Clostridioides difficile canonical L,D-transpeptidases catalyze a novel type of peptidoglycan cross-links and are not required for beta-lactam resistance.

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.

Repeated COVID-19 vaccination to maximum antibody response yields very low mortality and hospitalisation rates in patients with CLL and MBL.

We analysed COVID-19 infection outcomes of 129/241 chronic lymphocytic leukaemia (CLL) (53.9%) and 22/55 monoclonal B-lymphocytosis (MBL) (40.0%) patients following multiple vaccine doses aiming for maximum measured anti-spike antibody response. Throughout the pandemic to date, there were 8/129 CLL (6.2%) patients hospitalised, with one death (0.8%). No MBL patients were hospitalised or died. CLL patients with COVID-19 had lower anti-spike levels (3778.8 AU/mL) than those without (13 486.8 AU/mL; p = 0.0061). Anti-nucleocapsid antibody was detected in 29.8% within 2 months and 17.5% >6 months. Of COVID-19-infected CLL patients, 47.3% received anti-viral therapy. A multiple vaccine dosing strategy to achieve measured maximum antibody is highly effective in preventing severe COVID-19.

Multiple COVID-19 vaccine doses in CLL and MBL improve immune responses with progressive and high seroconversion.

Patients with chronic lymphocytic leukemia (CLL) or monoclonal B-lymphocytosis (MBL) have impaired response to COVID-19 vaccination. A total of 258 patients (215 with CLL and 43 with MBL) had antispike antibody levels evaluable for statistical analysis. The overall seroconversion rate in patients with CLL was 94.2% (antispike antibodies ≥50 AU/mL) and 100% in patients with MBL after multiple vaccine doses. After 3 doses (post-D3) in 167 patients with CLL, 73.7% were seropositive, 17.4% had antispike antibody levels between 50 and 999 AU/mL, and 56.3% had antispike antibody levels ≥1000 AU/mL, with a median rise from 144.6 to 1800.7 AU/mL. Of patients who were seronegative post-D2, 39.7% seroconverted post-D3. For those who then remained seronegative after their previous dose, seroconversion occurred in 40.6% post-D4, 46.2% post-D5, 16.7% post-D6, and 0% after D7 or D8. After seroconversion, most had a progressive increase in antispike antibody levels. Neutralization was associated with higher antispike antibody levels, more vaccine doses, and earlier severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants; neutralizing antibody against early clade D614G was detected in 65.3%, against Delta in 52.0%, and against Omicron in 36.5%. SARS-CoV-2-specific T-cell production of interferon γ and interleukin 2 occurred in 73.9% and 60.9%, respectively, of 23 patients tested. After multiple vaccine doses, by multivariate analysis, immunoglobulin M ≥0.53 g/L, immunoglobulin subclass G3 ≥0.22 g/L and absence of current CLL therapy were independent predictors of positive serological responses. Multiple sequential COVID-19 vaccination significantly increased seroconversion and antispike antibody levels in patients with CLL or MBL.

In vivo emergence of a still uncommon resistance to fidaxomicin in the urgent antimicrobial resistance threat Clostridioides difficile.

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.

COVID-19 vaccine failure in chronic lymphocytic leukaemia and monoclonal B-lymphocytosis; humoural and cellular immunity.

Chronic lymphocytic leukaemia (CLL) is associated with immunocompromise and high risk of severe COVID-19 disease and mortality. Monoclonal B-cell lymphocytosis (MBL) patients also have immune impairment. We evaluated humoural and cellular immune responses in 181 patients with CLL (160) and MBL (21) to correlate failed seroconversion [<50 AU/ml SARS-CoV-2 II IgG assay, antibody to spike protein; Abbott Diagnostics)] following each of two vaccine doses with clinical and laboratory parameters. Following first and second doses, 79.2% then 45% of CLL, and 50% then 9.5% of MBL patients respectively remained seronegative. There was significant association between post dose two antibody level with pre-vaccination reduced IgM (p < 0.0001), IgG2 (p < 0.035), and IgG3 (p < 0.046), and CLL therapy within 12 months (p < 0.001) in univariate analysis. By multivariate analysis, reduced IgM (p < 0.0002) and active therapy (p < 0.0002) retained significance. Anti-spike protein levels varied widely and were lower in CLL than MBL patients, and both lower than in normal donors. Neutralisation activity showed anti-spike levels <1000 AU/ml were usually negative for both an early viral clade and the contemporary Delta variant and 72.9% of CLL and 53.3% of MBL failed to reach levels ≥1000 AU/ml. In a representative sample, ~80% had normal T-cell responses. Failed seroconversion occurred in 36.6% of treatment-naïve patients, in 78.1% on therapy, and in 85.7% on ibrutinib.

