the activity of the antibiotic against gut flora, but also its activity against C. difficile. The increased activity of new fluoroquinolones against anaerobes may therefore be both an advantage and a disadvantage. Superior anti-C. difficile activity must be balanced against the possible perturbation of the anaerobic gut flora. In this context, it should be noted that clindamycin-associated C. difficile diarrhoea has been shown recently to be associated with an epidemic clindamycin-resistant C. difficile strain.24
High faecal concentrations (>1000 µg/g) are obtained with the fluoroquinolones studied in the present study, with the exception of levofloxacin (up to 100 µg/g) given its predominant excretion via the urinary tract. However, questions remain concerning the absolute bioavailability of fluoroquinolones in the gut lumen given the effects of drug metabolism, low pH, anaerobiasis, bacterial inoculum size and antibiotic binding to organic matter. For example, it is known that faecal concentrations of ciprofloxacin may reach 20003000 µg/g, and yet while the MICs of most gut anaerobes do not exceed 64128 mg/L, there is little or no reduction in the numbers of anaerobic flora following ciprofloxacin administration.25 Hence, the activity of fluoroquinolones on gut commensals or pathogens is probably much lower than expected from antibiotic concentration data alone.
This study was conceived before the market withdrawals on safety grounds of grepafloxacin and trovafloxacin. Pre-licensing trials have shown no evidence of an increased predisposition towards C. difficile infection in patients receiving moxifloxacin.26 It remains to be seen whether this observation, taken with the enhanced activity of moxifloxacin in comparison with other fluoroquinolones against C. difficile, means that this new generation fluoroquinolone is unlikely to be associated with C. difficile infection. One retrospective study, presented only in abstract form, documented an increased risk of C. difficile colitis associated with trovafloxacin use.27 It will therefore be important to monitor the use of new generation fluoroquinolones in elderly hospitalized patients where C. difficile is endemic. In particular, the reduced antibiotic susceptibility to all fluoroquinolones of the readily transmissible C. difficile strain documented here warrants further study.
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Acknowledgments |
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Notes |
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References |
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2 . Clostridium difficile in England and Wales: weeks 2753/98. (1999). Communicable Disease Report CDR Weekly 9, 59.
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Received 14 February 2000; returned 15 May 2000; revised 23 May 2000; accepted 26 June 2000