Glycopyrronium 320 µg/mL in children and adolescents with severe sialorrhoea and neurodisabilities: A randomized, double-blind, placebo-controlled trial.

AIM: To investigate the efficacy, safety, and impact on quality of life (QoL) of an oral formulation of 320 µg/mL glycopyrronium designed for children. METHOD: A double-blind, placebo-controlled SALIVA (Sialanar plus orAl rehabiLitation against placebo plus oral rehabilitation for chIldren and adolescents with seVere sialorrhoeA and neurodisabilities) trial was conducted. Children (3-17 years) with neurodisabilities and severe sialorrhoea (modified Teachers Drooling Scale ≥6) were randomized to 320 µg/mL glycopyrronium or placebo, in addition to non-pharmacological standard care. RESULTS: Of 87 participants, 44 were aged 10 years or under and 43 had cerebral palsy. The primary endpoint, change in total Drooling Impact Scale (DIS) score from baseline to day 84, was significantly greater (improved) with 320 µg/mL glycopyrronium versus placebo (median [quartile 1, quartile 3] -29.5 [-44.5, 0] vs -1 [-16, 5]; p < 0.001), an effect also observed at day 28 (median - 25 vs -2; p < 0.01). Significant reduction in bibs/clothes used per day was seen with glycopyrronium versus placebo at day 84 (median - 2 vs 0; p < 0.01). Glycopyrronium significantly improved DIS items 9 and 10 related to the extent that drooling affects the child's and family's life (p ≤ 0.03). Adverse events were reported by 77.3% and 69.8% of children with glycopyrronium and placebo respectively; the most common treatment-related adverse event was constipation (20.5% and 16.3%). INTERPRETATION: The formulation of 320 µg/mL glycopyrronium significantly improved drooling and reduced its impact on QoL, with good tolerability in children with neurodisabilities. WHAT THIS PAPER ADDS: The formulation of 320 µg/mL glycopyrronium significantly improved Drooling Impact Scale score versus placebo at day 84. The formulation reduced the impact of drooling on the child's and family's quality of life. There were no safety or tolerability concerns with this specific formulation.

Mucosal Immunization Has Benefits over Traditional Subcutaneous Immunization with Group A Streptococcus Antigens in a Pilot Study in a Mouse Model.

Group A Streptococcus (GAS) is a major human pathogen for which there is no licensed vaccine. To protect against infection, a strong systemic and mucosal immune response is likely to be necessary to prevent initial colonization and any events that might lead to invasive disease. A broad immune response will be necessary to target the varied GAS serotypes and disease presentations. To this end, we designed a representative panel of recombinant proteins to cover the stages of GAS infection and investigated whether mucosal and systemic immunity could be stimulated by these protein antigens. We immunized mice sublingually, intranasally and subcutaneously, then measured IgG and IgA antibody levels and functional activity through in vitro assays. Our results show that both sublingual and intranasal immunization in the presence of adjuvant induced both systemic IgG and mucosal IgA. Meanwhile, subcutaneous immunization generated only a serum IgG response. The antibodies mediated binding and killing of GAS cells and blocked binding of GAS to HaCaT cells, particularly following intranasal and subcutaneous immunizations. Further, antigen-specific assays revealed that immune sera inhibited cleavage of IL-8 by SpyCEP and IgG by Mac/IdeS. These results demonstrate that mucosal immunization can induce effective systemic and mucosal antibody responses. This finding warrants further investigation and optimization of humoral and cellular responses as a viable alternative to subcutaneous immunization for urgently needed GAS vaccines.

Randomised, double-blind, placebo-controlled trial of glycopyrronium in children and adolescents with severe sialorrhoea and neurodisabilities: protocol of the SALIVA trial.

