Antimicrobial and quorum sensing inhibitory activity of epiphytic bacteria isolated from the red alga Halymenia durvillei

  1. Mary Hannah Rose Padayao
  2. Francis Reuben Paul Padayao
  3. Jenny Marie Patalinghug
  4. Gem Stephen Raña
  5. Jonie Yee
  6. Paul John Geraldino
  7. Norman Quilantang

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Abstract

Halymenia durvillei is a red alga that is commonly utilized in the Philippines as food and as a source of high-value natural products for industrial applications. However, there are no studies regarding the microbial community associated with H. durvillei and its potential applications. This study aimed to isolate and identify the epiphytic bacteria of H. durvillei and determine their antimicrobial and quorum sensing inhibitory (QSI) effects. The thalli of H. durvillei were collected at the shores of Santa Fe, Bantayan, Cebu, Philippines. Bacterial isolates were identified using 16S rRNA, and their ethyl acetate (EtOAc) extracts were subjected to antimicrobial susceptibility tests against representative species of yeast and Gram-negative and Gram-positive bacteria. Their QSI activity against Chromobacterium violaceum was also determined. Fourteen distinct bacterial colonies belonging to four genera, namely Alteromonas (3), Bacillus (5), Oceanobacillus (1) and Vibrio (5), were successfully isolated and identified. All 14 bacterial isolates exhibited antibacterial effects. EPB9, identified as Bacillus safensis , consistently showed the strongest inhibition against Escherichia coli , Staphylococcus aureus and Staphylococcus epidermidis , with minimum inhibitory concentrations (MICs) ranging from 0.0625 to 1.0 mg ml −1 . In contrast, all 14 isolates showed weak antifungal effects. Both B. safensis (EPB9) and Bacillus australimaris (EPB15) exhibited QSI effects at 100 mg ml −1 , showing opaque zones of 3.1±0.9 and 3.8±0.4 mm, respectively. This study is the first to isolate and identify the distinct microbial epiphytic bacterial community of H. durvillei and its potential as an abundant resource for new antibacterial and QSI bioactives.

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  1. Version published to 10.1099/acmi.0.000563.v4 on Access Microbiology
  3. Version published to 10.1099/acmi.0.000563.v3 on Access Microbiology
  5. Access Microbiology

    Comments to Author

    * The authors have addressed the comments of the reviewers successfully. But few errors are still observed. eg: The word 'Gram' starting from simple letter Line 72: previously unused abbreviation QSI * Discussion could be improved further focusing to the main findings of the study * The limitations of the research could have mentioned and discussed in the discussion section

    Please rate the manuscript for methodological rigour

    Satisfactory

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

    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?

    Yes

  6. Access Microbiology

    Comments to Author

    Review comments have been addressed.

    Please rate the manuscript for methodological rigour

    Good

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

    Good

    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?

    Yes

  7. Version published to 10.1099/acmi.0.000563.v2 on Access Microbiology
  9. Access Microbiology

    Comments to Author

    1. Methodological rigour, reproducibility and availability of underlying data: Molecular identification by 16S rDNA to the strain level by BLASTN to NCBI reference sequences carries limitations. The underlying data is publicly available and when tested, mere nucleotides separate top matches. How can the authors be confident that EPB9 is a different species or strain different than the identities of the next top two BLAST matches? It would help to confirm the species identity by another molecular or biochemical method. Can the EPb9 isolate be compared to other isolates from the same geographical area? 2. Presentation of results: Figure 1C- Please add a scale bar. Figure 2A,B,C- The text within the images is very small. Can it be enlarged or can larger letters and a key be provided? 3. How the style and organization of the paper communicates and represents key findings: The paper title mentions B. safensis EPB9 and not the other isolates. Therefore, one expects that B. safensis EPB9 is the focus of the study. Instead, this bacterium is one of fourteen isolates and many sections in the paper do not reflect the title focus. The introduction study aims are completely different from the paper title. Please formulate a manuscript title consistent with the findings shown in the paper. 4. Literature analysis or discussion: The final statement does not relate very well to the title of the paper. Is there literature precedent for B. safensis as a macoralgae symbiont? Has B. safensis been isolated in the same geographical area as the isolate in the manuscript? 5. Any other relevant comments: Lines 17, 20- Unusual sentence structures Line 39- There are no plural subjects. The use of the word "they" is unclear.' Line 184- Unusual wording

