Infectious Diseases Society of America.

Anti–PD-1–based immunotherapy has had a major impact on cancer treatment but has only benefited a subset of patients. Among the variables that could contribute to interpatient heterogeneity is differential composition of the patients’ microbiome, which has been shown to affect antitumor immunity and immunotherapy efficacy in preclinical mouse models. We analyzed baseline stool samples from metastatic melanoma patients before immunotherapy treatment, through an integration of 16 S ribosomal RNA gene sequencing, metagenomic shotgun sequencing, and quantitative polymerase chain reaction for selected bacteria. A significant association was observed between commensal microbial composition and clinical response. Bacterial species more abundant in responders included Bifidobacterium longum , Collinsella aerofaciens , and Enterococcus faecium. Reconstitution of germ-free mice with fecal material from responding patients could lead to improved tumor control, augmented T cell responses, and greater efficacy of anti–PD-L1 therapy. Our results suggest that the commensal microbiome may have a mechanistic impact on antitumor immunity in human cancer patients.

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The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients

Butyrate-producing bacteria play a key role in colonic health in humans. This review provides an overview of the current knowledge of the diversity, metabolism and microbial ecology of this functionally important group of bacteria. Human colonic butyrate producers are Gram-positive firmicutes, but are phylogenetically diverse, with the two most abundant groups related to Eubacterium rectale/Roseburia spp. and to Faecalibacterium prausnitzii. Five different arrangements have been identified for the genes of the central pathway involved in butyrate synthesis, while in most cases butyryl-CoA : acetate CoA-transferase, rather than butyrate kinase, appears to perform the final step in butyrate synthesis. Mechanisms have been proposed recently in non-gut Clostridium spp. whereby butyrate synthesis can result in energy generation via both substrate-level phosphorylation and proton gradients. Here we suggest that these mechanisms also apply to the majority of butyrate producers from the human colon. The roles of these bacteria in the gut community and their influence on health are now being uncovered, taking advantage of the availability of cultured isolates and molecular methodologies. Populations of F. prausnitzii are reported to be decreased in Crohn's disease, for example, while populations of Roseburia relatives appear to be particularly sensitive to the diet composition in human volunteer studies.

Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine.

The human gut microbiota ferments dietary non-digestible carbohydrates into short-chain fatty acids (SCFA). These microbial products are utilized by the host and propionate and butyrate in particular exert a range of health-promoting functions. Here an overview of the metabolic pathways utilized by gut microbes to produce these two SCFA from dietary carbohydrates and from amino acids resulting from protein breakdown is provided. This overview emphasizes the important role played by cross-feeding of intermediary metabolites (in particular lactate, succinate and 1,2-propanediol) between different gut bacteria. The ecophysiology, including growth requirements and responses to environmental factors, of major propionate and butyrate producing bacteria are discussed in relation to dietary modulation of these metabolites. A detailed understanding of SCFA metabolism by the gut microbiota is necessary to underpin effective strategies to optimize SCFA supply to the host.

https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/1462-2920.13589

Formation of propionate and butyrate by the human colonic microbiota

In vertebrates, including humans, individuals harbor gut microbial communities whose species composition and relative proportions of dominant microbial groups are tremendously varied. Although external and stochastic factors clearly contribute to the individuality of the microbiota, the fundamental principles dictating how environmental factors and host genetic factors combine to shape this complex ecosystem are largely unknown and require systematic study. Here we examined factors that affect microbiota composition in a large (n = 645) mouse advanced intercross line originating from a cross between C57BL/6J and an ICR-derived outbred line (HR). Quantitative pyrosequencing of the microbiota defined a core measurable microbiota (CMM) of 64 conserved taxonomic groups that varied quantitatively across most animals in the population. Although some of this variation can be explained by litter and cohort effects, individual host genotype had a measurable contribution. Testing of the CMM abundances for cosegregation with 530 fully informative SNP markers identified 18 host quantitative trait loci (QTL) that show significant or suggestive genome-wide linkage with relative abundances of specific microbial taxa. These QTL affect microbiota composition in three ways; some loci control individual microbial species, some control groups of related taxa, and some have putative pleiotropic effects on groups of distantly related organisms. These data provide clear evidence for the importance of host genetic control in shaping individual microbiome diversity in mammals, a key step toward understanding the factors that govern the assemblages of gut microbiota associated with complex diseases.