Haem is crucial for medium-dependent metronidazole resistance in clinical isolates of Clostridioides difficile.

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.

Five-year Pan-European, longitudinal surveillance of Clostridium difficile ribotype prevalence and antimicrobial resistance: the extended ClosER study.

Clostridium difficile infection (CDI) has been primarily treated with metronidazole or vancomycin. High recurrence rates, the emergence of epidemic PCR ribotypes (RTs) and the introduction of fidaxomicin in Europe in 2011 necessitate surveillance of antimicrobial resistance and CDI epidemiology. The ClosER study monitored antimicrobial susceptibility and geographical distribution of C. difficile RTs pre- and post-fidaxomicin introduction. From 2011 to 2016, 28 European countries submitted isolates or faecal samples for determination of PCR ribotype, toxin status and minimal inhibitory concentrations (MICs) of metronidazole, vancomycin, rifampicin, fidaxomicin, moxifloxacin, clindamycin, imipenem, chloramphenicol and tigecycline. RT diversity scores for each country were calculated and mean MIC results used to generate cumulative resistant scores (CRSs) for each isolate and country. From 40 sites, 3499 isolates were analysed, of which 95% (3338/3499) were toxin positive. The most common of the 264 RTs isolated was RT027 (mean prevalence 11.4%); however, RT prevalence varied greatly between countries and between years. The fidaxomicin geometric mean MIC for years 1-5 was 0.04 mg/L; only one fidaxomicin-resistant isolate (RT344) was submitted (MIC ≥ 4 mg/L). Metronidazole and vancomycin geometric mean MICs were 0.46 mg/L and 0.70 mg/L, respectively. Of prevalent RTs, RT027, RT017 and RT012 demonstrated resistance or reduced susceptibility to multiple antimicrobials. RT diversity was inversely correlated with mean CRS for individual countries (Pearson coefficient r = - 0.57). Overall, C. difficile RT prevalence remained stable in 2011-2016. Fidaxomicin susceptibility, including in RT027, was maintained post-introduction. Reduced ribotype diversity in individual countries was associated with increased antimicrobial resistance.

Humoral immune failure defined by immunoglobulin class and immunoglobulin G subclass deficiency is associated with shorter treatment-free and overall survival in Chronic Lymphocytic Leukaemia.

Immune dysfunction attributed to hypogammaglobulinaemia is common in chronic lymphocytic leukaemia (CLL) and infection is a major contributor to morbidity and mortality. A higher incidence of multiple immunoglobulin and immunoglobulin G (IgG) subclass deficiency was associated with more advanced disease (P < 0·001 and P < 0·001, respectively) in a cohort of 147 CLL patients. Multiple immunoglobulin and IgG subclass deficiency were significantly associated with shorter treatment-free survival (TFS) (P < 0·001 and P = 0·006, respectively). The association between disease stage and immune dysfunction demonstrated by these data suggest aspects of immune deficiency correlate with disease severity and may be associated with shorter TFS in CLL.

In Vitro Activities of MCB3681 and Eight Comparators against Clostridium difficile Isolates with Known Ribotypes and Diverse Geographical Spread.

Treatments for Clostridium difficile infection remain limited, despite the introduction of fidaxomicin, and development of new agents is necessary. We determined the in vitro susceptibilities of 199 prevalent or emerging Clostridium difficile PCR ribotypes to MCB3681, a novel investigational quinolonyl-oxazolidinone, and 8 comparators (metronidazole, vancomycin, fidaxomicin, moxifloxacin, ciprofloxacin, clindamycin, tigecycline, and linezolid). MCB3681 showed good activity against C. difficile with no evidence of MCB3681 resistance in isolates showing either moxifloxacin or linezolid resistance or both moxifloxacin and linezolid resistance.

Role of cephalosporins in the era of Clostridium difficile infection.