INTRODUCTION: Severe sialorrhoea is a common, distressing problem in children/adolescents with neurodisabilities, which has adverse health and social consequences. The SALIVA trial is designed to evaluate the efficacy and safety of a paediatric-specific oral solution of glycopyrronium along with its impact on quality-of-life (QoL), which has been lacking from previous trials of sialorrhoea treatments. METHODS AND ANALYSIS: A double-blind, placebo-controlled, randomised phase IV trial is ongoing in several centres across France. Eighty children aged 3-17 years with severe sialorrhoea (≥6 on the modified Teachers Drooling Scale) related to chronic neurological disorders in whom non-pharmacological standard of care has already been implemented or has failed, will be recruited. Patients will be randomised 1:1 to receive a 2 mg/5 mL solution of glycopyrronium bromide (Sialanar 320 µg/mL glycopyrronium) or placebo three times daily during a 3-month blinded period. After Day 84, participants will be invited into a 6-month, open-label study extension period, where they will all receive glycopyrronium. The primary endpoint of the double-blind period will be the change from baseline to Day 84 in the Drooling Impact Scale (DIS), a validated measure to assess sialorrhoea. A series of secondary efficacy endpoints involving change in total DIS, specific DIS items and response (DIS improvement ≥13.6 points) will be analysed in a prespecified hierarchy. QoL data will be collected from parents, caregivers and patients where possible using specific DIS questions and DISABKIDS questionnaires. Safety endpoints, including adverse events, will be assessed throughout the trial periods. ETHICS AND DISSEMINATION: In total, 87 children have been recruited and recruitment is now complete. Final results are expected by the end of 2023. Findings will be presented at conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: EudraCT 2020-005534-15.

Streptococcus pyogenes Colonization in Children Aged 24-59 Months in the Gambia: Impact of Live Attenuated Influenza Vaccine and Associated Serological Responses.

BACKGROUND: Immunity to Streptococcus pyogenes in high burden settings is poorly understood. We explored S. pyogenes nasopharyngeal colonization after intranasal live attenuated influenza vaccine (LAIV) among Gambian children aged 24-59 months, and resulting serological response to 7 antigens. METHODS: A post hoc analysis was performed in 320 children randomized to receive LAIV at baseline (LAIV group) or not (control). S. pyogenes colonization was determined by quantitative polymerase chain reaction (qPCR) on nasopharyngeal swabs from baseline (day 0), day 7, and day 21. Anti-streptococcal IgG was quantified, including a subset with paired serum before/after S. pyogenes acquisition. RESULTS: The point prevalence of S. pyogenes colonization was 7%-13%. In children negative at day 0, S. pyogenes was detected at day 7 or 21 in 18% of LAIV group and 11% of control group participants (P = .12). The odds ratio (OR) for colonization over time was significantly increased in the LAIV group (day 21 vs day 0 OR, 3.18; P = .003) but not in the control group (OR, 0.86; P = .79). The highest IgG increases following asymptomatic colonization were seen for M1 and SpyCEP proteins. CONCLUSIONS: Asymptomatic S. pyogenes colonization appears modestly increased by LAIV, and may be immunologically significant. LAIV could be used to study influenza-S. pyogenes interactions. Clinical Trials Registration. NCT02972957.

Progress towards a glycoconjugate vaccine against Group A Streptococcus.

The Group A Carbohydrate (GAC) is a defining feature of Group A Streptococcus (Strep A) or Streptococcus pyogenes. It is a conserved and simple polysaccharide, comprising a rhamnose backbone and GlcNAc side chains, further decorated with glycerol phosphate on approximately 40% GlcNAc residues. Its conservation, surface exposure and antigenicity have made it an interesting focus on Strep A vaccine design. Glycoconjugates containing this conserved carbohydrate should be a key approach towards the successful mission to build a universal Strep A vaccine candidate. In this review, a brief introduction to GAC, the main carbohydrate component of Strep A bacteria, and a variety of published carrier proteins and conjugation technologies are discussed. Components and technologies should be chosen carefully for building affordable Strep A vaccine candidates, particularly for low- and middle-income countries (LMICs). Towards this, novel technologies are discussed, such as the prospective use of bioconjugation with PglB for rhamnose polymer conjugation and generalised modules for membrane antigens (GMMA), particularly as low-cost solutions to vaccine production. Rational design of "double-hit" conjugates encompassing species specific glycan and protein components would be beneficial and production of a conserved vaccine to target Strep A colonisation without invoking an autoimmune response would be ideal.

Multicomponent Vaccines against Group A Streptococcus Can Effectively Target Broad Disease Presentations.

