JAC Advance Access published online on August 7, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn298
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Original research |
Orally administered β-lactamase enzymes represent a novel strategy to prevent colonization by Clostridium difficile
1 Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, OH 44106, USA 2 Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA 3 Ipsat Therapies LtdTM, Helsinki Business and Science Park, Viikinkaari 4, FI-00790 Helsinki, Finland
* Correspondence address. Louis Stokes Cleveland Veterans Affairs Medical Center, Infectious Diseases Section, 10701 East Boulevard, Cleveland, OH 44106, USA. Tel: +1-216-791-3800 ext. 4788; Fax: +1-216-231-3482; E-mail: stiefel{at}medscape.com
Received 18 April 2008; returned 16 May 2008; revised 27 June 2008; accepted 30 June 2008
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Abstract |
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Objectives: Antibiotics that are excreted into the intestinal tract and that disrupt the indigenous microbiota may promote infection by Clostridium difficile. We previously demonstrated that oral administration of a proteolysis-resistant, recombinant class A β-lactamase inactivates ampicillin or piperacillin excreted into the small intestine during parenteral treatment. We hypothesized that oral administration of this β-lactamase in conjunction with parenteral ampicillin or piperacillin would preserve the colonic microbiota, thus preventing the overgrowth of and toxin production by C. difficile in mice.
Methods: Subcutaneous ampicillin, subcutaneous piperacillin or either of these plus oral β-lactamase or either of these plus tazobactam-inactivated oral β-lactamase were administered to mice 24 and 12 h prior to harvest of caecal contents. Contents were inoculated with one of four strains of C. difficile, and growth and toxin production were assessed after 24 h of incubation under anaerobic conditions. To assess changes in stool microbiota, denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal RNA genes was performed.
Results: Mice treated with ampicillin, piperacillin or either of these plus tazobactam-inactivated oral β-lactamase developed high-density colonization with C. difficile, whereas those treated with ampicillin or piperacillin plus the β-lactamase did not. DGGE demonstrated that antibiotic treatment resulted in significant alteration of the indigenous stool microbiota, whereas antibiotic plus β-lactamase treatment did not.
Conclusions: Administration of oral recombinant β-lactamase preserved the colonic microbiota of mice during parenteral β-lactam antibiotic treatment and prevented the overgrowth of and toxin production by C. difficile in caecal contents. Oral β-lactamase therapy may represent a novel approach towards preventing C. difficile infections in healthcare settings.
Key Words: hospital infections , antibiotic-associated diarrhoea , infection control , intestinal flora , β-lactams
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Introduction |
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Recently, both rising rates and increased severity of Clostridium difficile-associated disease (CDAD) have been implicated as causes of escalating disease-related mortality in healthcare facilities throughout the USA.1 The correlation between these events and the emergence of the so-called ‘epidemic strain’ of this pathogen over the last several years has also been widely remarked.1,2 Although the pharmaceutical industry has continued to pursue the development of new therapeutic agents for the treatment of established CDAD, prevention of disease in the healthcare setting has become of vital importance, especially in the outbreak setting.
The single most important risk factor for the development of CDAD is the administration of antibiotics,3 as these may interfere with the indigenous intestinal microbiota. β-Lactam or penicillin-type antibiotics are among the most commonly used antibiotics worldwide. We have previously shown that oral administration of recombinant, proteolysis-resistant β-lactamase enzymes that inactivate β-lactam antibiotics is able to protect the indigenous intestinal microbiota and preserve colonization resistance against vancomycin-resistant enterococci in mice treated with parenteral β-lactam antibiotics.4,5 Here, we hypothesized that administration of such an enzyme could limit the overgrowth of and toxin production by C. difficile in the caecal contents of antibiotic-treated mice.
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Materials and methods |
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C. difficile strains
Two non-epidemic strains (strains 9 and 11) and two epidemic strains (strains 17 and 20) of C. difficile were studied. Strain 9 is ATCC no. 9689. The remaining three strains are clinical isolates from Cleveland: strain 11 was typed as J29 or J30 by restriction endonuclease analysis (REA) (performed in Dr Dale Gerding's Laboratory), and strains 17 and 20 are epidemic strains typed as BI9 and BI6-8-17 by REA typing. All the strains produce toxins A and B. Furthermore, the two epidemic strains are moxifloxacin-resistant and have positive PCRs for the binary toxin gene cdtB. The C. difficile strains were prepared for inoculation into mouse caecal contents by serially diluting 24 h broth cultures in sterile pre-reduced PBS.
