Difficile Spores Research Articles

Soluble Non-Starch Polysaccharides From Plantain (Musa x paradisiaca L.) Diminish Epithelial Impact of Clostridioides difficile

Clostridioides difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea. Adhesion of this Gram-positive pathogen to the intestinal epithelium is a crucial step in CDI, with recurrence and relapse of disease dependent on epithelial interaction of its endospores. Close proximity, or adhesion of, hypervirulent strains to the intestinal mucosa are also likely to be necessary for the release of C. difficile toxins, which when internalized, result in intestinal epithelial cell rounding, damage, inflammation, loss of barrier function and diarrhoea. Interrupting these C. difficile-epithelium interactions could therefore represent a promising therapeutic strategy to prevent and treat CDI. Intake of dietary fibre is widely recognised as being beneficial for intestinal health, and we have previously shown that soluble non-starch polysaccharides (NSP) from plantain banana (Musa spp.), can block epithelial adhesion and invasion of a number of gut pathogens, such as E. coli and Salmonellae. Here, we assessed the action of plantain NSP, and a range of alternative soluble plant fibres, for inhibitory action on epithelial interactions of C. difficile clinical isolates, purified endospore preparations and toxins. We found that plantain NSP possessed ability to disrupt epithelial adhesion of C. difficile vegetative cells and spores, with inhibitory activity against C. difficile found within the acidic (pectin-rich) polysaccharide component, through interaction with the intestinal epithelium. Similar activity was found with NSP purified from broccoli and leek, although seen to be less potent than NSP from plantain. Whilst plantain NSP could not block the interaction and intracellular action of purified C. difficile toxins, it significantly diminished the epithelial impact of C. difficile, reducing both bacteria and toxin induced inflammation, activation of caspase 3/7 and cytotoxicity in human intestinal cell-line and murine intestinal organoid cultures. Dietary supplementation with soluble NSP from plantain may therefore confer a protective effect in CDI patients by preventing adhesion of C. difficile to the mucosa, i.e. a “contrabiotic” effect, and diminishing its epithelial impact. This suggests that plantain soluble dietary fibre may be a therapeutically effective nutritional product for use in the prevention or treatment of CDI and antibiotic-associated diarrhoea.

1037. Efficacy of Germinants and Omadacycline for Preventing Clostridioides difficile Relapse in a Murine Model

Background Clostridioides difficile is labeled one of five urgent pathogens by the CDC. The urgency is related to the high burden of disease, limited effective antimicrobials, and recurrent C. difficile infections (rCDI) from residual spores (Fig. 1). Impervious to antibiotics, C. difficile spores could be induced into vegetative cells by germinants, namely taurocholate, for antibiotic targeting. This study aims to evaluate spore reservoir eradication through applying germinants with antibiotics.Figure 1. Schematic of the infectious life cycle of C. difficile and treatment opportunities Noah Budi, Nasia Safdar, Warren E Rose, Treatment issues in recurrent Clostridioides difficile infections and the possible role of germinants, FEMS Microbes, Volume 1, Issue 1, September 2020, xtaa001, https://doi.org/10.1093/femsmc/xtaa001MethodsA published murine model of rCDI using C57BL/6 mice and 1 x 105C. difficile spores (VPI 10463) with modification was used (Fig. 2). Six hours after inoculation, mice received 1.5 mg vancomycin (VAN, n=10) or 0.25 mg omadacycline (OMC, n=10) daily by oral gavage until day 4 or either with germinant (G) solution (8 mg of sodium taurocholate, 10 mg of taurine, 0.2 mg of sodium docusate, and 1.72 mg of calcium gluconate) given concomitantly on days 1 to 3 (OMC+G, n=9 and VAN+G, n=8). As a positive control, five mice did not receive antibiotics after spores. To induce rCDI, clindamycin was given on days 10 to 12. Survival, clinical scoring (CS), and weight loss (WL) were recorded until day 15. Fecal samples were taken to measure toxin production and spore shedding. Mice that died prior to day 15, were too sick to provide samples, or had positive stool culture were considered positive for day 15 spore shedding. Fisher’s exact test was used.Figure 2: Experimental Design Antibiotic water consisted of kanamycin 0.4 mg/ml, gentamicin 0.035 mg/mL, colistin 850 U/mL, metronidazole 0.215 mg/mL, and vancomycin 0.045 mg/mL given noon to noon on specified days. Clindamycin IP injections given as weight based dose of 10 mg/kg.ResultsSurvival is summarized in Figure 3. Both OMC and VAN had 60% survival by day 15 while OMC+G and VAN+G had 100% (p=0.004). Germinant CS and WL were similar to respective antibiotic alone groups until day 8; OMC overall had less severe disease than VAN (Figure 4). Toxin production on day 10 was lower in OMC than VAN, but absent from OMC+G and VAN+G. On day 15, 100% of VAN mice were spore positive compared to 60% with OMC (p=0.087). No mice receiving germinants (OMC+G or VAN+G) were spore positive (p< 0.0001). Figure 3. Survival Percentage Figure 4. Clinical Scoring and Weight Loss ConclusionGerminant/antibiotic combinations improved survival in a rCDI mouse model compared to antibiotics alone. Germinants did not induce toxin production when combined with OMC or VAN and eliminated the spore reservoir at the end of treatment. This provides basis for further study of germinants combined with antibiotics to reduce rCDI. Disclosures Warren Rose, PharmD, MPH, Merck (Grant/Research Support)Paratek (Grant/Research Support, Advisor or Review Panel member)

