Background Infections with Clostridioides difficile remain a significant global healthcare problem for which novel (preventive) treatment strategies are urgently needed. Controlled human infection models (CHIMs) can contribute to a better understanding of the disease and accelerate (novel) medicinal product development. Methods A robust production process for C difficile spores for experimental human administration was implemented. Direct-release capsules containing viable C difficile spores were formulated. The manufacturing process aligns with Good Manufacturing Practice (GMP) guidelines, including quality controls and release by a Qualified Person (QP). Results Selected nontoxigenic and toxigenic C difficile strains were used to create a master cell bank and working cell bank from which multiple batches of purified active substance were produced. Substance was then used to manufacture the final active product. The active product and its intermediate products passed all quality controls for identity, potency, purity and safety, and were individually released by the QP. Stability of active substance and product is confirmed up to 12 months. Conclusions Although there are no clear standardized international guidelines for challenge material production, we demonstrate that it is feasible to produce C difficile human challenge material for small-scale application in CHIM trials. This application of GMP principles to an unconventional production process of a bacterial spore-forming anaerobic challenge agent is an example for the production of challenge material as described in the auxiliary medicinal product guidelines of the European Medicines Agency and the US Food and Drug Administration.

Clostridioides difficile infection (CDI) is a toxin-mediated gastro-intestinal disease. Yet, C. difficile is a phylogenetically diverse species that includes many non-toxigenic strains. In general, these are understudied, despite having significant potential impact for our understanding of the colonization process and as therapeutic modalities. Here, we present an in-depth characterization—including the complete genome sequence—of the non-toxigenic C. difficile strain L-NTCD03. This strain belongs to PCR ribotype 416, clade 4 and multilocus sequence type 39. It is resistant to multiple antimicrobials, but not those used for treatment of CDI. We validated the relevance of the cfr(B) gene from this strain in antimicrobial resistance to clindamycin, linezolid, retapamulin and streptogramin A. We found the L-NTCD03 strain to be non-toxic in various assays. Altogether, L-NTCD03 is a promising candidate for developing into a live biotherapeutic product.

Objectives: Increasing resistance to antimicrobials used for the treatment of Clostridioides difficile infections necessitates reproducible antimicrobial susceptibility testing. Current guidelines take a one-sizefits-all approach and/or offer limited guidance. We investigated how the choice of medium affects measured MIC values across two sites. Methods: We determined MIC values for the antimicrobials fidaxomicin, metronidazole, and vancomycin for a representative collection of European C. difficile strains (n = 235) using agar dilution on three different media: Brucella Blood Agar (BBA), Fastidious Anaerobe Agar supplemented with horse blood (FAA-HB), and Wilkins-Chalgren (WC) agar. The study was conducted at two sites to compare reproducibility. Usability (ease of preparation of the media as well as read-out of the assay) was assessed through a survey. Results: We found that all media result in highly consistent aggregated MIC data for all antibiotics, with MIC50 and MIC90 within two-fold of each other across sites. For fidaxomin, MIC values on WC were lower than on the other media (MIC90: WC = 0.125-0.25 mg/L; BBA and FAA-HB = 0.5 mg/L). Metronidazole showed the lowest MIC on BBA and the highest on WC (MIC90: WC = 2 mg/L; BBA = 0.5-1 mg/L; FAAHB: 1-2 mg/L). For vancomycin, MIC values were similar across media (MIC90: all media = 1-2 mg/L). Though absolute values for individual isolates differed between sites, identified resistant isolates were similar. Results obtained on FAA-HB were most consistent between sites and results obtained on WC showed the most divergence. FAA-HB was positively evaluated in the usability survey. Discussion: This study shows medium-dependent differences in C. difficile MICs for at least two antimicrobials across two sites. We suggest the use of FAA-HB to align with general European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations for susceptibility testing of anaerobic bacteria and deposited reference strains for standard susceptibility testing of C. difficile to increase interlaboratory reproducibility. Jane Freeman, Clin Microbiol Infect 2025;31:1011 (c) 2025 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/).

