Decoding past microbial life and antibiotic resistance in İnonü Cave’s archaeological soil

Sukran Ozturk; F.Gülden Ekmen; Hamza Ekmen; Esra Mine Ünal; Ayşegül Er; Emre Keskin; Benjamin Stanley Arbuckle

doi:10.1371/journal.pone.0326358

Peer Review History

Original SubmissionDecember 4, 2024
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Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: renamed_ba4a1.docx https://doi.org/10.1371/journal.pone.0326358.r001
Revision 1
18 Mar 2025 Author Response Dear Reviewers; This article brings together archaeology and microbiology and reveals bacterial populations and antibiotic resistance genes identified by examining soil microbiota detected in ancient times, and provides remarkable results in shedding light on the subject of ONE HEALTH. It is an interdisciplinary study covering different topics and raising awareness about antibiotic resistance. All revisions have been completed in line with your suggestions, and we would like to thank you, Dear Editor, and the referees who contributed to our article. We believe that our article is worthy of publication in PLOS ONE and we kindly request you and the team to give this chance. We believe that this article, which will be a guide for future projects, will also close the gaps in the literature on the subject. We kindly request that our article be evaluated positively Respectfully Attachments Attachment Submitted filename: Response to Reviewers1.docx https://doi.org/10.1371/journal.pone.0326358.r002
Decision Letter - Peter F. Biehl, Editor Dear Dr. Ozturk, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Please address the comments in detail before resubmission ============================== Please submit your revised manuscript by May 25 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). 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Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Peter F. Biehl, PhD Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments : Please address the comments in detail before resubmission [Note: HTML markup is below. 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If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org Attachments Attachment Submitted filename: renamed_b31ce.pdf https://doi.org/10.1371/journal.pone.0326358.r003
Revision 2
22 May 2025 Author Response PONE-D-24-53725R1 Decoding Past Microbial Life and Antibiotic Resistance in İnonü Cave’s Archaeological Soil PLOSONE Response to Reviewers'. �A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. -Response: - The required document is attached. �An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. -Response : - The required document is attached. Journal Requirements: Please review your reference list to ensure that it is complete and accurate. If you have cited retracted articles, please include the reason for doing so in the text of the manuscript or remove these references and replace them with relevant, up-to-date references. Any changes to the reference list should be noted in the rebuttal letter accompanying your revised manuscript. If you must cite a retracted article, indicate the retraction status of the article in the References list and also include a citation and full reference for the retraction notice. -Response: There have been changes in the references because there were reference additions. All changes are shown in the text, in the references section and on the renamed b31 document. 1.The introduction was well expanded and in its current state adds a lot more relevant context to the study. We would like to highlight again that every statement (especially referring to previous studies) requires a reference. Please revise the text with this in mind. Here are a few examples: The field of ancient DNA (aDNA) and microbial communities in archaeological contexts has received considerable attention in recent years. These investigations offer a distinct viewpoint on human behavior, health, and interactions with the environment in the past. By examining microbial DNA found in archeological sites, scientists can extract significant insights regarding the microbiomes of ancient civilizations, their food patterns, and the occurrence of contagious Illnesses (REF) -Response: - Appropriate reference added as suggested. (Higuchi, R.; Bowman, B.; Freiberger, M.; Ryder, O.A.; Wilson, A.C. DNA sequences from the quagga, an extinct member of the horse family. Nature 1984, 312, 282–284. [CrossRef] [PubMed] 3. Pääbo, S. Molecular cloning of Ancient Egyptian mummy DNA. Nature 1985, 314, 644–645. [CrossRef] [PubMed]). 2.Furthermore, examining ancient microbial communities allows us to comprehend the historical influence of human actions on environmental microbial ecosystems. Recent interdisciplinary studies have shown that the materials contained in archaeological sediments provide information beyond just defining culture (REF). -Response: Appropriate reference added as suggested.- Veeramah, K.R. Primate paleogenomics. Paleogenomics Genome-Scale Anal. Anc. DNA 2018, 353–373. [CrossRef] 3.In this context, organic remains dating back thousands of years play an important role. While research on organic remains found in archaeological sites has gained momentum in recent years, the number of studies, especially on microbial diversity and change, still needs to be increased. High-throughput sequencing technologies have opened new frontiers in microbial community analysis and have become widely used in assessing the diversity of bacterial components in soil (REF). - Response: Appropriate reference added as suggested -Orlando, L.; Allaby, R.; Skoglund, P.; Sarkissian, C.; Stockhammer, P.W.; Ávila-Arcos, M.C.; Fu, Q.; Krause, J.; Willerslev, E.; Stone, A.C.; et al. Ancient DNA analysis. Nat. Rev. Methods Primers 2021, 1, 14. [CrossRef]) 4.The recent application of next-generation sequencing methods such as Illumina and Roche provides reliable and accurate resultsin detecting microbial taxa. 16S rRNA studies are preferred as an excellent phylogenetic method to obtain important information about investigating and detecting microbial taxa and antibiotic resistance genes in soil samples from archaeological excavations (REF). -Response: Appropriate references added as suggested �McVean, G. A Genealogical Interpretation of Principal Components Analysis. PLoS Genet. 