Patients with Clostridioides difficile infection (CDI) with either eosinopenia or infected with a binary toxin strain have increased likelihood of mortality. However, the relationship between binary toxin and eosinopenia to synergistically increase mortality has not been studied in humans. We hypothesized that patients with CDI due to binary toxin strains and concomitant peripheral eosinopenia would have a higher likelihood of inpatient mortality. This multicenter, retrospective cohort study included adult patients with CDI of known ribotypes stratified by eosinopenia, defined as an absence of eosinophils in the peripheral blood (Houston cohort). The primary outcome was inpatient mortality. Results were supported by a separate national cohort of veterans with CDI (Veterans’ cohort). In the Houston cohort, a total of 688 patients from 13 institutions in 6 cities were included. Of these, 132 (19%) had an eosinophil count of 0.0 cells/µL (0.0 cells*10⁹/L) and 109 (16%) were infected with a binary toxin strain. After adjusting for covariates, the combination of eosinopenia and infection with a binary toxin strain was an independent predictor of inpatient mortality (odds ratio [OR], 7.8; 95% confidence interval [CI], 1.9–33.2; P = .005). In the separate Veterans’ cohort (n = 790), this combination was also a significant predictor of inpatient mortality (OR, 6.1; 95% CI, 1.5–23.9; P = .009). In conclusion, the combination of eosinopenia and CDI due to a binary toxin strain was correlated with increased mortality in hospitalized patients from 2 independent cohorts. Prospective studies should further study this important subset of patients at the time of CDI diagnosis.

Omadacycline is a potent aminomethylcycline with in vitro activity against Gram-positive, Gram-negative, and anaerobic bacteria. Preliminary data demonstrated that omadacycline has in vitro activity against Clostridioides difficile ; however, large-scale in vitro studies have not been done. The purpose of this study was to assess the in vitro susceptibility of omadacycline and comparators on a large biobank of clinical C. difficile isolates.

Fidaxomicin has novel pharmacologic effects on C. difficile spore formation including outgrowth inhibition and persistent spore attachment. However, the mechanism of fidaxomicin attachment on spores has not undergone rigorous microscopic studies. Fidaxomicin attachment to C. difficile spores of three distinct ribotypes and C. difficile mutant spores with inactivation of exosporium or spore-coat protein-coding genes were visualized using confocal microscopy with a fidaxomicin-bodipy compound (green fluorescence). The pharmacologic effect of the fidaxomicin-bodipy compound was determined. Confocal microscopy experiments included direct effect on C. difficile wild-type and mutant spores, effect of exosporium removal, and direct attachment to a comparator spore forming organism, Bacillus subtilis. The fidaxomicin-bodipy compound MIC was 1 mg/L compared to 0.06 mg/L for unlabeled fidaxomicin, a 16-fold increase. Using confocal microscopy, the intracellular localization of fidaxomicin into vegetative C. difficile cells was observed consistent with its RNA polymerase mechanism of action and inhibited spore outgrowth. The fidaxomicin-bodipy compound was visualized outside of the core of C. difficile spores with no co-localization with the membrane staining dye FM4-64. Exosporium removal reduced fidaxomicin-bodipy association with C. difficile spores. Reduced fidaxomicin-bodipy was observed in C. difficile mutant spores for the spore surface proteins CdeC and CotE. This study visualized a direct attachment of fidaxomicin to C. difficile spores that was diminished with mutants of specific exosporium and spore coat proteins. These data provide advanced insight regarding the anti-spore properties of fidaxomicin.