End 2023, the UK Health Security Agency sent an alert about a new hypervirulent Clostridioides difficile PCR ribotype, ribotype 955 (RT955), causing slowly progressing infection clusters in hospitals in the Midlands. Between March 2018 and February 2022, surveillance of Clostridioides difficile infections (CDI) was performed in southern Serbia with centres providing medical services for approximately 750,000 inhabitants. Using the ECDC recommended protocol, clinical, epidemiological and microbiological data were collected. C. difficile RT955 was identified in 27 (7%) of 383 surveyed patients with CDI. Of 27 patients, 16 (59%) was older than 60 years and 19 (70%) were male. CDI was always associated with previous antibiotic therapy and had a hospital onset in 23 (85%) patients. The clinical presentation was milder than reported in UK. All sequenced strains belonged to multilocus sequence type (ST) 1 and were highly similar, with 0-1 alleles differences in a core genome multilocus sequence typing analysis. The strains differed clearly from the UK RT955 outbreak strain by whole genome sequencing and phenotypic susceptibility to metronidazole, lincosamides and rifampicin. Interestingly, a high-level erythromycin resistance was observed associated with the presence of the mrmA gene. Both the UK and Serbian RT955 strains contained gyrA_p.T82I associated with resistance to fluoroquinolone antimicrobials and carried the PnimBG promoter mutation, suggestive for haem-dependent metronidazole resistance. We conclude that C. difficile RT955 is present in southern Serbia since 2018. The Serbian RT955 strains differed clearly from a representative UK cluster strain.

IntroductionToxigenic strains of Clostridioides difficile are the leading cause of healthcare-associated bacterial infections in Poland. In the Silesian Voivodeship, with 4.5 million inhabitants, the incidence of C. difficile infection (CDI) in 2023 reached 65.1/100,000, slightly down from 70.0/100,000 in 2022. This study aimed to investigate the occurrence of toxigenic C. difficile ribotypes in hospitalized patients in Silesia.Materials and methodsA total of 130 stool samples from patients with confirmed antibiotic-associated diarrhea were tested. Multiplex PCR detected genes for GDH, toxins A/B, binary toxin CDT, and 16S rDNA. Ribotyping was performed by capillary PCR, and antibiotic susceptibility was tested with ETEST for 10 antibiotics.ResultsMultiplex PCR confirmed the presence of all toxins (A+B+CDT+) in 97 isolates. Six strains (6%) represented ribotype 955 (RT955), first reported in Poland in 2023. RT027 remained dominant (60%, n = 78). All isolates were susceptible to vancomycin, while 4% (5/130) showed metronidazole resistance.ConclusionRT027 remains prevalent among CDI cases in Silesia. The emergence of RT955, closely related to a UK epidemic strain, suggests a possible shared origin and epidemiological link.

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Background: Clostridioides difficile is a leading cause of infectious diarrhea in both humans and livestock. In particular, C. difficile strains belonging to sequence type (ST) 11 are common enteropathogens. The aim of this study was to determine the presence and genetic relatedness of C. difficile types in dairy cattle and calves. Method: Dutch dairy farms were visited between February and December 2021. Feces was collected from adult dairy cattle and calves of two age categories ( <4 weeks and 4 weeks -4 months). Fecal samples were also requested from dairy farmers, family members and employees. Fecal samples were cultured in an enrichment medium for 10 -15 days and subcultured on solid media for capillary PCR ribotyping and whole genome sequencing. Results: C. difficile was detected on 31 out of 157 (19.8%) dairy farms. The highest prevalence was found in calves <4 weeks (17.5%). None of the 99 human samples collected were positive. Thirty-seven cultured isolates belonged to 11 different PCR ribotypes (RT) of which RT695 (56.8%) and RT078/126 (16.2%) were most abundant. In the database of the Netherlands National Expertise Centre for C. difficile infections (CDI, >10.000 patient isolates), RT695 was found in only two patients with hospital -onset CDI, diagnosed in 2020 and 2021. Sequence analysis of 21 C. difficile RT695 from cattle revealed that all isolates belonged to clade 5, ST11 and contained genes encoding toxin A, toxin B and binary toxin. RT695 strains carried antimicrobial resistance genes typically found in clade 5 C. difficile . Groups of genetically related RT695 isolates were found between dairy farms, whereas identical strains were only present in individual farms. Conclusions: C. difficile was found in similar to 20% of dairy farms with a predominance of the relatively unknown RT695. Isolates of RT695 belonged to the same clade and sequence type as RT078/126, which is recognized as an important zoonotic type.

