Clostridioides difficile infections; new treatments and future perspectives.

PURPOSE OF REVIEW: As a significant cause of global morbidity and mortality, Clostridioides difficile infections (CDIs) are listed by the Centres for Disease Control and prevention as one of the top 5 urgent threats in the USA. CDI occurs from gut microbiome dysbiosis, typically through antibiotic-mediated disruption; however, antibiotics are the treatment of choice, which can result in recurrent infections. Here, we highlight new treatments available and provide a perspective on different classes of future treatments. RECENT FINDINGS: Due to the reduced risk of disease recurrence, the microbiome-sparing antibiotic Fidaxomicin has been recommended as the first-line treatment for C. difficile infection. Based on the success of faecal microbiota transplantations (FMT) in treating CDI recurrence, defined microbiome biotherapeutics offer a safer and more tightly controlled alterative as an adjunct to antibiotic therapy. Given the association between antibiotic-mediated dysbiosis of the intestinal microbiota and the recurrence of CDI, future prospective therapies aim to reduce the dependence on antibiotics for the treatment of CDI. SUMMARY: With current first-in-line antibiotic therapy options associated with high levels of recurrent CDI, the availability of new generation targeted therapeutics can really impact treatment success. There are still unknowns about the long-term implications of these new CDI therapeutics, but efforts to expand the CDI treatment toolbox can offer multiple solutions for clinicians to treat this multifaceted infectious disease to reduce patient suffering.

MiGut: A scalable in vitro platform for simulating the human gut microbiome-Development, validation and simulation of antibiotic-induced dysbiosis.

In vitro models of the human colon have been used extensively in understanding the human gut microbiome (GM) and evaluating how internal and external factors affect the residing bacterial populations. Such models have been shown to be highly predictive of in vivo outcomes and have a number of advantages over animal models. The complexity required by in vitro models to closely mimic the physiology of the colon poses practical limits on their scalability. The scalable Mini Gut (MiGut) platform presented in this paper allows considerable expansion of model replicates and enables complex study design, without compromising on in vivo reflectiveness as is often the case with other model systems. MiGut has been benchmarked against a validated gut model in a demanding 9-week study. MiGut showed excellent repeatability between model replicates and results were consistent with those of the benchmark system. The novel technology presented in this paper makes it conceivable that tens of models could be run simultaneously, allowing complex microbiome-xenobiotic interactions to be explored in far greater detail, with minimal added resources or complexity. This platform expands the capacity to generate clinically relevant data to support our understanding of the cause-effect relationships that govern the GM.

Profiling the Effects of Systemic Antibiotics for Acne, Including the Narrow-Spectrum Antibiotic Sarecycline, on the Human Gut Microbiota.

Treatment for moderate-to-severe acne vulgaris relies on prolonged use of oral tetracycline-class antibiotics; however, these broad-spectrum antibiotics are often associated with off-target effects and negative gastrointestinal sequelae. Sarecycline is a narrow-spectrum antibiotic treatment option. Here, we investigated the effect of prolonged sarecycline exposure, compared with broad-spectrum tetracyclines (doxycycline and minocycline) upon the colonic microbiota. Three in vitro models of the human colon were instilled with either minocycline, doxycycline or sarecycline, and we measured microbiota abundance and diversity changes during and after antibiotic exposure. Significant reductions in microbial diversity were observed following minocycline and doxycycline exposure, which failed to recover post antibiotic withdrawal. Specifically, minocycline caused a ~10% decline in Lactobacillaceae and Bifidobacteriaceae abundances, while doxycycline caused a ~7% decline in Lactobacillaceae and Bacteroidaceae abundances. Both minocycline and doxycycline were associated with a large expansion (>10%) of Enterobacteriaceae. Sarecycline caused a slight decline in bacterial diversity at the start of treatment, but abundances of most families remained stable during treatment. Ruminococcaceae and Desulfovibrionaceae decreased 9% and 4%, respectively, and a transient increased in Enterobacteriaceae abundance was observed during sarecycline administration. All populations recovered to pre-antibiotic levels after sarecycline exposure. Overall, sarecycline had minimal and transient impact on the gut microbiota composition and diversity, when compared to minocycline and doxycycline.

