Transition of Staphylococcus aureus tetracycline resistance plasmid pT181 from independent multicopy replicon to predominantly integrated chromosomal element over 65 years

  1. Megan A Phillips
  2. Robert A Petit
  3. Daniel B Weissman
  4. Timothy D Read

  • Curated by eLife

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    eLife Assessment

    Using genome databases, the authors performed solid bioinformatic analyses to trace the genomic history of the clinically relevant Staphylococcus aureus tetracycline resistance plasmid pT181 over the last seven decades. They discovered that this element has transitioned from a multicopy plasmid to a chromosomally integrated element, and the work represents a valuable demonstration of the use of publicly available data to investigate plasmid biology and inform clinical epidemiology. This work will appeal to researchers interested in staphylococcal evolution, plasmid biology, antimicrobial resistance and bacterial genome structure.

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Abstract

Mobile genetic elements (MGEs), including plasmids, phages and genome islands, are major sources of bacterial genetic diversity. The small plasmid pT181 confers tetracycline resistance in bacterial pathogen Staphylococcus aureus via an efflux pump, TetK. pT181 was one of the earliest sequenced S. aureus plasmids and has been isolated in both clinical and livestock-associated strains for decades, both as an independent replicon and integrated in the chromosome as part of staphylococcal cassette chromosome mec (SCCmec). Bacterial genome analysis tools and high-quality sequences with metadata are publicly available, but these resources remain underleveraged for examining historical data, especially when studying the spread of MGEs across a species and over time. Using publicly available reads and metadata, we explored the evolution of pT181 over nearly seven decades of samples to identify temporal trends in sequence evolution, copy number changes, and spread across S. aureus and beyond. pT181 was prevalent across S. aureus (found in 9.5% of 83,366 genomes tested), with a conserved sequence outside of three hypervariable regions. The history of pT181 since 1954 was characterized by spread across strains, significant variation in plasmid copy number of the independent replicon, and increasing frequency of integration of the plasmid into the S. aureus chromosome. We identified multiple chromosomal integration locations of the plasmid, including outside of the previously characterized SCCmec. We found that pT181 was transferred across Staphylococcaceae and into a Gram-negative species. The repeated integration of pT181 into the chromosome may indicate coevolution of the plasmid and the host, potentially to facilitate increased antibiotic resistance.

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  1. eLife

    eLife Assessment

    Using genome databases, the authors performed solid bioinformatic analyses to trace the genomic history of the clinically relevant Staphylococcus aureus tetracycline resistance plasmid pT181 over the last seven decades. They discovered that this element has transitioned from a multicopy plasmid to a chromosomally integrated element, and the work represents a valuable demonstration of the use of publicly available data to investigate plasmid biology and inform clinical epidemiology. This work will appeal to researchers interested in staphylococcal evolution, plasmid biology, antimicrobial resistance and bacterial genome structure.

  2. eLife

    Reviewer #1 (Public review):

    [Editor's note: this version has been assessed by the Reviewing Editor without further input from the original reviewers.]

    The study provides a robust bioinformatic characterization of the evolution of pT181. My main criticism of the work is the lack of experimental validation for the hypotheses proposed by the authors.

    Previous comments on the study:

    (1) One potential reason for the decline in pT181 copy number over time may be a high cost associated with the multicopy state. In this sense, it would be interesting if the authors could use (or construct) isogenic strains differing only in the state of the plasmid (multicopy/integrated). With this system, the authors could measure the fitness of the strains in the presence and absence of tetracycline, and they could be able to understand the benefit associated with the plasmid transition. The authors discuss these ideas, but it would be nice to test them.

    (2) It would be interesting to know the transfer frequencies of the multicopy mobilizable pT181 plasmid, compared to the transfer frequency of the plasmid integrated into the SSCmec element (which can be co-transferred, integrated in conjugative plasmids, or by transduction).

