Abstract
Background Clostridium difficile (CD), a spore-forming and toxin-producing bacterium, is the main cause for antibiotic-associated diarrhea in the elderly. Here we investigated CD colonization in novel in vitro fermentation models inoculated with immobilized elderly fecal microbiota and the effects of antibiotic treatments.MethodsTwo continuous intestinal PolyFermS models inoculated with different immobilized elder microbiota were used to investigate selected factors of colonization of CD in proximal (PC, model 1) and transverse-distal (TDC, model 1 and 2) colon conditions. Colonization of two CD strains of different PCR ribotypes, inoculated as vegetative cells (ribotype 001, model 1) or spores (ribotypes 001 and 012, model 2), was tested. Treatments with two antibiotics, ceftriaxone (daily 150 mg L−1) known to induce CD infection in vivo or metronidazole (twice daily 333 mg L−1) commonly used to treat CD, were investigated in TDC conditions (model 2) for their effects on gut microbiota composition (qPCR, 16S pyrosequencing) and activity (HPLC), CD spore germination and colonization, and cytotoxin titer (Vero cell assay).ResultsCD remained undetected after inoculating vegetative cells in PC reactors of model 1, but was shown to colonize TDC reactors of both models, reaching copy numbers of up to log10 8 mL−1 effluent with stable production of toxin correlating with CD cell numbers. Ceftriaxone treatment in TDC reactors showed only small effects on microbiota composition and activity and did not promote CD colonization compared to antibiotic-free control reactor. In contrast, treatment with metronidazole after colonization of CD induced large modifications in the microbiota and decreased CD numbers below the detection limit of the specific qPCR. However, a fast CD recurrence was measured only 2 days after cessation of metronidazole treatment.ConclusionsUsing our in vitro fermentation models, we demonstrated that stable CD colonization in TDC reactors can be induced by inoculating CD vegetative cells or spores without the application of ceftriaxone. Treatment with metronidazole temporarily reduced the counts of CD, in agreement with CD infection recurrence in vivo. Our data demonstrate that CD colonized an undisturbed microbiota in vitro, in contrast to in vivo observations, thus suggesting an important contribution of host-related factors in the protection against CD infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13099-016-0144-y) contains supplementary material, which is available to authorized users.
Highlights
Clostridium difficile (CD), a spore-forming and toxin-producing bacterium, is the main cause for antibiotic-associated diarrhea in the elderly
Continuous fermentation models mimicking the elderly colon Testing of CD colonization and antibiotic treatments was performed in PolyFermS continuous fermentation models inoculated with fecal beads immobilizing elderly gut microbiota, and operated with conditions mimicking the elderly proximal (PC) and transverse-distal colon (TDC) [19]
In conclusion, our study provides evidence to support the use of continuous intestinal models for the in vitro investigation of CD colonization dynamics and antibiotic treatments on elderly gut microbiota independent of host factors
Summary
Clostridium difficile (CD), a spore-forming and toxin-producing bacterium, is the main cause for antibiotic-associated diarrhea in the elderly. Clostridium difficile (CD) was first identified in the 1970s as the causative agent of antibiotic-associated pseudomembranous colitis and is the leading cause of hospital-acquired diarrhea [1]. CD is a gram-positive anaerobic bacterium harboring several virulence factors such as the ability to form spores and produce toxins [2]. CD is part of the normal gut microbiota in 25–80% of infants but usually does not cause disease despite the finding that a significant fraction of the CD strains are toxin producers [3]. Following ingestion of CD spores, germination into the vegetative form is necessary for colonization in the gut, with subsequent toxin production leading to clinical manifestations [5]
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