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JAC Advance Access originally published online on October 8, 2006
Journal of Antimicrobial Chemotherapy 2006 58(5):1062-1065; doi:10.1093/jac/dkl364
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Tigecycline does not induce proliferation or cytotoxin production by epidemic Clostridium difficile strains in a human gut model

Simon D. Baines1, Katie Saxton1, Jane Freeman2 and Mark H. Wilcox1,2,*

1 Department of Microbiology, Institute of Molecular and Cellular Biology, University of Leeds Leeds LS2 9JT, UK 2 Department of Microbiology, The General Infirmary, Old Medical School Leeds LS1 3EX, UK

*Correspondence address. Department of Microbiology, The General Infirmary, Old Medical School, Leeds LS1 3EX, UK. Tel: +44-113-3926818; Fax: +44-113-3435649; E-mail: mark.wilcox{at}leedsth.nhs.uk

Received 2 June 2006; returned 12 July 2006; revised 9 August 2006; accepted 14 August 2006


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Objectives: Data on the risk of Clostridium difficile infection (CDI) associated with specific antibiotics are difficult to obtain because of confounding clinical factors. It is particularly important to evaluate the propensity of new antibiotics to induce CDI. We have examined the propensity of tigecycline to induce CDI using a human gut model.

Methods: We used a three-stage chemostat human gut model to study the effects of tigecycline on indigenous gut microflora and C. difficile. Two epidemic C. difficile were studied in separate experiments: PCR ribotype 001 (UK, CD001) and PCR ribotype 027 (North America, CD027). Tigecycline MICs for 39 C. difficile representing 19 distinct PCR ribotypes were also determined.

Results: Tigecycline MICs were 0.06 mg/L for all the C. difficile strains. Peak tigecycline concentrations in the gut model were 10.9 and 11.7 mg/L in CD027 and CD001 experiments, respectively. Tigecycline instillation invoked marked decreases in numbers of bacteroides and bifidobacteria (107–108 cfu/mL) and lesser reductions in facultative anaerobes. Despite markedly altered gut microflora, CD001 and CD027 remained as spores for the duration of the experiment, with no evidence of proliferation or cytotoxin production.

Conclusions: Tigecycline exposure did not induce C. difficile proliferation or cytotoxin production despite reduced competing microflora. The potency of tigecycline against C. difficile may contribute to the low risk of CDI induction. Factors other than gut microflora colonization resistance may be important in preventing C. difficile spore germination, proliferation and cytotoxin production.

Keywords: colonization resistance , ribotype 001 , ribotype 027


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Clostridium difficile is a major cause of morbidity among elderly, hospitalized patients. It is the cause of virtually all cases of pseudomembranous colitis and ~20–30% of episodes of antibiotic-associated diarrhoea.1 C. difficile infection (CDI) has been thought to arise following antibiotic-induced impairment of host colonization resistance within the colon. Other factors likely to be important in the development and severity of CDI include the virulence of the infecting C. difficile strain and host immune response to the microorganism and its toxins. Antibiotics with an increased propensity to induce CDI include broad-spectrum agents such as clindamycin, cephalosporins (particularly third generation) and aminopenicillins.2 Although fluoroquinolones have historically been associated with a low propensity to induce CDI,2 recent epidemics in North America and Canada identified prior fluoroquinolone administration as a significant risk factor for disease.3 However, the spectrum of activity of an antibiotic does not necessarily predict the predisposition to induce CDI. For example, piperacillin/tazobactam, a ureidopenicillin/ß-lactamase-inhibitor combination, is active against the predominant members of the colonic microflora but is less likely to induce CDI than cefotaxime in vivo and in vitro.4–6 The results of previous studies suggest that certain antimicrobials may possess a low propensity to induce CDI despite good activity against C. difficile and a markedly deleterious effect on the gut microflora.4–7 Therefore a simple relationship between antibiotic-mediated depletion of the colonic microflora and induction of CDI is not indicated. A more complex relationship between gut concentrations of antimicrobials, their inhibitory effects against the colonic microflora and the susceptibility of C. difficile may exist, which may be more relevant in determining the propensity of an antibiotic to induce CDI. Key factors associated with antimicrobial predisposition to induce CDI remain unclear, not least because of potential confounding factors in clinical studies, including polypharmacy, duration of therapy and exposure to C. difficile. Furthermore, the propensity of new antimicrobial agents to induce CDI is often unclear given the limitations of pre-licensing studies.