    (3) One important limitation of the study that should be mentioned is that inferring pT181 PCN from whole-genome data can be problematic. For example, some DNA extraction methods may underestimate the copy number of small plasmids because the small, circular plasmids are preferentially depleted during the process (see, for example, https://www.nature.com/articles/srep28063).

    Comment from the Reviewing Editor:

    In the revised version the authors have addressed comment 3 from Reviewer #1 through additional analysis, finding that the decline in copy number over time remains significant after controlling for instrument used, type of DNA extraction technique, and sequencing centre. The suggestion in Comment 1 from Reviewer #1 has not been acted upon as the authors state that this additional experimental work "goes beyond the scope of the paper" and is being explored for a future study. Similarly, the suggestion in Comment 2 from Reviewer #1 has not been acted upon as the authors state that "inferring these rates from natural sequence data is not feasible in this case given the low heterogeneity of the plasmid sequence. A laboratory-based experimental study could not address the real transfers we observe over the course of decades, as in vitro S. aureus transfer rates are often not good proxies for in vivo".

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    The authors performed bioinformatic analyses to trace the genomic history of the clinically relevant pT181 plasmid. Specifically, they:

    (1) tracked the presence of pT181 across different S. aureus strain backgrounds through time. It was first found in one, later multiple strains, though this may reflect changes in sampling over time.

    (2) estimated the mutation rate of the chromosome and plasmid.

    (3) estimated the plasmid copy number of pT181, and found that it decreased over time. The latter was supported by two sets of statistical analyses, first showing that the number of single-copy isolates increased over time, and second, that the multicopy isolates demonstrated a lower PCN over time.

    (4) reported the different integration sites at which pT181 integrated into the genome.

    As a caveat, they mentioned that identical plasmid sequences have variable plasmid copy numbers across different genomes in their dataset.

    Strengths:

    This is a very solid, well-considered bioinformatic study on publicly available data. I greatly appreciate the thoughtful approach the authors have taken to their subject matter, neither over- nor underselling their results. It is a strength that the authors focussed on a single plasmid in a single bacterial species, as it allowed them to take into account unique knowledge about the biology of this system and really dive deep into the evolution of this specific plasmid. It makes for a compelling case study. At the same time, I think the introduction and discussion can be strengthened to demonstrate what lessons might be drawn from this case study for other plasmids.

    Weaknesses:

    The finding that the pT181 copy number declined over time is the most interesting claim of the paper to me, and not something that I have seen done before. While the authors have looked at some confounders in this analysis, I think this could be strengthened further in a revision.

    For the flow of the storyline, I also think the estimation of mutation rates (starting L181) and integration into the chromosome (starting L255) could be moved to the supplement or a later position in the main text.

    Clearly, the use of publicly available data prevents the authors from controlling the growth and sequencing conditions of the isolates. It is striking that they observe a clear signal in spite of this, but I would have loved to see more discussion of the metadata that came with the publicly available sequences and even more use of that metadata to control for confounding.

    Comment from the Reviewing Editor:

    Reviewer #2's comment about the weakness due to "confounders in this analysis" has been addressed in the revision through additional analysis using metadata to control for confounding.

  4. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    eLife Assessment:

    Using genome databases, the authors performed solid bioinformatic analyses to trace the genomic history of the clinically relevant Staphylococcus aureus tetracycline resistance plasmid pT181 over the last seven decades. They discovered that this element has transitioned from a multicopy plasmid to a chromosomally integrated element, and the work represents a valuable demonstration of the use of publicly available data to investigate plasmid biology and inform clinical epidemiology. This work will appeal to researchers interested in staphylococcal evolution and plasmid biology.

    Thank you, we agree with this overview. We also think this work is interesting to people interested in antimicrobial resistance and bacterial genome structure.

    Public Reviews:

    Reviewer #1 (Public review):

    The study provides a robust bioinformatic characterization of the evolution of pT181. My main criticism of the work is the lack of experimental validation for the hypotheses proposed by the authors.

