BACKGROUND:CRS3123 is a potent inhibitor of protein synthesis in bacteria that express type 1 methionyl-tRNA ligase, resulting in selective antibacterial activity that holds promise as a novel treatment for Clostridioides difficile infection (CDI). The purpose of this study was to evaluate the safety and efficacy of CRS3123 in adults with a primary episode or first recurrence of CDI. METHODS:This multicentre, randomised, double-blind, vancomycin-controlled, phase 2 study was conducted across 14 enrolling sites in the USA and Canada. Enrolled patients were 18 years or older, with a clinical diagnosis of CDI, including diarrhoea (at least three unformed bowel movements in the 24 h before randomisation) and C difficile toxin A or B, or both, detected in stools. Patients were excluded if they had more than one episode of CDI within the past 3 months or more than two within the past 12 months. Patients were randomly assigned (1:1:1) to receive 10 days of treatment with one of two dose regimens of CRS3123 (200 mg and 400 mg orally twice daily) or oral vancomycin (125 mg orally four times daily). Randomisation used an interactive response technology system and was stratified by history of CDI (first episode vs first recurrence within the past 3-12 months). Masking was maintained by use of matching dummy capsules. Safety was a primary endpoint and was assessed in patients who received at least one dose of study drug. The primary efficacy endpoint was clinical cure (survival and resolution of diarrhoea) at the test-of-cure (TOC) visit (day 12 up to day 15) in the intention-to-treat (ITT) population. The rate of CDI recurrence was a crucial secondary outcome measure assessed at study days 40 and 70 in the microbiological ITT population, which included all patients in the ITT population who tested positively for C difficile toxin at screening or day 1 and who had C difficile isolated in culture. This trial was registered with ClinicalTrials.gov (NCT04781387) and is complete. FINDINGS:Between May 12, 2021, and April 26, 2024, 58 individuals were assessed for eligibility, 43 of whom were recruited and randomly assigned: 14 (33%) to the CRS3123 200 mg group, 15 (35%) to the CRS3123 400 mg group, and 14 (33%) to the vancomycin group. The mean age of patients was 58·4 years (SD 18·0), 33 (77%) patients were female, and ten (23%) patients were male. 31 (72%) patients had a primary episode of CDI and 12 (28%) patients had a first recurrence of CDI. All patients received at least one dose of the assigned drug. Treatment-emergent adverse events (TEAEs) were mild to moderate in severity and similar across treatment groups. TEAEs considered possibly related to study drug (all grade 1 or 2) were reported in one patient in the CRS3123 200 mg group (dry mouth, asthenia and gastro-oesophageal reflux disease, nausea, vomiting, increased alanine aminotransferase, and increased aspartate aminotransferase), three patients in the CRS3123 400 mg group (one with increases in alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase; one with malaise, nausea, and feeling abnormal; and one with headache); and in no patients in the vancomycin group. The only serious adverse event reported, pneumonia, was in the vancomycin group and was considered unrelated to the study drug. 13 (93%) of 14 patients in the CRS3123 200 mg group, all 15 (100%) patients in the CRS3123 400 mg group, and 13 (93%) of 14 patients in the vancomycin group had clinical cure at the TOC visit. No patient had clinical failure at the TOC visit; two patients (one each in the CRS3123 200 mg and vancomycin groups) had indeterminate responses due to missing data. Recurrence through day 40 occurred in one (7%) of 14 patients in the CRS3123 400 mg group, none of the 13 patients in the CRS3123 200 mg group, and three (23%) of 13 patients in the vancomycin group, and there was an additional recurrence on day 70 in the CRS3123 400 mg group. INTERPRETATION:Both doses of CRS3123 were deemed safe and well tolerated and showed efficacy similar to vancomycin at the TOC visit, with lower rates of recurrence. Together, these data support further development of CRS3123. FUNDING:US National Institute of Allergy and Infectious Diseases at the National Institutes of Health, Department of Health and Human Services.