Can SARS-CoV-2 be transmitted via faeces?

PURPOSE OF REVIEW: COVID-19 patients can present gastrointestinal symptoms, being diarrhoea one of the most frequent, suggesting intestinal health can be impacted by COVID-19. Here, we will discuss whether there is a correlation between the presence of SARS-CoV-2 RNA in faeces and diarrhoea, the relevance of gastrointestinal symptoms in disease diagnosis and transmission, and how COVID-19 can impact the gut microbial balance. RECENT FINDINGS: SARS-CoV-2 RNA has been reported in faeces or rectal swabs of COVID-19 patients with and without diarrhoea, suggesting faecal shedding can occur independently of gastrointestinal symptoms. However, the presence of the virus in the intestine can persist beyond its presence in the respiratory tract, with some reports suggesting that SARS-CoV-2 in the faeces can be infectious.COVID-19 can impact the gut microbiota causing an enhancement of biosynthesis pathways that favour the expansion of bacterial pathogens in the inflamed gut, and causing a decline in commensals involved in the human immune response. SUMMARY: Gastrointestinal symptoms may be the first indication of COVID-19. SARS-CoV-2 in faeces can potentiate routes of disease transmission, particularly as the high viral loads reported in patients with severe illness suggest virus replication in the intestine may be possible.

The potential of microbiome replacement therapies for Clostridium difficile infection.

PURPOSE OF REVIEW: There is a paradox when treating Clostridium difficile infection (CDI); treatment antibiotics reduce C. difficile colonization but cause further microbiota disruption and can lead to recurrent disease. The success of faecal microbiota transplants (FMT) in treating CDI has become a new research area in microbiome restorative therapies but are they a viable long-term treatment option? RECENT FINDINGS: C. difficile displays metabolic flexibility to use different nutritional sources during CDI. Using microbiome therapies for the efficient restoration of bile homeostasis and to reduce the bioavailability of preferential nutrients will target the germination ability of C. difficile spores and the growth rate of vegetative cells. Several biotechnology companies have developed microbiome therapeutics for treating CDI, which are undergoing clinical trials. SUMMARY: There is confidence in using restorative microbiome therapies for treating CDI after the demonstrated efficacy of FMT, where several biotechnology companies are aiming to supply what would be a 'first in class' treatment option. Efficient removal of C. difficile from the different intestinal biogeographies should be considered in future microbiome therapies. With the gut microbiota implicated in different diseases, more work is needed to assess the long-term consequences of microbiome therapies.

The use of first-generation cephalosporin antibiotics, cefalexin and cefradine, is not associated with induction of simulated Clostridioides difficile infection.

OBJECTIVES: The use of broad-spectrum cephalosporins is associated with induction of Clostridioides difficile infection (CDI). Recent knowledge on the importance of the healthy microbiota in preventing pathogen colonization/outgrowth highlights the caution needed when prescribing broad-spectrum antibiotics. The use of historical narrow-spectrum antibiotics, such as first-generation cephalosporins, is gaining increased attention once more as they have a reduced impact on the microbiota whilst treating infections. Here, the effects of two first-generation cephalosporins, compared with a third-generation cephalosporin, on the human microbiota were investigated and their propensity to induce simulated CDI. METHODS: Three in vitro chemostat models, which simulate the physiochemical conditions of the human colon, were seeded with a human faecal slurry and instilled with either narrow-spectrum cephalosporins, cefalexin and cefradine, or a broad-spectrum cephalosporin, ceftriaxone, at concentrations reflective of colonic levels. RESULTS: Instillation of cefalexin was associated with reduced recoveries of Bifidobacterium and Enterobacteriaceae; however, Clostridium spp. recoveries remained unaffected. Cefradine exposure was associated with decreased recoveries of Bifidobacterium spp., Bacteroides spp. and Enterobacteriaceae. These changes were not associated with induction of CDI, as we observed a lack of C. difficile spore germination/proliferation, thus no toxin was detected. This is in contrast to a model exposed to ceftriaxone, where CDI was observed. CONCLUSIONS: These model data suggest that the minimal impact of first-generation cephalosporins, namely cefalexin and cefradine, on the intestinal microbiota results in a low propensity to induce CDI.

