Earliest observation of the tetracycline destructase tet(X3)

ABSTRACT Tigecycline is an antibiotic of last resort for infections with carbapenem-resistant Acinetobacter baumannii. Plasmids harboring variants of the tetracycline destructase gene tetX promote rising tigecycline resistance rates. We report the earliest observation of tet(X3) in a clinical strain predating tigecycline’s commercialization, suggesting selective pressures other than tigecycline contributed to its emergence. IMPORTANCE We present the earliest observation of a tet(X3)-positive bacterial strain, predating by many years the earliest reports of this gene so far. This finding is significant as tigecycline is an antibiotic of last resort for carbapenem-resistant Acinetobacter baumannii (CRAB), which the World Health Organization ranks as one of its top three critical priority pathogens, and tet(X3) variants have become the most prevalent genes responsible for enabling CRAB to become tigecycline resistant. Moreover, the tet(X3)-positive strain we report is the first and only to be found that predates the commercialization of tigecycline, an antibiotic that was thought to have contributed to the emergence of this resistance gene. Understanding the factors contributing to the origin and spread of novel antibiotic resistance genes is crucial to addressing the major global public health issue, which is antimicrobial resistance.

Lines 113-115: Have the authors checked if there are gene promoters upstream of tet(X3) in the reported strain?Is the present tet(X3) variant usually associated with tigecycline, doxycycline, or minocycline resistance?What is the copy number of tet(X3) in the reported strain?I think this paragraph needs to be edited based on the results of the authors' analysis of the genome.
Line 117: Please add a reference to this info "Although cases of tet(X3)-positive Acinetobacter spp.have been isolated on all continents, ..." Lines 122-127: This paragraph includes a summary of the results from one study.The discussion is not comprehensive and suddenly terminated.Either delete this part or add more data from other studies.

Reviewer #2 (Comments for the Author):
The study is technically sound.

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Dear Editor,
We would like to thank both reviewers for having taken the time to read and comment our manuscript.Below, please find point-by-point responses to the issues raised by Reviewer # 1.As you will see, we agreed with all the points raised by Reviewer # 1 and have addressed all of them in a manner we believe is consistent with Reviewer # 1's intents.
We hope that with these modifications, the manuscript will be deemed satisfactory to be published in Microbiology Spectrum.
With best wishes, Louis-Patrick Haraoui, MD, MSc, FRCPC (on behalf of all co-authors) Associate Professor, Department of Microbiology and Infectious Diseases Faculty of Medicine and Health Sciences, Université de Sherbrooke Reviewer # 1 (Comments for the author) The study reported on the earliest occurrence of the tet(X3) gene in an Acinetobacter junii strain, obtained in 2004.Overall, the study was well designed and performed.The methodology was appropriate and the early detection of tet(X3) was interesting.However, there are a number minor and major comments (listed below) that need to be addressed.

Major comments
Line 94: No origin of replication was identified.What methods were used to detect the origin of replication?I would encourage the authors to double check the accuracy of this info.An annotation of the whole plasmid could be added (as a supplementary table ).
Response: No origin of replication was identified using PlasmidFinder 2.1 and MOB-suite 3.1.7and also none were annotated in the similar plasmids pAJ_351_2 and CP104336.1.MOB-suite did output genes for Mating pair formation (MPF) type: MPF_T.A map of pTet(X3)-Ajun-H1-2 was added to the manuscript as the new Figure 1.Given the size of the plasmid, we only annotated the antibiotic resistance genes and the MPF_T genes.The remainder of the annotation is accessible through the submitted GenBank data.The manuscript was modified to reflect these additional methods and results.

