Derivatization of pBACpAK Entrapment Vectors for Enhanced Mobile Genetic Elements Transposition Detection in Multidrug-Resistant Escherichia coli
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Aim: Antimicrobial resistance (AMR) poses a critical global health threat, driven by the dissemination of resistance genes via mobile genetic elements (MGEs). This study aims to enhance the detection of MGEs insertions in multidrug-resistant Escherichia coli by derivatizing the pBACpAK entrapment vector. Methods and results: Three derivatives were constructed with additional nucleotides upstream of the cI repressor gene, based on conserved regions identified from GenBank sequences containing known IS26 and IS1 insertions. Using colony PCR, intracellular transposition screening was performed on 194 tetracycline-resistant colonies from four E. coli ESI123 strains carrying different pBACpAK constructs. The derivatives showed increased MGE capture rates (10.7–73.1%) compared to the wild-type vector (3.75%), leading to the identification of multiple MGEs, including the novel composite transposon Tn7824. Tn7824 harbors the blaOXA-181 carbapenem resistance gene and the qnrS1 quinolone resistance gene, highlighting the clinical relevance of these findings. Long-read sequencing of transposants confirmed the accuracy of MGE identification and structural characterization, which also revealed chromosomal integration events of the pBACpAK derivatives mediated by flanking insertion sequences. Conclusions: The modifications introduced in the pBACpAK derivatives could increase the detection of transposition events by alleviating spatial constraints, allowing for more robust MGE detection. Impact statement: These findings highlight the utility of entrapment vectors for studying MGE-associated AMR dissemination in clinical and environmental settings. By improving the detection of novel and clinically relevant MGEs, such as Tn7824, this approach contributes to a better understanding of resistance gene mobility and may aid future AMR surveillance efforts.
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I thank the authors for revising this manuscript in line with the reviewer comments. I have reviewed the revised submission which I am satisfied that all points have been addressed, and new analysis included to strengthen the study’s findings. Therefore, I would like to congratulate you on acceptance of this manuscript and hope you will consider submitting to Access Microbiology again in the future.
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Dear authors, thank you for submitting your manuscript to Access Microbiology. It has now been reviewed by three experts in the field, whose comments are attached at the bottom of this email. The manuscript requires additional rationale for the study in the introduction. It has also been noted that additional citations are required to back up claims made in the discussion. I welcome the authors to address these concerns as well as all other author comments that will strengthen this timely and important research.
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Comments to Author
This is a well written manuscript that describes the development and experimental functional testing of variants of the transposon trapping plasmid vector pBACpAK. These new variants carry extra DNA sequences between the cI repressor and the tetA gene compared to the original pBACpAK plasmid. The authors rigorously test and clearly demonstrate that the plasmid variants that they constructed that had an extra 16bp or 26bp DNA sequences engineered upstream of the cI gene (Is26 insertion site, IS1 insertion site) were more efficient at trapping mobile elements than the original pBACpAK plasmid. The Results are clearly presented. I recommend 1) a few sentences in the Introduction that more explicitly state the idea behind engineering these particular sequences in and why the particular sequences were …
Comments to Author
This is a well written manuscript that describes the development and experimental functional testing of variants of the transposon trapping plasmid vector pBACpAK. These new variants carry extra DNA sequences between the cI repressor and the tetA gene compared to the original pBACpAK plasmid. The authors rigorously test and clearly demonstrate that the plasmid variants that they constructed that had an extra 16bp or 26bp DNA sequences engineered upstream of the cI gene (Is26 insertion site, IS1 insertion site) were more efficient at trapping mobile elements than the original pBACpAK plasmid. The Results are clearly presented. I recommend 1) a few sentences in the Introduction that more explicitly state the idea behind engineering these particular sequences in and why the particular sequences were chosen to put into pBACpAK, and 2) inclusion of some discussion about whether the vectors will trap mobile genetic elements that are not related to ISi and IS26 would be good to include.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly 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
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Comments to Author
1. Importance of the Manuscript for the Scientific Community This manuscript presents a timely and important contribution to microbial genetics and antimicrobial resistance surveillance by improving the pBACpAK entrapment vector system for enhanced detection of mobile genetic elements (MGEs) in Escherichia coli. Given the global concern over the spread of multidrug resistance via horizontal gene transfer, especially in clinical and environmental strains, this work offers a refined molecular tool that could accelerate the study of transposable elements. The improvements made to the vector system have clear practical implications for AMR monitoring and research. Overall, this study is both methodologically novel and scientifically relevant. 2. Title Evaluation The title, "Derivatization of pBACpAK …
Comments to Author