Trehalose-Induced Remodelling of the Human Microbiota Affects Clostridioides difficile Infection Outcome in an In Vitro Colonic Model: A Pilot Study.

Within the human intestinal tract, dietary, microbial- and host-derived compounds are used as signals by many pathogenic organisms, including Clostridioides difficile. Trehalose has been reported to enhance virulence of certain C. difficile ribotypes; however, such variants are widespread and not correlated with clinical outcomes for patients suffering from C. difficile infection (CDI). Here, we make preliminary observations on how trehalose supplementation affects the microbiota in an in vitro model and show that trehalose-induced changes can reduce the outgrowth of C. difficile, preventing simulated CDI. Three clinically reflective human gut models simulated the effects of sugar (trehalose or glucose) or saline ingestion on the microbiota. Models were instilled with sugar or saline and further exposed to C. difficile spores. The recovery of the microbiota following antibiotic treatment and CDI induction was monitored in each model. The human microbiota remodelled to utilise the bioavailable trehalose. Clindamycin induction caused simulated CDI in models supplemented with either glucose or saline; however, trehalose supplementation did not result in CDI, although limited spore germination did occur. The absence of CDI in trehalose model was associated with enhanced abundances of Finegoldia, Faecalibacterium and Oscillospira, and reduced abundances of Klebsiella and Clostridium spp., compared with the other models. Functional analysis of the microbiota in the trehalose model revealed differences in the metabolic pathways, such as amino acid metabolism, which could be attributed to prevention of CDI. Our data show that trehalose supplementation remodelled the microbiota, which prevented simulated CDI, potentially due to enhanced recovery of nutritionally competitive microbiota against C. difficile.

Biofilms harbour Clostridioides difficile, serving as a reservoir for recurrent infection.

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.

Eravacycline, a novel tetracycline derivative, does not induce Clostridioides difficile infection in an in vitro human gut model.

OBJECTIVES: The approval of new antibiotics is essential to combat infections caused by antimicrobial-resistant pathogens; however, such agents should be tested to determine their effect on the resident microbiota and propensity to select for opportunistic pathogens, such as Clostridioides difficile. Eravacycline is a new antibiotic for the treatment of complicated intra-abdominal infections. Here, we determined the effects of eravacycline compared with moxifloxacin on the microbiota and if these were conducive to induction of C. difficile infection (CDI). METHODS: We seeded in vitro chemostat models, which simulate the physiological conditions of the human colon, with a human faecal slurry and instilled gut-reflective concentrations of either eravacycline or moxifloxacin. RESULTS: Eravacycline instillation was associated with decreased Bifidobacterium, Lactobacillus and Clostridium species, which recovered 1 week after exposure. However, Bacteroides spp. levels decreased to below the limit of detection and did not recover prior to the end of the experiment. Post-eravacycline, a bloom of aerobic bacterial species occurred, including Enterobacteriaceae, compared with pre-antibiotic, which remained high for the duration of the experiment. These changes in microbiota were not associated with induction of CDI, as we observed a lack of C. difficile spore germination and thus no toxin was detected. Moxifloxacin exposure sufficiently disrupted the microbiota to induce simulated CDI, where C. difficile spore germination, outgrowth and toxin production were seen. CONCLUSIONS: These model data suggest that, despite the initial impact of eravacycline on the intestinal microbiota, similar to clinical trial data, this novel tetracycline has a low propensity to induce CDI.

Is there a causal relationship between trehalose consumption and Clostridioides difficile infection?