Group A Streptococcus (GAS) is an important global human pathogen, with a wide range of disease presentations, from mild mucosal infections like pharyngitis to invasive diseases such as toxic shock syndrome. The effect on health and mortality from GAS infections is substantial worldwide, particularly from autoimmune sequelae-like rheumatic heart disease (RHD), and there is currently no licenced vaccine. We investigated protein antigens targeting a broad range of GAS disease presentations as vaccine components in individual and combination formulations. The potency and functional immunity generated were evaluated and compared between groups. Antibodies against all components were found in pooled human IgG (IVIG) and an immune response generated following the subcutaneous immunisation of mice. A combination immunisation showed a reduction in IgG response for SpyCEP but an increase for Cpa and Mac-1 (IdeS). An opsonophagocytosis assay (OPA) showed the killing of GAS with immune sera against M protein and combination groups, with a lower killing activity observed for immune sera against other individual antigens. Specific antigen assays showed functional immunity against SpyCEP and Mac-1 from both individual and combination immunisations, with the activity correlating with antibody titres. However, efficient blocking of the binding activity of Cpa to collagen I and fibronectin could not be demonstrated with immune sera or purified IgG. Our data indicate that combination immunisations, while effective at covering a broader range of virulence factors, can also affect the immune response generated. Further, our results showed that an OPA alone is inadequate for understanding protection from vaccination, particularly when considering protection from immune evasion factors and evaluation of the colonisation leading to pharyngitis.

Extracellular DNA, cell surface proteins and c-di-GMP promote biofilm formation in Clostridioides difficile.

Clostridioides difficile is the leading cause of nosocomial antibiotic-associated diarrhoea worldwide, yet there is little insight into intestinal tract colonisation and relapse. In many bacterial species, the secondary messenger cyclic-di-GMP mediates switching between planktonic phase, sessile growth and biofilm formation. We demonstrate that c-di-GMP promotes early biofilm formation in C. difficile and that four cell surface proteins contribute to biofilm formation, including two c-di-GMP regulated; CD2831 and CD3246, and two c-di-GMP-independent; CD3392 and CD0183. We demonstrate that C. difficile biofilms are composed of extracellular DNA (eDNA), cell surface and intracellular proteins, which form a protective matrix around C. difficile vegetative cells and spores, as shown by a protective effect against the antibiotic vancomycin. We demonstrate a positive correlation between biofilm biomass, sporulation frequency and eDNA abundance in all five C. difficile lineages. Strains 630 (RT012), CD305 (RT023) and M120 (RT078) contain significantly more eDNA in their biofilm matrix than strains R20291 (RT027) and M68 (RT017). DNase has a profound effect on biofilm integrity, resulting in complete disassembly of the biofilm matrix, inhibition of biofilm formation and reduced spore germination. The addition of exogenous DNase could be exploited in treatment of C. difficile infection and relapse, to improve antibiotic efficacy.

Clostridium difficile clade 3 (RT023) have a modified cell surface and contain a large transposable island with novel cargo.

The major global pathogen Clostridium difficile (recently renamed Clostridioides difficile) has large genetic diversity including multiple mobile genetic elements. In this study, whole genome sequencing of 86 strains from the poorly characterised clade 3, predominantly PCR ribotype (RT)023, of C. difficile revealed distinctive surface architecture characteristics and a large mobile genetic island. These strains have a unique sortase substrate phenotype compared with well-characterised strains of C. difficile, and loss of the phage protection protein CwpV. A large genetic insertion (023_CTnT) comprised of three smaller elements (023_CTn1-3) is present in 80/86 strains analysed in this study, with genes common among other bacterial strains in the gut microbiome. Novel cargo regions of 023_CTnT include genes encoding a sortase, putative sortase substrates, lantibiotic ABC transporters and a putative siderophore biosynthetic cluster. We demonstrate the excision of 023_CTnT and sub-elements 023_CTn2 and 023_CTn3 from the genome of RT023 reference strain CD305 and the transfer of 023_CTn3 to a non-toxigenic C. difficile strain, which may have implications for the use of non-toxigenic C. difficile strains as live attenuated vaccines. Finally, we show that the genes within the island are expressed in a regulated manner in C. difficile RT023 strains conferring a distinct "niche adaptation".

The Streptococcos suis sortases SrtB and SrtF are essential for disease in pigs.

The porcine pathogen Streptococcus suis colonizes the upper respiratory tracts of pigs, potentially causing septicaemia, meningitis and death, thus placing a severe burden on the agricultural industry worldwide. It is also a zoonotic pathogen that is known to cause systemic infections and meningitis in humans. Understanding how S. suis colonizes and interacts with its hosts is relevant for future strategies of drug and vaccine development. As with other Gram-positive bacteria, S. suis utilizes enzymes known as sortases to attach specific proteins bearing cell wall sorting signals to its surface, where they can play a role in host-pathogen interactions. The surface proteins of bacteria are often important in adhesion to and invasion of host cells. In this study, markerless in-frame deletion mutants of the housekeeping sortase srtA and the two pilus-associated sortases, srtB and srtF, were generated and their importance in S. suis infections was investigated. We found that all three of these sortases are essential to disease in pigs, concluding that their cognate-sorted proteins may also be useful in protecting pigs against infection.