The incidence of Clostridium difficile infection (CDI) in Europe has increased markedly since 2000. Previous meta-analyses have suggested a strong association between cephalosporin use and CDI, and many national programmes on CDI control have focused on reducing cephalosporin usage. Despite reductions in cephalosporin use, however, rates of CDI have continued to rise. This review examines the potential association of CDI with cephalosporins, and considers other factors that influence CDI risk. EUCLID (the EUropean, multicentre, prospective biannual point prevalence study of CLostridium difficile Infection in hospitalized patients with Diarrhoea) reported an increase in the annual incidence of CDI from 6.6 to 7.3 cases per 10 000 patient bed-days from 2011-12 to 2012-13, respectively. While CDI incidence and cephalosporin usage varied widely across countries studied, there was no clear association between overall cephalosporin prescribing (or the use of any particular cephalosporin) and CDI incidence. Moreover, variations in the pharmacokinetic and pharmacodynamic properties of cephalosporins of the same generation make categorization by generation insufficient for predicting impact on gut microbiota. A multitude of additional factors can affect the risk of CDI. Antibiotic choice is an important consideration; however, CDI risk is associated with a range of antibiotic classes. Prescription of multiple antibiotics and a long duration of treatment are key risk factors for CDI, and risk also differs across patient populations. We propose that all of these are factors that should be taken into account when selecting an antibiotic, rather than focusing on the exclusion of individual drug classes.

Susceptibility of Clostridium difficile Isolates of Varying Antimicrobial Resistance Phenotypes to SMT19969 and 11 Comparators.

We determined the in vitro activity of SMT19969 and 11 comparators, including metronidazole, vancomycin, and fidaxomicin, against 107 C. difficile isolates of different antimicrobial resistance phenotypes. Fidaxomicin and SMT19969 were the most active. The fidaxomicin and SMT19969 geometric mean MICs were highest in ribotypes known to show multiple resistance. Coresistance to linezolid and moxifloxacin was evident in ribotypes 001, 017, 027, and 356. The high-level ceftriaxone resistance in ribotypes 356 and 018 was location linked.

Recurrence of dual-strain Clostridium difficile infection in an in vitro human gut model.

BACKGROUND: Clostridium difficile infection (CDI) is still a major challenge to healthcare facilities. The detection of multiple C. difficile strains has been reported in some patient samples during initial and recurrent CDI episodes. However, the behaviour of individual strains and their contribution to symptomatic disease is unclear. METHODS: An in vitro human gut model was used to investigate the germination and proliferation of two distinct C. difficile strains during initial and recurrent simulated CDI, as well as their response to vancomycin treatment. The gut model was inoculated with a pooled human faecal emulsion and indigenous gut microbiota, C. difficile populations (vegetative and spore forms), cytotoxin levels and antimicrobial activity were monitored throughout the experiment. RESULTS: Both C. difficile strains germinated and proliferated in response to ceftriaxone instillation, with cytotoxin detected during the peak vegetative growth. Vancomycin instillation resulted in a rapid decline in the vegetative forms of both strains, with only spores remaining 2 days after the start of dosing. A recrudescence of both strains occurred following the cessation of vancomycin installation, although this was observed more quickly, and to a greater extent, in one strain than the other. CONCLUSIONS: Within a human gut model, multiple C. difficile strains are able to germinate and proliferate concurrently in response to antibiotic challenge (the onset of simulated CDI). Similarly, more than one strain can proliferate during simulated recurrent CDI, although with differences in germination and growth rate and timing. It appears probable that multiple strains can contribute to CDI within an individual patient, with possible implications for management and bacterial transmission.

Comparison of planktonic and biofilm-associated communities of Clostridium difficile and indigenous gut microbiota in a triple-stage chemostat gut model.

BACKGROUND: Biofilms are characteristic of some chronic or recurrent infections and this mode of growth tends to reduce treatment efficacy. Clostridium difficile infection (CDI) is associated with a high rate of recurrent symptomatic disease. The presence and behaviour of C. difficile within intestinal biofilms remains largely unexplored, but may factor in recurrent infection. METHODS: A triple-stage chemostat gut model designed to facilitate the formation of intestinal biofilm was inoculated with a pooled human faecal emulsion. Bacterial populations were allowed to equilibrate before simulated CDI was induced by clindamycin (33.9 mg/L, four times daily, 7 days) and subsequently treated with vancomycin (125 mg/L, four times daily, 7 days). Indigenous gut microbiota, C. difficile total viable counts, spores, cytotoxin and antimicrobial activity in planktonic and biofilm communities were monitored during the 10 week experimental period. RESULTS: Vancomycin successfully treated the initial episode of simulated CDI, but ∼18 days after therapy cessation, recurrent infection occurred. Germination, proliferation and toxin production were evident within planktonic communities in both initial and recurrent CDI. In contrast, sessile C. difficile remained in dormant spore form for the duration of the experiment. The effects of and recovery from clindamycin and vancomycin exposure for sessile populations was delayed compared with responses for planktonic bacteria. CONCLUSIONS: Intestinal biofilms provide a potential reservoir for C. difficile spore persistence, possibly facilitating their dispersal into the gut lumen after therapeutic intervention, leading to recurrent infection. Therapeutic options for CDI could have increased efficacy if they are more effective against sessile C. difficile.