Background: Current recommended C. difficile infection (CDI) treatment with vancomycin (VAN) exacerbates dysbiosis leading to recurrent CDI (rCDI), while fidaxomicin (FDX) is expensive. Evidence for the current VAN dosage is lacking. We used a well-validated, clinically-reflective in vitro gut model to design and test VAN dosages to treat CDI but spare the gut microbiota. Methods: We performed time-kill studies on C. difficile PCR ribotypes 027, 014 and 078 for VAN concentrations from 2-2000 mg/L to inform investigations on VAN dose, frequency and duration against standard VAN (SVAN: 125mg/L qds/ 10d) in gut models. Simulated CDI was induced by clindamycin. We examined multiple VAN dosages against SVAN in GMs to produce an optimised VAN dosage (OVAN: 35mg/L qds 1d, then bd/30d). OVAN was tested in models against standard FDX (SFDX) (200mg/L bd/10d and FDX Extend (FDXX) (200mg/L bd (D1-5) then once daily on alternate days (D7-25). Models were monitored by viable counting and 16s microbiome analysis until 28d post-end of treatment to account for rCDI. Findings: VAN killing kinetics demonstrated no additional benefit with increasing concentrations beyond 2 x MIC. Gut-reflective concentrations (100-2000mg/L) showed reduced killing effects (< log10cfu/mL) consistent with Eagle Effect. All VAN dosages treated simulated primary CDI in gut models, but all led to rCDI by 28d post-end of treatment except SFDX, FDXX or OVAN. Culture and Shannon Diversity metrics indicated OVAN had comparable effects on the gut microbiome to FDX or FDXX. Interpretation: Eagle Effect may contribute to C. difficile survival in the gut and potentially to rCDI. OVAN treated simulated primary CDI and did not elicit rCDI in a gut model and showed similar microbiome effects to SFDX and FDXX. OVAN may be an effective strategy for treating primary CDI and reducing recurrence risk but requires clinical evaluation.

Background: This study aims to address a critical gap in diagnostic testing capabilities across sub-Saharan Africa by developing a comprehensive decision tool designed to guide laboratories in selecting optimal diagnostic modalities, appropriate to their unique contexts, clinical needs, and available resources. Methods:Evidence from a thematic framework analysis of stakeholder engagement from multiple African countries and global health institutions, and from a scoping review of grey literature on best practice for the design and selection of laboratory tests in resource poor-settings, was used to develop the decision tool in a decision tree format. Findings: Stakeholder engagement revealed needs for enhanced data-sharing mechanisms, clear frameworks for diagnostic test selection and implementation, strengthened intra- and inter-country networking, and greater integration of user groups at both local and regional levels. The scoping review identified 39 records mapped to five themes, namely intended use, categories of test (underlying technology), laboratory capacity, commercial availability, and performance evaluation. The characteristics of three test modalities, specifically assay format, multiple analytes, and instruments were summarised based on three factors: cost (including cost of performance evaluation), diagnostic accuracy, and turnaround time. Findings informed the design of the decision tool into an intuitive framework, utilising a simple decision tree structure with visualisations to clearly illustrate the trade-offs between cost, accuracy, and turnaround time. Interpretation: The decision tool enables stakeholders to make informed, context-specific decisions, contributing to expanding diagnostic readiness, local resilience and enhanced health outcomes across sub-Saharan Africa. The decision tool components are applicable worldwide.