PURPOSE OF REVIEW: Trehalose metabolism appears to play a role in the pathogenicity of some microbes. It has been claimed that trehalose consumption may be a risk factor for Clostridioides difficile infection (CDI), but the evidence for a causal link is contentious. RECENT FINDINGS: Epidemic ribotypes of C. difficile harbour mutations or have acquired extra genes that mean these strains can utilize lower concentrations of bioavailable trehalose, providing a competitive metabolic advantage in some CDI animal models. By contrast, evidence has emerged to show that trehalose-induced microbiota changes can help protect/reduce CDI in other models. In addition, C. difficile trehalose metabolic variants are widespread among epidemic and nonepidemic ribotypes alike, and the occurrence of these trehalose variants was not associated with increase disease severity or mortality. SUMMARY: Currently, there is no proven causal association between the incidence or severity of human CDI and the presence of trehalose metabolism variants. Furthermore, microbial metabolism reduces trehalose bioavailability, potentially removing this competitive advantage for C. difficile trehalose metabolism variants. Taken together, trehalose consumed as part of a normal diet has no increased risk of CDI.

A Novel, Orally Delivered Antibody Therapy and Its Potential to Prevent Clostridioides difficile Infection in Pre-clinical Models.

Clostridioides difficile infection (CDI) is a toxin-mediated infection in the gut and a major burden on healthcare facilities worldwide. We rationalized that it would be beneficial to design an antibody therapy that is delivered to, and is active at the site of toxin production, rather than neutralizing the circulating and luminal toxins after significant damage of the layers of the intestines has occurred. Here we describe a highly potent therapeutic, OraCAb, with high antibody titers and a formulation that protects the antibodies from digestion/inactivation in the gastrointestinal tract. The potential of OraCAb to prevent CDI in an in vivo hamster model and an in vitro human colon model was assessed. In the hamster model we optimized the ratio of the antibodies against each of the toxins produced by C. difficile (Toxins A and B). The concentration of immunoglobulins that is effective in a hamster model of CDI was determined. A highly significant difference in animal survival for those given an optimized OraCAb formulation versus an untreated control group was observed. This is the first study testing the effect of oral antibodies for treatment of CDI in an in vitro gut model seeded with a human fecal inoculum. Treatment with OraCAb successfully neutralized toxin production and did not interfere with the colonic microbiota in this model. Also, treatment with a combination of vancomycin and OraCAb prevented simulated CDI recurrence, unlike vancomycin therapy alone. These data demonstrate the efficacy of OraCAb formulation for the treatment of CDI in pre-clinical models.

Method comparison for the direct enumeration of bacterial species using a chemostat model of the human colon.

BACKGROUND: Clostridioides difficile infection (CDI) has a high recurrent infection rate. Faecal microbiota transplantation (FMT) has been used successfully to treat recurrent CDI, but much remains unknown about the human gut microbiota response to replacement therapies. In this study, antibiotic-mediated dysbiosis of gut microbiota and bacterial growth dynamics were investigated by two quantitative methods: real-time quantitative PCR (qPCR) and direct culture enumeration, in triple-stage chemostat models of the human colon. Three in vitro models were exposed to clindamycin to induce simulated CDI. All models were treated with vancomycin, and two received an FMT. Populations of total bacteria, Bacteroides spp., Lactobacillus spp., Enterococcus spp., Bifidobacterium spp., C. difficile, and Enterobacteriaceae were monitored using both methods. Total clostridia were monitored by selective culture. Using qPCR analysis, we additionally monitored populations of Prevotella spp., Clostridium coccoides group, and Clostridium leptum group. RESULTS: Both methods showed an exacerbation of disruption of the colonic microbiota following vancomycin (and earlier clindamycin) exposure, and a quicker recovery (within 4 days) of the bacterial populations in the models that received the FMT. C. difficile proliferation, consistent with CDI, was also observed by both qPCR and culture. Pearson correlation coefficient showed an association between results varying from 98% for Bacteroides spp., to 62% for Enterobacteriaceae. CONCLUSIONS: Generally, a good correlation was observed between qPCR and bacterial culture. Overall, the molecular assays offer results in real-time, important for treatment efficacy, and allow the monitoring of additional microbiota groups. However, individual quantification of some genera (e.g. clostridia) might not be possible without selective culture.

Clostridium difficile trehalose metabolism variants are common and not associated with adverse patient outcomes when variably present in the same lineage.