Disparate subcellular location of putative sortase substrates in Clostridium difficile.

Clostridium difficile is a gastrointestinal pathogen but how the bacterium colonises this niche is still little understood. Sortase enzymes covalently attach specific bacterial proteins to the peptidoglycan cell wall and are often involved in colonisation by pathogens. Here we show C. difficile proteins CD2537 and CD3392 are functional substrates of sortase SrtB. Through manipulation of the C-terminal regions of these proteins we show the SPKTG motif is essential for covalent attachment to the cell wall. Two additional putative substrates, CD0183 which contains an SPSTG motif, and CD2768 which contains an SPQTG motif, are not cleaved or anchored to the cell wall by sortase. Finally, using an in vivo asymmetric cleavage assay, we show that despite containing a conserved SPKTG motif, in the absence of SrtB these proteins are localised to disparate cellular compartments.

Draft Genome Sequence of Robinsoniella peoriensis 6600698, a Confounder of Clostridium difficile Diagnosis.

Robinsoniella peoriensis is a Gram-positive, strictly anaerobic, spore-forming, rod-shaped organism. Here, we report the draft genome of R. peoriensis 6600698, initially classified as Clostridium difficile due to growth on selective agar, a fecal gdh PCR-positive result, and clinical symptoms. R. peoriensis is a potential confounder of C. difficile diagnosis.

Review of Antimicrobial Resistance in the Environment and Its Relevance to Environmental Regulators.

The environment is increasingly being recognized for the role it might play in the global spread of clinically relevant antibiotic resistance. Environmental regulators monitor and control many of the pathways responsible for the release of resistance-driving chemicals into the environment (e.g., antimicrobials, metals, and biocides). Hence, environmental regulators should be contributing significantly to the development of global and national antimicrobial resistance (AMR) action plans. It is argued that the lack of environment-facing mitigation actions included in existing AMR action plans is likely a function of our poor fundamental understanding of many of the key issues. Here, we aim to present the problem with AMR in the environment through the lens of an environmental regulator, using the Environment Agency (England's regulator) as an example from which parallels can be drawn globally. The issues that are pertinent to environmental regulators are drawn out to answer: What are the drivers and pathways of AMR? How do these relate to the normal work, powers and duties of environmental regulators? What are the knowledge gaps that hinder the delivery of environmental protection from AMR? We offer several thought experiments for how different mitigation strategies might proceed. We conclude that: (1) AMR Action Plans do not tackle all the potentially relevant pathways and drivers of AMR in the environment; and (2) AMR Action Plans are deficient partly because the science to inform policy is lacking and this needs to be addressed.

Cyclic diGMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage.

In Gram-positive pathogens, surface proteins may be covalently anchored to the bacterial peptidoglycan by sortase, a cysteine transpeptidase enzyme. In contrast to other Gram-positive bacteria, only one single sortase enzyme, SrtB, is conserved between strains of Clostridium difficile. Sortase-mediated peptidase activity has been reported in vitro, and seven potential substrates have been identified. Here, we demonstrate the functionality of sortase in C. difficile. We identify two sortase-anchored proteins, the putative adhesins CD2831 and CD3246, and determine the cell wall anchor structure of CD2831. The C-terminal PPKTG sorting motif of CD2831 is cleaved between the threonine and glycine residues, and the carboxyl group of threonine is amide-linked to the side chain amino group of diaminopimelic acid within the peptidoglycan peptide stem. We show that CD2831 protein levels are elevated in the presence of high intracellular cyclic diGMP (c-diGMP) concentrations, in agreement with the control of CD2831 expression by a c-diGMP-dependent type II riboswitch. Low c-diGMP levels induce the release of CD2831 and presumably CD3246 from the surface of cells. This regulation is mediated by proteolytic cleavage of CD2831 and CD3246 by the zinc metalloprotease ZmpI, whose expression is controlled by a type I c-diGMP riboswitch. These data reveal a novel regulatory mechanism for expression of two sortase substrates by the secondary messenger c-diGMP, on which surface anchoring is dependent.

Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII.

Gram-positive surface proteins can be covalently or non-covalently anchored to the cell wall and can impart important properties on the bacterium in respect of cell envelope organisation and interaction with the environment. We describe here a mechanism of protein anchoring involving tandem CWB2 motifs found in a large number of cell wall proteins in the Firmicutes. In the Clostridium difficile cell wall protein family, we show the three tandem repeats of the CWB2 motif are essential for correct anchoring to the cell wall. CWB2 repeats are non-identical and cannot substitute for each other, as shown by the secretion into the culture supernatant of proteins containing variations in the patterns of repeats. A conserved Ile Leu Leu sequence within the CWB2 repeats is essential for correct anchoring, although a preceding proline residue is dispensable. We propose a likely genetic locus encoding synthesis of the anionic polymer PSII and, using RNA knock-down of key genes, reveal subtle effects on cell wall composition. We show that the anionic polymer PSII binds two cell wall proteins, SlpA and Cwp2, and these interactions require the CWB2 repeats, defining a new mechanism of protein anchoring in Gram-positive bacteria.

CbpA: a novel surface exposed adhesin of Clostridium difficile targeting human collagen.

Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudomembranous colitis. While the role of toxins in pathogenesis has been extensively described, the contribution of surface determinants to intestinal colonization is still poorly understood. We focused our study on a novel member of the MSCRAMM family, named CbpA (Collagen binding protein A), for its adhesive properties towards collagen. We demonstrate that CbpA, which carries an LPXTG-like cell wall anchoring domain, is expressed on the bacterial surface of C. difficile and that the recombinant protein binds at high affinity to collagens I and V (apparent Kd in the order of 10(-9 ) M). These findings were validated by confocal microscopy studies showing the colocalization of the protein with type I and V collagen fibres produced by human fibroblasts and mouse intestinal tissues. However, the collagen binding activity of the wild-type C. difficile 630 strain was indistinguishable to the cbpA knock-out strain. To overcome this apparent clostridial adherence redundancy, we engineered a Lactococcus lactis strain for the heterologous expression of CbpA. When exposed on the surface of L. lactis, CbpA significantly enhances the ability of the bacterium to interact with collagen and to adhere to ECM-producing cells. The binding activity of L. lactis-CbpA strain was prevented by an antiserum raised against CbpA, demonstrating the specificity of the interaction. These results suggest that CbpA is a newsurface-exposed adhesin contributing to the C. difficile interaction with the host.

Prognostic factors in cutaneous desmoplastic melanoma: a study of 252 patients.

BACKGROUND: Desmoplastic melanoma (DM) is a rare subtype of melanoma that is characterized by malignant spindle cells separated by prominent, fibrocollagenous stroma. Primary melanomas either may be entirely desmoplastic or almost entirely desmoplastic (pure DM [pDM]) or may exhibit a desmoplastic component admixed with a nondesmoplastic component (combined DM [cDM]). METHODS: Patients who were diagnosed between 1993 and 2007 at a single institution with clinically localized, primary cutaneous melanoma (PCM) that contained a desmoplastic component and who underwent sentinel lymph node (SLN) biopsy were identified. Clinical and pathologic features of the primary tumors were correlated with DM type, SLN status, and patient outcome. RESULTS: Two hundred fifty-two patients (167 men, 85 women) were identified (median age, 61 years). The median tumor thickness was 2.0 mm. One hundred twenty-three patients (48.8%) had pDM, and 129 patients (51.2%) had cDM. Overall, 17 patients (6.7%) had positive SLN status, including 12 patients with cDM and 5 patients with pDM. Because of the low SLN-positive rate, a statistically significant difference in SLN status between patients with cDM (8.5%) and patients with pDM (4.9%; P = .25) could not be demonstrated. Older patient age, being a man, positive SLN status, and increasing tumor thickness were associated significantly with poorer disease-free survival (P < .05), although only the latter 2 variables were independently predictive. In addition, cDM type (P = .017) was associated significantly and independently with a shorter time to recurrence. CONCLUSIONS: In this largest study to date of patients with DM who underwent SLN biopsy, the SLN-positive rate in patients with DM was lower than that in patients with conventional melanoma. The results indicated that DM type is associated significantly and independently with the time to recurrence and should be evaluated routinely in all patients with PCM. Cancer 2010. (c) 2010 American Cancer Society.