Introduction Acute uncomplicated urinary tract infection (UTI) is a common condition with potentially serious sequelae that is mostly diagnosed and managed in primary care settings. Around half of all women have a UTI in their lifetime, and a quarter experience an infection caused by organisms resistant to more than one antibiotic. Reducing inappropriate prescribing of antibiotics is a core tenet of antimicrobial stewardship. However, current diagnostics for UTI are unfit for purpose in acute (highest prescribing) settings, being too slow to inform the required immediate decision-making and often confounded by sample contamination.Rapid point-of-care diagnostic tests (POCTs) that facilitate timely decision-making are potential solutions to this problem. Several such tests have reached advanced stages of technology readiness, but their diagnostic performance has not been evaluated in primary care with clinical users. To progress novel tests towards implementation, a diagnostic field study is required, to allow for parallel and sequential evaluation of multiple tests in a primary care population.Methods and analysis We will recruit participants assigned female at birth from primary care clinics in England who contact their clinic with symptoms of acute uncomplicated UTI. Eligible participants will complete a short questionnaire to capture symptoms and symptom severity and will provide a urine sample. Samples will be split and initially tested using novel index tests (POCTs) and conventional urinalysis ‘dipstick’ at the primary care clinic. The second part of the sample will be processed at a National Health Service-based reference laboratory using a modified reference standard including microscopy, microbiological culture, pathogen speciation and antimicrobial susceptibility testing. The UTI reference standard culture, although based on the national methods, is modified to provide accurate bacterial counts, better to define a microbiological diagnosis of UTI. Susceptibility testing will be performed using ‘gold-standard’ methods, not usually performed in diagnostic laboratories. The primary outcome will be the diagnostic performance (sensitivity, specificity, positive and negative predictive values) of POCTs for detection of UTI and antimicrobial susceptibility for POCTs that include antimicrobial susceptibility testing. Secondary outcomes will include the symptom profile of patients presenting with uncomplicated UTI, a theoretical determination of how use of POCT results might change prescribing, an understanding of POCT failure rate and qualitative capture of the experiences of those using the POCT to deliver the study in primary care clinics.Ethics and dissemination Ethical approval was received from the London Central Research Ethics Committee (23/LO/0371) and the UK Health Research Authority. We will publish the findings of The plaTform fOr Urinary tract infection diagnostiC evAluatioN evaluations in peer-reviewed medical journals and more broadly following a dissemination plan formulated by a communications specialist in consultation with patients and the public.Trial registration number ISRCTN80937472.

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/).

Clostridioides difficile infection (CDI) continues to be a notable burden worldwide, both in terms of patient mortality and morbidity, and the economic costs associated with treatment, diagnosis and management. The epidemiology of C. difficile has changed markedly over the decades, with high CDI rates driven by clinical pressures exacerbated by the severe acute respiratory syndrome coronavirus 2 pandemic, antibiotic resistance and selective pressures caused by antimicrobial use. C. difficile is challenging to diagnose and treat as it forms spores and can persist asymptomatically within the gut. Some strains express multiple virulence factors, including adhesins and toxins. The gut microbiota is crucially important in CDI, as a healthy microbiota is resistant to colonization with C. difficile. Dysbiosis, often caused by antimicrobial exposure, enables C. difficile spores to germinate and produce toxin, causing symptoms that can range from mild diarrhoea to fulminant colitis and death. This Review describes changes in epidemiology and effects on diagnosis, discusses recent breakthroughs in the understanding of pathogenesis and antibiotic resistance and explores the role of microbiota dysbiosis in CDI and novel microbiota therapies in CDI treatment. Clostridioides difficile infection is challenging to diagnose and treat and is associated with considerable mortality, morbidity and economic costs worldwide. In this Review, Chilton et al. discuss changes in global epidemiology, breakthroughs in pathogenesis and antibiotic resistance, the role of microbiota dysbiosis and the potential for microbiota-based therapeutics for Clostridioides difficile infection.

OBJECTIVES:Reconstruct the phylogenetic status of a collection of historical Clostridioides difficile isolates and evaluate the congruence of their evolutionary trajectories with established molecular clock models. METHODS:Phylogenetic analysis was performed on Illumina sequence reads from previously analysed historic C. difficile isolates (1980-86; n = 75) demonstrating multiple antimicrobial resistances. Data was grouped by ribotype (RT), including comparators from European surveillance (2012-13) and phylogenetic studies (1985-2010). Reads were mapped to CD630/CD196 reference genomes and compared using recombination-adjusted maximum likelihood trees. Prediction intervals for expected SNP differences by age were calculated using a Poisson distribution and molecular clock estimates (0.74 SNPs per genome/per year). Root-to-tip analysis was performed to determine the date of most common recent ancestor of genomes sharing a ribotype. RESULTS:Moxifloxacin-resistant (>16 mg/L) RT027 isolate JV67 (1986) was two SNPs distinct from a 2006 genome, fewer than the expected lower estimate (4.4 SNPs) under current molecular clock calculations; (p = 3.93x10-5). For isolate JV02 (1981), the 13 SNP divergence from a 2008 isolate was consistent with expectations (5.9 SNPs; p = 0.07). JV73 (1983) demonstrated an 8 SNP difference, which although above the expected lower limit (5.5 SNPs), was outside the 95 % prediction interval; (p = 4.51x10-3). Only sixty-nine percent of historical genomes fit within the prediction interval for the number of SNPs expected compared to recent isolates, with fewer SNPs observed more frequently than expected. Root-to-tip analysis demonstrated a weak linear correlation. CONCLUSIONS:C. difficile molecular clock estimations may be more complex than previously considered, with periods of spore quiescence potentially complicating analyses.