761. Host Intestinal Defenses Against Clostridioides difficile Infection in Chemotherapy Patients

Abstract Background Clostridioides difficile infection (CDI) is a common complication in patients undergoing cancer treatment with cytotoxic chemotherapy. Exposure to antibiotics or chemotherapy disrupts the microbiome by killing protective intestinal flora which consequently promotes C. difficile spore germination and disease. The host defense against CDI includes colonization resistance conferred by the healthy microbiome and innate defenses provided by intestinal epithelial cells. One protective factor secreted by Paneth cells of the intestinal epithelium is lysozyme, an enzyme that degrades the cell walls of Gram-positive bacteria such as C. difficile. We hypothesized that chemotherapy-induced mucosal barrier injury and the resultant death of Paneth cells leads to decreased production of lysozyme. We thus sought to examine changes in lysozyme concentration in stools of chemotherapy patients. Methods We collected stool samples from six patients undergoing cancer treatment at four different time points. The first stool sample corresponded to the day prior to the start of chemotherapy (day zero). We then performed ELISA assays to determine the lysozyme concentration for each stool sample. Results On day zero, the lysozyme levels (n=6) averaged 268.1 ± 131.7 ng/mL. Over the course of chemotherapy, the lysozyme levels decreased 78.70 ± 24.19% from the starting value. The lowest values were observed around days 5 through 11 for most patients, coinciding with when they were most neutropenic around day 11. One of the patients developed CDI on day 5 and experienced more fluctuating lysozyme levels thereafter. On the day that the patient developed CDI, lysozyme was measured as 6.63 ng/mL. Throughout treatment, 3/6 patients showed recovery of lysozyme production with white blood cell recovery. Conclusion Our data indicate that chemotherapy causes decreased concentrations of lysozyme in stool. Low lysozyme levels could in part account for the increased susceptibility to CDI during chemotherapy. Future experiments will include bioinformatics analyses to determine how the microbiome changes in response to chemotherapy. Together, these experiments will inform our approach to determining patient susceptibility to chemotherapy-associated CDI. Disclosures All Authors: No reported disclosures

Inhibition of spores to prevent the recurrence of Clostridioides difficile infection - A possibility or an improbability?

Clostridioides difficile is one of the most common nosocomial gastrointestinal pathogens, and recurrence is a problematic issue because approximately 20-30% of patients experience at least one episode of recurrence, even after treatment with a therapeutic drug of choice for C. difficile infection (CDI), such as vancomycin. CDI recurrence has a multifactorial complex mechanism, in which gut microbiota disruption coincident with viable C. difficile spores, is considered the most important factor. The effectiveness of an anti-C. difficile antimicrobial agent against CDI cannot guarantee its inhibitory effect on C. difficile spores and vice versa. However, an antimicrobial agent, such as fidaxomicin, which has a good inhibitory effect on both C. difficile vegetative cells and spores is assumed to not only treat CDI but also prevent its recurrence. Prolonged adherence to the exosporium has been proposed as a possible mechanism of inhibiting spores, and as a result, redesigning anti-C. difficile antimicrobial agents with the ability to adhere to the exosporium may provide another pathway for the development of anti-C. difficile spore agents. For example, vancomycin lacks an inhibitory effect against C. difficile spores, but a vancomycin-loaded spore-targeting iron oxide nanoparticle that selectively binds to C. difficile spores has been developed to successfully delay spore germination. Some new antimicrobial agents in phase II clinical trials, including cadazolid and ridinilazole, have shown exceptional anti-C. difficile and spore-inhibiting effects that can be expected to not only treat CDI but also prevent its recurrence in the future.