    Comments on the study:

    (1) One potential reason for the decline in pT181 copy number over time may be a high cost associated with the multicopy state. In this sense, it would be interesting if the authors could use (or construct) isogenic strains differing only in the state of the plasmid (multicopy/integrated). With this system, the authors could measure the fitness of the strains in the presence and absence of tetracycline, and they could be able to understand the benefit associated with the plasmid transition. The authors discuss these ideas, but it would be nice to test them.

    We agree that the relative fitness of integrated versus multicopy plasmids is interesting and a costly multicopy state could explain the transition of independent pT181 replicons to chromosomal integration. This is a project we are exploring for a future study. However, we think that this additional experimental work goes beyond the scope of the paper.

    (2) It would be interesting to know the transfer frequencies of the multicopy mobilizable pT181 plasmid, compared to the transfer frequency of the plasmid integrated into the SSCmec element (which can be co-transferred, integrated in conjugative plasmids, or by transduction).

    We agree with the reviewer that this is an interesting question. However, we think inferring these rates from natural sequence data is not feasible in this case given the low heterogeneity of the plasmid sequence. A laboratory-based experimental study could not address the real transfers we observe over the course of decades, as in vitro S. aureus transfer rates are often not good proxies for in vivo (McCarthy et al.). In addition, we do not know what is moving the integrated plasmid. pT181 could be moved by a phage or plasmid, so we are uncertain what the correct experiment would be to explore this.

    (3) One important limitation of the study that should be mentioned is that inferring pT181 PCN from whole genome data can be problematic. For example, some DNA extraction methods may underestimate the copy number of small plasmids because the small, circular plasmids are preferentially depleted during the process (see, for example, https://www.nature.com/articles/srep28063).

    We have further investigated the role of technical variation in influencing measured PCN. The decline in copy number over time within our longest-collected strain remains significant after controlling for a number of technical variables: instrument used, type of DNA extraction technique, and sequencing center. In addition, plasmid copy number estimations between extraction techniques are not significant when comparing contemporaneously collected samples. We have added these supporting analyses to our results (L328-333) and methods (L612-634).

    Reviewer #2 (Public review):

    Summary:

    The authors performed bioinformatic analyses to trace the genomic history of the clinically relevant pT181 plasmid. Specifically, they:

    (1) tracked the presence of pT181 across different S. aureus strain backgrounds through time. It was first found in one, later multiple strains, though this may reflect changes in sampling over time.

    (2) estimated the mutation rate of the chromosome and plasmid.

    (3) estimated the plasmid copy number of pT181, and found that it decreased over time. The latter was supported by two sets of statistical analyses, first showing that the number of single-copy isolates increased over time, and second, that the multicopy isolates demonstrated a lower PCN over time.

    (4) reported the different integration sites at which pT181 integrated into the genome.

    As a caveat, they mentioned that identical plasmid sequences have variable plasmid copy numbers across different genomes in their dataset.

    Strengths:

    This is a very solid, well-considered bioinformatic study on publicly available data. I greatly appreciate the thoughtful approach the authors have taken to their subject matter, neither over- nor underselling their results. It is a strength that the authors focussed on a single plasmid in a single bacterial species, as it allowed them to take into account unique knowledge about the biology of this system and really dive deep into the evolution of this specific plasmid. It makes for a compelling case study. At the same time, I think the introduction and discussion can be strengthened to demonstrate what lessons might be drawn from this case study for other plasmids.

    Weaknesses:

    The finding that the pT181 copy number declined over time is the most interesting claim of the paper to me, and not something that I have seen done before. While the authors have looked at some confounders in this analysis, I think this could be strengthened further in a revision.

    We have investigated this issue further in revisions. The decline in copy number over time within our longest-collected strain remains significant after controlling for a number of technical variables. In addition, plasmid copy number estimations between extraction techniques are not significant. We have mentioned these updates in the results (L327-333) and provided more explanation for the extraction technique in the methods (L612 - 634).

    For the flow of the storyline, I also think the estimation of mutation rates (starting L181) and integration into the chromosome (starting L255) could be moved to the supplement or a later position in the main text.

