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Biofilms harbour Clostridioides difficile, serving as a reservoir for recurrent infection Charmaine Normington 1, Ines B. Moura 1, Jessica A. Bryant 2, Duncan J. Ewin1, Emma V. Clark1, Morgan J. Kettle1, Hannah C. Harris 1, William Spittal1, Georgina Davis1, Matthew R. Henn 2, Christopher B. Ford2, Mark H. Wilcox1 and Anthony M. Buckley 1✉

C. difficile infection (CDI) is a worldwide healthcare problem with ~30% of cases failing primary therapy, placing a burden on healthcare systems and increasing patient morbidity. We have little understanding of why these therapies fail. Here, we use a clinically validated in vitro gut model to assess the contribution of biofilms towards recurrent disease and to investigate biofilm microbiota-C. difficile interactions. Initial experiments show that C. difficile cells became associated with the colonic biofilm microbiota and are not depleted by vancomycin or faecal microbiota transplant therapies. We observe that transferring biofilm encased C. difficile cells into a C. difficile naïve but CDI susceptible model induces CDI. Members of the biofilm community can impact C. difficile biofilm formation by acting either antagonistically or synergistically. We highlight the importance of biofilms as a reservoir for C. difficile, which can be a cause for recurrent infections.

npj Biofilms and Microbiomes (2021) 7:16 ; https://doi.org/10.1038/s41522-021-00184-w

INTRODUCTION Clostridioides difficile is the leading cause of infective antibiotic- associated diarrhoea worldwide and a significant cause of morbidity and mortality; the burden of healthcare costs are estimated to be over €3B in Europe and $4.8B in USA1–3. Antibiotics deplete the intestinal microbiota which allows the germination of C. difficile spores followed by C. difficile cell proliferation and toxin production. Toxins A (TcdA) and B (TcdB) are responsible for the clinical manifestations of C. difficile infection (CDI)4,5. The primary treatment option is antibiotic therapy, with either metronidazole, vancomycin or fidaxomicin; however, antibiotic therapy further exacerbates intestinal dysbio- sis and potentiates recurrent infection6. Approximately 30% of primary CDI cases recur after antibiotic treatment for primary inflection7, after which, patients are at an increased risk of further treatment failures. The risk of a second and third recurrent episode increases to 45% and 64%, respectively, known as a ‘recurrence escalator’8. Recurrent CDI is particularly problematic for the patient and the healthcare system, increasing patient morbidity, extend- ing the number of bed days and requiring more therapy, thus increasing the cost of treatment3. The majority of recurrent episodes are attributed to the

original strain/ribotype9, suggesting that C. difficile can evade antibiotic treatment, possibly by occupying a protective niche within the intestine where antibiotic therapy is ineffective. Incorporation of C. difficile into intestinal biofilms, a known driver of chronic infection10, could function as a protective niche where C. difficile cells are protected from the effects of antibiotic therapy. In vitro, C. difficile forms aggregates enclosed in an extracellular matrix11–14 and can interact with other bacterial species found within the intestine to enhance biofilm formation11,15. Biofilm-associated C. difficile cells undergo metabolic remodelling compared with planktonic-associated cells and have a different array of cell-surface proteins/ organelles compared with luminal cells16. Indeed, biofilm

structures composed of C. difficile cells have been observed adjacent to epithelial cells in in vivo models of CDI17–20, where damaged and necrotic microvilli have been observed21. These biofilm cells are enclosed in a glycan-rich extracellular matrix that helps protect against antibiotic exposure22. However, little is known about this potential reservoir, the contribution towards disease recurrence and how other members of the biofilm community interact with C. difficile. We have previously developed a successful in vitro triple-stage