We have therefore examined the effect of exposure to the new broad-spectrum glycylcycline tigecycline on C. difficile and the gut microflora using a three-stage human gut model. Two epidemic C. difficile strains, from the UK (PCR ribotype 001, CD001) and North America (PCR ribotype 027, CD027), were evaluated in separate experiments. In addition, the susceptibilities of 39 C. difficile strains to tigecycline were determined.


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C. difficile strains

CD001 was isolated from a patient with CDI at the Leeds General Infirmary (Leeds, UK). CD027 was isolated during an outbreak of CDI at the Maine Medical Centre (Portland, MA, USA) and was obtained via Dr Robert Owens. A total of 39 C. difficile strains representing 19 distinct PCR ribotypes were used for MIC testing; these included a total of 15 PCR ribotype 027 strains. The strains were obtained from patient faecal (n = 28) and environmental (n = 11) samples in Leeds.

Three-stage chemostat gut model

We have previously described the use of a three-stage chemostat gut model to study the interplay among antimicrobial agents, the gut microflora and C. difficile.4,6,7 The gut model was validated against physicochemical and microbiological measurements from the intestinal contents of sudden-death victims. This in vitro model is, however, limited by its inability to mimic immunological or secretory events within the human colon. The gut model comprises three fermentation vessels top-fed by growth medium at a controlled rate (D = 0.015 h–1). The constituents and preparation of the growth medium and operating conditions of the gut model were as previously reported.6,7 The gut model was primed with faecal emulsion prepared from pooled C. difficile-negative faeces4,6 (10% w/v in pre-reduced PBS) of five healthy elderly volunteers (no antimicrobial therapy within 3 months).

Enumeration of gut bacteria and C. difficile

Gut bacterial populations and C. difficile were enumerated as previously described.7 C. difficile cytotoxin titres (relative units, RU) were determined using a Vero cell cytotoxicity assay as previously described.7

Experimental design

Following inoculation of the gut model with faecal emulsion, the media pump was started (day 0) and no further interventions were made for 13 days (period A, Figure 1). Gut microflora were enumerated every 2 days. C. difficile spores (~107 cfu) were prepared as described previously4,6,7 and added to vessel 1 on day 14 (period B, Figure 1); bacterial populations were enumerated daily. A further inoculum of C. difficile spores (~107 cfu) was instilled to vessel 1 (day 21) in addition to tigecycline twice daily for 7 days (period C, Figure 1). Tigecycline was instilled to reflect the mean concentration observed in faeces after standard therapy (6 mg/L).8 Gut bacterial populations and C. difficile were enumerated for a further 14 days (period D, Figure 1).


Figure 1
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  		Figure 1. Mean (±SE) viable counts (log10 cfu/mL) of selected gut bacterial populations in vessel 3 of the gut model in the experiment evaluating CD001. Vertical lines indicate the final day of each experimental time period.

 
Antimicrobial assay and MIC determination

Samples from the gut model were centrifuged (16 000 g), and the supernatants were removed and frozen at –20°C. Tigecycline concentrations were determined using an in-house large-plate bioassay on antibiotic medium #1 (pH 5.6–5.7) (Oxoid, Basingstoke, UK) supplemented with 30 g/L NaCl (Sigma, UK) with an indicator organism, Staphylococcus aureus NCTC 6571. Culture supernatants and tigecycline calibrators (0.06–64 mg/L) were sterilized by filtration and randomly assigned to bioassay plate wells (9 mm). Bioassay plates were incubated at 37°C overnight. Tigecycline MICs for 39 C. difficile were determined using agar incorporation (Wilkins Chalgren, Oxoid).9