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea worldwide with significant morbidity and mortality. This organism is naturally resistant to several beta-lactam antibiotics that inhibit the polymerization of peptidoglycan, an essential component of the bacteria cell envelope. Previous work has revealed that C. difficile peptidoglycan has an unusual composition. It mostly contains 3-3 cross-links, catalyzed by enzymes called L,D-transpeptidases (Ldts) that are poorly inhibited by beta-lactams. It was therefore hypothesized that peptidoglycan polymerization by these enzymes could underpin antibiotic resistance. Here, we investigated the catalytic activity of the three canonical Ldts encoded by C. difficile (LdtCd1, LdtCd2, and LdtCd3) in vitro and explored their contribution to growth and antibiotic resistance. We show that two of these enzymes catalyze the formation of novel types of peptidoglycan cross-links using meso-diaminopimelic acid both as a donor and an acceptor, also observed in peptidoglycan sacculi. We demonstrate that the simultaneous deletion of these three genes only has a minor impact on both peptidoglycan structure and resistance to beta-lactams. This unexpected result therefore implies that the formation of 3-3 peptidoglycan cross-links in C. difficile is catalyzed by as yet unidentified noncanonical Ldt enzymes.

Objectives:Clostridioides difficile epidemiology is evolving with country-associated emerging and resistant ribotypes (RT). Antimicrobial susceptibility testing of C. difficile isolated from clinical and animal samples collected across Europe in 2018 was performed to provide antimicrobial resistance data and according to C. difficile RTs and source. Methods:Samples were cultured for C. difficile and isolates PCR ribotyped. Metronidazole, vancomycin, fidaxomicin, moxifloxacin, clindamycin, imipenem, tigecycline, linezolid, rifampicin and meropenem minimum inhibitory concentrations (MICs) for 280 clinical and 126 animal isolates were determined by Wilkins-Chalgren agar dilution. Results:Fidaxomicin was the most active antimicrobial (all isolates geometric mean MIC = 0.03 mg/L) with no evidence of reduced susceptibility. Metronidazole MICs were elevated among RT027 (1.87 mg/L) and RT181 clinical isolates (1.03 mg/L). RT027 and RT181 had elevated geometric mean moxifloxacin MICs (14.49 mg/L, 16.88 mg/L); clindamycin (7.5 mg/L, 9.1 mg/L) and rifampicin (0.6 mg/L, 21.5 mg/L). Five isolates (RT002, RT010 and RT016) were metronidazole resistant (MIC = 8 mg/L) and 10 (RT027; RT198) had intermediate resistance (4 mg/L). Metronidazole MICs were not elevated in animal isolates. Increased geometric mean vancomycin MICs were observed among RT078, mostly isolated from animals, but there was no resistance (MIC ≥ 4 mg/L). Clinical and animal isolates of multiple RTs showed resistance to moxifloxacin and clindamycin. No resistance to imipenem or meropenem was observed. Conclusion:Increased antimicrobial resistance was detected in eastern Europe and mostly associated with RT027 and related emerging RT181, while clinical isolates from northern and western Europe had the lowest general levels of resistance.

The aim of this thematic evidence review is to identify and provideguidance on the application of different laboratory tests modalities with afocus on Low- or Middle-Income Country (LMIC)

Diagnostic tools are key to guiding patient management and informing public health policies to control infectious diseases. However, many diseases still do not have effective diagnostics and much of the global population faces restricted access to reliable, affordable testing. This limitation underscores the urgent need for innovation to enhance diagnostic availability and effectiveness. Developing diagnostics presents distinct challenges, especially for innovators and regulators. Unlike medicines, regulatory pathways for diagnostics are often less defined and more complex due to their diverse risk profiles and wide range of products. These challenges are amplified in low-income and middle-income countries, which often do not have regulatory frameworks for this specific purpose. In the UK, initiatives aim to support innovation by providing clearer regulatory pathways and ensuring that diagnostics are safe and effective. Regulators are also collaborating internationally to expedite diagnostics for high-need regions. Harmonised standards, regulatory frameworks, and approval processes are essential to ensure consistent quality and safety across regions and facilitate faster development and global access. This Series paper explores the regulatory challenges in infectious disease and antimicrobial resistance diagnostics, focusing on the UK's response and the broader global efforts to address these issues.