Clostridioides difficile strains of sequence type (ST) 37, primarily including PCR ribotype (RT) 017, are prevalent in mainland China. Our study aimed to compare the major virulence factors of an epidemic C. difficile isolate of ST37 type (Xy06) from China with the well-characterized C. difficile reference strains R20291 (RT027) and CD630E (ST54), as well as a Chinese ST54 strain (Xy07) isolated from the same hospital. The Xy06 genome was predicted to harbor two complete prophages and several transposon-like elements. Comparative analysis of PaLoc revealed a truncated tcdA gene, a functional tcdB gene, a functional tcdC gene, and well-conserved tcdR and tcdE genes. Phenotypic comparisons showed that Xy06 was a robust producer of TcdB, readily sporulated and germinated, and strongly bound to human gut epithelial cells. In a mouse model of C. difficile infection, Xy06 was more virulent than strains CD630E and Xy07 and was comparable to strain R20291 in virulence. Our data suggest the potential threat of the epidemic ST37 strains in China.

Aim: The aim was to highlight the incidence and epidemiology of C. difficile infections (CDI) in a tertiary Greek hospital during the COVID-19 pandemic.Methods: A single-center prospective observational cohort study was conducted (October 2021 until April 2022). 125 C. difficile isolates were cultured from hospitalized patients stool samples and screened by PCR for toxin A (tcdA), toxin B (tcdB), binary toxin (cdtA and cdtB) genes and the regulating gene of tcdC.Results: The incidence of CDI increased to 13.1 infections per 10,000 bed days. The most common PCR ribotypes identified included hypervirulent RT027-related RT181 (73.6%), presumably hypervirulent RT126 (8.0%) and toxin A negative RT017 (7.2%).Conclusion: Although the incidence of CDI increased significantly, the CDI epidemiology remained stable.

ABSTRACTClostridioides difficilePCR ribotype (RT) 002 is the second most prevalent RT in the Netherlands. In 2019, an increase inC. difficileinfections (CDI) due to a clonal RT002 strains was reported in Ireland. In retrospect, a significant increase in the proportion of CDI due to RT002 was detected in the Netherlands between May 2017-May 2018 (11.9% of 683 cases vs 6.5% of 865 in the previous year). Our aim was to analyze and characterize CDI due to RT002 over time and to assess whether RT002 isolates in the Netherlands were related to the Irish RT002 isolates.Over the period May 2009-April 2021, Dutch patients with CDI due to RT002 showed less severe CDI (22%) in comparison with hypervirulent RTs (RT027; 29% and RT078; 25%), similar to grouped data from other non-hypervirulent ribotypes (21%). However, RT002-associated CDI prior to the increased proportion (2009-2017) showed CDI severity similar to CDI associated with hypervirulent types, whereas this was no longer the case after the increase (2017-2021). Using core genome MLST, Dutch isolates were found not to be related to the Irish outbreak strain. Several Dutch strains clustered together without an evident epidemiological link, suggesting recent emergence.ImportanceClostridioides difficileis a significant cause of hospital-associated infections, making it relevant to understand the emergence and spread of different ribotypes. This study highlights a rise in cases of PCR ribotype 002 (RT002) in the Netherlands, suggesting changes in its prevalence. Despite the increase in RT002 infections, the severity of disease appears to have decreased and is lower compared to more virulent ribotypes. Further research is needed to understand the factors driving this change. Additionally, this study underscores some limitations of core genome multi-locus sequence typing (cgMLST). cgMLST is valuable for identifying genetic relationships, however it cannot always distinguish between widespread recently emerged strains and local transmission chains, making it challenging to determine the exact sources of infection. Continued surveillance and genetic characterization ofC. difficileare essential to better understand these trends and improve control measures.

