DNA targeting and interference by a bacterial Argonaute nuclease.

Members of the conserved Argonaute protein family use small RNA guides to locate their mRNA targets and regulate gene expression and suppress mobile genetic elements in eukaryotes1,2. Argonautes are also present in many bacterial and archaeal species3-5. Unlike eukaryotic proteins, several prokaryotic Argonaute proteins use small DNA guides to cleave DNA, a process known as DNA interference6-10. However, the natural functions and targets of DNA interference are poorly understood, and the mechanisms of DNA guide generation and target discrimination remain unknown. Here we analyse the activity of a bacterial Argonaute nuclease from Clostridium butyricum (CbAgo) in vivo. We show that CbAgo targets multicopy genetic elements and suppresses the propagation of plasmids and infection by phages. CbAgo induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. The loading of CbAgo with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interaction was reported for the acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes that encode Ago nucleases are enriched in CRISPR-Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest that the recognition of foreign nucleic acids by prokaryotic defence systems involves common principles.

Pangenome analyses of Clostridium butyricum provide insights into its genetic characteristics and industrial application.

Clostridium butyricum is a Gram-positive anaerobic bacterium known for its ability to produce butyate. In this study, we conducted whole-genome sequencing and assembly of 14C. butyricum industrial strains collected from various parts of China. We performed a pan-genome comparative analysis of the 14 assembled strains and 139 strains downloaded from NCBI. We found that the genes related to critical industrial production pathways were primarily present in the core and soft-core gene categories. The phylogenetic analysis revealed that strains from the same clade of the phylogenetic tree possessed similar antibiotic resistance and virulence factors, with most of these genes present in the shell and cloud gene categories. Finally, we predicted the genes producing bacteriocins and botulinum toxins as well as CRISPR systems responsible for host defense. In conclusion, our research provides a desirable pan-genome database for the industrial production, food application, and genetic research of C. butyricum.

Clostridium butyricum Bacteremia Associated with Probiotic Use, Japan.

Clostridium butyricum, a probiotic commonly prescribed in Asia, most notably as MIYA-BM (Miyarisan Pharmaceutical Co., Ltd.; https://www.miyarisan.com), occasionally leads to bacteremia. The prevalence and characteristics of C. butyricum bacteremia and its bacteriologic and genetic underpinnings remain unknown. We retrospectively investigated patients admitted to Osaka University Hospital during September 2011-February 2023. Whole-genome sequencing revealed 5 (0.08%) cases of C. butyricum bacteremia among 6,576 case-patients who had blood cultures positive for any bacteria. Four patients consumed MIYA-BM, and 1 patient consumed a different C. butyricum-containing probiotic. Most patients had compromised immune systems, and common symptoms included fever and abdominal distress. One patient died of nonocclusive mesenteric ischemia. Sequencing results confirmed that all identified C. butyricum bacteremia strains were probiotic derivatives. Our findings underscore the risk for bacteremia resulting from probiotic use, especially in hospitalized patients, necessitating judicious prescription practices.

Gut microbiota dynamics and fecal SCFAs after colonoscopy: accelerating microbiome stabilization by Clostridium butyricum.

BACKGROUND: Colonoscopy is a classic diagnostic method with possible complications including abdominal pain and diarrhoea. In this study, gut microbiota dynamics and related metabolic products during and after colonoscopy were explored to accelerate gut microbiome balance through probiotics. METHODS: The gut microbiota and fecal short-chain fatty acids (SCFAs) were analyzed in four healthy subjects before and after colonoscopy, along with seven individuals supplemented with Clostridium butyricum. We employed 16S rRNA sequencing and GC-MS to investigate these changes. We also conducted bioinformatic analysis to explore the buk gene, encoding butyrate kinase, across C. butyricum strains from the human gut. RESULTS: The gut microbiota and fecal short-chain fatty acids (SCFAs) of four healthy subjects were recovered on the 7th day after colonoscopy. We found that Clostridium and other bacteria might have efficient butyric acid production through bioinformatic analysis of the buk and assessment of the transcriptional level of the buk. Supplementation of seven healthy subjects with Clostridium butyricum after colonoscopy resulted in a quicker recovery and stabilization of gut microbiota and fecal SCFAs on the third day. CONCLUSION: We suggest that supplementation of Clostridium butyricum after colonoscopy should be considered in future routine clinical practice.

Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination.

BACKGROUND: Clostridium spp. has demonstrated therapeutic potential in cancer treatment through intravenous or intratumoral administration. This approach has expanded to include non-pathogenic clostridia for the treatment of various diseases, underscoring the innovative concept of oral-spore vaccination using clostridia. Recent advancements in the field of synthetic biology have significantly enhanced the development of Clostridium-based bio-therapeutics. These advancements are particularly notable in the areas of efficient protein overexpression and secretion, which are crucial for the feasibility of oral vaccination strategies. Here, we present two examples of genetically engineered Clostridium candidates: one as an oral cancer vaccine and the other as an antiviral oral vaccine against SARS-CoV-2. RESULTS: Using five validated promoters and a signal peptide derived from Clostridium sporogenes, a series of full-length NY-ESO-1/CTAG1, a promising cancer vaccine candidate, expression vectors were constructed and transformed into C. sporogenes and Clostridium butyricum. Western blotting analysis confirmed efficient expression and secretion of NY-ESO-1 in clostridia, with specific promoters leading to enhanced detection signals. Additionally, the fusion of a reported bacterial adjuvant to NY-ESO-1 for improved immune recognition led to the cloning difficulties in E. coli. The use of an AUU start codon successfully mitigated potential toxicity issues in E. coli, enabling the secretion of recombinant proteins in C. sporogenes and C. butyricum. We further demonstrate the successful replacement of PyrE loci with high-expression cassettes carrying NY-ESO-1 and adjuvant-fused NY-ESO-1, achieving plasmid-free clostridia capable of secreting the antigens. Lastly, the study successfully extends its multiplex genetic manipulations to engineer clostridia for the secretion of SARS-CoV-2-related Spike_S1 antigens. CONCLUSIONS: This study successfully demonstrated that C. butyricum and C. sporogenes can produce the two recombinant antigen proteins (NY-ESO-1 and SARS-CoV-2-related Spike_S1 antigens) through genetic manipulations, utilizing the AUU start codon. This approach overcomes challenges in cloning difficult proteins in E. coli. These findings underscore the feasibility of harnessing commensal clostridia for antigen protein secretion, emphasizing the applicability of non-canonical translation initiation across diverse species with broad implications for medical or industrial biotechnology.

Programmable cleavage of linear double-stranded DNA by combined action of Argonaute CbAgo from Clostridium butyricum and nuclease deficient RecBC helicase from E. coli.

Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Currently, the use of mesophilic pAgos as programmable endonucleases is hampered by their limited action on double-stranded DNA (dsDNA). We demonstrate here that efficient cleavage of linear dsDNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated in vitro via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecBexo-C). Properties of CbAgo and characteristics of simultaneous cleavage of DNA strands in concurrence with DNA strand unwinding by RecBexo-C were thoroughly explored using 0.03-25 kb dsDNAs. When combined with RecBexo-C, CbAgo could cleave targets located 11-12.5 kb from the ends of linear dsDNA at 37°C. Our study demonstrates that CbAgo with RecBexo-C can be programmed to generate DNA fragments with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. The combination of CbAgo and RecBexo-C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for in vitro use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of linear dsDNA at any desired location.

Diversity within the species Clostridium butyricum: pan-genome, phylogeny, prophage, carbohydrate utilization, and antibiotic resistance.

AIMS: Clostridium butyricum has been recognized as a strong candidate for the "next generation of probiotics" due to its beneficial roles on humans. Owing to our current understanding of this species is limited, it is imperative to unveil the genetic variety and biological properties of C. butyricum on sufficient strains. METHODS AND RESULTS: We isolated 53 C. butyricum strains and collected 25 publicly available genomes to comprehensively assess the genomic and phenotypic diversity of this species. Average nucleotide identity and phylogeny suggested that multiple C. butyricum strains might share the same niche. Clostridium butyricum genomes were replete with prophage elements, but the CRISPR-positive strain efficiently inhibited prophage integration. Clostridium butyricum utilizes cellulose, alginate, and soluble starch universally, and shows general resistance to aminoglycoside antibiotics. CONCLUSIONS: Clostridium butyricum exhibited a broad genetic diversity from the extraordinarily open pan-genome, extremely convergent core genome, and ubiquitous prophages. In carbohydrate utilization and antibiotic resistance, partial genotypes have a certain guiding significance for phenotypes.