Diagnostic tools are key to guiding patient management and informing public health policies to control infectious diseases. However, many diseases still do not have effective diagnostics and much of the global population faces restricted access to reliable, affordable testing. This limitation underscores the urgent need for innovation to enhance diagnostic availability and effectiveness. Developing diagnostics presents distinct challenges, especially for innovators and regulators. Unlike medicines, regulatory pathways for diagnostics are often less defined and more complex due to their diverse risk profiles and wide range of products. These challenges are amplified in low-income and middleincome countries, which often do not have regulatory frameworks for this specific purpose. In the UK, initiatives aim to support innovation by providing clearer regulatory pathways and ensuring that diagnostics are safe and effective. Regulators are also collaborating internationally to expedite diagnostics for high-need regions. Harmonised standards, regulatory frameworks, and approval processes are essential to ensure consistent quality and safety across regions and facilitate faster development and global access. This Series paper explores the regulatory challenges in infectious disease and antimicrobial resistance diagnostics, focusing on the UK's response and the broader global efforts to address these issues.

Severe outbreaks and deaths have been linked to the emergence and global spread of fluoroquinolone-resistant Clostridioides difficile over the past two decades. At the same time, metronidazole, a nitro-containing antibiotic, has shown decreasing clinical efficacy in treating C. difficile infection (CDI). Most metronidazole-resistant C. difficile exhibit an unusual resistance phenotype that can only be detected in susceptibility tests using molecularly intact heme. Here, we describe the mechanism underlying this trait. We find that most metronidazole-resistant C. difficile strains carry a T-to-G mutation (which we term P nimB G ) in the promoter of gene nimB , resulting in constitutive transcription. Silencing or deleting nimB eliminates metronidazole resistance. NimB is related to Nim proteins that are known to confer resistance to nitroimidazoles. We show that NimB is a heme-dependent flavin enzyme that degrades nitroimidazoles to amines lacking antimicrobial activity. Furthermore, occurrence of the P nimB G mutation is associated with a Thr82Ile substitution in DNA gyrase that confers fluoroquinolone resistance in epidemic strains. Our findings suggest that the pandemic of fluoroquinolone-resistant C. difficile occurring over the past few decades has also been characterized by widespread resistance to metronidazole.

BACKGROUND:Fidaxomicin is a first-line treatment for Clostridioides difficile infections (CDIs). Fidaxomicin resistance has rarely been reported in this urgent antimicrobial resistance threat as defined by the CDC.OBJECTIVES:To report a case of fidaxomicin-resistant C. difficile isolation in a patient treated by fidaxomicin, characterize the genetic determinant for resistance and the consequences on pathophysiological traits, and review the literature.PATIENT AND METHODS:A 38-year-old male patient with several risk factors for CDI experienced three episodes of hospital-acquired CDI and received fidaxomicin for the first episode. The successive isolates were subjected to phenotypic characterization (antimicrobial susceptibility, growth, sporulation ability and toxin production) and WGS analysis to evaluate clonality and modifications associated with resistance.RESULTS:Resistance to fidaxomicin arose in isolates from the recurrences of CDI (MIC: 16 mg/L). WGS analysis showed a close genetic link between strains suggestive of relapses in this patient. A T3428G mutation in the rpoB gene might be associated with fidaxomicin resistance. The resistance was associated with defects in growth, sporulation and production of toxins. A review of the literature found only three previous fidaxomicin-resistant C. difficile clinical strains.CONCLUSIONS:Although rarely reported, resistance to fidaxomicin may quickly emerge in vivo after a single course of treatment. This observation supports the need for prospective surveillance of the susceptibility of C. difficile to treatment antibiotics. However, the clinical relevance of fidaxomicin resistance still needs to be elucidated, particularly due to its apparent rareness and associated fitness cost.