2009, 5, e1000686. [CrossRef] �Patterson, N.; Price, A.L.; Reich, D. Population Structure and Eigenanalysis. PLoS Genet. 2006, 2, e190. [CrossRef] 5.Now, more than three decades later, scientists and researchers are actively engaged in ongoing research to solve various puzzles related to the origin of humanity, migration patterns, the emergence of infectious diseases, and their spread among ancient populations. aDNA analysis has emerged as a state-of-the-art genetic tool gaining momentum worldwide, changing our comprehensive understanding of the past. For example, in a recent groundbreaking study, aDNA analysis of seven individuals revealed the origin of the plague strain that caused the Black Death in present-day (2) (3). Are you sure (2) is the appropriate reference here? -Response: Necessary investigations were made and reference number 2, Dalal V, Pasupuleti N, Chaubey G, Rai N, Shinde V. Advancements and Challenges in Ancient DNA Research: Bridging the Global North–South Divide. Genes. 2023;14(2):479. was confirmed to be relevant to this topic. However, since references were added to previous sections based on your suggestions, its number was changed to ... . -Other added references were marked and indicated in the text and references section. 6.More broadly, aDNA data have revolutionized our knowledge and curiosity and have led to the publication of an enormous amount of literature. The latest technological innovations (If you mention innovations, make sure to name them! have proven to be an extraordinary tool for scientists and researchers in the race to obtain the 'gold' (aDNA) I would paraphrase this or leave it out as it is neither common nor scientific to refer to aDNA as “gold”.. In the present research, we aim to broaden the scope of the ongoing conversation in the field of aDNA by reporting relevant literature published worldwide and detailing advances and challenges.(Put aims at the end of the introduction rather than in the middle. Further, it seems obsolete to mention you’re including a state of the art literature review as this is just common scientific practice in a paper. I would remove this sentence.) -Response: It has been reviewed in line with your suggestions and these statements have been removed upon your suggestion. 7. While significant microbial abundance and diversity were found in soil samples from four archaeological levels, our study does not reveal differences in microbial community structure and diversity. These differences may be related to various consumer, disease, and nutritional factors. These microarchaeological findings and microbial characteristics may be attributed to different microbial responses to specific consumer habits. Hence, different consumption habits, patterns, and disease profiles lead to different compositions of the generated wastes and thus leave different microbial traces. This statement includes results and conclusions and does not really fit in the introduction. - Response: This section has been moved to the results section for your recommendations and manuscript layout. 8.Site (İnönü Cave) and Samples İnönü Cave is a cave of volcanic origin located in the Cambu geological formation. This volcanic cave, located on the Western Black Sea coast of Turkiye, developed in several stages (Fig 1) 5 Fig 3: Number of Identified Specimens (%) (17) Why is there a change of reference style (superscript)? -Response: (Fig 1)5 Fig 3: Number of Identified Specimens (%) (17) -Reference numbers 5 and 17 have been converted from superscript to normal notation. 9. Bioinformatics analysis identified the presence of a wide range of taxa, including Cyanobacteria, the Acidobacteriota phylum, the Actinobacteriota phylum, Bacteroidota, Chloroflexi, Firmicutes, Myxococcota, Proteobacteria, Cyanobacteria, and Nitrospirota species (Tabel 2). Typo: Table -Response: (Tabel 2). Typo: Table expression has been updated to Table. 10. Material and Method (Rather use the plural Materials and Methods) -Response : It has been corrected as Materials and Methods 11. Assoc analyzed the defined fillings analogically. Prof. Dr. Hamza Ekmen and Assoc. Prof. Dr. F. Gülden Ekmen and confirmed with 14 C analyses, were taken with sterile materials (Eppendorfs, falcon tubes, steel spatulas, steel collecting spoons) by pharmaceutical microbiologist Assoc. Prof. Dr. Sukran Ozturkfrom the Faculty of Pharmacy. (Paraphrase these sentences, the structure is not correct) The materials were sterilized in an autoclave (brand Nüve) at 121° C for 15 minutes in the Pharmaceutical Microbiology Laboratory of the Beun Faculty of Pharmacy and transported to the excavation site (Figures 3-4). -Response: This paragraph has been revised as follows. The fillings of the four archaeological levels were verified with C14 carbon analyses and evaluated by Assoc. Prof. Dr. Hamza Ekmen and Assoc. Prof. Dr. F. Gülden Ekmen. Soil samples were taken from these layers under the control of the excavation head Assoc. Prof. Dr. Hamza Ekmen and the responsible researcher Assoc. Prof. Dr. Gülden Ekmen, using materials prepared by Beun Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Head of the Department of Pharmaceutical Microbiology, Assoc. Prof. Dr. Şükran Öztürk, sterilized in an autoclave (Nüve) at 121° C for 15 minutes in the Pharmaceutical Microbiology Laboratory of Beun Faculty of Pharmacy, and considering antisepsis conditions (Eppendorfs, falcon tubes, steel spatulas, steel collecting spoons) and transferred to the laboratory environment under sterile conditions (Figure 3-4). 