    We have chosen to keep the structure of the results largely the same.

    Clearly, the use of publicly available data prevents the authors from controlling the growth and sequencing conditions of the isolates. It is striking that they observe a clear signal in spite of this, but I would have loved to see more discussion of the metadata that came with the publicly available sequences and even more use of that metadata to control for confounding.

    We have further investigated the role of technical variation in influencing measured PCN. The decline in copy number over time within our longest-collected strain remains significant after controlling for a number of technical variables: instrument used, type of DNA extraction technique, and sequencing center. In addition, plasmid copy number estimations between extraction techniques are not significant when comparing contemporaneously collected samples. We have added this to our results section (L327-333) and explain further in the methods (L612 - 634). Unfortunately, the metadata is incomplete for many isolates, limiting our ability to control for other possible confounders.

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    I would recommend doing some experiments to test the fitness effects of pT181 in multicopy and integrated into an isogenic S. aureus strain. Simple growth curves with and without tetracycline would be extremely informative.

    We think that this is an interesting scientific question but is beyond the scope of this paper.

    Reviewer #2 (Recommendations for the authors):

    Major:

    (1) L160 and Figure 1:

    To what extent is this decrease in pT181 prevalence related to a delay between isolate collection and sequence upload to public databases? In my own work, I have found an average of 2 years difference in the sequence collection and sequence upload date of GenBank sequences. This could explain the decreasing numbers in recent years and may possibly skew relative proportions of pT181 containing/or plasmid-free strains.

    There are fluctuations in collection rates over the years which definitely do not correspond to actual fluctuations in S. aureus prevalence in the population. We would expect delays in uploads in both plasmid-containing and plasmid-free strains, so we would not anticipate a decline in estimated plasmid prevalence due to this delay; however, it could be somewhat noisy for more recent years.

    The large peak of S99.5_27 around 2009 seems oddly reminiscent of targeted sampling during a hospital outbreak, or so? Did you do any checks on where these genomes came from? Could this affect any of the downstream results?

    The 2008 peak in S99.5_27 comes from a single BioProject (PRJEB4140), which contributed approximately 1,000 samples to our analysis. Based on the BioProject description, this BioProject aimed to characterize diversity in S. aureus ST239 diversity in Thailand. We performed the single-copy analysis with this strain excluded and still got the same result that integration has increased over time, so we feel that this is acceptable and should not unduly impact downstream results. We have clarified the results section to more explicitly state that this is the sampling peak that was excluded (L306-311).

    (2) Figure 3: If I understand the analysis correctly, all mutation rate and phylogenetic analyses were conducted based on comparison to a single reference genome. This can bias results: https://academic.oup.com/mbe/article/31/5/1077/2925634 . Please comment.

    Yes, SNPs were calculated relative to a reference genome. We understand this could bias results. However, it is standard practice in the field and we do not think it changes the main findings of the paper.

    We have taken the reference-free SKA alignment and the SKA alignment mapping to a reference plasmid and calculated the SNP distances between all plasmid pairs with the tool snp-dists (Seemann et al.). For given plasmid pairs, the distributions are different by Wilcoxon signed-rank test with continuity correction (p < 2.2 x 10-16) though this is due in part to the large sample size for pairwise comparison. The median and modal difference between methods is 0 SNPs detected. The average is 0.1 more SNPs detected in the reference-free method than the mapping method. We have added this analysis to the methods section (L505-511).

    (3) I am not fully convinced by the analysis in Figure 6 (i.e. that the pT181 copy number has declined over time). It is rather confusing how the authors account for strain and bioproject-related influences on their primary regression (L298-306).

    We have edited the explanation of the GLMM in the results section (L320-323). We have moved some of the explanation to the methods section (L592-605).

    (a) What does this analysis look like without S99.5.2?

    The slope is still negative and significant with S99.5_2 excluded. We have added this to the results (L327-328).

