Fehlbaum Sophie, Chassard Christophe, Poeker Sophie Annick, Derrien Muriel, Fourmestraux Candice, Lacroix Christophe
Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich, Switzerland.
Danone Nutricia Research, Palaiseau Cédex, France.
Gut Pathog. 2016 Dec 1;8:63. doi: 10.1186/s13099-016-0144-y. eCollection 2016.
(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.
Two 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) known to induce CD infection in vivo or metronidazole (twice daily 333 mg L) 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).
CD 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 log 8 mL 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.
Using 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.
艰难梭菌（CD）是一种产芽孢和毒素的细菌，是老年人抗生素相关性腹泻的主要病因。在此，我们研究了在接种固定化老年粪便微生物群的新型体外发酵模型中CD的定殖情况以及抗生素治疗的效果。
使用两个接种了不同固定化老年微生物群的连续肠道PolyFermS模型，研究在近端结肠（PC，模型1）和横结肠远端（TDC，模型1和2）条件下CD定殖的相关因素。测试了接种不同PCR核糖体分型的两种CD菌株的定殖情况，一种以营养细胞形式接种（核糖体分型001，模型1），另一种以芽孢形式接种（核糖体分型001和012，模型2）。在TDC条件下（模型2）研究了两种抗生素的治疗效果，即已知在体内可诱导CD感染的头孢曲松（每日150 mg/L）或常用于治疗CD的甲硝唑（每日两次，333 mg/L），观察其对肠道微生物群组成（qPCR、16S焦磷酸测序）和活性（HPLC）、CD芽孢萌发和定殖以及细胞毒素滴度（Vero细胞测定）的影响。
在模型1的PC反应器中接种营养细胞后未检测到CD，但在两个模型的TDC反应器中均显示CD能够定殖，流出物中的拷贝数可达每毫升对数8，毒素稳定产生且与CD细胞数量相关。与无抗生素对照反应器相比，TDC反应器中的头孢曲松治疗对微生物群组成和活性影响较小，且未促进CD定殖。相比之下，在CD定殖后用甲硝唑治疗会使微生物群发生较大改变，并使CD数量降至特异性qPCR检测限以下。然而，仅在甲硝唑治疗停止后2天就检测到CD快速复发。
使用我们构建的体外发酵模型，我们证明了在不应用头孢曲松的情况下，通过接种CD营养细胞或芽孢可在TDC反应器中诱导CD稳定定殖。甲硝唑治疗可暂时减少CD数量，这与体内CD感染复发情况一致。我们的数据表明，与体内观察结果不同，CD在体外定殖于未受干扰的微生物群中，因此提示宿主相关因素在预防CD感染方面具有重要作用。
