Antibacterial activity of ibezapolstat against antimicrobial-resistant clinical strains of Clostridioides difficile.

Antimicrobial resistance is emerging in clinical strains of Clostridioides difficile. Ibezapolstat (IBZ) is a DNA polymerase IIIC inhibitor that has completed phase II clinical trials. IBZ has potent in vitro activity against wild-type, susceptible strains but its effect on C. difficile strains with reduced susceptibility to metronidazole (MTZ), vancomycin (VAN), or fidaxomicin (FDX) has not been tested. The primary objective of this study was to test the antibacterial properties of IBZ against multidrug-resistant C. difficile strains. The in vitro activity, bactericidal, and time-kill activity of IBZ versus comparators were evaluated against 100 clinical strains of which 59 had reduced susceptibility to other C. difficile antibiotics. Morphologic changes against a multidrug resistance strain were visualized by light and scanning electron microscopy. The overall IBZ MIC50/90 values (µg/mL) for evaluated C. difficile strains were 4/8, compared with 2/4 for VAN, 0.5/1 for FDX, and 0.25/4 for MTZ. IBZ MIC50/90 values did not differ based on non-susceptibility to antibiotic class or number of classes to which strains were non-susceptible. IBZ bactericidal activity was similar to the minimum inhibitory concentration (MIC) and maintained in wild-type and non-susceptible strains. Time-kill assays against two laboratory wild-type and two clinical non-susceptible strains demonstrated sustained IBZ activity despite reduced killing by comparator antibiotics for IBZ and VAN non-susceptible strains. Microscopy visualized increased cell lengthening and cellular damage in multidrug-resistant strains exposed to IBZ sub-MIC concentrations. This study demonstrated the potent antibacterial activity of IBZ against a large collection of C. difficile strains including multidrug-resistant strains. This study highlights the therapeutic potential of IBZ against multidrug-resistant strains of C. difficile.

The Novel DNA Binding Mechanism of Ridinilazole, a Precision Clostridiodes difficile Antibiotic.

Clostridioides difficile infection (CDI) causes substantial morbidity and mortality worldwide with limited antibiotic treatment options. Ridinilazole is a precision bisbenzimidazole antibiotic being developed to treat CDI and reduce unacceptably high rates of infection recurrence in patients. Although in late clinical development, the precise mechanism of action by which ridinilazole elicits its bactericidal activity has remained elusive. Here, we present conclusive biochemical and structural data to demonstrate that ridinilazole has a primary DNA binding mechanism, with a co-complex structure confirming binding to the DNA minor groove. Additional RNA-seq data indicated early pleiotropic changes to transcription, with broad effects on multiple C. difficile compartments and significant effects on energy generation pathways particularly. DNA binding and genomic localization was confirmed through confocal microscopy utilizing the intrinsic fluorescence of ridinilazole upon DNA binding. As such, ridinilazole has the potential to be the first antibiotic approved with a DNA minor groove binding mechanism of action.

Fusobacteria behaving badly: Masquerading strains of strictly anaerobic Escherichiacoli misidentified due to the deletion of the hemB gene.

Three strictly anaerobic strains of Escherichia coli were misidentified as Fusobacterium mortiferum, due to a deletion of the hemB gene which is involved in anaerobic respiration. An unusual antimicrobial susceptibility pattern sparked the further diagnostic strategies that eventually identified these strains as true anaerobic E. coli This phenomenon is more common than appreciated and can have an impact on clinical practice including persistent and relapsing infections.

Visualization of fidaxomicin association with the exosporium layer of Clostridioides difficile spores.

BACKGROUND: 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. MATERIALS & METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Fusobacteriumnucleatum Adheres to Clostridioides difficile via the RadD Adhesin to Enhance Biofilm Formation in Intestinal Mucus.

