The origin and evolution of IncF33 plasmids based on large-scale data sets

ABSTRACT To comprehensively understand the formation and evolution of IncF33 plasmids, a global collection of whole-genome sequences of 863 strains positive for IncF33 plasmid replicons was analyzed. The results showed that the IncF33 plasmids were mainly identified in Enterobacterales, of which Escherichia coli (86.44%) was the dominant host, followed by Salmonella (8.57%) and Klebsiella pneumoniae (3.48%). Salmonella ST11 and K. pneumoniae ST11 were common and mostly from humans. IncF33 plasmids were found worldwide but prevalent in Chinese farm animals, predominantly carrying antibiotic resistance genes such as bla CTX-M-55, fosA3, bla TEM-1B, rmtB, and floR. Comparative genomics analysis of 103 complete IncF33 plasmid sequences showed highly similar backbones except for 16 lacking partial backbone fragments. Variable regions were diverse, containing various antibiotic resistance genes, insertion sequences, or other plasmid fragments. They can be roughly divided into two sublines based on the production of different CTX-M enzymes. Similar IncF33 plasmids from different countries were identified. Some early IncF33 plasmids lacked part of their leader regions, which showed over 99% homology with early F2:A-:B- plasmids, indicating that leader regions of IncF33 likely came from F2:A-:B- plasmids. In addition, IncF33 plasmids cointegrating with other types of plasmids to form new cointegrate plasmids are increasing, making them more efficient in their dissemination and persistence in Enterobacterales, which could pose a significant threat to global public health. IMPORTANCE Plasmids that capture multiple antibiotic resistance genes are spreading widely, leading to the emergence and prevalence of multidrug-resistant bacteria. IncF33 plasmids are a newly emerged plasmid type highly prevalent in animal-source Enterobacterales in China, and they are important vectors for transmitting several clinically important antibiotic resistance genes. The study revealed that the IncF33 plasmid is mainly prevalent in China animal-derived Escherichia coli and has the potential for cointegration and intercontinental dissemination. Therefore, it is crucial to enhance surveillance and control measures to limit the spread of IncF33 plasmids and their associated antibiotic resistance genes.

• Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.
For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/mSystems/submission-review-process.Submission of a paper that does not conform to mSystems guidelines will delay acceptance of your manuscript.
Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by mSystems.
Major comments: 1.The authors analyzed the variable regions of IncF33 plasmids and detailly described the genome characters of different clusters.It is worth discussing the potential influence of diverse variable regions on plasmids and bacteria.In addition, among all clusters, cluster A contained the largest number of plasmids but only 8 plasmids.It is necessary to consider adjusting the parameters of CD-Hit to make this analysis more representative.2. The paragraphs in the part of results and discussion need to be well-organized to present the results logically.3. Line 351，There are two different types of cointegrates of IncF33 plasmids with other types of plasmids, and the author stated the second way resulted from various plasmid fragments insertion in the IncF33 variable regions was more common.However, is it possible that the first way fusion of IncF33 was underestimated, because you excluded a considerable proportion of plasmids carrying blaKPC genes plasmids in the analysis process? ( Line 400 "Since plasmids carrying blaKPC genes were formed by cointegrating IncF33 and R-type plasmids and were not typical IncF33 plasmids, the strains carrying the blaKPC genes were excluded from this study.")4. As for the clustering analysis of variable region sequence, six clusters containing 26 plasmids were emphasized.However, a majority of 59/65 clusters were not further mentioned.What does the author think about these scattered clusters for which containing only 1-2 samples? 5. How were core genes and varied region defined?The relevant methods are not described.6.The authors should have a paragraph to describe the limit of this study in the part of results and discussion.
In this manuscript, Gao et al. analyzed the origin and evolution of IncF33 plasmids based on a large number of genomes.It is always interesting and essential to study the characteristics and evolution of transferable plasmids carrying antibiotic resistance determinants.Overall, the analysis is thorough and present comprehensive knowledge about IncF33 plasmids.I have a few comments as described below.
Major comments: 1.The authors analyzed the variable regions of IncF33 plasmids and detailly described the genome characters of different clusters.It is worth discussing the potential influence of diverse variable regions on plasmids and bacteria.In addition, among all clusters, cluster A contained the largest number of plasmids but only 8 plasmids.It is necessary to consider adjusting the parameters of CD-Hit to make this analysis more representative.2. The paragraphs in the part of results and discussion need to be well-organized to present the results logically.
3. Line 351，There are two different types of cointegrates of IncF33 plasmids with other types of plasmids, and the author stated the second way resulted from various plasmid fragments insertion in the IncF33 variable regions was more common.However, is it possible that the first way fusion of IncF33 was underestimated, because you excluded a considerable proportion of plasmids carrying blaKPC genes plasmids in the analysis process? ( Line 400 "Since plasmids carrying blaKPC genes were formed by cointegrating IncF33 and R-type plasmids and were not typical IncF33 plasmids, the strains carrying the blaKPC genes were excluded from this study.")4. As for the clustering analysis of variable region sequence, six clusters containing 26 plasmids were emphasized.However, a majority of 59/65 clusters were not further mentioned.What does the author think about these scattered clusters for which containing only 1-2 samples? 5. How were core genes and varied region defined?The relevant methods are not described.6.The authors should have a paragraph to describe the limit of this study in the part of results and discussion.

