Background
Clostridium difficile (CD) was first identified in the 1970s as the causative agent of antibiotic-associated pseudomembranous colitis and is now 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]. Interestingly, 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]. One of the main functions of an undisturbed gut microbiota is resistance against colonization of pathogens [
4]. The disruption of the microbiota and thus the colonization resistance is usually the first step in the pathogenesis of CD infection (CDI). 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].
Treatment with broad-spectrum antibiotics, such as ampicillin, clindamycin and third-generation cephalosporins (ceftriaxone, cefotaxime and ceftazidime) are considered to be main risk factors for CDI [
6]. The elderly population is especially at risk of developing CDI, with CD colonization rates of up to 73% in inpatients above 65 years [
7]. Standard therapy for CDI is antibiotic treatment with metronidazole or vancomycin. However, efficacy of these antibiotics is limited, with recurrence being observed in 20–40% of cases, mainly due to development of antibiotic resistances and a loss of gut barrier function that allows residual CD to re-colonize the colon after the antibiotic treatment is cancelled [
1]. Indeed, the important role of an undisturbed microbiota that provides colonization resistance is reflected in the high success rate of fecal microbiota transplantation of up to circa 92% that is used in severe CDI cases [
8].
Various animal models have been used to study the mechanisms underlying CDI and investigate antibiotic treatments. However, the use of animals is limited, primarily owing to ethical and practical reasons and notable inter-species differences in susceptibility to CDI. In vitro gut fermentation models represent an innovative technological platform consisting of multiple model designs that permit investigations, including survival or mechanistic studies on commensal–pathogen interactions and drug testing [
9,
10]. Different in vitro intestinal models with CDI, from simple batch to multistage continuous systems, have also been reported [
11]. Batch cultures were mainly used to study CD interactions with fecal microbiota with the aim to test alternative treatments to antibiotics [
12‐
15]. A three-stage intestinal model which may be more representative for colonic conditions, inoculated with pooled fecal microbiota from elderly donors was applied in a series of studies with different CD ribotypes and antibiotic treatments, as reviewed by Best et al. [
11]. With this model CD spores were inoculated while the antibiotic clindamycin was used to induce CD spore germination [
16‐
18]. However, monitoring of intestinal microbiota composition was limited to culture-based methods targeting a restricted range of gut bacteria, and no metabolic assessment was reported. To date, no study has reported in-depth analysis of CD colonization ability in in vitro continuous fermentation models using molecular methods and next generation sequencing to describe microbiota effects.
We have recently developed a new PolyFermS continuous model platform inoculated with immobilized fecal microbiota that mimics different sections of the elderly colon [
19]. We showed that the models closely reproduce the gut microbiota composition, density and activity of the fecal donor. Furthermore, the PolyFermS platform allows simultaneous testing of several treatments compared to a control in parallel reactors inoculated with the same fecal microbiota generated in the upstream inoculum reactor (IR) seeded with fecal beads inoculated with single donor microbiota [
20,
21]. The aim of the present study was to investigate colonization of CD vegetative cells and spores (PCR ribotype 001 and 012) in the new PolyFermS model of elderly colonic fermentation. Further, the effects of two antibiotics, ceftriaxone and metronidazole, were tested on the composition (qPCR, 16S pyrosequencing) and activity (HPLC) of the gut microbiota. CD growth and toxin production were analyzed with specific qPCR and Vero cell assays, respectively.
Discussion
In vitro intestinal fermentation models are useful tools to investigate colonization of enteropathogens, such as Salmonella, and interactions with the human gut microbiota, independent of the effects of the host [
22‐
24]. The human colon can be approximated to a continuous fermenter, divided in regions (ascending, transverse and descending colonic) that are different in metabolic activity and microbial composition. A major advancement of in vitro gut fermentation models was the development of multistage continuous fermentation models which enable the simulation of horizontal colon processes [
25]. Investigation of CD colonization in intestinal models has been primarily performed with three-stage colonic models, inoculated with a liquid fecal suspension from pooled donors, using cultivation methods for microbiota analysis [
16,
18].
In a recent study we investigated novel in vitro continuous colonic fermentation models inoculated with immobilized fecal microbiota from single elderly donors and characterized in detail microbiota composition, diversity and activity [
19]. Immobilization of fresh gut microbiota reproduced both the planktonic (free-cell) and sessile (biofilm-associated) states of bacterial populations in the colon and yielded self-contained continuous fermentation system of high cell density, diversity and population stability, similar to what is observed in the human GI tract [
9]. In this study, we used two fermentation models to investigate the colonization of CD vegetative cells or spores, and the response of the microbiota to antibiotics known to induce or used to treat CDI.
It is suggested that the small intestine is the site of CD spores germination into the metabolically active vegetative cells [
26,
27], which then proliferate in the large intestine and cause symptoms typical for CDI [
5,
28]. Using PolyFermS models, we showed that CD vegetative cells or spores only colonize in TDC reactors after a time period of 7–10 days. Our data are in agreement with previous studies that reported an absence of CD colonization added as spores in the proximal colon section of a three stage colonic fermentation model [
29,
30], which may be explained by pH inhibition effect [
31].