BACKGROUND & AIMS: Although Clostridioides difficile infection (CDI) is known to involve the disruption of the gut microbiota, little is understood regarding how mucus-associated microbes interact with C difficile. We hypothesized that select mucus-associated bacteria would promote C difficile colonization and biofilm formation. METHODS: To create a model of the human intestinal mucus layer and gut microbiota, we used bioreactors inoculated with healthy human feces, treated with clindamycin and infected with C difficile with the addition of human MUC2-coated coverslips. RESULTS: C difficile was found to colonize and form biofilms on MUC2-coated coverslips, and 16S rRNA sequencing showed a unique biofilm profile with substantial cocolonization with Fusobacterium species. Consistent with our bioreactor data, publicly available data sets and patient stool samples showed that a subset of patients with C difficile infection harbored high levels of Fusobacterium species. We observed colocalization of C difficile and F nucleatum in an aggregation assay using adult patients and stool of pediatric patients with inflammatory bowel disease and in tissue sections of patients with CDI. C difficile strains were found to coaggregate with F nucleatum subspecies in vitro; an effect that was inhibited by blocking or mutating the adhesin RadD on Fusobacterium and removal of flagella on C difficile. Aggregation was shown to be unique between F nucleatum and C difficile, because other gut commensals did not aggregate with C difficile. Addition of F nucleatum also enhanced C difficile biofilm formation and extracellular polysaccharide production. CONCLUSIONS: Collectively, these data show a unique interaction of between pathogenic C difficile and F nucleatum in the intestinal mucus layer.

In vitro activity of eravacycline against common ribotypes of Clostridioides difficile.

BACKGROUND: Eravacycline is a novel synthetic fluorocycline antibacterial approved for complicated intra-abdominal infections. OBJECTIVES: The purpose of this study was to assess the in vitro activities of eravacycline and comparator antibiotics against contemporary clinical isolates of Clostridioides difficile representing common ribotypes, including isolates with decreased susceptibility to metronidazole and vancomycin. METHODS: Clinical C. difficile strains from six common or emerging ribotypes were used to test the in vitro activities of eravacycline and comparator antibiotics (fidaxomicin, vancomycin and metronidazole) by broth microdilution. In addition, MBC experiments, time-kill kinetic studies and WGS experiments were performed. RESULTS: A total of 234 isolates were tested, including ribotypes RT001 (n = 37), RT002 (n = 41), RT014-020 (n = 39), RT027 (n = 42), RT106 (n = 38) and RT255 (n = 37). MIC50/90 values were lowest for eravacycline (≤0.0078/0.016 mg/L), followed by fidaxomicin (0.016/0.063 mg/L), metronidazole (0.25/1.0 mg/L) and vancomycin (2.0/4.0 mg/L). MBCs were lower for eravacycline compared with vancomycin for all ribotypes tested. Both vancomycin and eravacycline demonstrated bactericidal killing, including for epidemic RT027. The presence of the tetM or tetW resistance genes did not affect the MIC of eravacycline. CONCLUSIONS: This study demonstrated potent in vitro activity of eravacycline against a large collection of clinical C. difficile strains that was not affected by ribotype, susceptibility to vancomycin or the presence of certain tet resistance genes. Further development of eravacycline as an antibiotic to be used in patients with Clostridioides difficile infection is warranted.

In Vitro Activity of Omadacycline, a New Tetracycline Analog, and Comparators against Clostridioides difficile.

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. In vitroC. difficile susceptibility to omadacycline and comparators (fidaxomicin, metronidazole, and vancomycin) was assessed using the broth microdilution method. Minimum bactericidal concentrations (MBCs) and time-kill assays were assessed for pharmacodynamics analysis, and whole-genome sequencing was performed in a subset of isolates to assess distribution of MICs and resistance determinants. Two hundred fifty clinical C. difficile isolates collected between 2015 and 2018 were tested for in vitro susceptibility of omadacycline and comparators. Ribotypes included F001 (n = 5), F002 (n = 56), F014-020 (n = 66), F017 (n = 8), F027 (n = 53), F106 (n = 45), and F255 (n = 17). Omadacycline demonstrated potent in vitro activity with an MIC range of 0.016 to 0.13 µg/ml, an MIC50 of 0.031 µg/ml, and an MIC90 of 0.031 µg/ml. No difference was observed for omadacycline MIC50 and MIC90 values stratified by ribotype, disease severity, or vancomycin susceptibility. Bactericidal activity was confirmed in time-kill studies. No difference was observed in MIC based on C. difficile phylogeny. Further development of omadacycline as an intravenous and oral antibiotic directed toward C. difficile infection is warranted.

