Novel, non-colonizing, single-strain live biotherapeutic product ADS024 protects against Clostridioides difficile infection challenge in vivo.

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 antibiotic-resistance 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.

Identification of ADS024, a newly characterized strain of Bacillus velezensis with direct Clostridiodes difficile killing and toxin degradation bio-activities.

Clostridioides difficile infection (CDI) remains a significant health threat worldwide. C. difficile is an opportunistic, toxigenic pathogen that takes advantage of a disrupted gut microbiome to grow and produce signs and symptoms ranging from diarrhea to pseudomembranous colitis. Antibiotics used to treat C. difficile infection are usually broad spectrum and can further disrupt the commensal gut microbiota, leaving patients susceptible to recurrent C. difficile infection. There is a growing need for therapeutic options that can continue to inhibit the outgrowth of C. difficile after antibiotic treatment is completed. Treatments that degrade C. difficile toxins while having minimal collateral impact on gut bacteria are also needed to prevent recurrence. Therapeutic bacteria capable of producing a range of antimicrobial compounds, proteases, and other bioactive metabolites represent a potentially powerful tool for preventing CDI recurrence following resolution of symptoms. Here, we describe the identification and initial characterization of ADS024 (formerly ART24), a novel therapeutic bacterium that can kill C. difficile in vitro with limited impact on other commensal bacteria. In addition to directly killing C. difficile, ADS024 also produces proteases capable of degrading C. difficile toxins, the drivers of symptoms associated with most cases of CDI. ADS024 is in clinical development for the prevention of CDI recurrence as a single-strain live biotherapeutic product, and this initial data set supports further studies aimed at evaluating ADS024 in future human clinical trials.

The Use of a Mini-Bioreactor Fermentation System as a Reproducible, High-Throughput ex vivo Batch Model of the Distal Colon.

Ex vivo colon fermentation systems are highly versatile as models for analyzing gastrointestinal tract microbiota composition and functionality. Ex vivo colon models range in size and functionality from bench-top micro fermenters to large units housed in individualized cabinets. The length of set-up time (including stabilization periods) for each fermentation system can range from hours to weeks to months. The aim of this study was to investigate a single-use cassette mini-fermentation system as a reproducible batch model of the colon. The online data log from the cassettes (triplicate wells across four different cassettes, n = 12) was sensitive enough to identify real-time changes in pH, temperature, dissolved oxygen or liquid addition (sodium hydroxide) during the runs which could be addressed if an alarm set-point was triggered. The alpha diversity indices also showed little variation between cassettes with the samples clustering around the mean. The weighted beta diversity PCoA analysis illustrated that 95% of the variance between the samples was accounted for by the time-point and not the fermentation run/cassette used. The variation in taxonomic diversity between cassettes was limited to less than 20 out of 115 genera. This study provides evidence that micro-bioreactors provide some very attractive advantages as batch models for the human colon. We show for the first time the use of the micro-Matrix a 24-well sophisticated parallel controlled cassette-based bioreactors as a batch colon model. We demonstrated a high level of reproducibility across fermentation cassettes when used in conjunction with a standardized fecal microbiota. The machine can operate 24 individual fermentations simultaneously and are relatively cost effective. Based on next generation sequencing analysis, the micro-bioreactors offer a high degree of reproducibility together with high-throughput capacity. This makes it a potential system for large screening projects that can then be scaled up to large fermenters or human/animal in vivo experiments.

Core fecal microbiota of domesticated herbivorous ruminant, hindgut fermenters, and monogastric animals.

In this pilot study, we determined the core fecal microbiota composition and overall microbiota diversity of domesticated herbivorous animals of three digestion types: hindgut fermenters, ruminants, and monogastrics. The 42 animals representing 10 animal species were housed on a single farm in Ireland and all the large herbivores consumed similar feed, harmonizing two of the environmental factors that influence the microbiota. Similar to other mammals, the fecal microbiota of all these animals was dominated by the Firmicutes and Bacteroidetes phyla. The fecal microbiota spanning all digestion types comprised 42% of the genera identified. Host phylogeny and, to a lesser extent, digestion type determined the microbiota diversity in these domesticated herbivores. This pilot study forms a platform for future studies into the microbiota of nonbovine and nonequine domesticated herbivorous animals.

Preparation of a standardised faecal slurry for ex-vivo microbiota studies which reduces inter-individual donor bias.

BACKGROUND: In-vitro gut fermentation systems provide suitable models for studying gut microbiota composition and functionality. However, such methods depend on the availability of donors and the assumption of reproducibility between microbial communities before experimental treatments commence. The aim of this study was to develop a frozen standardised inoculum (FSI) which minimizes inter-individual variation and to determine its stability over time using culture-dependent and culture-independent techniques. RESULTS: A method for the preparation difference of a FSI is described which involves pooling the faecal samples, centrifugation and pelleting of the cell biomass and finally homogenising the cell pellets with phosphate buffer and glycerol. Using this approach, no significant difference in total anaerobe cell viability was observed between the fresh standardised inoculum (before freezing) and the 12days post freezing FSI. Moreover, Quantitative PCR revealed no significant alterations in the estimated bacterial numbers in the FSI preparations for any of the phyla. MiSeq sequencing revealed minute differences in the relative abundance at phylum, family and genus levels between the FSI preparations. Differences in the microbiota denoted as significant were limited between preparations in the majority of cases to changes in percentage relative abundance of ±0.5%. The independently prepared FSIs revealed a high degree of reproducibility in terms of microbial composition between the three preparations. CONCLUSIONS: This study provides a method to produce a standardised human faecal inoculum suitable for freezing. Based on culture-dependent and independent analysis, the method ensures a degree of reproducibility between preparations by lessening the effect of inter-individual variation among the donors, thereby making the system more suitable for the accurate interpretation of the effects of experimental treatments.