/pdf/individuality-in-gut-microbiota-composition-is-a-complex-27tl8z3393.pdf

Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors

Bacterial butyryl-CoA CoA-transferase activity plays a key role in butyrate formation in the human colon, but the enzyme and corresponding gene responsible for this activity have not previously been identified. A novel CoA-transferase gene is described from the colonic bacterium Roseburia sp. A2-183, with similarity to acetyl-CoA hydrolase as well as 4-hydroxybutyrate CoA-transferase sequences. The gene product, overexpressed in an Escherichia coli lysate, showed activity with butyryl-CoA and to a lesser degree propionyl-CoA in the presence of acetate. Butyrate, propionate, isobutyrate and valerate competed with acetate as the co-substrate. Despite the sequence similarity to 4-hydroxybutyrate CoA-transferases, 4-hydroxybutyrate did not compete with acetate as the co-substrate. Thus the CoA-transferase preferentially uses butyryl-CoA as substrate. Similar genes were identified in other butyrate-producing human gut bacteria from clostridial clusters IV and XIVa, while other candidate CoA-transferases for butyrate formation could not be detected in Roseburia sp. A2-183. This suggests strongly that the newly identified group of CoA-transferases described here plays a key role in butyrate formation in the human colon.

A novel class of CoA-transferase involved in short-chain fatty acid metabolism in butyrate-producing human colonic bacteria.

The Family Coriobacteriaceae

The diversity of bacteria associated with inflamed mucosa was investigated by culturing ileal samples from TNF(deltaARE) mice on a selective medium containing mucin. Among eight isolates, two strains (Mt1B3 and Mt1B8(T)) belonged to bacterial groups not yet cultured from the mouse intestine. Whereas strain Mt1B3 was identified as a member of the family Planococcaceae and is closely related to Sporosarcina species and Filibacter limicola DSM 13886(T), strain Mt1B8(T) was a novel bacterium. Based on phylogenetic analysis, strain Mt1B8(T) is a member of the family Coriobacteriaceae. The closest relatives with validly published names were Asaccharobacter celatus, Adlercreutzia equolifaciens (<96 % similarity) and Eggerthella species (<92 %). With respect to Asaccharobacter celatus and Eggerthella, the phylogenetic position of strain Mt1B8(T) was confirmed at the chemotaxonomic level by Fourier-transform infrared spectroscopic analysis. The major fatty acid of strain Mt1B8(T) is C(16 : 0) (23.9 %). Menaquinones were monomethylated. DNA-DNA relatedness between strain Mt1B8(T) and Asaccharobacter celatus DSM 18785(T) was 28 %. Strain Mt1B8(T) is a Gram-positive-staining rod that does not form spores and has a high DNA G+C content (64.2 mol%). Cells are aerotolerant but grow only under strictly anoxic conditions. They are sensitive to cefotaxime, clarithromycin, erythromycin, metronidazole, tetracycline, tobramycin and vancomycin. API and VITEK analysis showed the ability of strain Mt1B8(T) to convert a variety of amino acid derivatives. According to these findings, it is proposed to create a novel genus and species, Enterorhabdus mucosicola gen. nov., sp. nov., to accommodate strain Mt1B8(T). The type strain of Enterorhabdus mucosicola is Mt1B8(T) (=DSM 19490(T) =CCUG 54980(T)).

Isolation of bacteria from the ileal mucosa of TNFdeltaARE mice and description of Enterorhabdus mucosicola gen. nov., sp. nov.