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Gut bacterial populations

Changes in gut bacterial populations were similar in all vessels of the gut model in both experiments, and therefore only those observed in vessel 3 of the CD001 experiment are shown in Figure 1 (results for CD027 not shown). Bacterial populations were largely stable throughout periods A and B, with bacteroides and bifidobacteria predominant (Figure 1). Tigecycline instillation (period C) promoted marked declines in numbers of bifidobacteria (107 cfu/mL) and bacteroides (108 cfu/mL) below the limits of detection within 5 days and 7 days, respectively (Figure 1). Other bacterial genera declined by 101–103 cfu/mL. Bacteroides populations remained undetectable in both CD001 and CD027 studies. Bifidobacteria recovered to steady-state levels in CD001 but not CD027 experiments. Facultative anaerobes generally recovered to, or exceeded, the bacterial concentrations in steady-state (period A) populations.

Tigecycline concentrations and MICs

Tigecycline MICs for all 39 tested C. difficile were uniformly 0.06 mg/L. Tigecycline concentrations in CD001 (CD027) experiments peaked at 11.74 (10.93), 9.21 (6.73) and 7.35 (4.73) mg/L in vessels 1, 2 and 3, respectively. Tigecycline concentrations were below the limits of detection (1 mg/L) within 6 days of cessation of instillation.

C. difficile populations and cytotoxin titres

C. difficile remained predominantly as spores for the duration of both experiments; results for CD001 are shown in Figure 2 (results for CD027 not shown). No germination, proliferation or cytotoxin production was detected in either experiment.


Figure 2
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  		Figure 2. Mean (±SE) CD001 total viable counts (TVC), spore counts (SP) (log10 cfu/mL), cytotoxin titres (CYT; relative units, RU) and tigecycline (TIG) concentrations (mg/L) in vessel 3 of the gut model. Vertical lines indicate the final day of each experimental time period.

 

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The propensity of certain antimicrobials to induce CDI is well accepted, yet the reasons for this association remain unclear. Previous studies of antimicrobial predisposition to CDI have employed either test-tube or hamster models, neither of which is truly reflective of the complex environmental conditions within the human colon. We previously demonstrated C. difficile proliferation and high-level cytotoxin production in a three-stage human gut model in response to administration of both cefotaxime4 and clindamycin.7 In contrast, instillation of piperacillin/tazobactam, an antimicrobial infrequently associated with CDI,5 did not induce proliferation of C. difficile or cytotoxin production.6 The gut model is therefore a reproducible, gut-reflective experimental system that we believe may indicate the relative risk of CDI in vivo following antimicrobial exposure.

Tigecycline, the first licensed glycylcycline antibiotic to be marketed, has broad-spectrum activity against both Gram-positive and Gram-negative facultative and obligate anaerobes, including those with tetracycline resistance determinants.10 The risk of tigecycline-associated CDI has not been specifically evaluated in vitro or in vivo. In the present studies tigecycline was inhibitory to all enumerated components of the gut microflora. The marked reductions observed in bifidobacteria and bacteroides (≥107 cfu/mL) during tigecycline administration were notable given the predominant bacteriostatic activity of this antibiotic, at least against facultative anaerobes. The changes in bacterial populations observed in our gut model experiments following tigecycline instillation largely reflected those reported in healthy human volunteer studies.8 However, we observed that bacteroides populations were severely affected following exposure to tigecycline and failed to recover during period D, in contrast to the minor reductions in counts reported by Nord et al.8 These authors recorded a range of tigecycline concentrations in faeces from volunteers,8 which may account for variability in bacteroides viable counts reported. In the present study a defined dosage of tigecycline yielded very similar inter-experimental antimicrobial concentrations in respective gut model vessels.

Despite a substantial decrease in counts of obligate anaerobes, and lesser reductions in facultative anaerobes, C. difficile remained as spores, with no evidence of either proliferation or cytotoxin production. Spore germination is a prerequisite for toxin production as this occurs immediately prior to sporulation. Factors other than colonization resistance are therefore likely to have been important in inhibiting C. difficile. The potent activity of tigecycline against C. difficile10 was reflected in the uniformly low MICs determined in this study using a large collection of isolates. It should be emphasized that unlike most MIC studies for C. difficile, where the potential for clonality of tested isolates has not been evaluated, our results include data on 19 distinct PCR ribotypes, as well as multiple isolates of the emerging PCR 027 ribotype.