Clostridioides difficile infection (CDI) is recognised by the CDC as an “urgent threat” in the USA, responsible for nearly 13,000 deaths, and carries an economic burden ranging from &5.4 to &6.3 billion per year. In a phase II study, ridinilazole was shown to be effective at treating CDI and decreasing subsequent recurrence compared to vancomycin. However, the precise mechanism of action of ridinilazole has yet to be fully elucidated. We now present data that reveals ridinilazole clearly co-localises with DNA in C. difficile and binds with high affinity to the minor groove of DNA. These interactions are predicted to have consequences on cellular functions within C. difficile. High resolution confocal microscopy was used to track the intracellular localisation of ridinilazole in C. difficile. Fluorescence intensity was used to characterise the DNA binding properties of ridinilazole; sequence specificity was demonstrated with AT- or GC-rich DNA polymers, and tight binding was shown using short double-stranded oligonucleotides. Hanging drop vapour diffusion enabled co-crystallisation and subsequent structural determination of DNA-bound ridinilazole. Confocal microscopy revealed clear co-localisation of ridinilazole to the DNA within C. difficile. Ridinilazole demonstrated a dose-dependent increase in fluorescence in response to increasing concentration of target DNA. Fluorescence binding studies revealed that ridinilazole shows a preference towards AT-rich DNA sequences. Tight binding characteristics were demonstrated by ridinilazole in complex with short double-stranded oligonucleotides, returning dissociation constants (K_d) of 20 – 50 nM. Crystallisation enabled co-structures of ridinilazole bound to the minor groove of double-stranded DNA oligonucleotides to be solved. Ridinilazole demonstrates tight binding with sequence specificity within the minor groove of DNA and co-localises with DNA in C. difficle. Further analysis is ongoing to fully understand this novel mechanism of action, the downstream consequences of these interactions and how they contribute to the bactericidal activity of ridinilazole. Clive Mason, PhD, Summit Therapeutics (Employee, Shareholder) Tim Avis, n/a, Summit therapeutics (Shareholder) Chris Coward, PhD, Summit Therapeutics (Employee, Scientific Research Study Investigator, Shareholder) David Powell, PhD, Summit Therapeutics (Employee) Kevin W. Garey, Pharm.D., M.S., FASHP, Summit Therapeutics (Research Grant or Support)

An Enterococcus faecalis strain SHH039 was isolated from a 68 year old man who was hospitalised with cholecystitis. The genomic sequence of this isolate which had a size of 2,990,081 bp and 2,663 proteins with functional assignments is presented here. Analysis of the genome revealed Enterococcus faecalis with multiple antibiotic resistance genes which may be associated with acute cholecystitis. It may be not clear if the infection symptoms are the consequence of enterococci manifestation. However, this opportunistic organism may play a minor role in the disease.

Antibiotics can negatively affect gut microbiome composition and function. However, preclinical evaluations of antibiotic effects on the microbiome are rarely undertaken. The purpose of this study was to characterize pharmacokinetics and metagenomic profiles of PolC inhibitor compounds compared to linezolid in a neutropenic murine thigh infection model. Neutropenic mice thighs were inoculated with methicillin-resistant Staphylococcus aureus (MRSA) and dosed orally starting 1 h later with ACX-728, ACX-801, ACX-978, or linezolid. Untreated control mice were euthanized at 1 h for baseline data, and a second untreated group were euthanized at 24 h after the start of dosing, when all remaining mice were euthanized. At euthanasia, thigh muscles were excised for MRSA quantification, and fecal pellets, colon tissue, and plasma samples were collected for drug concentration and microbiome analyses. All PolC inhibitors were detectable in plasma and feces at 25 h. Compared with linezolid, PolC inhibitors maintained greater gut microbial richness and diversity in this acute murine infection model. PolC-treated samples preserved overall community structure and Bacteroidota dominance, whereas linezolid induced marked disruption, with secondary expansion of Pseudomonadota , primarily Escherichia coli . Microbial pathway analysis showed preservation of community-level metabolic function for PolC inhibitors but marked disruption by linezolid. Compared to controls, linezolid produced a 4.4 log reduction in MRSA colony-forming unit per gram, while the PolC inhibitors produced a mean log reduction of 0.4–2.5 per group. In these exploratory studies, PolC inhibitor compounds preserved anaerobic community structure and fermentative capacity, whereas linezolid induced depletion of healthy commensals and expansion of aerotolerant taxa. These findings highlight the importance of microbiome-informed antibiotic evaluation in preclinical studies. Larger preclinical and clinical studies are warranted to confirm these findings. IMPORTANCE: This study evaluated the class effect of PolC inhibitors on the microbiome as part of the preclinical development plan for systemic PolC inhibitors. Similar to the non-absorbable antibiotic, ibezapolstat, a narrow effect on the microbiome was observed with these novel compounds.