    (b) In line 296, it is made clear that 2 separate analyses were carried out, but in the subsequent paragraphs (starting 298 and 315), it would be helpful to repeat that each looks at only one component.

    We have edited this section to explicitly state that separate regressions were carried out for each component (L298-300).

    (c) Figure 6 should include error bars that reflect the different number of samples per collection year (please also comment on how this influences the power/validity of the linear regression!).

    We have modified Fig. 6A to show samples collected per year as the color of the point. Samples collected per year can also be viewed in Fig. 1. In addition, we have changed to logistic regression to assess single-copyness as a function of collection year. The error bar on the regression curve shows the SE range. While there are many more samples collected in recent times than in the more distant past, we still think the signal is real. It does put more emphasis on each early sample. However, the genomic data matches early descriptions of tetracycline resistance in S. aureus being plasmid-mediated, not chromosomal (Gillespie et al.).

    (4) Strains that were isolated in the 60s were presumably sequenced at a much later point in time. Were they grown and isolated from their respective host bacteria following similar protocols as present-day isolates? Growth conditions can have massive effects on PCN, and selective PCR amplification on the relative number of plasmid-related reads. How do you account for this? [Figure 7/8].

    Yes, samples that were collected in the 1960s were sequenced roughly 50 years later. The date of collection could impact storage conditions for the isolate. However, they were grown up for sequencing and actually sequenced much closer together and should have the same growth conditions as isolates collected at a more recent date. Underlying our model of PCN is that it is a property of the isolate and will not fluctuate over time in storage.

    Minor:

    (1) This study is a nice demonstration of the type of genomic epidemiology that can be conducted now that genomes are sequenced and shared more widely. This is already somewhat common for clinically relevant sequence types of pathogenic bacteria, but I have not seen it much for plasmids. This could be nice to highlight. Stressing this more clearly in the abstract and introduction may broaden the audience of this work.

    We have described our approach as genomic epidemiology in the first paragraph of the introduction (L72).

    (2) More generally, the introduction is quite winding. A lot of interesting information is mentioned, but without a clear narrative structure as to why this is important for the study. Some editing may strengthen the message of the paper and invite more people to read it. For instance, starting each paragraph with a topical sentence about the general message the paragraph should convey. Yet, this is entirely a style/personal taste remark, and I leave it to the authors to decide whether to make these changes.

    We have made edits to restructure the text.

    (2a) Similar comment for the discussion (possibly some discussion could be put in the supplement too).

    We have made edits to restructure the text, but have chosen to leave the content in the Discussion section. Additional references to previous research have also been added (L386-388).

    (3) L125: Could you include a reference for this statement?

    We have added a reference for this statement (L109).

    There is interesting existing work on the evolution of genes on multi-copy plasmids (and the evolution of copy-number itself), which seems more pertinent here than the segregation mechanisms of low-copy-number plasmids.

    We have chosen to keep the general background on plasmid copy number regulation and segregation, to keep the paper approachable to readers who have no background on plasmid biology. We have added additional references to work on evolvability of multicopy plasmids and their copy number (L117-120).

    (4) The references are a bit of a mess. Certain entries are in all caps, others are missing titles, etc. I would recommend looking this over again carefully.

    We have resolved this issue.

    (5) L187 CDS is used in line 187 but not defined as a term until line 201.

    We have defined CDS with first usage (L188).

    (6) L189: Do you have any estimate of the error around 1.15 mutations/Mb/year from the linear regression?

    We have added standard error for the 1.15 mut/Mbp/year from the regression to the results (L191): SE = 0.3 mut/Mbp/year.

    (7) Figure 2: It would be helpful to have a visual indication of which areas the authors consider hypervariable.

    We have added a black dash underneath the hypervariable regions on the plot.

    (8) Figure 5: The two colors of grey are hard to distinguish.

    The grey for shared genes has been made darker to better distinguish them.

    (9) L285: Why do you write "could assume the copy number of the larger element"? What other options are possible?