[Effects of Peroxisome Proliferator-Activated Receptor γ on Modulating Angiopoietin-Like Protein 4 Synthesis in Caco-2 Cells Exposed to Clostridium butyricum].

Angiopoietin-like protein 4 (ANGPTL4) is considered to be one of the important circulating mediators linking intestinal microorganisms and host lipid metabolism. The objective of this study was to assess the effects of peroxisome proliferator-activated receptor у (PPARγ) on modulating ANGPTL4 synthesis in Caco-2 cells exposed to Clostridium butyricum. The viability of Caco-2 cells and the expression of PPARγ and ANGPTL4 in Caco-2 cells were detected after the Caco-2 cells were co-cultured with C. butyricum at the concentration of 1 x 10^(6), 1 x 10^(7) and 1 x 10^(8) CFU/mL. The results showed that cell viability was enhanced by C. butyricum. Besides, PPARγ and ANGPTL4 expression and secretion in Caco-2 cells was significantly increased by 1 x 10^(7) and 1 x 10^(8) CFU/mL of C. butyricum. Furthermore, the effects of PPARγ on modulating ANGPTL4 synthesis in Caco-2 cells regulated by 1 x 10^(8) CFU/mL of C. butyricum was also be expounded in PPARγ activation/inhibition model based on Caco-2 cells and via ChIP technique. It was found that C. butyricum promoted the binding of PPARγ to the PPAR binding site (chr19: 8362157-8362357, located upstream of the transcriptional start site of angptl4) of the angptl4 gene in Caco-2 cells. However, the PPARγ was not the only way for C. butyricum to stimulate ANGPTL4 production. Taken together, PPARγ played a role in the regulation of ANGPTL4 synthesis by C. butyricum in Caco-2 cells.

Commensal bacteria-derived extracellular vesicles suppress ulcerative colitis through regulating the macrophages polarization and remodeling the gut microbiota.

BACKGROUND: The extracellular vesicles (EVs) traffic constitutes an essential pathway of cellular communication. And the molecules in EVs produced by procaryotes help in maintaining homeostasis, addressing microbial imbalance and infections, and regulating the immune system. Despite the fact that Clostridium butyricum (C. butyricum) is commonly used for treating ulcerative colitis (UC), the potential role of C. butyricum-secreted EVs in commensals-host crosstalk remains unclear. RESULTS: Here, we performed flow cytometry, western blot, immunohistochemistry and 16S rRNA analysis to explore the role of C. butyricum-derived EVs on macrophage polarization and gut microbiota composition in a dextran sulfate sodium (DSS)-induced UC mouse model. The antibiotic cocktail-induced microbiome depletion and faecal transplantations were used to further investigate the mechanisms by which EVs regulate macrophage balance. Our findings showed that C. butyricum-derived EVs improved the remission of murine colitis and polarized the transformation of macrophages to the M2 type. Furthermore, C. butyricum-derived EVs restored gut dysbiosis and altered the relative abundance of Helicobacter, Escherichia-Shigella, Lactobacillus, Akkermansia and Bacteroides, which, in turn, faecal transplantations from EVs-treated mice relieved the symptoms of UC and improved the impact of EVs on the reprogramming of the M2 macrophages. CONCLUSION: C. butyricum-derived EVs could protect against DSS-induced colitis by regulating the repolarization of M2 macrophages and remodelling the composition of gut microbiota, suggesting the potential efficacy of EVs from commensal and probiotic Clostridium species against UC.

Novel Putative Transposable Element Associated with the Subtype E5 Botulinum Toxin Gene Cluster of Neurotoxigenic Clostridium butyricum Type E Strains from China.

Previously, a whole-genome comparison of three Clostridium butyricum type E strains from Italy and the United States with different C. botulinum type E strains indicated that the bont/e gene might be transferred between the two clostridia species through transposition. However, transposable elements (TEs) have never been identified close to the bont/e gene. Herein, we report the whole genome sequences for four neurotoxigenic C. butyricum type E strains that originated in China. An analysis of the obtained genome sequences revealed the presence of a novel putative TE upstream of the bont/e gene in the genome of all four strains. Two strains of environmental origin possessed an additional copy of the putative TE in their megaplasmid. Similar putative TEs were found in the megaplasmids and, less frequently, in the chromosomes of several C. butyricum strains, of which two were neurotoxigenic C. butyricum type E strains, and in the chromosome of a single C. botulinum type E strain. We speculate that the putative TE might potentially transpose the bont/e gene at the intracellular and inter-cellular levels. However, the occasional TE occurrence in the clostridia genomes might reflect rare transposition events.