Gut microbiota in burned patients with Clostridioides difficile infection

BackgroundThe survival rate of patients with severe burn is positively associated with increasing the incidence of the Clostridioides difficile (C. difficile) infection (CDI). The surviving rate of severe burn patients now has an improved but the incidence of Clostridioides difficile (C. difficile) infection (CDI) has been continues increasing during recent two decades.This study assessed the molecular typing and phenotypic characterization isolates of C. difficile in burn patients with diarrhea, as well as environmental and skin infections with C. difficile spores at a referral burn hospital in Isfahan, Iran. It mainly aimed to evaluate the dominant bacterial structure in the gut microbiome of burned subjects with and without CDI. MethodsIn general, 309 samples were collected from 189 burned patients with hospital-acquired diarrhea and 120 swabs were collected from the healthcare workers’ dominant hands, different sites of patients’ skin, and medical tools. In addition, C. difficile isolates were characterized considering the existence of antibiotic resistance and toxin genes. Clinical cultures with identification of organisms and antibiotic susceptibility were done. C. difficle isolates were then genotyped and compared to clinical outcomes. Finally, the clinical characteristics of the participants were gathered through their records, and the bacterial targets of the gut microbiome were detected using quantitative real-time polymerase chain reaction (PCR). ResultsBased on the findings, 51 C. difficile isolates were detected from 189 severe burn patients hospitalized in the hospital. Further, PCR amplification tcdB and tcdA showed 23 isolates (12.2%) as toxigenic. Overall, 18.3% (22/120) of skin and environment samples demonstrated a positive result for C. difficile colonization. A low concentration of metronidazole and vancomycin (MIC90, 0.5, and 1.2 mg/L) inhibited all toxigenic C. difficile strains. Moreover, these isolates represented the highest rates of resistance to moxifloxacin and clindamycin (MIC90, 0.5, and 1.6 mg/L). A significantly reduced abundance of Clostridium spp., Bacteroidetes, and Bifidobacterium and an increase in the quantity of Firmicutes was observed in the gastrointestinal microbiome of burn patients (P < 0.01). Burn patients with CDI showed a significant decrease in Faecalibacterium prausnitzii (F. prausnitzii) while higher Akkermansia muciniphila (A. muciniphila) loads in comparison with healthy controls (P < 0.001 and P < 0.05). Contrarily, burned cases displayed increased levels of opportunistic pathogenic bacteria including the members of Enterococcus spp. and Escherichia coli (P < 0.05). ConclusionsDespite appropriate infection control strategies in the burn intensive care unit, CDI remains prevalent in severe burn patients. Eventually, the overgrowth of A. muciniphila and the decreased abundance of F. prausnitzii in burn cases with CDI could be potential predictive microbiome biomarkers in burned patients.

Studies on the Importance of the 7α-, and 12α- hydroxyl groups of N-Aryl-3α,7α,12α-trihydroxy-5β-cholan-24-amides on their Antigermination Activity Against a Hypervirulent Strain of Clostridioides (Clostridium) difficile

Chenodeoxycholic acid (CDCA) is a natural germination inhibitor for C. difficile spores. In our previous study (J. Med. Chem., 2018, 61, 6759–6778), we identified N-phenyl-3α,7α,12α-trihydroxy-5β-cholan-24-amide as an inhibitor of C. difficile strain R20291 with an IC50 of 1.8 μM. Studies of bile salts on spore germination have shown that chenodeoxycholate, ursodeoxycholate and lithocholate are more potent inhibitors of germination compared to cholate. Given this, we created amide analogs of chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids using amines identified from our previous studies. We found that chenodeoxy- and deoxycholate derivatives were active with potencies equivalent to those for cholanamides. This indicates that only 2 out of the 3 hydroxyl groups are needed for activity and that the alpha stereochemistry at position 7 is required for inhibition of spore germination.