Whilst creating a bacterial collection of strains from the mouse intestine, we isolated a Gram-negative, spore-forming, non-motile and strictly anaerobic rod-shaped bacterium from the caecal content of a TNFdeltaARE mouse. The isolate, referred to as strain SRB-521-5-IT, was originally cultured on a reduced agar medium containing yeast extract, rumen fluid and lactic acid as energy and carbon sources. Phylogenetic analysis of partial 16S rRNA genes revealed that the species most closely related to strain SRB-521-5-IT (GenBank accession no. KC311367) were Flavonifractor plautii and Pseudoflavonifractor capillosus (<95 % sequence identity; 1436 bp). In contrast to F. plautii and P. capillosus, strain SRB-521-5-IT contained a substantial amount of C18:0 dimethylacetal. Additional major fatty acids were C14:0 methyl ester, C16:0 dimethylacetal and C18:0 aldehyde. Strain SRB-521-5-IT differed in its enzymatic profile from F. plautii and P. capillosus by being positive for dextrin, maltotriose, turanose, D,L-lactic acid and D-lactic acid methyl ester but negative for D-fructose. In reduced Wilkins-Chalgren-Anaerobe broth, strain SRB-521-5-IT produced approximately 8 mM butyrate and 4 mM acetate. In contrast to F. plautii, the strain did not metabolize flavonoids. It shows intermediate resistance towards the antibiotics ciprofloxacin, colistin and tetracycline. Based on genotypic and phenotypic characteristics, we propose to create the name Intestinimonas butyriciproducens gen. nov., sp. nov. to accommodate strain SRB-521-5-IT (= DSM 26588T = CCUG 63529T)

Intestinimonas butyriciproducens gen. nov., sp. nov., a butyrate-producing bacterium from the mouse intestine.

Background: There remains a critical need for more effective, safe, long-term treatments for cystic fibrosis (CF). Any successful therapeutic strategy designed to combat the respiratory pathology of this condition must address the altered lung physiology and recurrent, complex, polymicrobial infections and biofilms that affect the CF pulmonary tract. Cysteamine is a potential solution to these unmet medical needs and is described here for the first time as (Lynovex®) a single therapy with the potential to deliver mucoactive, antibiofilm and antibacterial properties; both in oral and inhaled delivery modes. Cysteamine is already established in clinical practice for an unrelated orphan condition, cystinosis, and is therefore being repurposed (in oral form) for cystic fibrosis from a platform of over twenty years of safety data and clinical experience. Methods: The antibacterial and antibiofilm attributes of cysteamine were determined against type strain and clinical isolates of CF relevant pathogens using CLSI standard and adapted microbiological methods and a BioFlux microfluidic system. Assays were performed in standard nutrient media conditions, minimal media, to mimic the low metabolic activity of microbes/persister cells in the CF respiratory tract and in artificial sputum medium. In vivo antibacterial activity was determined in acute murine lung infection/cysteamine nebulisation models. The mucolytic potential of cysteamine was assessed against DNA and mucin in vitro by semi-quantitative macro-rheology. In all cases, the ‘gold standard’ therapeutic agents were employed as control/comparator compounds against which the efficacy of cysteamine was compared. Results: Cysteamine demonstrated at least comparable mucolytic activity to currently available mucoactive agents. Cysteamine was rapidly bactericidal against both metabolically active and persister cells of Pseudomonas aeruginosa and also emerging CF pathogens; its activity was not sensitive to high ionic concentrations characteristic of the CF lung. Cysteamine prevented the formation of, and disrupted established P. aeruginosa biofilms. Cysteamine was synergistic with conventional CF antibiotics; reversing antibiotic resistance/insensitivity in CF bacterial pathogens. Conclusions: The novel mucolytic-antimicrobial activity of cysteamine (Lynovex®) provides potential for a much needed new therapeutic strategy in cystic fibrosis. The data we present here provides a platform for cysteamine’s continued investigation as a novel treatment for this poorly served orphan disease.

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Cedric Charrier

Papers

A novel class of CoA-transferase involved in short-chain fatty acid metabolism in butyrate-producing human colonic bacteria.

The Family Coriobacteriaceae

Isolation of bacteria from the ileal mucosa of TNFdeltaARE mice and description of Enterorhabdus mucosicola gen. nov., sp. nov.

Intestinimonas butyriciproducens gen. nov., sp. nov., a butyrate-producing bacterium from the mouse intestine.

Cysteamine (Lynovex®), a novel mucoactive antimicrobial & antibiofilm agent for the treatment of cystic fibrosis