    We have expanded on this section to elaborate on the ways that copy number could be changed by cointegration, and added an additional citation to support these claims (L276-280).

    (10) Figure S1: What does this scale mean? What are the units? This figure is uninformative as it stands. I would possibly consider labelling the tips.

    We have modified the figure and legend to indicate that the scale is in substitutions per site. We are providing these figures to give some idea of scale and shape, showing how star-like they are. Unfortunately, tip labels are illegible on these trees given the large number of samples.

    References

    Gillespie, M. T., et al. “Antibiotic Susceptibilities and Plasmid Profiles of Nosocomial Methicillin-Resistant Staphylococcus aureus: A Retrospective Study.” Journal of Medical Microbiology, vol. 17, no. 3, June 1984, pp. 295–310. DOI.org (Crossref), https://doi.org/10.1099/00222615-17-3-295.

    McCarthy, Alex J., et al. “Extensive Horizontal Gene Transfer during Staphylococcus aureus Co-Colonization in Vivo.” Genome Biology and Evolution, vol. 6, no. 10, 2014, pp. 2697–708, https://doi.org/10.1093/gbe/evu214.

    Seemann, Torsten, et al. Snp-Dists. https://github.com/tseemann/snp-dists.

  5. Version published to 10.7554/elife.109393.2 on eLife
  6. Version published to 10.7554/elife.109393 on eLife
  7. Version published to 10.1101/2025.09.14.675889v2 on bioRxiv
  8. eLife

    eLife Assessment

    Using genome databases, the authors performed solid bioinformatic analyses to trace the genomic history of the clinically relevant Staphylococcus aureus tetracycline resistance plasmid pT181 over the last seven decades. They discovered that this element has transitioned from a multicopy plasmid to a chromosomally integrated element, and the work represents a valuable demonstration of the use of publicly available data to investigate plasmid biology and inform clinical epidemiology. This work will appeal to researchers interested in staphylococcal evolution and plasmid biology.

  9. eLife

    Reviewer #1 (Public review):

    The study provides a robust bioinformatic characterization of the evolution of pT181. My main criticism of the work is the lack of experimental validation for the hypotheses proposed by the authors.

    Comments on the study:

    (1) One potential reason for the decline in pT181 copy number over time may be a high cost associated with the multicopy state. In this sense, it would be interesting if the authors could use (or construct) isogenic strains differing only in the state of the plasmid (multicopy/integrated). With this system, the authors could measure the fitness of the strains in the presence and absence of tetracycline, and they could be able to understand the benefit associated with the plasmid transition. The authors discuss these ideas, but it would be nice to test them.

    (2) It would be interesting to know the transfer frequencies of the multicopy mobilizable pT181 plasmid, compared to the transfer frequency of the plasmid integrated into the SSCmec element (which can be co-transferred, integrated in conjugative plasmids, or by transduction).

    (3) One important limitation of the study that should be mentioned is that inferring pT181 PCN from whole genome data can be problematic. For example, some DNA extraction methods may underestimate the copy number of small plasmids because the small, circular plasmids are preferentially depleted during the process (see, for example, https://www.nature.com/articles/srep28063).

  10. eLife

    Reviewer #2 (Public review):

    Summary:

    The authors performed bioinformatic analyses to trace the genomic history of the clinically relevant pT181 plasmid. Specifically, they:

    (1) tracked the presence of pT181 across different S. aureus strain backgrounds through time. It was first found in one, later multiple strains, though this may reflect changes in sampling over time.

    (2) estimated the mutation rate of the chromosome and plasmid.

    (3) estimated the plasmid copy number of pT181, and found that it decreased over time. The latter was supported by two sets of statistical analyses, first showing that the number of single-copy isolates increased over time, and second, that the multicopy isolates demonstrated a lower PCN over time.

    (4) reported the different integration sites at which pT181 integrated into the genome.

    As a caveat, they mentioned that identical plasmid sequences have variable plasmid copy numbers across different genomes in their dataset.