Exploiting heterologous and endogenous CRISPR-Cas systems for genome editing in the probiotic Clostridium butyricum.

Clostridium butyricum has been widely used as a probiotic for humans and food animals. However, the mechanisms of beneficial effects of C. butyricum on the host remain poorly understood, largely due to the lack of high-throughput genome engineering tools. Here, we report the exploitation of heterologous Type II CRISPR-Cas9 system and endogenous Type I-B CRISPR-Cas system in probiotic C. butyricum for seamless genome engineering. Although successful genome editing was achieved in C. butyricum when CRISPR-Cas9 system was employed, the expression of toxic cas9 gene result in really poor transformation, spurring us to develop an easy-applicable and high-efficient genome editing tool. Therefore, the endogenous Type I-B CRISPR-Cas machinery located on the megaplasmid of C. butyricum was co-opted for genome editing. In vivo plasmid interference assays identified that ACA and TAA were functional protospacer adjacent motif sequences needed for site-specific CRISPR attacking. Using the customized endogenous CRISPR-Cas system, we successfully deleted spo0A and aldh genes in C. butyricum, yielding an efficiency of up to 100%. Moreover, the conjugation efficiency of endogenous CRISPR-Cas system was dramatically enhanced due to the precluding expression of cas9. Altogether, the two approaches developed herein remarkably expand the existing genetic toolbox available for investigation of C. butyricum.

Analysis of the core genome and pangenome of Clostridium butyricum.

Clostridium butyricum is an anaerobic bacterium that inhabits broad niches. Clostridium butyricum is known for its production of butyrate, 1,3-propanediol, and hydrogen. This study aimed to present a comparative pangenome analysis of 24 strains isolated from different niches. We sequenced and annotated the genome of C. butyricum 3-3 isolated from the Chinese baijiu ecosystem. The pangenome of C. butyricum was open. The core genome, accessory genome, and strain-specific genes comprised 1011, 4543, and 1473 genes, respectively. In the core genome, Carbohydrate metabolism was the largest category, and genes in the biosynthetic pathway of butyrate and glycerol metabolism were conserved (in the core or soft-core genome). Furthermore, the 1,3-propanediol operon existed in 20 strains. In the accessory genome, numerous mobile genetic elements belonging to the Replication, recombination, and repair (L) category were identified. In addition, genome islands were identified in all 24 strains, ranging from 2 (strain KNU-L09) to 53 (strain SU1), and phage sequences were found in 17 of the 24 strains. This study provides an important genomic framework that could pave the way for the exploration of C. butyricum and future studies on the genetic diversification of C. butyricum.

DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute.

Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65°C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37°C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.

The Draft Genome Sequence of Clostridium butyricum QXYZ514, a Potent Bacterium for Converting Glycerol into Fuels and Bioproducts in the Waste-Based Biorefinery.

An anaerobic bacterium Clostridium buturicum QXYZ514 was isolated from a pond soil sample located neighboring to a biodiesel factory. This bacterium possesses excellent metabolic features for converting biodiesel-derived glycerol into various bioproducts, including 1,3-propanediol, butyrate, lactate, and acetate, and fuels, like ethanol and butanol. To further improve the yield of the target products and minimize the production of the by-products, the whole genome sequence of this multipurpose strain might provide necessary genetic information, and hence, the complete genome of QXYZ514 strain was sequenced using the PacBio RS II sequencing method. According to the complete genome sequence, the genome of QXYZ514 consisted of two circular chromosomes with a total of 4,636,461 bp, where GC content was found to be 28.76%. Major predicted features of the genome included a total of 4220 coding sequences (CDS), 87 tRNAs genes, and 36 rRNAs genes, which were annotated with the help of different databases for a better understanding of this strain. Six possible CRISPR components were also predicted in the genome. The exploration of the complete genome sequence of the QXYZ514 strain would have the potential to enrich the diversity of this species, and to recognize some significant hydrolytic enzymes, which could provide the references for overcoming the bottlenecks in the biorefinery usage of this bacterium in the valorization of biodiesel-derived glycerol.

A gene cluster for taurine sulfur assimilation in an anaerobic human gut bacterium.