Response to Reviewers Reviewer comments:
Reviewer #1 (Comments for the Author): Comments: Gao and coworkers did a nice job describing the evolution of IncF33 plasmids.They carefully analyzed the structure of a data set of 103 complete plasmids, identified the plasmid backbone, and analyzed variation within the structure of the plasmids.I liked the work done, and I'm sure that the paper will make a nice contribution to the field.I have a request: It is important to indicate which plasmids form cointegrates.Also, indicate if these cointegrates consist of two or more plasmids and mention which plasmid components are in these cointegrates.Finally, it is important to know if the replication modules (different from IncF33) have a narrow or wide host range.A table will be more than enough.
Answer: Thank you for your positive feedback and valuable suggestions.We agree that indicating which plasmids form cointegrates and providing details on the plasmid components involved will help readers better understand the evolution of IncF33 plasmids.To address your request, we have prepared Table S4 providing information on IncF33 cointegrate plasmids.
Reviewer #2 (Comments for the Author): Comment 1: The authors analyzed the variable regions of IncF33 plasmids and detailly described the genome characters of different clusters.It is worth discussing the potential influence of diverse variable regions on plasmids and bacteria.In addition, among all clusters, cluster A contained the largest number of plasmids but only 8 plasmids.It is necessary to consider adjusting the parameters of CD-Hit to make this analysis more representative.
Answer: Thank you for your thoughtful comments and valuable suggestions on our work.We have discussed the potential influence of diverse variable regions on plasmids and bacteria (Lines 344-347).Regarding CD-Hit parameters, we tested multiple cutoffs previously.By iteratively testing different parameters, we found the optimal value was 0.8 (i.e., 0.8 produced the most representative clustering with well-separated clusters), while lower cutoffs of 0.6-0.7 resulted in imprecise clustering (i.e., distinct variable regions grouped together).We therefore do not recommend lowering the cutoff further as it may introduce inaccuracies.
Comment 2: The paragraphs in the part of results and discussion need to be well-organized to present the results logically.
Answer: We agree with the reviewer.We have reorganized the Results and Discussion sections to make it more logical and clearer.
Comment 3: Line 351, There are two different types of cointegrates of IncF33 plasmids with other types of plasmids, and the author stated the second way resulted from various plasmid fragments insertion in the IncF33 variable regions was more common.However, is it possible that the first way fusion of IncF33 was underestimated, because you excluded a considerable proportion of plasmids carrying bla KPC genes plasmids in the analysis process? ( Line 400 "Since plasmids carrying bla KPC genes were formed by cointegrating IncF33 and R-type plasmids and were not typical IncF33 plasmids, the strains carrying the bla KPC genes were excluded from this study.")Answer: Thank you for pointing out this important issue regarding cointegrate plasmids.We agree that excluding plasmids carrying bla KPC genes may make the first way fusion of IncF33 underestimated.However, in this study, we mainly focused on typical IncF33 plasmids.We have reclassified the 292 plasmids (Lines 102-111), categorizing the bla KPC -positive cointegrated plasmids into a third class (Type III), and re-described the formation and proportions of Type I and Type II plasmids (Lines 365-370).At the same time, in the newly added Limitations section (Lines 388-394), we also acknowledged the limitation of excluding the bla KPC -positive cointegrated plasmids.
Comment 4: As for the clustering analysis of variable region sequence, six clusters containing 26 plasmids were emphasized.However, a majority of 59/65 clusters were not further mentioned.What does the author think about these scattered clusters for which containing only 1-2 samples?Answer: Thank you for raising this important issue.In our analysis, clusters with only 1-2 plasmids were indeed scattered and underrepresented.We did analyze plasmids in these clusters, and found most of them represented intermediate evolutionary forms.However, due to the need to summarize variable region characteristics and the word limitation, we could not provide detailed descriptions of these underrepresented clusters, and only focused on major clusters.We added brief descriptions of these scattered clusters in the Results and Discussion sections (Lines 336-340).
Comment 5: How were core genes and varied region defined?The relevant methods are not described.