12. Detailed information is shown in Table 1. (I don’t hink the NISP Figure 3 is relevant here) PCR and Bioinformatics Small 16S gene region primers (16SV3-F/16SV3-R) were used for the qPCR test to see if PCR inhibition happened 15 . Again superscript. -Response: The superscript has been corrected to normal spelling. (15) 13. Data availability Our data falls under the list of data types that must be deposited with you. We provide the relevant accession numbers, and the name of the database in which your data is stored is "The datasets generated and/or analyzed during the current study are available in the NCBI BioProject repository, BioProcecj ID: PRJNA1134133." Omit the first part, this is not necessary to mention and just state: “The datasets generated and/or analyzed during the current study are available in the NCBI BioProject repository, BioProcecj ID: PRJNA1134133.” -Response: The specified corrections were made in accordance with the suggestions. The name of the database in which your data is stored is “The datasets generated and/or analysed during the current study are available in the NCBI BioProject repository, BioProcecj ID: PRJNA1134133.” Results 14. We have found information supporting our study on these taxa (Fig 6a,b,c,d). The figures are not supporting this statement but your own results. Leave this sentence out. You are discussing your results in a later part of the results anyways. -Response: We have found information supporting our study on these taxa (Fig 6a,b,c,d). “This sentence has been removed based on your suggestions. 15. The MN NUCLEOSPIN Soil DNA Extraction Kit was chosen for its proven efficiency in isolating ancient DNA (aDNA) from complex soil matrices, which often contain high levels of PCR inhibitors such as humic acids. This kit's robust inhibitor removal technology ensures higher purity and yield of DNA, which is critical for downstream applications in ancient microbiome studies. Furthermore, its dual-buffer system (SL1 and SL2) optimized lysis efficiency across varied soil types, enhancing DNA recovery from both Gram-positive and Gram-negative bacteria (Fig 6a,b,c,d). This is a repetition form the Methods section. Shorten or leave out. Following this paragraph there is an icon of a file named “aDNA_silva_krona.html” in the text. This seems to be a mistake. -Response: This sentence has been removed based on your suggestions. “aDNA_silva_krona.html” -This link was shared so that you could access the metagenomic data. However, it was removed based on your suggestions since it was not possible to access it. 16. The Actinobacteriota phylum, or Actinobacteria, is a diverse group of bacteria found in various environments, including soil, water, and plants. They are characterized by their high G+C content in their genomic DNA and filamentous growth pattern. Actinobacteria are known for their ability to produce a wide range of bioactive compounds, such as antibiotics, antifungals, and antitumor agents. This has led to extensive research into their potential applications in medicine and biotechnology. Additionally, Actinobacteria play essential roles in soilecosystems, involving nutrient cycling and organic matter degradation (51). Another identified bacterial taxon is Bacteroidota. They are commonly found in aquatic environments, soil, and the guts of animals, including humans. Bacteroidota bacteria are essential in breaking down complex organic matter, such as polysaccharides, and play a vital role in the carbon cycle (25). These detailed descriptions of the phyla seems a bit repetitive as well. Shorten and focus on your results. -Response: This section has been shortened according to recommendations. 17. Lactobacillus species produce fermented foods such as yogurt and cheese (27, 28). Bacillus species are known for making antibiotics and enzymes, while Clostridium species are essential in producing biogas and other industrial products. Lactobacillus species produce fermented foods such as yogurt and cheese, while Staphylococcus aureus can cause human infections (27, 28). Repetitive. -Response: Lactobacillus species produce fermented foods such as yogurt and cheese, while Staphylococcus aureus can cause human infections -This section is edited according to recommendations. Note: The quality of the figures, even after downloading them, is so bad that they could not be evaluated by us. -Response: The quality of the figures has been revised. AConclusio: This revision added substantial value to the manuscript and after addressing our aforementioned comments, we recommend publication. - Thank you for your consideration. Respectively Assos.Prof. Sukran OZTURK PhD Attachments Attachment Submitted filename: Minor Rev_Response Reviewer 2_ rev tracks manuscript.docx https://doi.org/10.1371/journal.pone.0326358.r004
Decision Letter - Peter F. Biehl, Editor Decoding Past Microbial Life and Antibiotic Resistance in İnonü Cave’s Archaeological Soil PONE-D-24-53725R2 Dear Dr. Ozturk, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Peter F. Biehl, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0326358.r005
Formally Accepted
Acceptance Letter - Peter F. Biehl, Editor PONE-D-24-53725R2 PLOS ONE Dear Dr. Ozturk, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Peter F. Biehl Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0326358.r006

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