chemostat human gut model to evaluate the impact of antimicrobials on intestinal microbiome colonisation resistance to CDI23 (Supplementary Fig. 1A). Pooled human faeces are used to establish microbial populations within the gut model. C. difficile spores are then added but remain quiescent until the microbial populations and associated colonisation resistance is disrupted, i.e. following antibiotic instillation, which leads to C. difficile germination, outgrowth and toxin production (Fig. 1A). Data generated from in vitro gut models have been shown to be clinically reflective with respect to CDI. For example, antibiotics with a high propensity to induce CDI in patients also induce simulated CDI within the gut model24–26. Conversely, antibiotics with a lower in vitro propensity to induce simulated CDI are now recognised to have lower CDI risk23,27. Our in vitro model has been fitted with removable biofilm

support structures28 enabling us to independently delineate the microbiota dynamics of the biofilm and luminal populations. We have previously described and validated the use of our biofilm support structures in our in vitro model (Supplementary Fig. 2)28. In this study, we leverage these structures to investigate the role of biofilms in recurrent CDI. Here, we describe the biofilm- associated microbiota dynamics during simulated CDI and recurrent infections, and the interactions between C. difficile and members of the colonic biofilm microbiota.

1Healthcare-Associated Infections Group, Leeds Institute of Medical Research, Faculty of Medicine and Health, University of Leeds, Leeds LS1 9JT, UK. 2Microbiome Sciences, Seres Therapeutics Inc., Cambridge, MA, USA. ✉email: [email protected]

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RESULTS Vancomycin therapy and FMT installation are required to prevent recurrent CDI In two gut models, a CDI recurrence (rCDI) model and a faecal microbiota transplant (FMT) model, we simulated the induction of CDI through the instillation of an induction antibiotic, clindamycin, and administered a ‘treatment’ antibiotic, vancomycin, which is comparable to a clinical setting (Fig. 1). CDI induction was characterised by C. difficile spore germination, vegetative cell outgrowth and detection of toxin activity; peak toxin was detected on day 61 at 3.5 log10 reciprocal titre in both rCDI and FMT models. Vancomycin successfully reduced the luminal C. difficile recoveries to undetectable levels; however, similar to a clinical setting29, we detected recurrent CDI in the rCDI model. This was characterised by a second C. difficile outgrowth event and the

detection of further toxin activity after 28 days (day 100) after vancomycin administration with a peak toxin of 3 log10 reciprocal titre (Fig. 1B, red line)26. In the FMT treatment model, we sought to replicate FMT

therapy with a 10% w/v faecal slurry instillation from a single healthy donor, simulating the protocol used at the Leeds General Infirmary (U.K.), via the nasal-jejunal route of administration (Supplementary Fig. 1B). FMT therapy is an effective treatment for the resolution of recurrent CDI with a documented success rate of 76.1%29. Antibiotic bioassay determination showed an unde- tectable level of vancomycin in vessel 1 of the gut model at the time of FMT instillation. FMT instillation successfully prevented the recurrence of CDI up to 35 days following cessation of vancomycin (Fig. 1C, blue line). However, C. difficile spores were transiently

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Fig. 1 Efficacy of FMT to treat simulated recurrent CDI. A Timeline of two in vitro chemostat models that were used to simulate primary CDI and recurrence after vancomycin treatment (black) and vancomycin treatment followed by FMT instillation (green). Luminal C. difficile recoveries from the recurrence model (B) and from FMT model (C). Both figures show the total viable counts (red lines), spores (blue lines) and period of toxin detection (black arrows). Results are shown as mean log10 cfu/mL from two biological replicates, and three technical replicates from each. Error bars represent the standard deviation.

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detected post FMT but we did not detect germination or toxin activity.