    Please rate the manuscript for methodological rigour

    Satisfactory

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

    Good

    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

  10. Access Microbiology

    Comments to Author

    The work is interesting, especially in the era of antibiotic resistance, however significant changes must be made to make it scientifically sound. Abstract There is redundancy in the introduction part within the abstract, it should be concise and focused, better to move these highlights to the introduction section. Introduction: Authors should provide references of the fundamental work that sustain their statements, e.g. the use of C. violaceum to assess the antibiofilm efficiency, potential use of marine Bacillus safensis as antibiotic, in addition to the importance and accuracy of resazurin in detecting the MIC. Line#63-64: you mentioned that global warming can lead to rapid increases in AMR bacteria and environmental genes, give a brief explanation. Methods Elaborate on the molecular detection of bacterial isolates, e.g which genes were targeted? You should use the relevant appropriate and references for antimicrobial screening, MIC, MBC, and the quorum sensing inhibitory effect. What OD600 value you determined for standardising bacterial concentration for measuring the MIC. It is most important to mention the source of the bacterial and fungal test strains used in the study. Also show the if they are MDR or not. You should define their antimicrobial sensitivity to the control antibiotics used. Results You should describe the colonial morphology of bacterial isolates. Describe the SEM micrographs of bacterial isolates, especially the aggregates and biofilm structure. Line # 243, you have to distinguish the Firmicutes (Gram-positive) form the Proteobacteria (Gram negative). I suggest grouping the isolates belonging to Firmicutes together, and the Proteobacteria together, especially in tables 1-4 to make it easy for the reader to compare. Discussion Very important to provide mechanistic insights that could explain the anti quorum sensing activities of bacterial extract. In line# 235, you mentioned that" motility indicates that they form biofilms", what is the correlation between motility and biofilm formation? General Unify the referencing style The manuscript needs proofreading and editing; e.g. "of note" was repeated many times, "Gram" should always start with a capital letter,…etc.

    Please rate the manuscript for methodological rigour

    Good

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

    Very good

    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

  11. Access Microbiology

    Comments to Author

    Abstract should be summarized especially following " Seaweed-associated bacteria participate in complex interactions necessary for the health and defense of their host. It involves the production of antifouling metabolites and antimicrobials to prevent secondary colonization of pathogenic epibiota. Halymenia durvillei, commonly known as "red sea lettuce," is a red alga ubiquitously distributed in the Indo-Pacific region. It is cultivated in the Philippines due to its abundance in high21 value pigments such as phycoerythrin and r-phycocyanin. However, there are no studies regarding the microbial community associated with H. durvillei and its potentialpplications. This study aimed to isolate and 23 identify the epiphytic bacteria of H. durvillei and determine their antimicrobial and quorum-sensing inhibitory (QSI) effects. The thalli of H. durvillei were collected at the shores of Santa Fe, Bantayan, Cebu, Philippines" Avoid Un necessary use of "The" MIC (MIC) ranging from 0.0625 - 1.0 mg mL-1 is too high so how can it be regarded as ANTIBIOTIC. Also the zone of inhibition is 3mm with 100mg/mL???????? I do not consider it as promising. Why Violacine inhibition assay was not performed?? antibiotic effects at 100 mg mL-1 is too much = and can't be considered as promising

    Please rate the manuscript for methodological rigour

    Poor

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

    Satisfactory

    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?

    Yes

  12. Version published to 10.1099/acmi.0.000563.v1 on Access Microbiology