Background: Data from the past decade indicates that Clostridioides difficile infection (CDI) is not only a nosocomial infection but is also increasingly recognized as a disease in the community. Objective: We aimed to study community-onset (CO) CDI in the various age groups in south Serbia with its clinical characteristics, risk factors and microbiological characterization. Methods: The study group included 93 patients with CO-CDI (median age 62). The control group consisted of 186 patients with community-onset diarrhea and stool samples negative tested for CDI. Results: Of all CDI cases diagnosed with a community onset, 74.19% had a previous contact with a healthcare facility in the previous 12 weeks, but 34.40% have no record on hospitalization in the previous 12 months. Using a multivariate statistical regression model, the following risk factors for CO-CDI development were found; antacid usage (OR 1/4 0.267, 95%C.I.:0.10e0.291, p < 0.01), chronic kidney disease (OR = 0.234, 95%C.I.:0.10e0.51, p < 0.01) and antibiotic use during the prior 2 months (OR = 0.061, 95%C.I.:0.02e0.17, p < 0.01), especially tetracycline's (OR = 0.146, 95% C.I.:0.07e0.22, p < 0.01) and cephalosporin's (OR = 0.110, 95%C.I.:0.14e0.42, p < 0.01). The most common ribotypes (RTs) detected in patients with CO-CDI were RT001 (32.3%) and RT027 (24.7%). All tested toxin producing C. difficile isolates were sensitive to metronidazole, vancomycin and tigecycline. A high rate of resistance to moxifloxacin (73.11%) and rifampicin (23.65%) was found. Conclusion: Patients with CO-CDI had frequently contact with healthcare facility in the previous 12 weeks. Restriction of antacid usage and of high-risk antibiotics in the community may help reduce the incidence of CO-CDI. (c) 2022 Elsevier Ltd. All rights reserved.

The perpetually evolving antimicrobial resistance (AMR) of C. difficile is an important contributor to its epidemiology and is a grave concern to global public health. This exacerbates the challenge of treating the infections caused by this multidrug-resistant causative organism of potentially life-threatening diarrhea.

Clostridioides difficile infections (CDI) have a high morbidity and mortality rate and have always been considered a nosocomial disease. Nonetheless, the number of cases of community-acquired CDI is increasing, and new evidence suggests additional C. difficile reservoirs exist. Pathogenic C. difficile strains have been found in livestock, domestic animals, and meat, so a zoonotic transmission has been proposed. Objective: The goal of this study was to isolate C. difficile strains in dogs at a veterinary clinic in Rio de Janeiro, Brazil, and characterize clinical and pathological findings associated with lower gastrointestinal tract disorders. Methods: Fifty stool samples and biopsy fragments from dogs were obtained and cultured in the CDBA selective medium. All suggestive C. difficile colonies were confirmed by MALDI-TOF MS and PCR (tpi gene). Vancomycin, metronidazole, moxifloxacin, erythromycin, and rifampicin were tested for antibiotic susceptibility. Biofilm, motility assays, and a PCR for the toxins (tcdA, tcdB, and cdtB), as well as ribotyping, were also performed. Results: Blood samples and colonic biopsy fragments were examined in C. difficile positive dogs. Ten animals (20%) tested positive for C. difficile by using stool samples, but not from biopsy fragments. Most C. difficile strains were toxigenic: six were A+B+ belonging to RT106; two were A+B+ belonging to RT014/020; and two were AB- belonging to RT010. All strains were biofilm producers. In the motility test, 40% of strains were as motile as the positive control, CD630 (RT012). In the disc diffusion test, two strains (RT010) were resistant to erythromycin and metronidazole; and another to metronidazole (RT014/020). In terms of C. difficile clinicopathological correlations, no statistically significant morphological changes, such as pseudomembranous and "volcano" lesions, were observed. Regarding hematological data, dogs positive for C. difficile had leucopenia (p = 0.02) and lymphopenia (p = 0.03). There was a significant correlation between senility and the presence of C. difficile in the dogs studied (p = 0,02). Conclusions: Although C. difficile has not been linked to canine diarrheal disorders, it appears to be more common in dogs with intestinal dysfunctions. The isolation of ribotypes frequently involved in human CDI outbreaks around the world supports the theory of C. difficile zoonotic transmission.

Clostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenic C. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human recurrent CDI. In this study, we evaluated the efficacy of metronidazole-resistant NTCD-E4 in preventing CDI facilitated by a range of antimicrobials in an in vitro human gut model. NTCD-E4 spores (at a dose of 107) were instilled 7 days before a clinical ribotype (RT) 027 (at the same dose) strain (210). In separate experiments, four different antimicrobials were used to perturb gut microbiotas; bacterial populations and cytotoxin production were determined using viable counting and Vero cell cytotoxicity, respectively. RT027 and NTCD-E4 proliferated in the in vitro model when inoculated singly, with RT027 demonstrating high-level cytotoxin (3-5-log10-relative units) production. In experiments where the gut model was pre-inoculated with NTCD-E4, RT027 was remained quiescent and failed to produce cytotoxins. NTCD-E4 showed mutations in hsmA and a gene homologous to CD196-1331, previously linked to medium-dependent metronidazole resistance, but lacked other metronidazole resistance determinants. This study showed that RT027 was unable to elicit simulated infection in the presence of NTCD-E4 following stimulation by four different antimicrobials. These data complement animal and clinical studies in suggesting NTCD offer prophylactic potential in the management of human CDI.

A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential importance, C. difficile plasmids remain poorly characterized. Here, we provide the complete genome sequence of a human clinical isolate that carries three high-copy number plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile that is also compatible with the plasmid pCD630 that is found in many laboratory strains. Together, our data advance our understanding of C. difficile plasmid biology.

Objectives: To assess the value of screening for Clostridioides difficile colonization (CDC) at hospital admission in an endemic setting.Methods: A multi-centre study was conducted at four hospitals located across the Netherlands. Newly admitted patients were screened for CDC. The risk of development of Clostridioides difficile infection (CDI) during admission and 1-year follow-up was assessed in patients with and without colonization. C. difficile isolates from patients with colonization were compared with isolates from incident CDI cases using core genome multi-locus sequence typing to determine whether onwards transmission had occurred.Results: CDC was present in 108 of 2211 admissions (4.9%), whereas colonization with a toxigenic strain (toxigenic Clostridoides difficile colonization [tCDC]) was present in 68 of 2211 admissions (3.1%). Among these 108 patients with colonization, diverse PCR ribotypes were found and no 'hypervirulent' PCR ribotype 027 (RT027) was detected (95% CI, 0-0.028). None of the patients with colonization developed CDI during admission (0/49; 95% CI, 0-0.073) or 1-year follow-up (0/38; 95% CI, 0-0.93). Core genome multi-locus sequence typing identified six clusters with genetically related isolates from patients with tCDC and CDI; however, in these clusters, only one possible transmission event from a patient with tCDC to a patient with CDI was identified based on epidemiological data.Conclusion: In this endemic setting with a low prevalence of 'hypervirulent' strains, screening for CDC at admission did not detect any patients with CDC who progressed to symptomatic CDI and detected only one possible transmission event from a patient with colonization to a patient with CDI. Thus, screening for CDC at admission is not useful in this setting. Monique J.T. Crobach, Clin Microbiol Infect 2023;29:891 & COPY; 2023 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/).

ABSTRACTWe report a patient case with pseudomembranous colitis associated with a mono-toxin producingClostridioides difficilebelonging to the very rarely diagnosed PCR ribotype (RT) 151. The infection was difficult to diagnose, since the isolate and the feces sample tested negative for toxin-encoding genes using a routine commercial test. This prompted us to sequence n = 11 RT151s from various geographical regions to study their genomic characteristics and relatedness. By including whole genome sequence data from other sources, we could further place these isolates into the phylogenetic tree ofC. difficileand assign them to their respective clades. These analyses revealed that 1) RT151s are polyphyletic with isolates falling into clades 1, and cryptic clades C-I and C-II 2) RT151 contains both non-toxigenic and toxigenic isolates and 3) RT151 C-II isolates contained mono-toxin pathogenicity loci (PaLoc). Additional analysis with PacBio circular consensus sequencing revealed that the isolate from our patient case report contains a novel PaLoc insertion site, lackedtcdAand a had significantly divergenttcdBsequence that might explain the failure of the diagnostic test. The study is noteworthy as 1) RT151 encompasses both typical and cryptic clades and 2) conclusive evidence for CDI due to clade C-II isolates was hitherto lacking.