C. difficile is incorporated into the multispecies biofilm and is not depleted by vancomycin or FMT instillation We characterised the biofilm communities using 16Sv4 rRNA gene sequencing to investigate the effect of antibiotics on the sessile community, and whether the biofilms in our experiments could be a source of both transient C. difficile spore detection post FMT and the origin of the recurrent CDI observed in the rCDI model. Taxonomic analysis and visualisation of the biofilm community isolated from these support structures highlight a varied commu- nity enclosed in an extracellular matrix forming a complex structure (Supplementary Fig. 2)28. Bifidobacteriaceae, Lactobacil- laceae and Eubacteriaceae were the most abundant bacterial families present in the biofilm community prior to antibiotic exposure (Fig. 2). Post clindamycin exposure, an increase in the relative abundances of Enterobacteriaceae, Bacteroidaceae and Methanobacteriaceae were observed, which was accompanied by the decreased relative abundances of Bifidobacteriaceae and Eubacteriaceae. Vancomycin exposure, with no further interven- tion, was associated with the reduction in the abundance of several bacterial families; Bacteroidaceae, Eubacteriaceae, Lach- nospiraceae, Ruminoccocaceae and Comamonadaceae had lower abundances for the remainder of the experiment compared with their pre-antibiotic abundance (Fig. 2A). FMT instillation was associated with the recovery of these same bacterial families at either 2- or 3-week post FMT, except for Comamonadaceae which did not recover by the end of the experiment (Fig. 2B). Furthermore, by direct enumeration, we recovered several

different yeast species as part of the biofilm microbiota from both models, albeit at low levels. Upon the addition of C. difficile into the lumen of the model, the

bacterial spores became intimately associated with the biofilm structures present in all three vessels. During clindamycin induction and at peak CDI, the overall C. difficile levels recovered from the biofilm slightly decreased; however, the recovered C. difficile population was a mix of both spore cells and vegetative cells, ~1:3 ratio respectively. Sessile C. difficile cells accounted for approximately 0.007% of the total bacteria present in the biofilm community (Supplementary Fig. 3). Vancomycin therapy alone did not affect the recovery of C. difficile associated with the biofilm (Fig. 2C), nor was the instillation of FMT able to displace biofilm- associated C. difficile cells entirely (Fig. 2D).

Biofilm-associated C. difficile cells can cause simulated CDI Determining the role of biofilms in recurrent CDI has been particularly challenging with other in vitro and in vivo models of CDI as it has been difficult to independently delineate the luminal and planktonic populations. However, our model is ideally placed to investigate this question due to the accessibility of the biofilm support structures in our system. Here, we set up a biofilm transfer experiment, where a biofilm donor model (model D) underwent vancomycin ‘treatment’ of simulated CDI and the biofilm support structures from this model were transferred to a C. difficile-naïve recipient model (model R) and two independent biological replicates were performed (Fig. 3A). CDI was induced in model D following clindamycin exposure and at peak CDI, where C. difficile luminal recovery was 5.4 log10 cfu/mL (peak toxin was detected at 3 log10 reciprocal titre), vancomycin was instilled.

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Fig. 2 Changes in the biofilm-associated microbiota during CDI and recurrence. Percentage taxonomic abundance of bacterial families isolated from biofilm support structures taken from recurrence (rCDI) model (A) or the FMT model (B). Graphs constructed using mean (of least 3 support structures/time point) percent abundance of bacterial OTUs assigned to the family taxonomic level. Enumeration of biofilm- associated C. difficile (vegetative cells – red lines, spores – blue lines) from support structures from the recurrence (C) and FMT models (D). Results shown as mean log10 cfu/g wet biofilm mass from two biological replicates and at least four support structures. Error bars represent the standard deviation.

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Vancomycin treatment depleted the luminal-associated C. difficile population in the donor model to below the limit of detection (Supplementary Fig. 4AB) but, crucially, the biofilm-associated population remained present mostly as spores, as the vegetative cells were reduced (Supplementary Fig. 4CD). These biofilm support structures were then transferred to the recipient model. The mean C. difficile titre in the biofilm was assessed from two support structures at 3.8 log10 cfu/g wet biomass (Supplementary Fig. 4D). From this we estimate that a total of 4.1 log10 cfu C. difficile cells were transferred to the recipient model based on the number of support structures transferred and the average biofilm mass attached to each structure. The biofilm recipient model was exposed to clindamycin to create an environment conducive for