Eosinopenia and Binary Toxin Increase Mortality in Hospitalized Patients With Clostridioides difficile Infection.

BACKGROUND: 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. METHODS: 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). RESULTS: 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). CONCLUSIONS: 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.

Activity of Hospital Disinfectants against Vegetative Cells and Spores of Clostridioides difficile Embedded in Biofilms.

Clostridioides difficile spores can survive in the environment in either mono- or mixed-species biofilms. However, no previous studies have investigated chemical disinfection of C. difficile spores embedded in biofilms. Thus, the purpose of this study was to assess the in vitro effectiveness of hospital disinfectants against C. difficile spores embedded within biofilms. Five unique C. difficile strains embedded in three different biofilm types grown for 72 or 120 h were exposed to seven different hospital disinfectants. C. difficile abundance [as log(number of CFU/milliliter)] was calculated after manufacturer-determined contact times along with biofilm biomass and microscopy. The primary analysis compared differences between C. difficile vegetative cell and spore counts as well as amounts of biomass after exposure to disinfectants. C. difficile vegetative cells and spores were recovered from biofilms regardless of the type of biofilm growth or biofilm growth time. No disinfectant was able to completely eliminate C. difficile from the biofilms. Overall, Clorox, ortho-phthalaldehyde (OPA), and Virex were most effective at killing C. difficile spores regardless of biofilm age, ribotype, or wash conditions (whether biofilms are washed or unwashed) (P = 0.001, each). Clorox and OPA were also effective at killing total vegetative cell growth (P = 0.001, each), but Virex was found to be ineffective against vegetative cell growth in biofilms (P = 0.77). Clorox and Virex were most effective in reducing biomass, followed by Nixall, OPA, and Vital Oxide. No disinfectant was able to completely eliminate C. difficile embedded within biofilms although differences among disinfectants were noted. Future research will be required to determine methods to eradicate this persister reservoir.

Ridinilazole for the treatment of Clostridioides difficile infection.

INTRODUCTION: Ridinilazole is a novel antibiotic being developed for the treatment of Clostridioides difficile infection (CDI). Ridinilazole has completed two phase II trials and phase III trials which are denoted Ri-CoDIFy 1 and 2, are planned (ClinicalTrials.gov identifiers: NCT03595553 and NCT03595566). Areas covered: This article covers the chemistry, mechanism of action, in vitro microbiology versus C. difficile and host microbiota, pre-clinical and clinical efficacy, pharmacokinetics, pharmacodynamics and safety and tolerability of ridinilazole. Expert opinion: Ridinilazole is a novel antibiotic with ideal properties for the treatment of CDI. Given the promising results from the phase II clinical trial, ridinilazole may have the capability to lower the risk for CDI recurrence thus improving sustained clinical response rates - a current unmet medical need. Assuming a positive phase III trial, ridinilazole will enter a market with heightened awareness on the importance of prevention of CDI. This along with further research into the economic consequences and decreased patient quality of life associated with recurrent CDI, should provide clinicians with further evidence for the need for therapy that limits CDI recurrence and improves sustained clinical cure.

Epidemic Clostridioides difficile Ribotype 027 Lineages: Comparisons of Texas Versus Worldwide Strains.