Clostridioides difficile is an important health care-associated pathogen. The aim of this study was to analyze the antibiotic susceptibility of C. difficile isolates from feces of patients from 13 hospitals in Silesia, Poland. The incidence of CDI per 100.000 people in Silesia in 2018–2019 was higher than the average in Poland (39.3–38.7 vs. 30.2–29.5, respectively). The incidence doubled from 26.4 in 2020 to 55.1 in 2021. Two hundred and thirty stool samples tested positive for GDH (glutamate dehydrogenase) and toxins were cultured anaerobically for C. difficile. The isolates were characterized, typed, and tested for susceptibility to 11 antibiotics by E-test (EUCAST, 2021). The genes of toxins A/B and binary were detected by mPCR. Of 215 isolates, 166 (77.2%) were classified as RT 027 and 6 (2.8%) as related RT 176. Resistance to ciprofloxacin (96.7%), moxifloxacin (79.1%), imipenem (78.1%), penicillin (67%), and rifampicin (40.5%) was found. The ermB gene was detected in 79 (36.7%) strains. Multidrug resistance (MDR) was confirmed in 50 (23.3%) strains of RT 027 (94%). We concluded that a high prevalence of MDR among hypervirulent RT 027/176 C. difficile was found in the Silesian region of Poland, emphasizing the need to enhance regional infection control on CDI and antibiotic stewardships.

Objectives: The epidemiology of Clostridioides difficile infection (CDI) has undergone many changes since the beginning of this century and continues to evolve based on recent studies. Here, we performed a molecular analysis of C. difficile isolates in northern Greece across 10 health-care facilities, spanning from 2016 to 2019. Methods: 221 C. difficile isolates were cultured from stool samples of hospitalized patients with diarrhea and screened by PCR for the presence of the toxin A (tcdA), toxin B (tcdB), the binary toxin (cdtA and cdtB) genes and the regulating gene of tcdC. PCR ribotyping of the cultured isolates was performed by a standardized protocol for capillary gel-based PCR ribotyping and an international database with well documented reference strains. Results: Thirty-five different PCR ribotypes were identified. The most common RTs identified were: 181 (36%, 80/221), 017 (10%, 21/221), 126 (9%, 19/221), 078 (4%, 9/221) and 012 (4%, 8/221). Notably, the predominant RT181, with toxin profile tcdA thorn tcdB thorn cdtA thorn cdtB thorn , was identified in seven out of ten participating hospitals. Conclusions: Multiple C. difficile ribotypes have been circulating in the northern Greece region with RTs 181 (closely related to 027), 017, 126 and 078 being predominant. (C) 2022 Elsevier Ltd. All rights reserved.

Background During the COVID-19 pandemic, several factors, such as improved hand hygiene, social distancing, and restricted hospital referral, may have had an influence on the epidemiology of Clostridioides difficile infections (CDI). Methods The annual CDI incidence rate of nine hospitals participating in the Dutch sentinel CI surveillance with complete data was compared between 2020 and the previous five surveillance years. Trends in characteristics of hos-pitalised CDI patients in 21-24 participating hospitals were compared between the first (March 13-May 12, 2020) or second Dutch COVID-19 wave (September 17, 2020-January 1, 2021) and the same calendar periods in 2015 through 2019. All analyses were adjusted for trend changes over time. Findings The annual CDI incidence rate in 2020 was lower compared to previous years. During the second wave, the percentage of CDI patients with severe CDI was higher compared to earlier (25.8% in 2020 vs 17.9% in 2015-2019 (RR 1.6; 95%CI 1.1-2.3)). After adjustment for delayed C. difficile diagnostics (>= 8 days from start symptoms), the increase disappeared. Delayed C. difficile diagnostics was indeed more common during the second wave (RR 1.7; 95%CI 1.1-2.6), but only for community-onset CDI (CO-CDI). Interpretation This study shows that a higher percentage of severe CDI cases was observed during the second COVID-19 wave. This may partially be caused by delayed diagnostics, potentially due to decreased visits to a physi-cian or restricted hospital referral for CO-CDI patients. Funding Dutch ministry of Health. Copyright (C) 2022 The Authors. Published by Elsevier Ltd.