CDI prior to the transfer of the support structures. Post biofilm transfer, luminal-associated C. difficile vegetative cells were recovered 9 days post transfer and toxin production was detected by the end of the experiment at 1 log10 reciprocal titre (Fig. 3B, green lines/arrow). In parallel to the recipient model, we ran an experimental

control model (model C). The purposes of this model were to ensure colonisation resistance had established in both R and C models, and that clindamycin exposure was able to create the microbial niche needed for CDI progression (Supplementary Fig. 3AB). Colonisation resistance was confirmed when the C. difficile spore dose added to the control model did not show spore germination or outgrowth (Fig. 3B). To confirm a CDI susceptible

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Fig. 3 Contribution of biofilm-associated C. difficile to cause recurrent disease. A Timeline of events from the biofilm donor model (Model D, blue arrow) prior to support structure transfer, and two further models; a control model (Model C, red arrow) inoculated with C. difficile spores, and a biofilm recipient model (Model R, green arrow), which did not receive spores, but received the biofilm support structures from model D (broken blue arrow). B Recoveries of luminal C. difficile (total viable counts – TVC; spores – Sp) from model C (red line) and model R (green line) and subsequent periods of toxin detection (arrows). Solid lines are C. difficile total viable cells and broken lines are spore recoveries. Results expressed as mean log10 cfu/mL from two biological replicates. C Quantitative PCR enumeration of selected luminal microbiota populations from model C (whole lines) and model R (dotted lines) immediately prior to clindamycin instillation (pre-clindamycin), during clindamycin therapy and throughout CDI development and progression. Results expressed as mean log10 copy number per μL of luminal fluid from two biological replicates. All error bars represent the standard deviation.

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niche, clindamycin was instilled into the control model, alongside another inoculum of C. difficile spores and evidence of spore germination was detected 7 days post clindamycin, followed by vegetative outgrowth and toxin production, detected from day 41 onwards and at a peak of 3.0 log10 reciprocal titre (Fig. 3B, red lines/arrow). We monitored the microbiota dynamics and the effects of

clindamycin within the control and recipient models. The microbial populations enumerated by quantitative PCR from both models were similar immediately prior to antibiotic instillation (Fig. 3C and Supplementary Fig. 5). Clindamycin had a pleiotropic effect on the microbiota, causing an average decrease of at least 1 log10 copies/µL in Prevotella spp., Bifidobacterium spp. and Bacteroides spp., whilst Enterobacteriaceae and Enterococcus spp. increased by at least 1 log10 copies/µL in all models. The monitored microbiota recovered to pre-clindamycin levels by day 43.

Biofilm microbiota can affect C. difficile biofilm formation Following the findings that C. difficile cells associated with the biofilm were unaffected by either antibiotic therapy or FMT microbial therapy, having the potential to cause disease, we investigated the influence of other microbes on C. difficile biofilm formation in vitro. Microorganisms were cultured directly from the biofilm support structures in our gut model and identified to the species level by MALDI-TOF analysis (Supplementary Table 1).

These biofilm isolates were co-cultured with C. difficile and the effect on C. difficile biofilm formation was characterised as either antagonistic (the isolate reduced C. difficile biofilm formation), co- operative (summation of individual mono-species biomass is equal to that of the co-culture biofilm) or synergistic (the isolate enhanced C. difficile biofilm formation). A wide range of bacterial and yeast species were identified