BACKGROUND: Clostridium difficile ribotype-027, ribotype-078, and ribotype-017 are virulent and epidemic lineages. Trehalose metabolism variants in these ribotypes, combined with increased human trehalose consumption, have been hypothesised to have contributed to their emergence and virulence. METHODS: 5232 previously whole-genome sequenced C. difficile isolates were analysed. Clinical isolates were used to investigate the impact of trehalose metabolism variants on mortality. Import data were used to estimate changes in dietary trehalose. Ribotype-027 virulence was investigated in a clinically reflective gut model. FINDINGS: Trehalose metabolism variants found in ribotype-027 and ribotype-017 were widely distributed throughout C. difficile clade-2 and clade-4 in 24/29 (83%) and 10/11 (91%) of sequence types (STs), respectively. The four-gene trehalose metabolism cluster described in ribotype-078 was common in genomes from all five clinically-important C. difficile clades (40/167 [24%] STs). The four-gene cluster was variably present in 208 ribotype-015 infections (98 [47%]); 27/208 (13%) of these patients died within 30-days of diagnosis. Adjusting for age, sex, and infecting ST, there was no association between 30-day all-cause mortality and the four-gene cluster (OR 0.36 [95%CI 0.09-1.34, p = 0.13]). Synthetic trehalose imports in the USA, UK, Germany and the EU were  < 1 g/capita/year during 2000-2006, and  < 9 g/capita/year 2007-2012, compared with dietary trehalose from natural sources of ~100 g/capita/year. Trehalose supplementation did not increase ribotype-027 virulence in a clinically-validated gut model. INTERPRETATION: Trehalose metabolism variants are common in C. difficile. Increases in total dietary trehalose during the early-mid 2000s C. difficile epidemic were likely relatively minimal. Alternative explanations are required to explain why ribotype-027, ribotype-078 and ribotype-017 have been successful. FUNDING: National Institute for Health Research. Gut model experiments only: Hayashibara Co. Ltd.

Omadacycline Gut Microbiome Exposure Does Not Induce Clostridium difficile Proliferation or Toxin Production in a Model That Simulates the Proximal, Medial, and Distal Human Colon.

A clinically reflective model of the human colon was used to investigate the effects of the broad-spectrum antibiotic omadacycline on the gut microbiome and the subsequent potential to induce simulated Clostridium difficile infection (CDI). Triple-stage chemostat gut models were inoculated with pooled human fecal slurry from healthy volunteers (age, ≥60 years). Models were challenged twice with 107 CFU C. difficile spores (PCR ribotype 027). Omadacycline effects were assessed in a single gut model. Observations were confirmed in a parallel study with omadacycline and moxifloxacin. Antibiotic instillation was performed once daily for 7 days. The models were observed for 3 weeks postantibiotic challenge. Gut microbiota populations and C. difficile total viable and spore counts were enumerated daily by culture. Cytotoxin titers and antibiotic concentrations were also measured. Gut microbiota populations were stable before antibiotic challenge. Moxifloxacin instillation caused an ∼4 log10 CFU/ml decline in enterococci and Bacteroides fragilis group populations and an ∼3 log10 CFU/ml decline in bifidobacteria and lactobacilli, followed by simulated CDI (vegetative cell proliferation and detectable toxin). In both models, omadacycline instillation decreased populations of bifidobacteria (∼8 log10 CFU/ml), B. fragilis group populations (7 to 8 log10 CFU/ml), lactobacilli (2 to 6 log10 CFU/ml), and enterococci (4 to 6 log10 CFU/ml). Despite these microbial shifts, there was no evidence of C. difficile bacteria germination or toxin production. In contrast to moxifloxacin, omadacycline exposure did not facilitate simulated CDI, suggesting this antibiotic may have a low propensity to induce CDI in the clinical setting.

New class of precision antimicrobials redefines role of Clostridium difficile S-layer in virulence and viability.