Answer:
We have added the definition of core genes and variable regions in the Materials and Methods section (Lines 446-452).
Comment 6: The authors should have a paragraph to describe the limit of this study in the part of results and discussion.

Answer:
We have added a new paragraph in the Results and Discussion sections (Lines 388-394) to describe the limitations of our study.

Answer:
We have changed "domestic" into "China" throughout the manuscript, and "IncF2" has been modified to the more accurate designation "F2:A-:B-".Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.For your reference, ASM Journals' address is given below.Before it can be scheduled for publication, your manuscript will be checked by the mSystems production staff to make sure that all elements meet the technical requirements for publication.They will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.
As an open-access publication, mSystems receives no financial support from paid subscriptions and depends on authors' prompt payment of publication fees as soon as their articles are accepted.

Publication Fees:
We have partnered with Copyright Clearance Center to collect author charges.You will soon receive a message from no-reply@copyright.com with further instructions.For questions related to paying charges through RightsLink, please contact Copyright Clearance Center by email at ASM_Support@copyright.com or toll free at +1.877.622.5543.Hours of operation: 24 hours per day, 7 days per week.Copyright Clearance Center makes every attempt to respond to all emails within 24 hours.For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
If you would like to submit a potential Featured Image, please email a file and a short legend to msystems@asmusa.org.Please note that we can only consider images that (i) the authors created or own and (ii) have not been previously published.By submitting, you agree that the image can be used under the same terms as the published article.File requirements: square dimensions (4" x 4"), 300 dpi resolution, RGB colorspace, TIF file format.
For mSystems research articles, you are welcome to submit a short author video for your recently accepted paper.Videos are normally 1 minute long and are a great opportunity for junior authors to get greater exposure.Importantly, this video will not hold up the publication of your paper, and you can submit it at any time.
Details of the video are: • Minimum resolution of 1280 x 720 • .movor .mp4.video format • Provide video in the highest quality possible, but do not exceed 1080p • Provide a still/profile picture that is 640 (w) x 720 (h) max • Provide the script that was used We recognize that the video files can become quite large, and so to avoid quality loss ASM suggests sending the video file via https://www.wetransfer.com/.When you have a final version of the video and the still ready to share, please send it to mSystems staff at msystems@asmusa.org.
-23R1 (The origin and evolution of IncF33 plasmids based on large-scale datasets) Dear Prof. Jian-Hua Liu: Thank you for submitting your paper to mSystems.