Lactobacillus ruminis strains cluster according to their mammalian gut source.

BACKGROUND: Lactobacillus ruminis is a motile Lactobacillus that is autochthonous to the human gut, and which may also be isolated from other mammals. Detailed characterization of L. ruminis has previously been restricted to strains of human and bovine origin. We therefore sought to expand our bio-bank of strains to identify and characterise isolates of porcine and equine origin by comparative genomics. RESULTS: We isolated five strains from the faeces of horses and two strains from pigs, and compared their motility, biochemistry and genetic relatedness to six human isolates and three bovine isolates including the type strain 27780(T). Multilocus sequence typing analysis based on concatenated sequence data for six individual loci separated the 16 L. ruminis strains into three clades concordant with human, bovine or porcine, and equine sources. Sequencing the genomes of four additional strains of human, bovine, equine and porcine origin revealed a high level of genome synteny, independent of the source animal. Analysis of carbohydrate utilization, stress survival and technological robustness in a combined panel of sixteen L. ruminis isolates identified strains with optimal survival characteristics suitable for future investigation as candidate probiotics. Under laboratory conditions, six human isolates of L. ruminis tested were aflagellate and non-motile, whereas all 10 strains of bovine, equine and porcine origin were motile. Interestingly the equine and porcine strains were hyper-flagellated compared to bovine isolates, and this hyper-flagellate phenotype correlated with the ability to swarm on solid medium containing up to 1.8% agar. Analysis by RNA sequencing and qRT-PCR identified genes for the biosynthesis of flagella, genes for carbohydrate metabolism and genes of unknown function that were differentially expressed in swarming cells of an equine isolate of L. ruminis. CONCLUSIONS: We suggest that Lactobacillus ruminis isolates have potential to be used in the functional food industry. We have also identified a MLST scheme able to distinguish between strains of L. ruminis of different origin. Genes for non-digestible oligosaccharide metabolism were identified with a putative role in swarming behaviour.

The Genome of the Predominant Equine Lactobacillus Species, Lactobacillus equi, Is Reflective of Its Lifestyle Adaptations to an Herbivorous Host.

We report the draft genome sequence of Lactobacillus equi strain DPC6820, isolated from equine feces. L. equi is a predominant Lactobacillus species in the horse hindgut microbiota. An examination of the genome identified genes and enzymes highlighting L. equi adaptations to the herbivorous gastrointestinal tract of the horse, including fructan hydrolases. This genome sequence may help us further understand the microbial ecology of the equine hindgut and the influence lactobacilli have on it.

Catabolic flexibility of mammalian-associated lactobacilli.

Metabolic flexibility may be generally defined as "the capacity for the organism to adapt fuel oxidation to fuel availability". The metabolic diversification strategies used by individual bacteria vary greatly from the use of novel or acquired enzymes to the use of plasmid-localised genes and transporters. In this review, we describe the ability of lactobacilli to utilise a variety of carbon sources from their current or new environments in order to grow and survive. The genus Lactobacillus now includes more than 150 species, many with adaptive capabilities, broad metabolic capacity and species/strain variance. They are therefore, an informative example of a cell factory capable of adapting to new niches with differing nutritional landscapes. Indeed, lactobacilli naturally colonise and grow in a wide variety of environmental niches which include the roots and foliage of plants, silage, various fermented foods and beverages, the human vagina and the mammalian gastrointestinal tract (GIT; including the mouth, stomach, small intestine and large intestine). Here we primarily describe the metabolic flexibility of some lactobacilli isolated from the mammalian gastrointestinal tract, and we also describe some of the food-associated species with a proven ability to adapt to the GIT. As examples this review concentrates on the following species - Lb. plantarum, Lb. acidophilus, Lb. ruminis, Lb. salivarius, Lb. reuteri and Lb. sakei, to highlight the diversity and inter-relationships between the catabolic nature of species within the genus.

Effect of Lactobacillus salivarius bacteriocin Abp118 on the mouse and pig intestinal microbiota.