BACKGROUND: The epidemic Clostridioides difficile ribotype 027 strain resulted from the dissemination of 2 separate fluoroquinolone-resistant lineages: FQR1 and FQR2. Both lineages were reported to originate in North America; however, confirmatory large-scale investigations of C difficile ribotype 027 epidemiology using whole genome sequencing has not been undertaken in the United States. METHODS: Whole genome sequencing and single-nucleotide polymorphism (SNP) analysis was performed on 76 clinical ribotype 027 isolates obtained from hospitalized patients in Texas with C difficile infection and compared with 32 previously sequenced worldwide strains. Maximum-likelihood phylogeny based on a set of core genome SNPs was used to construct phylogenetic trees investigating strain macro- and microevolution. Bayesian phylogenetic and phylogeographic analyses were used to incorporate temporal and geographic variables with the SNP strain analysis. RESULTS: Whole genome sequence analysis identified 2841 SNPs including 900 nonsynonymous mutations, 1404 synonymous substitutions, and 537 intergenic changes. Phylogenetic analysis separated the strains into 2 prominent groups, which grossly differed by 28 SNPs: the FQR1 and FQR2 lineages. Five isolates were identified as pre-epidemic strains. Phylogeny demonstrated unique clustering and resistance genes in Texas strains indicating that spatiotemporal bias has defined the microevolution of ribotype 027 genetics. CONCLUSIONS: Clostridioides difficile ribotype 027 lineages emerged earlier than previously reported, coinciding with increased use of fluoroquinolones. Both FQR1 and FQR2 ribotype 027 epidemic lineages are present in Texas, but they have evolved geographically to represent region-specific public health threats.

Environmental transmission of Clostridioides difficile ribotype 027 at a long-term care facility; an outbreak investigation guided by whole genome sequencing.

OBJECTIVE: This article describes a CDI outbreak in a long-term care (LTC) facility that used molecular typing techniques and whole-genome sequencing to identify widespread dissemination of the clonal strain in the environment which was successfully removed after terminal cleaning. SETTING: This study was conducted in a long-term care facility in Texas. METHODS: A recently hospitalized LTC patient was diagnosed with CDI followed shortly thereafter by 7 subsequent CDI cases. A stool specimen was obtained from each patient for culturing and typing. An environmental point-prevalence study of the facility was conducted before and after terminal cleaning of the facility to assess environmental contamination. Cultured isolates were typed using ribotyping, multilocus variant analysis, and whole-genome sequencing. RESULTS: Stool samples were available for 5 of 8 patients; of these specimens, 4 grew toxigenic C. difficile ribotype 027. Of 50 environmental swab samples collected throughout the facility prior to the facility-wide terminal cleaning, 19 (38%) grew toxigenic C. difficile (most commonly ribotype 027, 79%). The terminal cleaning was effective at reducing C. difficile spores in the environment and at eradicating the ribotype 027 strain (P<.001). Using multilocus variance analysis and whole-genome sequencing, clinical and environmental strains were highly related and, in some cases, were identical. CONCLUSION: Using molecular typing techniques, we demonstrated reduced environmental contamination with toxigenic C. difficile and the eradication of a ribotype 027 clone. These techniques may help direct infection control efforts and decrease the burden of CDI in the healthcare system.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment.

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (> 50% reduction in cell length for each antibiotic; P< 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug's MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

Community Environmental Contamination of Toxigenic Clostridium difficile.