Aminoethylsulfonate (taurine) is widespread in the environment and highly abundant in the human body. Taurine and other aliphatic sulfonates serve as sulfur sources for diverse aerobic bacteria, which carry out cleavage of the inert sulfonate C-S bond through various O2-dependent mechanisms. Taurine also serves as a sulfur source for certain strict anaerobic fermenting bacteria. However, the mechanism of C-S cleavage by these bacteria has long been a mystery. Here we report the biochemical characterization of an anaerobic pathway for taurine sulfur assimilation in a strain of Clostridium butyricum from the human gut. In this pathway, taurine is first converted to hydroxyethylsulfonate (isethionate), followed by C-S cleavage by the O2-sensitive isethionate sulfo-lyase IseG, recently identified in sulfate- and sulfite-reducing bacteria. Homologs of the enzymes described in this study have a sporadic distribution in diverse strict and facultative anaerobic bacteria, from both the environment and the taurine-rich human gut, and may enable sulfonate sulfur acquisition in certain nutrient limiting conditions.

Electrospun Solid Formulation of Anaerobic Gut Microbiome Bacteria.

A model anaerobic bacterium strain from the gut microbiome (Clostridium butyricum) producing anti-inflammatory molecules was incorporated into polymer-free fibers of a water-soluble cyclodextrin matrix (HP-ß-CD) using a promising scaled-up nanotechnology, high-speed electrospinning. A long-term stability study was also carried out on the bacteria in the fibers. Effect of storage conditions (temperature, presence of oxygen) and growth conditions on the bacterial viability in the fibers was investigated. The viability of the sporulated anaerobic bacteria in the fibers was maintained during 12 months of room temperature storage in the presence of oxygen. Direct compression was used to prepare tablets from the produced bacteria-containing fibers after milling (using an oscillating mill) and mixing with tableting excipients, making easy oral administration of the bacteria possible. No significant decrease was observed in bacterial viability following the processing of the fibers (milling and tableting).

Synergistic anti-tumour effects of Clostridium butyricum in combination with apatinib in CT26 colorectal tumour-bearing mice.

To investigate the synergistic antitumour effect of Clostridium butyricum combined with apatinib on colorectal cancer in mice. Murine colorectal carcinoma cell line CT26.WT cells were xenografted into the skin of BALB/c mice. Tumour-bearing mice were randomly divided into four groups, and given different treatment options (PBS control; C. butyricum; apatinib; C. butyricum + apatinib). Real-time PCR was used to detect C. butyricum content in the intestine of mice given C. butyricum. The effects of various regimens on tumour growth were monitored, and CD31, proliferating cell nuclear antigen (PCNA), Bcl-2 and cleaved caspase-3 expressions in tumour were analysed by immunohistochemistry. C. butyricum combined with apatinib significantly inhibits tumour growth with decreased CD31, PCNA and Bcl-2 expressions, and increased cleaved caspase-3 expressions. Our study confirms that C. butyricum combined with apatinib in the treatment of xenografted colon tumour in mice can significantly inhibit tumour growth and promote cell apoptosis than apatinib alone treatments, providing the reference for clinical treatments.

Cell factories converting lactate and acetate to butyrate: Clostridium butyricum and microbial communities from dark fermentation bioreactors.

BACKGROUND: Interactions between microorganisms during specific steps of anaerobic digestion determine metabolic pathways in bioreactors and consequently the efficiency of fermentation processes. This study focuses on conversion of lactate and acetate to butyrate by bacteria of dark fermentation. The recently recognized flavin-based electron bifurcation as a mode of energy coupling by anaerobes increases our knowledge of anaerobic lactate oxidation and butyrate formation. RESULTS: Microbial communities from dark fermentation bioreactors or pure culture of Clostridium butyricum are able to convert lactate and acetate to butyrate in batch experiments. The ability of C. butyricum to transform lactate and acetate to butyrate was shown for the first time, with ethanol identified as an additional end product of this process. A search for genes encoding EtfAB complexes and their gene neighbourhood in C. butyricum and other bacteria capable of lactate and acetate conversion to butyrate as well as butyrate-producers only and the lactate oxidiser Acetobacterium woodii, revealed that the Etf complexes involved in (i) lactate oxidation and (ii) butyrate synthesis, form separate clusters. There is a more extent similarity between Etf subunits that are involved in lactate oxidation in various species (e.g. A. woodii and C. butyricum) than between the different etf gene products within the same species of butyrate producers. A scheme for the metabolic pathway of lactate and acetate transformation to butyrate in C. butyricum was constructed. CONCLUSIONS: Studies on the conversion of lactate and acetate to butyrate by microbial communities from dark fermentation bioreactors or Clostridium butyricum suggest that a phenomenon analogous to cross-feeding of lactate in gastrointestinal tract also occurs in hydrogen-yielding reactors. A scheme of lactate and acetate transformation pathway is proposed, based on the example of C. butyricum, which employs flavin-based electron bifurcation. This process utilizes electron-transferring flavoprotein (Etf) complexes specific for (i) lactate oxidation and (ii) butyrate formation. Phylogenetic analysis revealed that such complexes are encoded in the genomes of other bacteria capable of lactate and acetate conversion to butyrate. These findings contribute significantly to our understanding of the metabolic pathways and symbiotic interactions between bacteria during the acidogenic step of anaerobic digestion.