The potential of microbiome replacement therapies for Clostridium difficile infection.

There is a paradox when treating Clostridium difficile infection (CDI); treatment antibiotics reduce C. difficile colonization but cause further microbiota disruption and can lead to recurrent disease. The success of faecal microbiota transplants (FMT) in treating CDI has become a new research area in microbiome restorative therapies but are they a viable long-term treatment option? C. difficile displays metabolic flexibility to use different nutritional sources during CDI. Using microbiome therapies for the efficient restoration of bile homeostasis and to reduce the bioavailability of preferential nutrients will target the germination ability of C. difficile spores and the growth rate of vegetative cells. Several biotechnology companies have developed microbiome therapeutics for treating CDI, which are undergoing clinical trials. There is confidence in using restorative microbiome therapies for treating CDI after the demonstrated efficacy of FMT, where several biotechnology companies are aiming to supply what would be a 'first in class' treatment option. Efficient removal of C. difficile from the different intestinal biogeographies should be considered in future microbiome therapies. With the gut microbiota implicated in different diseases, more work is needed to assess the long-term consequences of microbiome therapies.

A lipoprotein allosterically activates the CwlD amidase during Clostridioides difficile spore formation.

Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. During gemination, spores must degrade their cortex layer, which is a thick, protective layer of modified peptidoglycan. Cortex degradation depends on the presence of the spore-specific peptidoglycan modification, muramic-∂-lactam (MAL), which is specifically recognized by cortex lytic enzymes. In C. difficile, MAL production depends on the CwlD amidase and its binding partner, the GerS lipoprotein. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind Zn2+ stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to Zn2+, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of Zn2+ co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.

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

ObjectivesThe 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.MethodsThree 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.ResultsInstillation 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.ConclusionsThese 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.

The small acid-soluble proteins of Clostridioides difficile are important for UV resistance and serve as a check point for sporulation.

Clostridioides difficile is a nosocomial pathogen which causes severe diarrhea and colonic inflammation. C. difficile causes disease in susceptible patients when endospores germinate into the toxin-producing vegetative form. The action of these toxins results in diarrhea and the spread of spores into the hospital and healthcare environments. Thus, the destruction of spores is imperative to prevent disease transmission between patients. However, spores are resilient and survive extreme temperatures, chemical exposure, and UV treatment. This makes their elimination from the environment difficult and perpetuates their spread between patients. In the model spore-forming organism, Bacillus subtilis, the small acid-soluble proteins (SASPs) contribute to these resistances. The SASPs are a family of small proteins found in all endospore-forming organisms, C. difficile included. Although these proteins have high sequence similarity between organisms, the role(s) of the proteins differ. Here, we investigated the role of the main α/β SASPs, SspA and SspB, and two annotated putative SASPs, CDR20291_1130 and CDR20291_3080, in protecting C. difficile spores from environmental insults. We found that SspA is necessary for conferring spore UV resistance, SspB minorly contributes, and the annotated putative SASPs do not contribute to UV resistance. In addition, the SASPs minorly contribute to the resistance of nitrous acid. Surprisingly, the combined deletion of sspA and sspB prevented spore formation. Overall, our data indicate that UV resistance of C. difficile spores is dependent on SspA and that SspA and SspB regulate/serve as a checkpoint for spore formation, a previously unreported function of SASPs.

Managing microbes

Managing microbes

PHI-004 - Effectiveness of ozone generated by dielectric barrier discharge plasma reactor against multidrug-resistant bacteria and Clostridium difficile spore

PHI-004 - Effectiveness of ozone generated by dielectric barrier discharge plasma reactor against multidrug-resistant bacteria and Clostridium difficile spore

Clostridioides difficile infection induces a rapid influx of bile acids into the gut during colonization of the host.