Fluoroquinolone and cephalosporin use in healthcare settings has triggered outbreaks of high-mortality, multidrug-resistant C. difficile infection. Here, we identify a mechanism associated with raised cephalosporin MICs in C. difficile comprising amino acid substitutions in two cell wall transpeptidase enzymes (penicillin binding proteins).

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.

Clostridioides difficile infection (CDI) is a major cause of hospital-acquired diarrhoea affecting considerable numbers of people each year. CDI can cause a debilitating illness, which impacts on people and their families and the economic resources of health care systems. Despite this, and the increasing acceptance and promotion of patient and public involvement (PPI) in health care research, PPI in CDI research is uncommon.

Background: Until recently, metronidazole was the first-Line treatment for Clostridioides difficile infection and it is still commonly used. Though resistance has been reported due to the plasmid pCD-METRO, this does not explain all cases. Objectives: To identify factors that contribute to plasmid-independent metronidazole resistance of C. difficile. Methods: Here, we investigate resistance to metronidazole in a collection of clinical isolates of C. difficile using a combination of antimicrobial susceptibility testing on different solid agar media and WGS of selected isolates. Results: We find that nearly all isolates demonstrate a haem-dependent increase in the MIC of metronidazole, which in some cases Leads to isolates qualifying as resistant (MIC >2 mg/L). Moreover, we find an SNP in the haem-responsive gene hsmA, which defines a metronidazole-resistant Lineage of PCR ribotype 010/MLST ST15 isolates that also includes pCD-METRO-containing strains. Conclusions: Our data demonstrate that haem is crucial for medium-dependent metronidazole resistance in C. difficile.

OBJECTIVES:Clostridioides difficile (CD) is widely reported as one of the most prevalent multi-drug resistant (MDR) organisms. Assessment of temporally disparate isolate collections can give valuable epidemiological data to further the understanding of antimicrobial resistance progression. METHODS:A collection of 75 CD isolates (1980-86) was characterised by PCR ribotyping, cell cytotoxicity assay and susceptibility testing with a panel of 16 antimicrobials and compared to a modern surveillance collection consisting of 416 UK isolates (2012-2016). Agar-incorporation was performed to ascertain susceptibility data for vancomycin, metronidazole, rifampicin, fidaxomicin, moxifloxacin, clindamycin, imipenem, chloramphenicol, tigecycline, linezolid, ciprofloxacin, piperacillin/tazobactam, ceftriaxone, amoxicillin, tetracycline and erythromycin. Genomes were obtained using Illumina HiSeq3000 sequencing and assembled using CLC Genomics Workbench. Resistance genes were identified using the Comprehensive Antibiotic Research Database's Resistance Gene Identifier and ResFinder3.0. RESULTS:Twenty-six known and one previously unobserved ribotype (RT) were detected. RT015 and RT020 dominated; 21.3% and 17.3%, respectively. Three moxifloxacin resistant (16-32 mg/L) RT027 isolates were recovered, pre-dating the earliest reports of this phenotype/genotype. Phenotypic resistance was observed to moxifloxacin (9.3% of isolates), ciprofloxacin (100%), erythromycin (17.3%), tetracycline (9.3%), linezolid and chloramphenicol (4.0%). Phenotypic comparisons with modern strains revealed increasing minimum inhibitory concentrations (MIC), with MIC50 elevations of one doubling-dilution for the majority of compounds, excluding clindamycin and imipenem. Moxifloxacin MIC90 comparisons revealed a two doubling-dilution increase between temporal isolate collections. Historical genomes revealed twenty different resistance determinants, including ermB (8.0% of isolates), tetM (9.3%), cfr (5.3%) and gyrA substitution Thr-82→Ile (9.3%). Seventeen isolates (22.7%) were resistant to ≥3 compounds (MDR), demonstrating ten different combinations. Intra-RT diversity was observed. CONCLUSIONS:Antibiotic resistance in CD has increased since the early 1980s, across the majority of classes. Moxifloxacin resistance determinants may pre-date its introduction.