    Strengths:

    This is a very solid, well-considered bioinformatic study on publicly available data. I greatly appreciate the thoughtful approach the authors have taken to their subject matter, neither over- nor underselling their results. It is a strength that the authors focussed on a single plasmid in a single bacterial species, as it allowed them to take into account unique knowledge about the biology of this system and really dive deep into the evolution of this specific plasmid. It makes for a compelling case study. At the same time, I think the introduction and discussion can be strengthened to demonstrate what lessons might be drawn from this case study for other plasmids.

    Weaknesses:

    The finding that the pT181 copy number declined over time is the most interesting claim of the paper to me, and not something that I have seen done before. While the authors have looked at some confounders in this analysis, I think this could be strengthened further in a revision.

    For the flow of the storyline, I also think the estimation of mutation rates (starting L181) and integration into the chromosome (starting L255) could be moved to the supplement or a later position in the main text.

    Clearly, the use of publicly available data prevents the authors from controlling the growth and sequencing conditions of the isolates. It is striking that they observe a clear signal in spite of this, but I would have loved to see more discussion of the metadata that came with the publicly available sequences and even more use of that metadata to control for confounding.

  11. eLife

    Author response:

    eLife Assessment

    Using genome databases, the authors performed solid bioinformatic analyses to trace the genomic history of the clinically relevant Staphylococcus aureus tetracycline resistance plasmid pT181 over the last seven decades. They discovered that this element has transitioned from a multicopy plasmid to a chromosomally integrated element, and the work represents a valuable demonstration of the use of publicly available data to investigate plasmid biology and inform clinical epidemiology. This work will appeal to researchers interested in staphylococcal evolution and plasmid biology.

    Thank you, we agree with this overview. We also think this work is interesting to people interested in antimicrobial resistance and bacterial genome structure.

    Public Reviews:

    Reviewer #1 (Public review):

    The study provides a robust bioinformatic characterization of the evolution of pT181. My main criticism of the work is the lack of experimental validation for the hypotheses proposed by the authors.

    Comments on the study:

    (1) One potential reason for the decline in pT181 copy number over time may be a high cost associated with the multicopy state. In this sense, it would be interesting if the authors could use (or construct) isogenic strains differing only in the state of the plasmid (multicopy/integrated). With this system, the authors could measure the fitness of the strains in the presence and absence of tetracycline, and they could be able to understand the benefit associated with the plasmid transition. The authors discuss these ideas, but it would be nice to test them.

    We agree that the relative fitness of integrated versus multicopy plasmids is interesting and a costly multicopy state could explain the transition of independent pT181 replicons to chromosomal integration. This is a project we are exploring for a future study. However, we think that this additional experimental work goes beyond the scope of the paper.

    (2) It would be interesting to know the transfer frequencies of the multicopy mobilizable pT181 plasmid, compared to the transfer frequency of the plasmid integrated into the SSCmec element (which can be co-transferred, integrated in conjugative plasmids, or by transduction).

    We agree with the reviewer that this is an interesting question. However, we think inferring these rates from natural sequence data is not feasible in this case given the low heterogeneity of the plasmid sequence. A laboratory-based experimental study could not address the real transfers we observe over the course of decades, as in vitro S. aureus transfer rates are often not good proxies for in vivo (McCarthy et al., 2014). In addition, we do not know what is moving the integrated plasmid. pT181 could be moved by a phage or plasmid, so we are uncertain what the correct experiment would be to explore this.

    (3) One important limitation of the study that should be mentioned is that inferring pT181 PCN from whole genome data can be problematic. For example, some DNA extraction methods may underestimate the copy number of small plasmids because the small, circular plasmids are preferentially depleted during the process (see, for example, https://www.nature.com/articles/srep28063).

    We will investigate this issue further in the revisions. The kits used to extract DNA for the earlier-collected samples may possibly yield more plasmid DNA relative to the chromosome compared to newer ones on average; however, we think this is not driving the decline that we observe in multicopy pT181 copy number. Multiple BioProjects find the same result, where earlier samples have higher copy number compared to later samples. We expect extraction methods to be consistent within a BioProject, suggesting that this decline is genuine and not technical. In revisions, we intend to evaluate the effect of date of sequencing and additional metadata on copy number.