SUMMARYClostridioides difficile is the leading cause of nosocomial intestinal infections in the United States. Ingested C. difficile spores encounter host bile acids and other cues that are necessary for germinating into toxin-producing vegetative cells. While gut microbiota disruption (often by antibiotics) is a prerequisite for C. difficile infection (CDI), the mechanisms C. difficile employs for colonization remain unclear. Here, we pioneered the application of imaging mass spectrometry to study how enteric infection changes gut metabolites. We find that CDI induces an influx of bile acids into the gut within 24 h of the host ingesting spores. In response, the host reduces bile acid biosynthesis gene expression. These bile acids drive C. difficile outgrowth, as mice receiving the bile acid sequestrant cholestyramine display delayed colonization and reduced germination. Our findings indicate that C. difficile may facilitate germination upon infection and suggest that altering flux through bile acid pathways can modulate C. difficile outgrowth in CDI-prone patients.

Clostridioides difficile SpoVAD and SpoVAE Interact and Are Required for Dipicolinic Acid Uptake into Spores.

Clostridioides difficile spores, like the spores from most endospore-forming organisms, are a metabolically dormant stage of development with a complex structure that conveys considerable resistance to environmental conditions, e.g., wet heat. This resistance is due to the large amount of dipicolinic acid (DPA) that is taken up by the spore core, preventing rotational motion of the core proteins. DPA is synthesized by the mother cell, and its packaging into the spore core is mediated by the products of the spoVA operon, which has a variable number of genes, depending on the organism. C. difficile encodes 3 spoVA orthologues, spoVAC, spoVAD, and spoVAE. Prior work has shown that C. difficile SpoVAC is a mechanosensing protein responsible for DPA release from the spore core upon the initiation of germination. However, the roles of SpoVAD and SpoVAE remain unclear in C. difficile. In this study, we analyzed the roles of SpoVAD and SpoVAE and found that they are essential for DPA uptake into the spore, similar to SpoVAC. Using split luciferase protein interaction assays, we found that these proteins interact, and we propose a model where SpoVAC/SpoVAD/SpoVAE proteins interact at or near the inner spore membrane, and each member of the complex is essential for DPA uptake into the spore core. IMPORTANCE C. difficile spore heat resistance provides an avenue for it to survive the disinfection protocols in hospital and community settings. The spore heat resistance is mainly the consequence of the high DPA content within the spore core. By elucidating the mechanism by which DPA is taken up by the spore core, this study may provide insight into how to disrupt the spore heat resistance with the aim of making the current disinfection protocols more efficient at preventing the spread of C. difficile in the environment.

Pragmatic Strategy for Fecal Specimen Storage and the Corresponding Test Methods for Clostridioides difficile Diagnosis.

The quality of fecal specimens is one of the factors responsible for successful Clostridioides difficile infection (CDI) diagnosis. The quality depends largely on the storage conditions, including the temperature and time period. In this study, we organized the outputs of previous studies, filled experimental gaps in the knowledge of storage conditions, and introduced a pragmatic strategy for fecal storage for CDI diagnosis. A 5-step pathway was adopted to develop the fecal specimen storage strategy as follows: step 1, bibliomic analysis; step 2, experimental gap-filling; step 3, comparative evaluation; step 4, strategy development; step 5, internal review. Step 1 identified eight articles providing experimental information on the effects of fecal specimen storage conditions on the effectiveness of C. difficile detection methods. Step 2 provided additional quantitative data on C. difficile vegetative and spore cell viability and DNA stability. All previous and current results were compared (step 3). In step 4, fir general and nine special strategies were developed, followed by an internal review of the overall approaches (step 5). It is recommended to separate fecal samples into aliquots before testing and storing them. It is particularly recommended that fecal specimen samples be stored for CDI diagnosis at 4 °C for up to 60 days for all test methods.

Teicoplanin Suppresses Vegetative Clostridioides difficile and Spore Outgrowth.

In recent decades, the incidence of Clostridioides difficile infection (CDI) has remained high in both community and health-care settings. With the increasing rate of treatment failures and its ability to form spores, an alternative treatment for CDI has become a global priority. We used the microdilution assay to determine minimal inhibitory concentrations (MICs) of vancomycin and teicoplanin against 30 distinct C. difficile strains isolated from various host origins. We also examined the effect of drugs on spore germination and outgrowth by following the development of OD600. Finally, we confirmed the spore germination and cell stages by microscopy. We showed that teicoplanin exhibited lower MICs compared to vancomycin in all tested isolates. MICs of teicoplanin ranged from 0.03–0.25 µg/mL, while vancomycin ranged from 0.5–4 µg/mL. Exposure of C. difficile spores to broth supplemented with various concentrations of antimicrobial agents did not affect the initiation of germination, but the outgrowth to vegetative cells was inhibited by all test compounds. This finding was concordant with aberrant vegetative cells after antibiotic treatment observed by light microscopy. This work highlights the efficiency of teicoplanin for treatment of C. difficile through prevention of vegetative cell outgrowth.