We previously demonstrated germination and proliferation of C. difficile spores only after clindamycin concentrations decreased below the MIC for CD001;7 the same phenomenon was seen with CD027 after clindamycin exposure (S. D. Baines, J. Freeman, W. N. Fawley and M. H. Wilcox, unpublished data). Inhibitory levels of tigecycline, below the limits of bioassay detection (1 mg/L), may have persisted within the gut model during period D and prevented germination and proliferation of C. difficile spores. However, the rate of decline of tigecycline concentrations measured (Figure 2) makes it likely that antibiotic levels would have fallen beneath the MIC for C. difficile at least 1 week before the end of each experiment. Alternatively, components of the gut microflora important in colonization resistance to CDI may have recovered to sufficient population densities at tigecycline concentrations above the MIC for C. difficile and promoted a nutrient-limited environment not conducive to germination, proliferation and cytotoxin production. These data indicate that tigecycline may have a low propensity to induce CDI despite its broad spectrum of activity and consequent marked inhibition of gut microflora.


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M. H. W. has received honoraria for consultancy work, financial support to attend meetings and research funding from Astra-Zeneca, Bayer, Genzyme, Pfizer and Wyeth.


   Acknowledgements
 
We thank Dr Warren N. Fawley for performing PCR ribotyping. Tigecycline was supplied by Wyeth-Ayerst, who also provided a grant to support this work.


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1 George WL, Rolfe RD, Finegold SM. (1982) Clostridium difficile and its cytotoxin in feces of patients with antimicrobial agent-associated diarrhea and miscellaneous conditions. J Clin Microbiol 15:1049–53.[Abstract/Free Full Text]

2 Freeman J and Wilcox MH. (1999) Antibiotics and Clostridium difficile. Microbes Infect 1:377–84.[CrossRef][Web of Science][Medline]

3 Pepin J, Saheb N, Coulombe MA, et al. (2005) Emergence of fluoroquinolones as the predominant risk factor for Clostridium difficile-associated diarrhea: a cohort study during an epidemic in Quebec. Clin Infect Dis 41:1254–60.[CrossRef][Web of Science][Medline]

4 Freeman J, O'Neill FJ, Wilcox MH. (2003) Effects of cefotaxime and desacetylcefotaxime upon Clostridium difficile proliferation and toxin production in a triple-stage chemostat model of the human gut. J Antimicrob Chemother 52:96–102.[Abstract/Free Full Text]

5 Settle CD, Wilcox MH, Fawley WN, et al. (1998) Prospective study of the risk of Clostridium difficile diarrhoea in elderly patients following treatment with cefotaxime or piperacillin-tazobactam. Aliment Pharmacol Ther 12:1217–23.[CrossRef][Web of Science][Medline]

6 Baines SD, Freeman J, Wilcox MH. (2005) Effects of piperacillin/tazobactam on Clostridium difficile growth and toxin production in a human gut model. J Antimicrob Chemother 55:974–82.[Abstract/Free Full Text]

7 Freeman J, Baines SD, Jabes D, et al. (2005) Comparison of the efficacy of ramoplanin and vancomycin in both in vitro and in vivo models of clindamycin-induced Clostridium difficile infection. J Antimicrob Chemother 56:717–25.[Abstract/Free Full Text]

8 Nord CE, Sillerstrom E, Wahlund E, et al. (2006) Ecological impact of tigecycline on the normal oropharyngeal and intestinal microflora. Sixteenth European Congress of Clinical Microbiology and Infectious Diseases, Nice, France(Blackwell Publishing, Oxford, UK) pp. 1779.

9 National Committee for Clinical Laboratory Standards. (1997) Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Fourth Edition: Approved Standard M11-A4.(NCCLS, Wayne, PA, USA).

10 Petersen PJ, Jacobus NV, Weiss WJ, et al. (1999) In vitro and in vivo antibacterial activities of a novel glycylcycline, the 9-t-butylglycylamido derivative of minocycline (GAR-936). Antimicrob Agents Chemother 43:738–44.[Abstract/Free Full Text]


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