    Reviewer #2 (Public review):

    Summary:

    The authors performed bioinformatic analyses to trace the genomic history of the clinically relevant pT181 plasmid. Specifically, they:

    (1) Tracked the presence of pT181 across different S. aureus strain backgrounds through time. It was first found in one, later multiple strains, though this may reflect changes in sampling over time.

    (2) Estimated the mutation rate of the chromosome and plasmid.

    (3) Estimated the plasmid copy number of pT181, and found that it decreased over time. The latter was supported by two sets of statistical analyses, first showing that the number of single-copy isolates increased over time, and second, that the multicopy isolates demonstrated a lower PCN over time.

    (4) Reported the different integration sites at which pT181 integrated into the genome.

    As a caveat, they mentioned that identical plasmid sequences have variable plasmid copy numbers across different genomes in their dataset.

    Strengths:

    This is a very solid, well-considered bioinformatic study on publicly available data. I greatly appreciate the thoughtful approach the authors have taken to their subject matter, neither over- nor underselling their results. It is a strength that the authors focused on a single plasmid in a single bacterial species, as it allowed them to take into account unique knowledge about the biology of this system and really dive deep into the evolution of this specific plasmid. It makes for a compelling case study. At the same time, I think the introduction and discussion can be strengthened to demonstrate what lessons might be drawn from this case study for other plasmids.

    Weaknesses:

    The finding that the pT181 copy number declined over time is the most interesting claim of the paper to me, and not something that I have seen done before. While the authors have looked at some confounders in this analysis, I think this could be strengthened further in a revision.

    In the revisions, we will further explore the impact that technical variation could have in contributing to copy number variation and update our claims for the decline in copy number of the independent replicon over time and variation for the same plasmid sequence accordingly. Multiple BioProjects show earlier samples have higher copy number compared to later samples; we expect extraction methods to be consistent within a BioProject, supporting our initial findings that this decline over time is not due to technical variation.

    For the flow of the storyline, I also think the estimation of mutation rates (starting L181) and integration into the chromosome (starting L255) could be moved to the supplement or a later position in the main text.

    We will revisit the text organization for flow and clarity of storyline.

    Clearly, the use of publicly available data prevents the authors from controlling the growth and sequencing conditions of the isolates. It is striking that they observe a clear signal in spite of this, but I would have loved to see more discussion of the metadata that came with the publicly available sequences and even more use of that metadata to control for confounding.

    In revisions, we will further investigate possible contributors to the observed decline in copy number of multicopy pT181 over time. We have incorporated the date of sample collection and BioProject in our analysis, but not the date of sequencing or extraction technique.

    References

    McCarthy, A. J., Loeffler, A., Witney, A. A., Gould, K. A., Lloyd, D. H., & Lindsay, J. A. (2014). Extensive horizontal gene transfer during Staphylococcus aureus co-colonization in vivo. Genome Biology and Evolution, 6(10), 2697–2708. https://doi.org/10.1093/gbe/evu214

  12. Version published to 10.7554/elife.109393.1 on eLife
  13. Mentioned by @biorxiv-genomic.bsky.social on Bluesky

    Transition of Staphylococcus aureus tetracycline resistance plasmid pT181 from independent multicopy replicon to predominantly integrated chromosomal element over 65 years https://www.biorxiv.org/content/10.1101/2025.09.14.675889v1

  14. Mentioned by @biorxivpreprint.bsky.social on Bluesky

    Transition of Staphylococcus aureus tetracycline resistance plasmid pT181 from independent multicopy replicon to predominantly integrated chromosomal element over 65 years https://www.biorxiv.org/content/10.1101/2025.09.14.675889v1

  15. Version published to 10.1101/2025.09.14.675889v1 on bioRxiv