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/xtaa001 Methods A 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. Results Survival 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 Conclusion Germinant/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)

Omadacycline compared to vancomycin when combined with germinants to disrupt the life cycle of Clostridioides difficile

Clostridioides difficile (C. difficile) infections (CDI) are commonly treated with antibiotics that do not impact the dormant spore form of the pathogen. CDI-directed antibiotics, such as vancomycin and metronidazole, can destroy the vegetative form of C. difficile and protective microbiota. After treatment, spores can germinate into vegetative cells causing clinical disease relapse and further spore shedding. This in vitro study compares the combination of germinants with vancomycin or omadacycline to antibiotics alone in eradicating C. difficile spores and vegetative cells. Among the four strains in this study, omadacycline minimum inhibitory concentrations (0.031-0.125 mg/L) were lower than vancomycin (1-4 mg/L). Omadacycline nor vancomycin in media alone reduced spore counts. In three of the four strains, including the epidemic ribotype 027, spore eradication with germinants was 94.8-97.4% with vancomycin and 99.4-99.8% with omadacycline (p<0.005). In ribotype 012, either antibiotic combined with germinants resulted in 100% spore eradication at 24 hours. The addition of germinants with either antibiotic did not result in significant toxin A or B production, which were below the limit of detection (<1.25 ng/mL) by 48 hours. Limiting the number of spores present in patient GI tracts at the end of therapy may be effective at preventing recurrent CDI and limiting spore shedding in the healthcare environment. These results with germinants warrant safety and efficacy evaluations in animal models.

Encephalitozoon cuniculi infection in cats: European guidelines from the ABCD on prevention and management

Overview: Encephalitozoon cuniculi is a common obligate intracellular microsporidian parasite of rabbits that is increasingly recognised as a pathogen of cats and other mammalian species. These guidelines aim to review the literature on feline E cuniculi infection and provide recommendations on prevention and management. Infection in cats: E cuniculi infection should be considered as a differential diagnosis in cases of feline uveitis and cataract formation. It is not significantly associated with either chronic kidney disease or meningoencephalitis. E cuniculi infection is more common in stray or feral cats than in pet cats. Diagnosis and treatment: Serological tests for antibody detection in the blood are easy to perform and can be useful for diagnosis, but their specificity is low as antibodies have been found in apparently healthy cats. PCR appears to be more sensitive than histopathology for diagnosis, and is more sensitive when performed on cataractous lenses compared with aqueous humour, although ease of sampling is an obvious limitation. Treatment is with fenbendazole for 3 weeks and phacoemulsification to remove microsporidia from cataractous lenses. Zoonotic risk: E cuniculi is a potential zoonotic agent, and there is a particular risk to immunocompromised humans posed by infected rabbits. Albeit infrequent, spore shedding has been identified in cats, so care should be taken around infected cats.

Encephalitozoon intestinalis: A new target for auranofin in a mice model

Despite the fact that many approaches have been developed over years to find efficient and well-tolerated therapeutic regimens for microsporidiosis, the effectiveness of current drugs remains doubtful, and effective drugs against specific targets are still scarce. The present study is the first that was designed to evaluate the potency of auranofin, an anti-rheumatoid FDA approved drug, against intestinal Encephalitozoon intestinalis. Evaluation of the drug was achieved through counting of fecal and intestinal spores, studying the intestinal histopathological changes, measuring of intestinal hydrogen peroxide level, and post therapy follow-up of mice for 2 weeks for detection of relapse. Results showed that auranofin has promising anti-microsporidia potential. It showed a promising efficacy in mice experimentally infected with E. intestinalis. It has revealed an obvious reduction in fecal spore shedding and intestinal tissue spore load, amelioration of intestinal tissue pathological changes, and improvement of the local inflammatory infiltration without significant changes in hydrogen peroxide level. Interestingly, auranofin prevented the relapse of infection. Thus, considering the results of the present work, auranofin could be considered a therapeutic alternative for the gold standard drug ‘albendazole’ against the intestinal E. intestinalis infection especially in relapsing cases.

Spore shedding pattern of Enterocytozoon bieneusi in asymptomatic children

Stool samples from seven human immunodeficiency virus (HIV)-negative and two HIV-positive children with asymptomatic Enterocytozoon bieneusi infections were daily examined to quantify spore shedding using Gram-chromotrope staining under light microscopy. The spore shedding pattern and intensity in these children was variable. Mean spore concentrations in the stool samples from these children ranged from 2.4 × 102 to 1.2 × 105 spores per gram. Light microscopy could detect spores in stool specimens for 9–33 days, while PCR was able to detect E. bieneusi in stool specimens for 3–40 days longer. This suggests that light microscopy may not detect low levels of spore shedding. Considering that the asymptomatic group are a potential source of infection, detection methods with a higher sensitivity should be used.

Isolation and characterization of an avian isolate of Encephalitozoon hellem

Members of the phylum Microspora are a group of unusual, obligate intracellular eukaryotic parasites that infect a wide range of hosts. However, there are a limited number of microsporidial infections reported in avian hosts, and no parasite species has been defined as an avian pathogen. A microsporidian organism was recovered from the droppings of a clinically normal peach-faced lovebird (Agapornis roseicollis) and established in in vitro culture. Intermittent parasite spore shedding was documented over a 2-month period using calcofluor M2R staining of cloacal swabs. The organism was identified as Encephalitozoon hellem based on protein and antigenic profiles and molecular sequencing of the small subunit and internal transcribed spacer regions of the ribosomal RNA gene.