Lactobacilli are gram-positive bacteria that are a subdominant element in the human gastrointestinal microbiota, and which are commonly used in the food industry. Some lactobacilli are considered probiotic, and have been associated with health benefits. However, there is very little culture-independent information on how consumed probiotic microorganisms might affect the entire intestinal microbiota. We therefore studied the impact of the administration of Lactobacillus salivarius UCC118, a microorganism well characterized for its probiotic properties, on the composition of the intestinal microbiota in two model animals. UCC118 has anti-infective activity due to production of the bacteriocin Abp118, a broad-spectrum class IIb bacteriocin, which we hypothesized could impact the microbiota. Mice and pigs were administered wild-type (WT) L. salivarius UCC118 cells, or a mutant lacking bacteriocin production. The microbiota composition was determined by pyrosequencing of 16S rRNA gene amplicons from faeces. The data show that L. salivarius UCC118 administration had no significant effect on proportions of major phyla comprising the mouse microbiota, whether the strain was producing bacteriocin or not. However, L. salivarius UCC118 WT administration led to a significant decrease in Spirochaetes levels, the third major phylum in the untreated pig microbiota. In both pigs and mice, L. salivarius UCC118 administration had an effect on Firmicutes genus members. This effect was not observed when the mutant strain was administered, and was thus associated with bacteriocin production. Surprisingly, in both models, L. salivarius UCC118 administration and production of Abp118 had an effect on gram-negative microorganisms, even though Abp118 is normally not active in vitro against this group of microorganisms. Thus L. salivarius UCC118 administration has a significant but subtle impact on mouse and pig microbiota, by a mechanism that seems at least partially bacteriocin-dependent.

Carbohydrate catabolic flexibility in the mammalian intestinal commensal Lactobacillus ruminis revealed by fermentation studies aligned to genome annotations.

BACKGROUND: Lactobacillus ruminis is a poorly characterized member of the Lactobacillus salivarius clade that is part of the intestinal microbiota of pigs, humans and other mammals. Its variable abundance in human and animals may be linked to historical changes over time and geographical differences in dietary intake of complex carbohydrates. RESULTS: In this study, we investigated the ability of nine L. ruminis strains of human and bovine origin to utilize fifty carbohydrates including simple sugars, oligosaccharides, and prebiotic polysaccharides. The growth patterns were compared with metabolic pathways predicted by annotation of a high quality draft genome sequence of ATCC 25644 (human isolate) and the complete genome of ATCC 27782 (bovine isolate). All of the strains tested utilized prebiotics including fructooligosaccharides (FOS), soybean-oligosaccharides (SOS) and 1,3:1,4-ß-D-gluco-oligosaccharides to varying degrees. Six strains isolated from humans utilized FOS-enriched inulin, as well as FOS. In contrast, three strains isolated from cows grew poorly in FOS-supplemented medium. In general, carbohydrate utilisation patterns were strain-dependent and also varied depending on the degree of polymerisation or complexity of structure. Six putative operons were identified in the genome of the human isolate ATCC 25644 for the transport and utilisation of the prebiotics FOS, galacto-oligosaccharides (GOS), SOS, and 1,3:1,4-ß-D-Gluco-oligosaccharides. One of these comprised a novel FOS utilisation operon with predicted capacity to degrade chicory-derived FOS. However, only three of these operons were identified in the ATCC 27782 genome that might account for the utilisation of only SOS and 1,3:1,4-ß-D-Gluco-oligosaccharides. CONCLUSIONS: This study has provided definitive genome-based evidence to support the fermentation patterns of nine strains of Lactobacillus ruminis, and has linked it to gene distribution patterns in strains from different sources. Furthermore, the study has identified prebiotic carbohydrates with the potential to promote L. ruminis growth in vivo.

Genome sequences and comparative genomics of two Lactobacillus ruminis strains from the bovine and human intestinal tracts.

BACKGROUND: The genus Lactobacillus is characterized by an extraordinary degree of phenotypic and genotypic diversity, which recent genomic analyses have further highlighted. However, the choice of species for sequencing has been non-random and unequal in distribution, with only a single representative genome from the L. salivarius clade available to date. Furthermore, there is no data to facilitate a functional genomic analysis of motility in the lactobacilli, a trait that is restricted to the L. salivarius clade. RESULTS: The 2.06 Mb genome of the bovine isolate Lactobacillus ruminis ATCC 27782 comprises a single circular chromosome, and has a G+C content of 44.4%. In silico analysis identified 1901 coding sequences, including genes for a pediocin-like bacteriocin, a single large exopolysaccharide-related cluster, two sortase enzymes, two CRISPR loci and numerous IS elements and pseudogenes. A cluster of genes related to a putative pilin was identified, and shown to be transcribed in vitro. A high quality draft assembly of the genome of a second L. ruminis strain, ATCC 25644 isolated from humans, suggested a slightly larger genome of 2.138 Mb, that exhibited a high degree of synteny with the ATCC 27782 genome. In contrast, comparative analysis of L. ruminis and L. salivarius identified a lack of long-range synteny between these closely related species. Comparison of the L. salivarius clade core proteins with those of nine other Lactobacillus species distributed across 4 major phylogenetic groups identified the set of shared proteins, and proteins unique to each group. CONCLUSIONS: The genome of L. ruminis provides a comparative tool for directing functional analyses of other members of the L. salivarius clade, and it increases understanding of the divergence of this distinct Lactobacillus lineage from other commensal lactobacilli. The genome sequence provides a definitive resource to facilitate investigation of the genetics, biochemistry and host interactions of these motile intestinal lactobacilli.