BACKGROUND: Clostridium difficile infection is often considered to result from recent acquisition of a C difficile isolate in a healthcare setting. However, C difficile spores can persist for long periods of time, suggesting a potentially large community environmental reservoir. The objectives of this study were to assess community environmental contamination of toxigenic C difficile and to assess strain distribution in environmental versus clinical isolates. METHODS: From 2013 to 2015, we collected community environmental swabs from homes and public areas in Houston, Texas to assess C difficile contamination. All positive isolates were tested for C difficile toxins A and B, ribotyped, and compared with clinical C difficile isolates obtained from hospitalized patients in Houston healthcare settings. RESULTS: A total of 2538 environmental samples were collected over the study period. These included samples obtained from homes (n = 1079), parks (n = 491), chain stores (n = 225), fast food restaurants (n = 123), other commercial stores (n = 172), and hospitals (n = 448). Overall, 418 environmental isolates grew toxigenic C difficile (16.5%; P < .001) most commonly from parks (24.6%), followed by homes (17.1%), hospitals (16.5%), commercial stores (8.1%), chain stores (7.6%), and fast food restaurants (6.5%). A similar distribution of ribotypes was observed between clinical and environmental isolates with the exception that ribotype 027 was more common in clinical isolates compared with environmental isolates (P < .001). CONCLUSIONS: We identified a high prevalence of toxigenic C difficile from community environs that were similar ribotypes to clinical isolates. These findings suggest that interventions beyond isolation of symptomatic patients should be targeted for prevention of C difficile infection.

Cadazolid for the treatment of Clostridium difficile.

INTRODUCTION: Antibiotic development goals for CDI include potent antimicrobial effect against C. difficile, limited killing of host microbiota, potential effect on spores, and ability to interfere with toxin production. Cadazolid, a novel, non-absorbable hybrid antibiotic has many of these criteria. In phase I and II clinical trials, cadazolid was shown to be safe, well tolerated, and efficacious positioning itself as a potential future viable therapeutic option for CDI. Areas covered: This review provides an in-depth evaluation of the chemistry, microbiology, pharmacodynamics, pharmacokinetics, and clinical trial results for cadazolid. Clinical therapeutic outcomes are compared between cadazolid, fidaxomicin, and surotomycin. Expert opinion: Preclinical and early clinical studies demonstrated that cadazolid has unique properties that will likely be valuable to treat CDI and reduce recurrent infection. With compelling phase II clinical results, results from the ongoing phase III trial will better define the role of cadazolid for treating CDI in the future.

Novel antibiotics in development to treat Clostridium difficile infection.

PURPOSE OF REVIEW: Clostridium difficile infections (CDI) remain a challenge to treat clinically due primarily to limited number of antibiotics available and unacceptably high recurrence rates. Because of this, there has been significant demand for creating innovative therapeutics, which has resulted in the development of several novel antibiotics. RECENT FINDINGS: This review updates seven different antibiotics that are currently in development to treat CDI including fidaxomicin, surotomycin, ridinilazole, ramoplanin, cadazolid, LFF571, and CRS3123. Available preclinical and clinical data are compared between these antibiotics. SUMMARY: Many of these new antibiotics display almost ideal properties for antibiotics directed against CDI. Despite these properties, not all clinical development of these compounds has been successful. These studies have provided key insights into the pathogenesis of CDI and will continue to inform future drug development. Successful phase III clinical trials should result in several new and novel antibiotics to treat CDI.

A novel method for imaging the pharmacological effects of antibiotic treatment on Clostridium difficile.

Clostridium difficile is a significant cause of nosocomial-acquired infection that results in severe diarrhea and can lead to mortality. Treatment options for C. difficile infection (CDI) are limited, however, new antibiotics are being developed. Current methods for determining efficacy of experimental antibiotics on C. difficile involve antibiotic killing rates and do not give insight into the drug's pharmacologic effects. Considering this, we hypothesized that by using scanning electron microscopy (SEM) in tandem to drug killing curves, we would be able to determine efficacy and visualize the phenotypic response to drug treatment. To test this hypothesis, supraMIC kill curves were conducted using vancomycin, metronidazole, fidaxomicin, and ridinilazole. Following collection, cells were either plated or imaged using a scanning electron microscope (SEM). Consistent with previous reports, we found that the tested antibiotics had significant bactericidal activity at supraMIC concentrations. By SEM imaging and using a semi-automatic pipeline for image analysis, we were able to determine that vancomycin and to a lesser extent fidaxomicin and ridinilazole significantly affected the cell wall, whereas metronidazole, fidaxomicin, and ridinilazole had significant effects on cell length suggesting a metabolic effect. While the phenotypic response to drug treatment has not been documented previously in this manner, the results observed are consistent with the drug's mechanism of action. These techniques demonstrate the versatility and reliability of imaging and measurements that could be applied to other experimental compounds. We believe the strategies laid out here are vital for characterizing new antibiotics in development for treating CDI.