There is a medical need for antibacterial agents that do not damage the resident gut microbiota or promote the spread of antibiotic resistance. We recently described a prototypic precision bactericidal agent, Av-CD291.2, which selectively kills specific Clostridium difficile strains and prevents them from colonizing mice. We have since selected two Av-CD291.2-resistant mutants that have a surface (S)-layer-null phenotype due to distinct point mutations in the slpA gene. Using newly identified bacteriophage receptor binding proteins for targeting, we constructed a panel of Avidocin-CDs that kills diverse C. difficile isolates in an S-layer sequence-dependent manner. In addition to bacteriophage receptor recognition, characterization of the mutants also uncovered important roles for S-layer protein A (SlpA) in sporulation, resistance to innate immunity effectors, and toxin production. Surprisingly, S-layer-null mutants were found to persist in the hamster gut despite a complete attenuation of virulence. These findings suggest antimicrobials targeting virulence factors dispensable for fitness in the host force pathogens to trade virulence for viability and would have clear clinical advantages should resistance emerge. Given their exquisite specificity for the pathogen, Avidocin-CDs have substantial therapeutic potential for the treatment and prevention of C. difficile infections.

Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains.

The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment.

Bacteriophage Combinations Significantly Reduce Clostridium difficile Growth In Vitro and Proliferation In Vivo.

The microbiome dysbiosis caused by antibiotic treatment has been associated with both susceptibility to and relapse of Clostridium difficile infection (CDI). Bacteriophage (phage) therapy offers target specificity and dose amplification in situ, but few studies have focused on its use in CDI treatment. This mainly reflects the lack of strictly virulent phages that target this pathogen. While it is widely accepted that temperate phages are unsuitable for therapeutic purposes due to their transduction potential, analysis of seven C. difficile phages confirmed that this impact could be curtailed by the application of multiple phage types. Here, host range analysis of six myoviruses and one siphovirus was conducted on 80 strains representing 21 major epidemic and clinically severe ribotypes. The phages had complementary coverage, lysing 18 and 62 of the ribotypes and strains tested, respectively. Single-phage treatments of ribotype 076, 014/020, and 027 strains showed an initial reduction in the bacterial load followed by the emergence of phage-resistant colonies. However, these colonies remained susceptible to infection with an unrelated phage. In contrast, specific phage combinations caused the complete lysis of C. difficile in vitro and prevented the appearance of resistant/lysogenic clones. Using a hamster model, the oral delivery of optimized phage combinations resulted in reduced C. difficile colonization at 36 h postinfection. Interestingly, free phages were recovered from the bowel at this time. In a challenge model of the disease, phage treatment delayed the onset of symptoms by 33 h compared to the time of onset of symptoms in untreated animals. These data demonstrate the therapeutic potential of phage combinations to treat CDI.

LOV-based reporters for fluorescence imaging.

Chromophore-binding domains from plant and bacterial photoreceptor proteins have recently gathered increasing attention as new sources of genetically encoded fluorescent proteins (FPs). In particular, FPs based on the flavin-binding LOV (light, oxygen, or voltage sensing) domain offer advantages over green fluorescent protein (GFP) owing to their smaller size, pH and thermal stability, utility under anaerobic conditions and their ability to generate reactive oxygen species. This review focuses on the potential applications of this emerging class of fluorescent reporters, discusses the advantages and limitations of LOV-based FPs, whilst offering insights regarding the further development of this technology for bioimaging and photodynamic therapy.

The post-translational modification of the Clostridium difficile flagellin affects motility, cell surface properties and virulence.

Clostridium difficile is a prominent nosocomial pathogen, proliferating and causing enteric disease in individuals with a compromised gut microflora. We characterized the post-translational modification of flagellin in C. difficile 630. The structure of the modification was solved by nuclear magnetic resonance and shown to contain an N-acetylglucosamine substituted with a phosphorylated N-methyl-l-threonine. A reverse genetics approach investigated the function of the putative four-gene modification locus. All mutants were found to have truncated glycan structures by LC-MS/MS, taking into account bioinformatic analysis, we propose that the open reading frame CD0241 encodes a kinase involved in the transfer of the phosphate to the threonine, the CD0242 protein catalyses the addition of the phosphothreonine to the N-acetylglucosamine moiety and CD0243 transfers the methyl group to the threonine. Some mutations affected motility and caused cells to aggregate to each other and abiotic surfaces. Altering the structure of the flagellin modification impacted on colonization and disease recurrence in a murine model of infection, showing that alterations in the surface architecture of C. difficile vegetative cells can play a significant role in disease. We show that motility is not a requirement for colonization, but that colonization was compromised when the glycan structure was incomplete.