In a beagle dog model, an orally administered recombinant class A β-lactamase including amino acids 41–268 of Bacillus licheniformis 749/C (Ipsat Therapies LtdTM, Espoo, Finland) has been shown to resist proteolysis and degrade parenteral ampicillin in the small intestine in a dose-dependent manner, without affecting serum ampicillin concentrations.6 Furthermore, we have demonstrated that oral administration of this penicillinase (6 g/kg) preserves colonization resistance of piperacillin-treated mice against nosocomial pathogens, including vancomycin-resistant Enterococcus.4 In the current experiments, the recombinant β-lactamase enzyme was used at the same dose.
Rodent doses of β-lactam antibiotics used, calculated according to the method of Freireich et al.,7 were equivalent to the usual human doses administered over a 24 h period (mg of antibiotic/g of body weight) and were as follows: ampicillin, 5.3 mg/dose and piperacillin, 10.7 mg/dose.
A non-competitive inhibitor of class A β-lactamases, tazobactam, was used to confirm that the protective effects of the oral β-lactamase enzyme were specifically related to its β-lactamase activity. Tazobactam inactivation of the β-lactamase was achieved by enzyme pre-incubation with a 100-fold excess of tazobactam for at least 2 h prior to use in mice.
Mouse model of colonization resistance to C. difficile
A mouse model of colonization resistance was adapted from that utilized in hamsters by Borriello et al.8 We have previously found that this model yields similar results in mice and hamsters treated with clindamycin or aztreonam,8,9 and we have also subsequently used the model to evaluate the effect of fluoroquinolone treatment on C. difficile growth and toxin production in the caecal content of mice.10 The experimental protocol was approved by the Animal Care Committee of the Louis Stokes Cleveland Veterans Affairs Medical Center.
Female CF-1 mice weighing 25–30 g (Harlan Sprague-Dawley, Indianapolis, IN, USA) were housed in individual cages with plastic filter tops to prevent cross-contamination among animals. Subcutaneous injections (total volume of 0.2 mL) of normal saline, ampicillin, piperacillin or subcutaneous antibiotic plus oral β-lactamase (0.5 mL volume given by orogastric gavage) or antibiotic plus tazobactam-inactivated oral β-lactamase were administered to mice (eight per group, except four in the saline-treated group) 24 and 12 h prior to sacrifice and harvest of caecal contents. Stool pellets were also collected for denaturing gradient gel electrophoresis (DGGE) analysis immediately prior to sacrifice of the animals. After harvest, caecal contents were removed immediately to an anaerobic chamber (Coy Laboratories, Grass Lake, MI, USA) and inoculated under anaerobic conditions with one of the four strains of C. difficile, to give a final concentration of 104 cfu/mL. After incubation for 24 h, the samples were diluted in sterile PBS and plated on pre-reduced cefoxitin-cycloserine-fructose agar (Becton Dickinson, Cockeysville, MD, USA) containing 1% taurocholic acid sodium salt (Sigma, St Louis, MO, USA) to quantify C. difficile.9,10 For the piperacillin experiments, toxin production was also measured as described previously.9,10 To determine toxin production, a commercially available kit detecting cytopathic effect of C. difficile toxin B (Diagnostic Hybrids, Inc., Athens, OH, USA) was used, as recommended by the manufacturer. The caecal content supernatants were serially diluted 10-fold in specimen diluent. Following dilution, samples were added to microtitre plates containing human fibroblast cells and observed by a brightfield microscope, at 24 and 48 h, for evidence of C. difficile toxin cytopathic effect.
Denaturing gradient gel electrophoresis
To monitor antibiotic-induced changes in the indigenous microbiota, DGGE of PCR-amplified bacterial rRNA genes was performed on fresh stool pellets collected from mice immediately prior to sacrifice, according to our methods described previously.4 DGGE provides a ‘molecular fingerprint’ of the colonic bacterial flora for any particular mouse, with a banding pattern that is reflective of a variety of microbial species that are present. DGGE images were captured using Quantity One software (Version 4.6.2, Bio-Rad, Hercules, CA, USA) and visualized using a GelDoc 2000 transilluminator (Bio-Rad). Dendrogram analysis was conducted for the purposes of calculating similarity indices between the different treatment groups, using Fingerprinting II Informatix software (Version 3.0, Bio-Rad).
