Multi-Metal Resistant Staphylococcus warneri strain TWSL_1: Revealing Heavy Metal Resistant Genomic Features by Whole Genome Sequencing

  1. Dilani Dissanayake
  2. Naduviladath Chandrasekharan2
  3. Dilrukshi Wijayaratahna

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Abstract

2. Abstract This study investigates the genomic basis of heavy metal resistance in Staphylococcus warneri strain TWSL_1, isolated from the effluent of a textile dyeing industry in Sri Lanka. The strain exhibited substantial resistance to Cd2+, Pb2+, and Cu2+, with minimum inhibitory concentrations of 50 mg/L, 1200 mg/L, and 75 mg/L, respectively. Phylogenetic analysis of the 16S rRNA gene confirmed its placement within the Staphylococcus warneri clade. Whole genome sequencing revealed a 2,661,318 bp genome with 2,567 coding sequences and a 99.81% average nucleotide identity with S. warneri WS479. Key heavy metal resistance genes were identified through the comparative genomic analysis, including those encoding a cadmium efflux system accessory protein and a cadmium resistance protein uniquely present in TWSL_1 but absent in Staphylococcus aureus RF122. The strain also harbored genes associated with cobalt-zinc-cadmium resistance underscoring TWSL_1's adaptation to heavy metal stress. These findings highlight the comprehensive genetic toolkit of TWSL_1, enhancing its resilience in metal-contaminated environments and demonstrating its potential for bioremediation, supporting the development of eco-friendly remediation strategies.

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  1. Access Microbiology

    Thank you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field, whose comments are attached at the bottom of this email. They agree that this study of in interest, but needs major corrections and clarifications. Please provide a revised version of the manuscript (including a tracked changes document) along with a point-by-point response to the reviewer comments within 2 months.

  2. Access Microbiology

    Comments to Author

    I would like to thank the authors for putting this interesting study together. Below are my comments aimed at improving the quality and clarity of this manuscript: Major Comments: 1. Introduction: o The introduction is disorganized and incomplete. It needs to be extended and refined. For example: Which bacterial species are being researched to combat metal pollution? What genes have been identified and tested for metal resistance in Staphylococcus warneri or related species? Lines 117-118 mention "metal binding and detoxification proteins." Please elaborate, as this is currently unclear. 2. Sample Collection: Line 145: "An effluent sample was collected from a textile dyeing industry in the Western Province of Sri Lanka". Was the sample collected from a single location in the said industry, or were multiple sites sampled? 3. Selection of Strain: Lines 150-151: "The strain designated TWSL_1 demonstrated the most promising heavy metal resistance and was selected for further study." Is this conclusion based solely on growth in 20 mg/L metal plates? If so, what salts were used for each metal? Were all concentrations set at 20 mg/L? Please include these details. Including pictures of plates showing the growth of various strains on metal-amended media could be beneficial for readers. Was strain TWSL_1 the only isolate to exhibit growth in metal ammended plate? Were there other isolates that showed comparable growth? 4. Heavy Metal Removal Capacity and Tolerance: Were there biological replicates in the experiments? If so, please specify this in the methodology. Were the experiments repeated? If yes, how many times? If not, the experiments should be repeated for validation. Why wasn't a metal-sensitive strain included as a secondary control? Including a sensitive strain is advisable. 5. Biochemical Tests: Lines 171-173: The manuscript states that various biochemical tests were conducted. However, the results are not included. Please provide this data in the manuscript. 6. 16S rRNA Sequencing and Whole Genome Sequencing: Since whole-genome sequencing was performed, a core genome phylogenetic tree is recommended instead of 16S rRNA-based phylogeny. Refer to "https://academic.oup.com/bioinformatics/article/31/22/3691/240757" for guidance on using Roary for identifying core genes and subsequent analysis. Include Staphylococcus aureus RF122 in the phylogenetic tree for comparison. 7. Comparative Genomics: The manuscript discusses heavy metal resistance genes absent in Staphylococcus aureus RF122. However, the rationale for comparing these two strains is not provided. Please explain why this comparison was chosen. 8. Functional Validation: Are any of the identified genes functionally validated for their role in metal resistance? If so, please include this in the discussion. If not, it is incorrect to make definitive assumptions about their function. 9) Conclusion: Conclusion can be extended. line 409-411: Authors talk about functional analysis showing the role of genes in metal transport. This is not done in the study neither discussed in the manuscript Minor Revisions: 1. Title: Revise the title to better reflect the study's content. The manuscript primarily focuses on characterizing the strain, and none of the resistance genes are functionally proven for metal resistance in this study. 2. Subheadings: Line 306: "Genome features of the strain S. warneri TWSL_1." Add subheadings for genome statistics , ANI and subsystem classification for clarity. 3. Species Name: ANI is used for species determination. Use the species name only after presenting ANI results. Before that, refer to the isolate as strain TWSL_1. Avoid using 16S rRNA phylogeny for species determination, as it has limitations. 4. Editing for Clarity: Line 219: "Isolation and evaluation of the heavy metal resistance of the strain". Revise to: "Isolation and evaluation of the heavy metal resistance strain." I wish the authors all the best with their revisions and the successful publication of this 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