The impact of Clostridium butyricum MIYAIRI 588 on the murine gut microbiome and colonic tissue.

BACKGROUND: Clostridium butyricum MIYAIRI 588 (CBM 588) is a probiotic bacterium that is used as an anti-diarrheal medicine in Japan. However, the impact of this probiotic on the gut microbiome has not been fully elucidated, especially, when used with antimicrobials. MATERIAL AND METHODS: In an in vivo study, CBM 588 monotherapy, clindamycin monotherapy, CBM 588 and clindamycin (combination therapy), or normal saline (control) was orally administered to mice for 4 days, and fecal samples were collected for 18 days to enumerate C. butyricum. We also extracted DNA from these fecal samples for metagenomics analysis by amplification of the V3-V4 region of the bacterial 16S rRNA gene and MiSeq Illumina sequencing. In addition, the concentrations of some short chain fatty acids were assessed in the fecal samples. A histological analysis was also conducted. RESULTS: On day 4 (the last treatment day), there was no difference in the total counts of C. butyricum between the CBM 588 monotherapy and combination therapy groups (5.21 ±â€¯0.78 vs. 5.13 ±â€¯0.45 log10 cfu/g, p = 0.86). Clindamycin treatment resulted in dramatic increases in the phylum Firmicutes, especially Enterobacteriaceae, Clostridiaceae, Lactobacillus, and Enterococcus, compared with the other groups during the treatment period. CBM 588 treatment modified the bacterial community composition at lower phylogenetic levels. Some bacterial taxa, such as Bifidobacterium, Coprococcus, and Bacteroides, were significantly increased in the combination therapy group when compared with the other groups. In the metabolic analysis, CBM 588 enhanced lactic acid production. It also enhanced the efficiency of lactic acid use for the production of butyric acid. Only the clindamycin monotherapy group showed abnormal colon tissue, with superficial epithelial necrosis and the presence of inflammatory cells. CONCLUSION: CBM 588 treatment modulated the gut microbiota composition under dysbiosis due to the use of an antimicrobial with strong activity against anaerobes and significantly reduced epithelial damage.

Comparative phenotypic analysis of "Clostridium neonatale" and Clostridium butyricum isolates from neonates.

"Clostridium neonatale" was recently described as a new species within the Cluster I of the Clostridium genus sensu stricto. In this study, we characterized "C. neonatale" isolates (n = 42) and compared their phenotypic properties with those of Clostridium butyricum (n = 26), a close related species. Strains isolated from fecal samples of healthy neonates were tested for different phenotypic characteristics. Compared to C. butyricum, "C. neonatale" showed a significant higher surface hydrophobicity (p = 0.0047), exopolysaccharide production (p = 0.0069), aero-tolerance (p = 0.0222) and viability at 30 °C (p = 0.0006). A lower swimming ability (p = 0.0146) and tolerance against bile (0.3%) (p = 0.0494), acid (pH 4.5) (p < 0.0001), osmolarity (NaCl 5%, p = 0.0188) and temperature at 50 °C (p = 0.0013) characterized "C. neonatale" strains. Our results showed that "C. neonatale" behaves very differently from C. butyricum and suggests specific responses to environmental changes. Besides it is the first study on clinical isolates for these two anaerobic members of the newborns' gut microbiota and broadens our knowledge about their phenotypic traits.