Evaluating the Effects of Surotomycin Treatment on Clostridium difficile Toxin A and B Production, Immune Response, and Morphological Changes.

Surotomycin is a cyclic lipopeptide in development for Clostridium difficile-associated diarrhea. This study aimed to assess the impact of surotomycin exposure on C. difficile toxin A and B concentrations and the associated changes in immune response in comparison to vancomycin and metronidazole. Time-kill curve assays were performed using strain R20291 (BI/NAP1/027) at supra-MICs (4× and 40×) and sub-MICs (0.5×) of surotomycin and comparators. Following treatment, CFU counts, toxin A and B concentrations, and cellular morphological changes using scanning electron microscopy were examined. Inflammatory response was determined by measuring interleukin-8 (IL-8) concentrations from polarized Caco-2 cells exposed to antibiotic-treated C. difficile growth media. Supra-MICs (4× and 40×) of surotomycin resulted in a reduction of vegetative cells over 72 h (4-log difference, P < 0.01) compared to controls. These results correlated with decreases of 77% and 68% in toxin A and B production at 48 h, respectively (P < 0.005, each), which resulted in a significant reduction in IL-8 concentration compared to controls. Similar results were observed with comparator antibiotics. Bacterial cell morphology showed that the cell wall was broken apart by surotomycin treatment at supra-MICs while sub-MIC studies showed a "deflated" phenotype plus a rippling effect. These results suggest that surotomycin has potent killing effects on C. difficile that results in reduced toxin production and attenuates the immune response similar to comparator antibiotics. The morphological data also confirm observations that surotomycin is a membrane-active antibiotic.

Impact on toxin production and cell morphology in Clostridium difficile by ridinilazole (SMT19969), a novel treatment for C. difficile infection.

OBJECTIVES: Ridinilazole (SMT19969) is a narrow-spectrum, non-absorbable antimicrobial with activity against Clostridium difficile undergoing clinical trials. The purpose of this study was to assess the pharmacological activity of ridinilazole and assess the effects on cell morphology. METHODS: Antibiotic killing curves were performed using the epidemic C. difficile ribotype 027 strain, R20291, using supra-MIC (4× and 40×) and sub-MIC (0.125×, 0.25× and 0.5×) concentrations of ridinilazole. Following exposure, C. difficile cells were collected for cfu counts, toxin A and B production, and morphological changes using scanning electron and fluorescence microscopy. Human intestinal cells (Caco-2) were co-incubated with ridinilazole-treated C. difficile growth medium to determine the effects on host inflammatory response (IL-8). RESULTS: Treatment at supra-MIC concentrations (4× and 40× MIC) of ridinilazole resulted in a significant reduction in vegetative cells over 72 h (4 log difference, P < 0.01) compared with controls without inducing spore formation. These results correlated with a 75% decrease in toxin A production (P < 0.05) and a 96% decrease in toxin B production (P < 0.05). At sub-MIC levels (0.5× MIC), toxin A production was reduced by 91% (P < 0.01) and toxin B production was reduced by 100% (P < 0.001), which resulted in a 74% reduction in IL-8 release compared with controls (P < 0.05). Sub-MIC (0.5×)-treated cells formed filamentous structures ∼10-fold longer than control cells. Following fluorescence labelling, the cell septum was not forming in sub-MIC-treated cells, yet the DNA was dividing. CONCLUSIONS: Ridinilazole had robust killing effects on C. difficile that significantly reduced toxin production and attenuated the inflammatory response. Ridinilazole also elicited significant cell division effects suggesting a potential mechanism of action.