Response:
We agree that pTet(X3)-Ajun-H1-2 shares a part of its sequence with pAJ_351-2 and the 7 Unnamed plasmids isolated in A. baumannii in the USA in 2021.We have modified the manuscript accordingly.Figure 1 is now renamed Figure 2.
Lines 113-115: Have the authors checked if there are gene promoters upstream of tet(X3) in the reported strain?Is the present tet(X3) variant usually associated with tigecycline, doxycycline, or minocycline resistance?What is the copy number of tet(X3) in the reported strain?I think this paragraph needs to be edited based on the results of the authors' analysis of the genome.
Response: Only one copy of the tet(X3) gene was present in the genome.The gene was found on the pTet(X3)-Ajun-H1-2 plasmid.The sequencing coverage of the plasmid was in equal ratio to the chromosome (1:1).Hence, only one copy of this gene was found in each cell.
In the strain we report, tet(X3) was found on a 6,094 bp sequence bordered by two copies of ISVsa3.No gene promoter was found upstream of tet(X3).We compared the genetic surroundings of tet(X3) in our reported strain to the one found in AB34, a tetracycline/doxycycline/minocycline and tigecycline resistant A. baumannii.AB34 was the first strain reported to carry tet(X3).Compared to our strain, AB34 contains 3 identical copies of tet(X3), each in a similar 6,094 bp region as the one found in our strain.These 3 6,094 bp regions are aligned one after the other in AB34.It seems therefore possible that the unexpected resistance profile in our reported strain might be due to the presence of a single copy of tet(X3) when compared to a more broadly resistant strain containing three identical copies of the same gene.The manuscript was modified to reflect these additional results.
Line 117: Please add a reference to this info "Although cases of tet(X3)-positive Acinetobacter spp.have been isolated on all continents, ..." Response: we modified the sentence and added references.
Lines 122-127: This paragraph includes a summary of the results from one study.The discussion is not comprehensive and suddenly terminated.Either delete this part or add more data from other studies.
Response: we deleted this paragraph.

The quality of Table 1 is very bad!
Response: We included the relevant data from Table 1 into the new Figure 1 and removed the previously named Table 1 from the manuscript.The previously named Table 2 was renamed Table 1.The previously named Figure 1 was renamed Figure 2. Thank you for the privilege of reviewing your work.Below you will find my comments, instructions from the Spectrum editorial office, and the reviewer comments.
Please make a separate Data Availability statement.The Bioproject number PRJNA845765 is currently not available in NCBI.
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, notify me immediately so that the manuscript may be formally withdrawn from consideration by Spectrum.

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Thank you for submitting your paper to Spectrum.

Sincerely, Cheryl Andam Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): The revised manuscript is good.No further comments or suggestions.
1 Dear Editor, We would like to thank both reviewers for having taken the time to read and comment our manuscript.Below, please find point-by-point responses to the issues raised by Reviewer # 1.As you will see, we agreed with all the points raised by Reviewer # 1 and have addressed all of them in a manner we believe is consistent with Reviewer # 1's intents.
We hope that with these modifications, the manuscript will be deemed satisfactory to be published in Microbiology Spectrum.
With best wishes, Louis-Patrick Haraoui, MD, MSc, FRCPC (on behalf of all co-authors) Associate Professor, Department of Microbiology and Infectious Diseases Faculty of Medicine and Health Sciences, Université de Sherbrooke Reviewer # 1 (Comments for the author) The study reported on the earliest occurrence of the tet(X3) gene in an Acinetobacter junii strain, obtained in 2004.Overall, the study was well designed and performed.The methodology was appropriate and the early detection of tet(X3) was interesting.However, there are a number minor and major comments (listed below) that need to be addressed.

Major comments
Line 94: No origin of replication was identified.What methods were used to detect the origin of replication?I would encourage the authors to double check the accuracy of this info.An annotation of the whole plasmid could be added (as a supplementary table ).
Response: No origin of replication was identified using PlasmidFinder 2.1 and MOB-suite 3.1.7and also none were annotated in the similar plasmids pAJ_351_2 and CP104336.1.MOB-suite did output genes for Mating pair formation (MPF) type: MPF_T.A map of pTet(X3)-Ajun-H1-2 was added to the manuscript as the new Figure 1.Given the size of the plasmid, we only annotated the antibiotic resistance genes and the MPF_T genes.The remainder of the annotation is accessible through the submitted GenBank data.The manuscript was modified to reflect these additional methods and results.

Response:
We agree that pTet(X3)-Ajun-H1-2 shares a part of its sequence with pAJ_351-2 and the 7 Unnamed plasmids isolated in A. baumannii in the USA in 2021.We have modified the manuscript accordingly.Figure 1 is now renamed Figure 2.
• 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 -23R1 (Earliest observation of the tetracycline destructase tet(X3)) Dear Dr. Louis-Patrick Haraoui: