Identification of Mammaliicoccus fleurettii as the source of a methicillin-resistance gene in a First Nation reserve lake in Manitoba, Canada

  1. Sabrin Bashar
  2. Rakesh Patidar
  3. Alvan Wai
  4. Dawn White
  5. George R. Golding
  6. Annemieke Farenhorst
  7. Ayush Kumar

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Abstract

Our study aimed to identify the bacterial source of a previously detected mobile antibiotic-resistance gene, mecA, found in a lake that serves as a source to a water treatment plant operated by a First Nation reserve. Three methicillin-resistant presumptive Staphylococcus spp. isolated from the sample using selective media were verified as mecA positive by PCR (polymerase chain reaction). MALDI-TOF (matrix-assisted laser desorption ionization–time-of-flight mass spectrometry) and whole genome sequencing of each isolate confirmed that all three were Mammaliicoccus fleurettii. Antibiotic-resistance gene analysis of the assembled genomes predicted mecA with 99.7% sequence identity, and phylogenetic analysis grouped our three mecA genes with the mecA allele from a methicillin-resistant strain of Staphylococcus aureus. Identifying microbial species known to harbour mobile antibiotic-resistance elements can provide greater depth of information about drinking water, an especially essential need in First Nation reserves where water quality too frequently is poor.

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  2. Version published to 10.1099/acmi.0.000861.v2 on Access Microbiology
  4. Access Microbiology

    Comments to Author

    Dear Author, There are specific detailed comments that I have mentioned in the attached document for your attention and action aside the ones below. Design and Methodology Are the research objectives clearly stated and were they met? The article writing is well structured and the research objectives were clearly stated and met. Is there an adequate review of the published literature? To enhance the credibility and relevance of your work, I recommend you incorporate recent references. Your research will demonstrate its connection to current advances. Are the methods described in enough detail to permit someone to reproduce the experiments? To make the methods reproducible, the use of Gram Staining technique (i. e., the method used to confirm the isolates as Staphylococcus spp) and coagulase tests should be mentioned in the manuscript. This would strengthen the claim that the isolates were further confirmed before proceeding to molecular methods such as PCR etc. Are the methods appropriate to the research? Do they address the problem, and do they include relevant controls? Yes all the methods employed in the research were found relevant with adequate experimental controls. Have the authors used appropriate statistical tools to address the findings? Yes! Relevant and appropriate statistical tools were applied. Have the authors followed relevant ethical guidelines? Ethical clearance for the study if required or not was not disclosed by the author. I recommend that ethical adherence regarding experimental protocols and safety should be disclosed by the author Results and conclusions How significant are the findings described in the article, and do they represent an advance in knowledge and understanding? The findings are significant to solving the little public awareness of the poor water quality associated with people in developed countries. It also underscores the importance of understanding the full bacterial composition of lake and drinking waters to identify seemingly harmless bacteria harbouring transferable antibiotic-resistance genes which may reduce potentially serious human health issues. Have the results been interpreted properly? The figures and tables recommended should be added by the author Are the conclusions based on sound data, with reasonable reference to the published literature? The conclusions were based on sound and reasonable findings. Relevant recent reference should hereby be cited to support this. General Is the article well written and of an appropriate length? The article is well written for a short communication Is the number of tables and figures appropriate, or should some of the data be published as supplementary material? Figures of the whole genome sequencing diagram of the isolates should be included. Table showing the primers used with their expected base pairs. This could form a part of the supplementary reports Is the supplementary material relevant to the article? Yes Yes

    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?

    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

  5. Access Microbiology

    Comments to Author

    1. The sample was collected in 2019.At this point of time resistance has been reported among the fourth generation as well. So what was the specific need of highlighting this antibiotic resistance gene? 2. Other microbial species could have been targeted for a comprehensive understanding of the Antimicrobial resistance pattern. 3. It is suggested that the author will have to add the treatment pattern regarding the concerned bacterial species. 4. Scientific language could have been improved in the "impact statement" 5. The aims and objectives are ambiguous. There is also lack of coherence in introduction and Discussion. 6. Temporal period: The sample could have analysed in different time frames as well. (Only June is targeted) 7. Concentration of oxacillin for isolation (no refrence or CLSI guidelines mentioned). Further analysis of resistant methicillin strain was absent in the study.(6 microgram/mL was used in the study) 8. Replicates of antibiotics similar to oxacillin could be opted as well. (naficillin) 9. Comparison with previous published data regarding presence of ARG is absent. 10. M.fleurettii is not a natural lake water inhabitant. The author does not highlight the possible sources of Reserve contamination. (Livestock industry, agricultural runoff, sewage waste) 11. Visual representation of results could be improvised. 12. The author did not add the Limitations regarding the present study.

    Please rate the manuscript for methodological rigour

    Satisfactory

    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

  6. Version published to 10.1099/acmi.0.000861.v1 on Access Microbiology