Detection of butyrate from a Clostridium cluster I (Clostridium sensu stricto) strain, Clostridium tertium, under anaerobic condition

  1. Mei Miyazaki
  2. Nanako Kaneko
  3. Nicholas Hardy
  4. Hiroki Iwai

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Abstract

Lactic acid bacteria and bacillus bifidus are well-known as probiotics used as alternative drugs confronting infectious disease, however, butyric acid (butyrate) bacteria such as Clostridium butyricum are also useful probiotics but little-known in any part of the world. To discover a new bacteria species producing butyrate, we examined the production of organic acid including butyrate in Clostridium tertium and C. butyricum. Protein BLAST (BLASTP) searches on Butyrate kinase (BUK) proteins in the genomes of C. tertium have identified 2 proteins. To confirm their presence in the genome of C. tertium, we detected two buk genes by the polymerase chain reaction (PCR). As phosphotransbutyrylase (ptb), a buk gene adjacent gene, is also present in the announced genome of C. tertium, we confirmed its presence similarly by PCR. Next, we investigated the production of butyrate in C. tertium. Lactic acid (lactate), formic acid (formate), acetic acid (acetate) and butyrate were detected in both thioglycolate medium cultures. Thus, we conclude C. tertium produces butyrate. Our findings provide a new butyrate producing bacterium as a potential probiotic.

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  2. Access Microbiology

    Comments to Author

    The aim of the manuscript was to assess the production of butyrate by Clostridium tertium and C. butyricum. Key genes for the production of butyrate have been searched. The topic is potentially interesting but I cannot recommend publication since the experimental approach, at least in the way it is presented, has criticisms mostly in terms of structure and rationale of the study, and for the scarce novelty of the results. The production of butyrate by C. tertium is not a novelty, as claimed in the abstract (Candeliere et al. Genomic and functional analysis of the mucinolytic species Clostridium celatum, Clostridium tertium, and Paraclostridium bifermentans. Front Microbiol. 2024 Mar 6;15:1359726.) The setup of the experiments and their rationale are not clear. The presentation of the results starts with an in vivo study on mice that has not been presented earlier in the text. The Ethic Committee approval is not reported.The experimental design of this trial is not explained in Materials and Methods, and the results are presented in supplementary material. Several figures of the outcome of a metagenomic analysis are presented in supplementary material, without any reference to metagenome in any part of the text. Metagenome date are nos discussed in the results. Four genera of butyrate producers are presented in the results, but the authors do not explain whether and how they isolated colonies, nor how they resolved taxonomy. The sole unedited result is the detection by PCR of the butyrate kinase and phosphotransbutyrylase genes in a strain of C. tertium. Rigour of the scientific lexicon sometimes is improper and English is poor and unsuitable for a scientific journal. I suggest to reject the manuscript.

    Please rate the manuscript for methodological rigour

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Very poor

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    No: No information provided

  3. Access Microbiology

    Comments to Author

    The manuscript by Miyazaki et al describes the production of butyrate from a strain of Clostridium tertium, grown under anaerobic conditions. This finding is not novel as the authors actually state that butyrate has been found previously (refs 31 and 32). While novelty is not a requirement for this journal, lines 171-175 should be reworded to state that butyrate has been found previously in C. tertium, but the amounts produced not quantified (if that is indeed correct). Authors should cite papers that have found C. tertium associated with bacteraemia and septicaemia e.g. PMID: 30976516, PMID: 14032818 and PMID: 31245219 and therefore the attraction as a potential probiotic must be weighed against possible pathogenic status of the organism. The analysis of genomic information, the PCR analysis and the butyrate production all seem to have been carried out correctly and are reported well. The manuscript is however very long and the introduction could be shortened, particularly some of the descriptive taxonomy (lines 28 - 43). English usage needs improvement throughout the manuscript.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Poor

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  4. Version published to 10.1099/acmi.0.000940.v1 on Access Microbiology