  3. Access Microbiology

    Comments to Author

    Here, the authors present the discovery of a strain of Staphylococcus, which was isolated from industrial waste. This strain grows in high concentrations of heavy metals which prompted the authors to sequence and assemble its genome to gain insights into the genetic basis of its heavy metal resistance. They provide a draft genome assembly, which is used to delineate the strain as S. warneri primarily through analysis of pairwise ANI values. They annotate this genome assembly and give an overview the types of functions it encodes. Lastly, when compared to those of strains from other Staphylococcus species, this strain's genome is enriched with genes that encode heavy metal resistance. Methodological rigour, reproducibility and availability of underlying/supporting data. Data availability The authors provide accession numbers to genomes used in this study. The reads used to assemble the genome should be uploaded to SRA to enhance reproducibility. Methodology. Bacteriology methods were commendable, suitable, and well-applied. For genome sequencing and assembly, the current description of methods used are insufficient to result in the genome assembly of accession number CP135051. Unless this species has an unusually low number of repeat sequences, obtaining a circularised genome via de novo assembly of illumina short reads is rare (relatedly, the authors do not specify the insert size used for Illumina sequencing). Indeed, I searched the genomes of other strains of this species in Genbank, and ranked them according to their contig N50, and among the twenty with the least contig N50, none was assembled via Illumina reads only. As expected, most were assembled using a combination Illumina+PAcbio/ONP or ONP/PAcbio only demonstrating that it is rare to assemble circularised genomes from this species via short reads only. For the comparative genome analysis, would it not be better to compare this strain to another strain within the same species instead of to S. epidermidis and S. aureus: animal and human pathogens? The former type of comparison would allow the authors to investigate whether genes that encode heavy metal resistance are generally found within this species or are uniquely found in this strain that was isolated from industrial waste. Presentation of the results. Figure 1: The authors present p-values without having described anywhere in the entire manuscript, what specific hypothesis testing (e.g. t-test) was used to arrive at them. Figure 2. With a genome assembly in hand, I would not analyse a single gene phylogeny but rather a multigene or genome-based phylogeny, as the latter are more robust and accurate. Table 1. The assembly statistics provided are not for the CP135051. Figure 5. Remove all other genes and leave only those that encode heavy metal resistance since this is the main finding you are relaying from the comparative genome analysis. How the style and organization of the paper communicates and represents key findings, literature analysis or discussion. The paper in general is well-written and the authors have analysed the literature of Staphylococcus exceptionally well and presented it in a cohesive interesting manner. The results were well-placed in the context of current findings on heavy metal resistant bacteria and how they can be potentially applied for bioremediation. Of concern, is the high similarity score.

    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?

    Yes: Table 1. The assembly statistics provided are not for the genome assembly claimed to be from this study and deposited in Genbank (CP135051). These statistics are for a draft genome whereas the genome that the authors assembled and deposited is a circularised genome (CP135051).

    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.0.000954.v1 on Access Microbiology