Antimicrobial susceptibility in Clostridioides difficile varies according to European region and isolate source.

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.

Evaluation of NVB302 versus vancomycin activity in an in vitro human gut model of Clostridium difficile infection.

OBJECTIVES: First-line treatment options for Clostridium difficile infection (CDI) are limited. NVB302 is a novel type B lantibiotic under evaluation for the treatment of CDI. We compared the responses to NVB302 and vancomycin when used to treat simulated CDI in an in vitro gut model. METHODS: We used ceftriaxone to elicit simulated CDI in an in vitro gut model primed with human faeces. Vancomycin and NVB302 were instilled into separate gut models and the indigenous gut microbiota and C. difficile total viable counts, spores and toxin levels were monitored throughout. RESULTS: Ceftriaxone instillation promoted C. difficile germination and high-level toxin production. Commencement of NVB302 and vancomycin instillation reduced C. difficile total viable counts rapidly with only C. difficile spores remaining within 3 and 4 days, respectively. Cytotoxin was reduced to undetectable levels 5 and 7 days after vancomycin and NVB302 instillation commenced in vessel 2 and 3, respectively, and remained undetectable for the remainder of the experiments. C. difficile spores were unaffected by the presence of vancomycin or NVB302. NVB302 treatment was associated with faster resolution of Bacteroides fragilis group. CONCLUSIONS: Both NVB302 and vancomycin were effective in treating simulated CDI in an in vitro gut model. C. difficile spore recrudescence was not observed following successful treatment with either NVB302 or vancomycin. NVB302 displayed non-inferiority to vancomycin in the treatment of simulated CDI, and had less deleterious effects against B. fragilis group. NVB302 warrants further clinical investigation as a potentially novel antimicrobial agent for the treatment of CDI.

Evaluation of antimicrobial activity of ceftaroline against Clostridium difficile and propensity to induce C. difficile infection in an in vitro human gut model.

OBJECTIVES: To examine the effects of exposure to ceftaroline or ceftriaxone on the epidemic Clostridium difficile strain PCR ribotype 027 and the indigenous gut microflora in an in vitro human gut model. Additionally, the MICs of ceftriaxone and ceftaroline for 60 C. difficile isolates were determined. METHODS: Two triple-stage chemostat gut models were primed with human faeces and exposed to ceftaroline (10 mg/L, twice daily, 7 days) or ceftriaxone (150 mg/L, once daily, 7 days). Populations of indigenous gut microorganisms, C. difficile total viable counts, spore counts, cytotoxin titres and antimicrobial concentrations were monitored throughout. MICs were determined by a standard agar incorporation method. RESULTS: In the gut model, both ceftaroline and ceftriaxone induced C. difficile spore germination, proliferation and toxin production, although germination occurred 5 days later in the ceftaroline-exposed model. Toxin detection was sustained until the end of the experimental period in both models. No active antimicrobial was detected in vessel 3 of either model, although inhibitory effects on microflora populations were observed. Ceftaroline was ∼8-fold more active against C. difficile than ceftriaxone (geometric mean MICs, 3.38 versus 28.18 mg/L; MIC90s, 4 versus 64 mg/L; and MIC ranges, 0.125-16 versus 8-128 mg/L). CONCLUSIONS: Ceftaroline, like ceftriaxone, can induce simulated C. difficile infection in a human gut model. However, low in vivo gut concentrations of ceftaroline and increased activity against C. difficile in comparison with ceftriaxone mean that the true propensity of this novel cephalosporin to induce C. difficile infection remains unclear.

Mixed infection by Clostridium difficile in an in vitro model of the human gut.

