BACKGROUND The Centers for Disease Control and Prevention estimate that Clostridioides difficile(C.difficile)causes half a million infections(CDI)annually and is a major cause of total infectious disease death in the ...BACKGROUND The Centers for Disease Control and Prevention estimate that Clostridioides difficile(C.difficile)causes half a million infections(CDI)annually and is a major cause of total infectious disease death in the United States,causing inflammation of the colon and potentially deadly diarrhea.We recently reported the isolation of ADS024,a Bacillus velezensis(B.velezensis)strain,which demonstrated direct in vitro bactericidal activity against C.difficile,with minimal collateral impact on other members of the gut microbiota.In this study,we hypothesized that in vitro activities of ADS024 will translate in vivo to protect against CDI challenge in mouse models.AIM To investigate the in vivo efficacy of B.velezensis ADS024 in protecting against CDI challenge in mouse models.METHODS To mimic disruption of the gut microbiota,the mice were exposed to vancomycin prior to dosing with ADS024.For the mouse single-dose study,the recovery of ADS024 was assessed via microbiological analysis of intestinal and fecal samples at 4 h,8 h,and 24 h after a single oral dose of 5×108 colony-forming units(CFU)/mouse of freshly grown ADS024.The single-dose study in miniature swine included groups that had been pre-dosed with vancomycin and that had been exposed to a dose range of ADS024,and a group that was not pre-dosed with vancomycin and received a single dose of ADS024.The ADS024 colonies[assessed by quantitative polymerase chain reaction(qPCR)using ADS024-specific primers]were counted on agar plates.For the 28-d miniature swine study,qPCR was used to measure ADS024 levels from fecal samples after oral administration of ADS024 capsules containing 5×109 CFU for 28 consecutive days,followed by MiSeq compositional sequencing and bioinformatic analyses to measure the impact of ADS024 on microbiota.Two studies were performed to determine the efficacy of ADS024 in a mouse model of CDI:Study 1 to determine the effects of fresh ADS024 culture and ADS024 spore preparations on the clinical manifestations of CDI in mice,and Study 2 to compare the efficacy of single daily doses vs dosing 3 times per day with fresh ADS024.C.difficile challenge was performed 24 h after the start of ADS024 exposure.To model the human distal colon,an anerobic fecal fermentation system was used.MiSeq compositional sequencing and bioinformatic analyses were performed to measure microbiota diversity changes following ADS024 treatment.To assess the potential of ADS024 to be a source of antibiotic resistance,its susceptibility to 18 different antibiotics was tested.RESULTS In a mouse model of CDI challenge,single daily doses of ADS024 were as efficacious as multiple daily doses in protecting against subsequent challenge by C.difficile pathogen-induced disease.ADS024 showed no evidence of colonization based on the observation that the ADS024 colonies were not recovered 24 h after single doses in mice or 72 h after single doses in miniature swine.In a 28-d repeat-dose study in miniature swine,ADS024 was not detected in fecal samples using plating and qPCR methods.Phylogenetic analysis performed in the human distal colon model showed that ADS024 had a selective impact on the healthy human colonic microbiota,similarly to the in vivo studies performed in miniature swine.Safety assessments indicated that ADS024 was susceptible to all the antibiotics tested,while in silico testing revealed a low potential for off-target activity or virulence and antibioticresistance mechanisms.CONCLUSION Our findings,demonstrating in vivo efficacy of ADS024 in protecting against CDI challenge in mouse models,support the use of ADS024 in preventing recurrent CDI following standard antibiotic treatment.展开更多
文摘BACKGROUND The Centers for Disease Control and Prevention estimate that Clostridioides difficile(C.difficile)causes half a million infections(CDI)annually and is a major cause of total infectious disease death in the United States,causing inflammation of the colon and potentially deadly diarrhea.We recently reported the isolation of ADS024,a Bacillus velezensis(B.velezensis)strain,which demonstrated direct in vitro bactericidal activity against C.difficile,with minimal collateral impact on other members of the gut microbiota.In this study,we hypothesized that in vitro activities of ADS024 will translate in vivo to protect against CDI challenge in mouse models.AIM To investigate the in vivo efficacy of B.velezensis ADS024 in protecting against CDI challenge in mouse models.METHODS To mimic disruption of the gut microbiota,the mice were exposed to vancomycin prior to dosing with ADS024.For the mouse single-dose study,the recovery of ADS024 was assessed via microbiological analysis of intestinal and fecal samples at 4 h,8 h,and 24 h after a single oral dose of 5×108 colony-forming units(CFU)/mouse of freshly grown ADS024.The single-dose study in miniature swine included groups that had been pre-dosed with vancomycin and that had been exposed to a dose range of ADS024,and a group that was not pre-dosed with vancomycin and received a single dose of ADS024.The ADS024 colonies[assessed by quantitative polymerase chain reaction(qPCR)using ADS024-specific primers]were counted on agar plates.For the 28-d miniature swine study,qPCR was used to measure ADS024 levels from fecal samples after oral administration of ADS024 capsules containing 5×109 CFU for 28 consecutive days,followed by MiSeq compositional sequencing and bioinformatic analyses to measure the impact of ADS024 on microbiota.Two studies were performed to determine the efficacy of ADS024 in a mouse model of CDI:Study 1 to determine the effects of fresh ADS024 culture and ADS024 spore preparations on the clinical manifestations of CDI in mice,and Study 2 to compare the efficacy of single daily doses vs dosing 3 times per day with fresh ADS024.C.difficile challenge was performed 24 h after the start of ADS024 exposure.To model the human distal colon,an anerobic fecal fermentation system was used.MiSeq compositional sequencing and bioinformatic analyses were performed to measure microbiota diversity changes following ADS024 treatment.To assess the potential of ADS024 to be a source of antibiotic resistance,its susceptibility to 18 different antibiotics was tested.RESULTS In a mouse model of CDI challenge,single daily doses of ADS024 were as efficacious as multiple daily doses in protecting against subsequent challenge by C.difficile pathogen-induced disease.ADS024 showed no evidence of colonization based on the observation that the ADS024 colonies were not recovered 24 h after single doses in mice or 72 h after single doses in miniature swine.In a 28-d repeat-dose study in miniature swine,ADS024 was not detected in fecal samples using plating and qPCR methods.Phylogenetic analysis performed in the human distal colon model showed that ADS024 had a selective impact on the healthy human colonic microbiota,similarly to the in vivo studies performed in miniature swine.Safety assessments indicated that ADS024 was susceptible to all the antibiotics tested,while in silico testing revealed a low potential for off-target activity or virulence and antibioticresistance mechanisms.CONCLUSION Our findings,demonstrating in vivo efficacy of ADS024 in protecting against CDI challenge in mouse models,support the use of ADS024 in preventing recurrent CDI following standard antibiotic treatment.