One-way analysis of variance was performed to compare C. difficile densities and toxin production among the treatment groups. P values were adjusted for multiple comparisons using the Scheffe correction. Computations were performed with the use of Stata software (version 5.0, Stata, College Station, TX, USA). P < 0.05 was considered significant. DGGE similarity indices were compared using Student's t-test.
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Results and discussion |
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Mice treated with parenteral ampicillin or piperacillin developed a massive overgrowth of C. difficile in their harvested caecal contents 1 day after inoculation of this pathogen, whereas saline controls and mice treated with either antibiotic in conjunction with the oral β-lactamase did not (P < 0.0001) (Figure 1a). The protective effect of the β-lactamase was abrogated when the enzyme had been pre-incubated with tazobactam, a β-lactamase inhibitor, demonstrating that enzymatic activity was necessary to confer protection. High levels of toxin were produced in the caecal contents of mice that received prior treatment with piperacillin or piperacillin plus tazobactam-inactivated oral β-lactamase, whereas toxin levels were low or undetectable in mice treated with saline or piperacillin plus oral β-lactamase (P < 0.01) (Figure 1b).
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2 log10 cfu/mL stool). Treatment with subcutaneous ampicillin (Amp) or piperacillin (Pip) resulted in high-density C. difficile colonization, whereas treatment with normal saline (NS) or subcutaneous antibiotic in conjunction with orogastric β-lactamase (BL) did not (P < 0.0001). Inactivation of the β-lactamase by pre-incubation with the β-lactamase inhibitor tazobactam (Inac BL) resulted in a loss of efficacy. (b) High levels of toxin were produced in the caecal contents of mice that received prior treatment with piperacillin or piperacillin plus tazobactam-inactivated oral β-lactamase, whereas toxin levels were low or undetectable in mice treated with saline or piperacillin plus oral β-lactamase (P < 0.01). The vertical bars represent standard errors.DGGE analysis showed that piperacillin caused a significant disruption of the murine indigenous microbiota, but that piperacillin in conjunction with oral β-lactamase treatment caused relatively minor alteration of the microbiota (mean similarity indices of mice treated with piperacillin, with piperacillin plus oral β-lactamase and with piperacillin plus inactivated oral β-lactamase in comparison with indices of saline controls were 16%, 56% and 7%, respectively; P < 0.0001 for differences between saline- and piperacillin-treated mice and for differences between saline-treated mice and mice treated with piperacillin plus inactivated oral β-lactamase) (Figure 2). Quantitative cultures of total enterococci and total Gram-negative bacteria were performed and significant decimation of the microbiota in the face of antibiotic pressure was also confirmed, which was not present when oral β-lactamase was used in conjunction with β-lactam antibiotics (1.72 log10 cfu/g more enterococci and 1.62 log10 cfu/g more Gram-negative bacteria in the stool contents of piperacillin-treated mice protected with oral β-lactamase when compared with mice treated with piperacillin alone, P < 0.0001).
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Here, we demonstrate that a proteolysis-resistant, enteral β-lactamase enzyme prevented the overgrowth of C. difficile, including epidemic C. difficile, in caecal contents of antibiotic-treated mice. Toxin production in caecal contents was also inhibited. The mechanism by which these phenomena occur appears to be via preservation of the normal intestinal microbiota in the face of β-lactam antibiotic pressure. Our findings suggest that oral β-lactamase treatment may represent a promising new strategy to limit colonization and dissemination of C. difficile in hospitalized patients. The use of multiple classes of antibiotics concomitantly in some patients, however, could diminish the utility of such an approach. This potential drawback will also require further investigation.
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Funding |
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This work was supported by a Research Career Development Award from the Department of Veterans Affairs to U.S. and by funds for research from Ipsat Therapies, LtdTM to C. J. D.
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Transparency declarations |
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C. J. D. has previously received funds for research from Ipsat Therapies, LtdTM and P. K. is an employee of Ipsat Therapies, LtdTM. Other authors: none to declare.
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Acknowledgements |
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The bulk of the findings described earlier were previously presented as an oral abstract at the Forty-seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007. We would like to thank Dr Robert Bonomo for helpful discussions of β-lactamase enzymology.
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References |
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