Sentinel node positive melanoma patients: prediction and prognostic significance of nonsentinel node metastases and development of a survival tree model.

BACKGROUND: Completion lymph node dissection (CLND) following positive sentinel node biopsy (SNB) for melanoma detects additional nonsentinel node (NSN) metastases in approximately 20% of cases. This study aimed to establish whether NSN status can be predicted, to determine its effect on survival, and to develop survival tree models for the sentinel node (SN) positive population. MATERIALS AND METHODS: Sydney Melanoma Unit (SMU) patients with at least 1 positive SN, meeting inclusion criteria and treated between October 1992 and June 2005, were identified from the Unit database. Survival characteristics, potential predictors of survival, and NSN status were assessed using the Kaplan-Meier method, Cox regression model, and logistic regression analyses, respectively. Classification tree analysis was performed to identify groups with distinctly different survival characteristics. RESULTS: A total of 323 SN-positive melanoma patients met the inclusion criteria. On multivariate analysis, age, gender, primary tumor thickness, mitotic rate, number of positive NSNs, or total number of positive nodes were statistically significant predictors of survival. NSN metastasis, found at CLND in 19% of patients, was only predicted to a statistically significant degree by ulceration. Multivariate analyses demonstrated that survival was more closely related to number of positive NSNs than total number of positive nodes. Classification tree analysis revealed 4 prognostically distinct survival groups. CONCLUSIONS: Patients with NSN metastases could not be reliably identified prior to CLND. Prognosis following CLND was more closely related to number of positive NSNs than total number of positive nodes. Classification tree analysis defined distinctly different survival groups more accurately than use of single-factor analysis.

Sentinel lymph node biopsy in pediatric and adolescent cutaneous melanoma patients.

BACKGROUND: The rarity of melanoma in young patients, particularly pediatric ones, has to date precluded any valid comparisons being made between young patients and adults undergoing sentinel lymph node biopsy (SLNB) for intermediate thickness localized melanoma. The present study takes advantage of the large Sydney Melanoma Unit (SMU) database to clarify this issue. MATERIALS AND METHODS: Clinical and pathologic data on pediatric and adolescent AJCC Stage I and II cutaneous melanoma patients aged <20 years undergoing SLNB at the SMU between January 1993 and February 2008 were reviewed. SLNB positivity rates and outcomes in these patients were compared with adult SMU patients. RESULTS: In 55 young patients, overall median tumor thickness was 1.7 mm (range, 0.6-5.2 mm) and overall SLNB positivity rate was 14 of 55 (25%), tumors tending to be thicker (median, 2.6 mm), and SLNB positivity rate higher (2 of 6; 33%) in patients aged <10 years. Of the 14 patients, 13 underwent immediate completion lymph node dissection (CLND); 2 patients had non-SLN metastases (15.4%). Only 0.7% of a total of 295 lymph nodes removed at CLND were involved with melanoma. In 14 SLNB-positive patients with follow-up data, 3 (21%) have died from melanoma after a median follow-up of 60 months, compared with 42% of 356 SLNB positive adults. CONCLUSIONS: Although the SLNB positivity rate was higher in pediatric and adolescent melanoma patients than in adults (25% vs. 17%, respectively), non-SLN positivity and melanoma-specific death rates were low.

A novel genetic switch controls phase variable expression of CwpV, a Clostridium difficile cell wall protein.

Clostridium difficile is a nosocomial pathogen that can cause severe gastrointestinal infections. C. difficile encodes a family of cell wall proteins, some of which are implicated in pathogenesis. Here we have characterized CwpV, the largest member of this family. CwpV is surface expressed and post-translationally processed in a manner analogous to the major S-layer protein SlpA. Expression of cwpV is phase variable, with approximately 5% of cells in a population expressing the protein under standard laboratory growth conditions. Upstream of cwpV, inverted repeats flank a 195 bp sequence which undergoes DNA inversion. Use of a gusA transcriptional reporter demonstrated that phase variation is mediated by DNA inversion; in one orientation cwpV is expressed while in the opposite orientation the gene is silent. The inversion region contains neither the promoter nor any of the open reading frame, therefore this system differs from previously described phase variation mechanisms. The cwpV promoter is located upstream of the inversion region and we propose a model of phase variation based on intrinsic terminator formation in the OFF transcript. A C. difficile site-specific recombinase able to catalyse the inversion has been identified.