Opportunities for Nanomedicine in Clostridioides difficile Infection.

Clostridioides difficile, a spore-forming bacterium, is a nosocomial infectious pathogen which can be found in animals as well. Although various antibiotics and disinfectants were developed, C. difficile infection (CDI) remains a serious health problem. C. difficile spores have complex structures and dormant characteristics that contribute to their resistance to harsh environments, successful transmission and recurrence. C. difficile spores can germinate quickly after being exposed to bile acid and co-germinant in a suitable environment. The vegetative cells produce endospores, and the mature spores are released from the hosts for dissemination of the pathogen. Therefore, concurrent elimination of C. difficile vegetative cells and inhibition of spore germination is essential for effective control of CDI. This review focused on the molecular pathogenesis of CDI and new trends in targeting both spores and vegetative cells of this pathogen, as well as the potential contribution of nanotechnologies for the effective management of CDI.

Efficacy and Safety of Investigational Microbiome Drug SER-109 for Treatment of Recurrent Clostridioides difficile Infection

Background: Antibiotics targeted against Clostridioides difficile bacteria are necessary, but insufficient, to achieve a durable clinical response because they have no effect on C. difficile spores that germinate within a disrupted microbiome. ECOSPOR-III evaluated SER-109, an investigational, biologically derived microbiome therapeutic of purified Firmicute spores for treatment of rCDI. Herein, we present the interim analysis in the ITT population at 8 and 12 weeks. Methods: Adults ≥18 years with rCDI (≥3 episodes in 12 months) were screened at 75 US and CAN sites. CDI was defined as ≥3 unformed stools per day for &lt;48 hours with a positive C. difficile assay. After completion of 10–21 days of vancomycin or fidaxomicin, adults with symptom resolution were randomized 1:1 to SER-109 (4 capsules × 3 days) or matching placebo and stratified by age (≥ or &lt;65 years) and antibiotic received. Primary objectives were safety and efficacy at 8 weeks. Primary efficacy endpoint was rCDI (recurrent toxin+ diarrhea requiring treatment); secondary endpoints included efficacy at 12 weeks after dosing. Results: Overall, 287 participants were screened and 182 were randomized (59.9% female; mean age, 65.5 years). The most common reason for screen failure was a negative C. difficile toxin assay. A significantly lower proportion of SER-109 participants had rCDI after dosing compared to placebo at week 8 (11.1% vs 41.3%, respectively; relative risk [RR], 0.27; 95% confidence interval [CI], 0.15–0.51; p-value &lt;0.001). Efficacy rates were significantly higher with SER-109 vs placebo in both stratified age groups (Figure 1). SER-109 was well-tolerated with a safety profile similar to placebo. The most common treatment-emergent adverse events (TEAEs) were gastrointestinal and were mainly mild to moderate. No serious TEAEs, infections, deaths, or drug discontinuations were deemed related to study drug. Conclusions: SER-109, an oral live microbiome therapeutic, achieved high rates of sustained clinical response with a favorable safety profile. By enriching for Firmicute spores, SER-109 achieves high efficacy while mitigating risk of transmitting infectious agents, beyond donor screening alone. SER-109 represents a major paradigm shift in the clinical management of patients with recurrent CDI. Clinicaltrials.gov Identifier NCT03183128. These data were previously presented as a late breaker at American College of Gastroenterology 2020.Funding: Seres TherapeuticsDisclosures: None

Medical Tape Contamination Study: Effect of Packaging on the Reduction of Cross Contamination

