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.

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.

A point-prevalence study on community and inpatient Clostridioides difficile infections (CDI): results from Combatting Bacterial Resistance in Europe CDI (COMBACTE-CDI), July to November 2018.

BackgroundThere is a paucity of data on community-based Clostridioides difficile infection (CDI) and how these compare with inpatient CDI.AimTo compare data on the populations with CDI in hospitals vs the community across 12 European countries.MethodsFor this point-prevalence study (July-November 2018), testing sites sent residual diagnostic material on sampling days to a coordinating laboratory for CDI testing and PCR ribotyping (n = 3,163). Information on whether CDI testing was requested at the original site was used to identify undiagnosed CDI. We used medical records to identify differences between healthcare settings in patient demographics and risk factors for detection of C. difficile with or without free toxin.ResultsThe CDI positivity rate was 4.4% (country range: 0-16.2) in hospital samples, and 1.3% (country range: 0-2.2%) in community samples. The highest prevalence of toxinotype IIIb (027, 181 and 176) was seen in eastern European countries (56%; 43/77), the region with the lowest testing rate (58%; 164/281). Different predisposing risk factors were observed (use of broad-spectrum penicillins in the community (OR: 8.09 (1.9-35.6), p = 0.01); fluoroquinolones/cephalosporins in hospitals (OR: 2.2 (1.2-4.3), p = 0.01; OR: 2.0 (1.1-3.7), p = 0.02)). Half of community CDI cases were undetected because of absence of clinical suspicion, accounting for three times more undiagnosed adults in the community compared with hospitals (ca 111,000 vs 37,000 cases/year in Europe).ConclusionThese findings support recommendations for improving diagnosis in patients presenting with diarrhoea in the community, to guide good practice to limit the spread of CDI.

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

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

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

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

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

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

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.

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

C. difficile infection (CDI) is a worldwide healthcare problem with ~30% of cases failing primary therapy, placing a burden on healthcare systems and increasing patient morbidity. We have little understanding of why these therapies fail. Here, we use a clinically validated in vitro gut model to assess the contribution of biofilms towards recurrent disease and to investigate biofilm microbiota-C. difficile interactions. Initial experiments show that C. difficile cells became associated with the colonic biofilm microbiota and are not depleted by vancomycin or faecal microbiota transplant therapies. We observe that transferring biofilm encased C. difficile cells into a C. difficile naïve but CDI susceptible model induces CDI. Members of the biofilm community can impact C. difficile biofilm formation by acting either antagonistically or synergistically. We highlight the importance of biofilms as a reservoir for C. difficile, which can be a cause for recurrent infections.

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

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