OBJECTIVES: Clostridium difficile infection (CDI) is still a major clinical challenge. Previous studies have demonstrated multiple distinct C. difficile strains in the faeces of patients with CDI; yet whether true mixed CDI occurs in vivo is unclear. In this study we evaluated whether two distinct C. difficile strains could co-germinate and co-proliferate in an in vitro human gut model. METHODS: An in vitro triple-stage chemostat was used to study the responses of two PCR ribotype 001 C. difficile strains following exposure to ceftriaxone at concentrations observed in vivo (7 days). C. difficile viable counts (vegetative and spore forms), cytotoxin titres and indigenous microflora viable counts were monitored throughout the experiment. RESULTS: Both C. difficile strains germinated and proliferated following exposure to ceftriaxone. Cytotoxin production was detected in the gut model following C. difficile spore germination and proliferation. Ceftriaxone elicited reduced viable counts of Bifidobacterium spp. and elevated viable counts of Enterococcus spp. CONCLUSIONS: These data suggest that multiple C. difficile strains are able to proliferate concurrently in an in vitro model reflective of the human colon. Previous studies in the gut model have reflected clinical observations so clinicians should be mindful of the possibility that multiple C. difficile strains may infect patients. These observations augment recent human epidemiological studies in this area.

Non-Toxigenic Clostridioides difficile Strain E4 (NTCD-E4) Prevents Establishment of Primary C. difficile Infection by Epidemic PCR Ribotype 027 in an In Vitro Human Gut Model.

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.

Penicillin Binding Protein Substitutions Cooccur with Fluoroquinolone Resistance in Epidemic Lineages of Multidrug-Resistant Clostridioides difficile.

Clostridioides difficile remains a key cause of healthcare-associated infection, with multidrug-resistant (MDR) lineages causing high-mortality (≥20%) outbreaks. Cephalosporin treatment is a long-established risk factor, and antimicrobial stewardship is a key control. A mechanism underlying raised cephalosporin MICs has not been identified in C. difficile, but among other species, this is often acquired via amino acid substitutions in cell wall transpeptidases (penicillin binding proteins [PBPs]). Here, we investigated five C. difficile transpeptidases (PBP1 to PBP5) for recent substitutions, associated cephalosporin MICs, and co-occurrence with fluoroquinolone resistance. Previously published genome assemblies (n = 7,096) were obtained, representing 16 geographically widespread lineages, including healthcare-associated ST1(027). Recent amino acid substitutions were found within PBP1 (n = 50) and PBP3 (n = 48), ranging from 1 to 10 substitutions per genome. ß-Lactam MICs were measured for closely related pairs of wild-type and PBP-substituted isolates separated by 20 to 273 single nucleotide polymorphisms (SNPs). Recombination-corrected phylogenies were constructed to date substitution acquisition. Key substitutions such as PBP3 V497L and PBP1 T674I/N/V emerged independently across multiple lineages. They were associated with extremely high cephalosporin MICs; 1 to 4 doubling dilutions >wild-type, up to 1,506 µg/mL. Substitution patterns varied by lineage and clade, showed geographic structure, and occurred post-1990, coincident with the gyrA and/or gyrB substitutions conferring fluoroquinolone resistance. In conclusion, recent PBP1 and PBP3 substitutions are associated with raised cephalosporin MICs in C. difficile. Their co-occurrence with fluoroquinolone resistance hinders attempts to understand the relative importance of these drugs in the dissemination of epidemic lineages. Further controlled studies of cephalosporin and fluoroquinolone stewardship are needed to determine their relative effectiveness in outbreak control. IMPORTANCE 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). The higher the number of substitutions, the greater the impact on phenotype. Dated phylogenies revealed that substitutions associated with raised cephalosporin and fluoroquinolone MICs were co-acquired immediately before clinically important outbreak strains emerged. PBP substitutions were geographically structured within genetic lineages, suggesting adaptation to local antimicrobial prescribing. Antimicrobial stewardship of cephalosporins and fluoroquinolones is an effective means of C. difficile outbreak control. Genetic changes associated with raised MIC may impart a "fitness cost" after antibiotic withdrawal. Our study therefore identifies a mechanism that may explain the contribution of cephalosporin stewardship to resolving outbreak conditions. However, due to the co-occurrence of raised cephalosporin MICs and fluoroquinolone resistance, further work is needed to determine the relative importance of each.

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile.

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 PnimBG) 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 PnimBG 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.