Background: Medical tape is used routinely for a variety of tasks across healthcare settings. The literature contains numerous publications in which common practices around medical tapes have been suspected to lead to infection transmission. Healthcare providers can turn to individually packaged single-patient-use medical tape rolls to help reduce cross-contamination risk by limiting exposure to environmental contaminants, minimizing contact with hospital surfaces and equipment, and minimizing exposure to healthcare workers’ hands and other patients. Methods: We evaluated the effect of individually packaged tape on cross contamination using a controlled laboratory assay. Ceramic tiles were inoculated with microorganisms evenly spread across the surface and allowed to air dry. Using gloves, packaged and unpackaged tapes were rolled over their entire outside circumference onto the contaminated tiles to simulate cross contamination. Using new gloves, the packaged tapes were then removed from their package with minimum contact. All cross-contaminated tape rolls were placed in phosphate-buffered water and mixed in a vortexer for bacterial recovery procedures. Serial dilutions were plated on appropriate media for bacterial enumeration. The average log10 colony-forming unit (CFU) recovery was measured for comparison. We used 4 types of tapes in this study (3M Micropore S Surgical Tape, 3M Medipore H Soft Cloth Surgical Tape, 3M Durapore Surgical Tape, and 3M Transpore White Surgical Tape). We used 4 different microorganisms as inoculates: Staphylococcus aureus (methicillin-resistant), Enterococcus faecium (vancomycin-resistant), Klebsiella pneumoniae (carbapenem-resistant), and Clostridium difficile (spore). Each test (tape and bacteria combination) was done in 3 or 6 replicates; each bacterial enumeration was the average of duplicate plates. The detection limit for this method is 8 CFU per sample, which is equivalent to 0.9 log10. Results: The results for all tapes tested showed a statistically significant lower mean log10 recovery of each of the microorganisms tested for packaged versus unpackaged tape (Figure 1). The mean differences of log10 recoveries from a packaged and unpackaged tape ranged from 2.51 log10 (for S. aureus on Micropore S) to 4.64 log10 (for K. pneumoniae on Medipore H). This is equivalent to 99%–99.99% cross-contamination protection from the 4 organisms tested. Conclusions: Individual packaging of medical tape rolls protects them from external contaminants. Even if the packaging becomes contaminated, the tape retrieved from the package will be significantly less contaminated than it would have been from exposure to the same contaminants without packaging.Funding: 3M CompanyDisclosures: None

Milk Exosomes Protect Human Microbiota Associated-Mice Against Clostridioides difficile Infection

ObjectivesExosomes are natural nanoparticles that facilitate cell-to-cell communication in animals and bacteria. Milk contains ∼1012 exosomes/mL. Evidence suggests that milk exosomes (MEs) contribute to maintaining a tight gut barrier function, bind toxins secreted by gut pathogens, and alter the growth of gut bacteria. Clostridioides difficile infection is a leading cause of health care-associated diarrhea in North America. Our objective of this study is to test if milk exosomes alter the severity of C. difficile infection. MethodsC57BL/6 mice stably colonized with a gut microbiome from a healthy elderly human (HMAmice) were fed ME- and RNA-sufficient (ERS) diet or ME- and RNA-depleted (ERD) diet for four weeks starting at age of three weeks (N = 20 in ERS, 17 in ERD). The content of ME and miRNA cargos in ERD is 15% and 1% of that in ERS. After four weeks, mice were treated with a combination of six antibiotics and challenged with 105C. difficile spores. Disease progression was monitored over time through measurement of weight loss, changes in behavior and stool consistency, and mortality. C. difficile colonization and toxin activity over time were assessed by colony forming units (CFU) and relative toxin units (RTU) in feces. A subset of mice was euthanized on day 3 to collect tissue samples for measurement of tissue damage and inflammation. Mann-Whitney test was used for statistical analysis and P < 0.05 was considered statistically significant. ResultsMice fed ERD diet exhibited more severe disease than mice fed ERS diet. Mice fed ERD diet had an increased mortality rate (58%) compared to mice fed ERS diet (25%, P = 0.0481) as well as 3-fold increase in weight loss two days following C. difficile infection compared to mice on an ERS diet (3.07 ± 3.77% (ERD) vs 1.08 ± 5.92%, P = 0.0488), The level of CFU and toxin activity was also higher in ERD-fed mice than that in ERS-fed mice. Finally, myeloperoxidase (MPO) activity assays (marker of neutrophil infiltration) and IL17a qRT-PCR provided evidence of higher immune activation in ERD-fed mice compared to ERS-fed mice. ConclusionsMEs decreased the severity of C. difficile infection in juvenile HMAmice. Funding SourcesNIH P20GM104320, NIFA 2016-67001-25301 and 2020-67017-30834, USDA W-4002.