associated with the biofilm support structures (Supplementary Table 1) removed at different time points throughout the recurrence and FMT gut models. Initially, these microbial species were individually co-cultured with C. difficile, where six microbial species were found to act antagonistically to significantly (p ≤ 0.05) reduce the biofilm biomass produced and four microbial species acted in a synergistic manner to significantly (p ≤ 0.05) increase the biomass produced in these biofilms (Fig. 4A and Supplementary Fig. 6). Additionally, two microbial species, Lactobacillus delbrueckii and Clostridium paraputrificum, were identified as co-operative species, as the sum of the biomass from individual biofilms was equal to that of the dual species’ biofilms with C. difficile. We determined if the reduced co-culture biomass from those ‘antagonistic’ species against C. difficile was a result of decreased biofilm matrix production or a reduced number of C. difficile cells within the biofilm. Direct enumeration of the C. difficile viable cells from co-culture biofilms showed significantly less C. difficile cells compared with monoculture biofilms (Fig. 4B). Interestingly, co-culture of C. difficile with Bifidobacterium breve also reduced the number of C. difficile

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Fig. 4 Biofilm-associated microbiota can affect C. difficile biofilm formation. A Biofilm formation of C. difficile when co-cultured with different microbial species, compared with the biofilm formed from a C. difficile monoculture. Blue or red bars indicate species that significantly (p ≤ 0.05) reduced or increased the biofilm formed and grey bars represent species that did not significantly affect biofilm formation. Results expressed as fold change of crystal violet absorption of the dual co-culture vs monoculture. B Antagonistic bacteria reduced C. difficile recoveries from biofilms. C. difficile recoveries from mono and dual culture biofilms and C the enhanced antagonistic effect of polymicrobial biofilms on C. difficile recoveries from biofilms. D Poly synergistic biofilms are greater than the sum of the individual monoculture biofilms. B–D Results expressed as box and whisker plots showing the median log10 cfu/mL, lower and upper quartile ranges, and the minimum and maximum results from at least four technical replicates from three biological repeats. False-coloured SEM of C. difficile (E, ×10,000, scale bar is 2 µm) and C. parapsilosis (F, ×5000, scale bar 5 µm) monoculture biofilms. Polymicrobial biofilms of C. difficile (red cells), S. warneri (blue cells) and C. parapsilosis (green cells) showing close interaction between the microbial cells (G, ×2500, scale bar 20 µm). Insert, zoomed section highlighting the extracellular matrix-like substance (light green colour) (H, ×10,000, scale bar 2 µm). White arrows denote what appears to be extracellular matrix in all images. Asterix denotes significantly difference with a p value < 0.01.

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biofilm cells, even though there was no significant difference in the biomass (Fig. 4AB). Lactobacillus rhamnosus, Bifidobacterium longum and B. breve all had a reductive effect of 3.3, 1.2 and 2.5 log10 cfu/mL, respectively, on C. difficile counts. These reductions in C. difficile cells within the biofilm coincided with reduced toxin activity detected, ~2 log10 reduction in toxin titre (from 3.5 to 1.5 log10 median toxin titre), in the medium (data not shown). Co- culturing L. rhamnosus and B. longum with C. difficile in a polymicrobial biofilm caused an additive antagonistic effect on the C. difficile biofilm formation, where a reduction of 4.4 log10 cfu/ mL was seen (Fig. 4C). Of those microbial species that were able to enhance C. difficile

biomass in dual cultures, none increased the number of C. difficile cells within the biofilm; however, they all increased the amount of biofilm biomass produced in dual co-culture biofilms (Supple- mentary Fig. 7C). Interestingly, when several of these microbial species were cultured together in a polymicrobial biofilm, the resulting biofilm biomass was greater than the sum of the individual monocultures (Fig. 4D). Scanning electron microscopic imaging of either C. difficile or Candida parapsilosis monoculture biofilms showed what appears to be the extracellular matrix produced by either species with distinctive physical character- istics. C. difficile produces a filamentous-like matrix (Fig. 4E)12,13

whereas C. parapsilosis produces a dense granular extracellular matrix (Fig. 4F)30; however, we did not determine the composition of the specific extracellular matrix from each species. In a polymicrobial biofilm of C. difficile, C. parapsilosis and Staphylo- coccus warneri, the individual microbial cells showed a close interaction with each other in a heter

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