The CD concentration determined in TDC reactors of our models (from 6.0 to 8.9 GC mL
−1) is within the range of in vivo qPCR data for feces of antibiotic-associated diarrhea patients (aged 3–89 years; from 5.6 to 11.2 cell equivalents log
10 g
−1 [
32]). Growth of CD in gut models upon inoculation of vegetative cells was only reported for batch fermentations, yielding cell counts up to 8 log
10 cfu mL
−1 after 24 h [
12,
13]. Although we used a high inoculum of vegetative cells (log
10 9.8 cfu) in model 1, CD cells were washed-out and remained undetected for a period of 6 days after which they were shown to colonize in TDC reactors.
The PolyFermS design of model 2 was therefore selected to mimic TDC conditions for CD colonization. We investigated the colonization of two PCR ribotypes of CD spores and the effects of two antibiotics, ceftriaxone and metronidazole, that are known to respectively promote and treat infection. CD remained undetected in all TRs for 5 days after which a repeated inoculation of spores was performed. This result suggests that either the spores were washed out of the system, or that they were able to grow, but remained below the detection limit of the qPCR method (4.4 GC mL
−1). A quiescent state of CD spores for a period of at least 7 days was previously reported in a three-stage model, and was tentatively explained by the absence of antibiotic treatment [
29,
30]. Our data demonstrate that application of antibiotic may not be required for CD spore germination and colonization of an in vitro continuous colonic fermentation model mimicking TDC conditions of an elderly microbiota. This is in contrast to in vivo data in humans and animal models suggesting that CD infection is promoted by a disturbed microbiota mainly due to antibiotic treatment [
6,
33]. This discrepancy between in vitro and in vivo CD colonization may be a consequence of host-specific mechanisms, including the epithelial cell layer and immune responses, both of which are lacking in in vitro models. Lawley et al. [
34] showed that CD can asymptomatically colonize mice in the absence of antibiotic treatment while antibiotics were necessary for CD to proliferate and sporulate. The study suggested that toll-like receptor signaling may protect against virulent CD overgrowth.
Ceftriaxone is a broad-spectrum antibiotic that is associated with a high risk of developing diarrhea due to CD colonization [
35,
36]. Pletz et al. [
37] reported that in the fecal microbiota of ten healthy adults, ceftriaxone treatment (2 g administered once daily for 7 days) induced a marked decrease in lactobacilli, bifidobacteria, clostridia and
Bacteroides, with an attendant increase in enterococci. In the present study, we observed only minor effects of ceftriaxone on the gut microbiota when added at 150 mg L
−1 to mimic ceftriaxone levels found in feces of volunteers from the study mentioned above [
37] on the 4th day of the treatment.
Bifidobacterium spp. was most affected, but diversity and metabolic activity was not changed compared to the control reactor. The detrimental effect of ceftriaxone on
Bifidobacterium spp. was also observed in the human gut model of Baines [
29] when the same antibiotic concentration was used to induce CD spore germination. In contrast to our study, Baines only used plating for gut microbiota enumeration (total facultative anaerobes, total anaerobes (facultative + obligate), lactose-fermenting
Enterobacteriaceae, enterococci, lactobacilli, bifidobacteria, total
Clostridium spp. and
Bacteroides fragilis group), and did not test diversity and metabolic effects.
Metronidazole is used in the treatment of anaerobic infections [
38], including cases of mild to moderate CDI [
39]. We measured an extreme shift of the gut microbiota composition during metronidazole treatment, with a very large increase of Actinobacteria at the expense of Bacteroidetes and sharp inhibition of butyrate production. A detrimental effect of metronidazole on Bacteroidetes was also observed in a previous single-stage human distal colon model which was used to investigate the activity of a bacteriocin versus antibiotics against CD, however, metronidazole mainly promoted growth of bacteria belonging to the phylum Proteobacteria [
13]. In previous in vitro studies which used concentrations varying greatly between 9.3 mg L
−1 every 8 h [
17] to 330 mg L
−1 per day [
14] no effect on CD counts and toxin production was observed with the low antibiotic level [
17]. However, CD eradication was reported for the higher concentration in a batch fermentation which contained a total anaerobes concentration of approximately log
10 10.3 cfu mL
−1, estimated with plate counts [
14]. In our model we chose to apply a higher antibiotic concentration (333 mg L
−1, twice daily) to account for the high total cell numbers tested in TDC (in the range of log
10 11.1–11.2 GC mL
−1 [
19]). Furthermore, we considered that the concentration of metronidazole entering the colon is higher than that tested in feces since the gut bacteria metabolize the drug [
40]. With our conditions, we observed a strong reduction of CD numbers while toxin production decreased below the detection limits.
An important issue to consider with treatment of CDI is the high recurrence rate after antibiotic treatment, typically around 27% after metronidazole treatment [
41]. Our data consistently demonstrate that a fast recurrence of CD occurred only 2 days after cessation of the metronidazole treatment, also supporting the applied metronidazole dosage. Gut bacterial groups that were mainly affected by metronidazole (
Clostridium cluster IV,
F. prausnitzii and
Roseburia spp.) showed only partial recovery to control levels after 10 days. To our knowledge, this is the first report CD recovery after metronidazole treatment in a gut fermentation model.
Authors’ contributions
SF participated in the design of the study, carried out the experiments and drafted the manuscript. CC assisted in the conception and design of the study, data collection and analyses. SAP provided support for the experiments and data analysis. MD carried out the sequence analyses and helped to draft the manuscript. CF contributed to data analysis and drafting the manuscript. CL participated in the conception and design of the study, interpretation of data and writing of the manuscript. All authors read and approve the final manuscript.