1. Importance of the Manuscript for the Scientific Community This manuscript presents a timely and important contribution to microbial genetics and antimicrobial resistance surveillance by improving the pBACpAK entrapment vector system for enhanced detection of mobile genetic elements (MGEs) in Escherichia coli. Given the global concern over the spread of multidrug resistance via horizontal gene transfer, especially in clinical and environmental strains, this work offers a refined molecular tool that could accelerate the study of transposable elements. The improvements made to the vector system have clear practical implications for AMR monitoring and research. Overall, this study is both methodologically novel and scientifically relevant. 2. Title Evaluation The title, "Derivatization of pBACpAK Entrapment Vectors for Enhanced Mobile Genetic Elements Transposition Detection in Multidrug-Resistant Escherichia coli," is accurate and descriptive. It clearly conveys the focus of the study and includes essential keywords (pBACpAK, MGEs, MDR E. coli). No changes are necessary. 3. Abstract Evaluation The abstract is comprehensive and well-structured, covering the background, objectives, methods, key findings, and significance. However, a few suggestions can improve clarity: * Mention the number of isolates tested, if applicable. * Specify the method used for transposition detection. * Consider including specific data to support key results. 4. Subsections and Structure The manuscript adheres to standard scientific formatting with clear and logical subsections (Introduction, Methods, Results, Discussion, Conclusion). Transitions between sections are smooth, and the structure enhances readability and comprehension. Figures and tables are appropriately integrated and supplement the text effectively. 5. Scientific Correctness The study is scientifically robust. It applies sound molecular biology techniques to modify and validate a functional genetic tool. The derivatized pBACpAK vectors were evaluated using multidrug-resistant clinical isolates, and transposition events were successfully captured and analyzed. The authors demonstrated a strong understanding of vector dynamics and MGE behavior. Proper controls were used, and the conclusions are logically supported by the experimental data. This research offers a practical application with long-term benefits for AMR tracking. 6. References The references are generally relevant and sufficient, with most citations drawn from credible journals. However, inclusion of a few recent publications (2022-2024) on MGEs and AMR surveillance technologies would strengthen the literature context 7. Language Quality The English language is of a high scholarly standard. The manuscript is clearly written, grammatically sound, and terminology is used appropriately. Minor editorial polishing could further enhance clarity, but no major issues were detected. 8. Ethical Issues No ethical concerns were identified. The study does not involve human or animal subjects, and proper biosafety considerations for handling multidrug-resistant bacteria appear to have been followed. 9. Competing Interests There are no disclosed conflicts of interest. The declaration is clear and complete. 10. Plagiarism The writing appears original, and data presentation is consistent with standard academic practices. A similarity check through plagiarism-detection software is still recommended for due diligence. Detailed Sectional Review Methods * The methods are technically sound and suitable for the study's objectives. * The derivatization and validation steps for pBACpAK are clearly explained, including use of E. coli strains, plasmid stability assays, and detection of insertion events. * The experimental setup allows for reproducibility. Additional details on reagent concentrations or incubation conditions in certain protocols could enhance reproducibility. * The literature background is sufficient to justify the approach. * While statistical methods are limited, data interpretation is methodical and appropriate. Results & Discussion * The data are well-controlled with adequate positive/negative comparisons. * Results show increased efficiency and sensitivity of the modified vectors, backed by experimental evidence. * Findings are significant, as they facilitate better detection of MGEs, which are crucial to AMR evolution and dissemination. * The discussion is based on empirical data and relevant literature. The authors maintain a balanced tone and avoid overstatements. Conclusion . * The conclusion is sound and reasonable, with adequate literature reference to situate their contribution in the broader context of AMR research and genetic surveillance. General recommendations to the authors. Insufficient Experimental Detail in Methods * Key experimental details, such as the precise conditions for transposition assays, concentrations of reagents, incubation times, and transformation conditions, are missing or vaguely described. * There is limited information on the selection markers used and how the authors ensured that observed events were indeed transposition events rather than random insertions or plasmid rearrangements. Suggestion: Add a detailed step-by-step description of the experimental procedures, especially the construction of the derivatized pBACpAK vectors, transformation protocols, selection steps, and transposition validation strategies. Lack of Quantitative and Statistical Analysis * The study lacks quantitative assessments of the performance of derivatized vectors compared to the original pBACpAK. For instance, what percentage increase in transposition detection efficiency was observed? * No statistical validation is provided to support the claim of "enhanced" detection. Suggestion: Include quantitative performance data (e.g., frequency of transposition events, number of unique insertions, comparative efficiencies) and apply appropriate statistical analyses to validate enhancement claims. Weak Discussion on Broader Applications and Limitations * The discussion lacks a deep exploration of how these vectors can be used in real-world settings, such as outbreak surveillance or environmental AMR tracking. * There is no critical analysis of limitations, such as potential biases introduced by the plasmid size, copy number, or host-range restrictions. Suggestion: Expand the discussion to reflect broader implications, real-world applications, and known limitations of the vector system.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly 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
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Comments to Author
In this manuscript, Tansirichaiya et al. present a series of pBACpAK-derived entrapment vectors for improved transposon detection and analysis. They demonstrate enhanced function of their vectors over the original vector for the capture of IS26, IS1 and IS186B from a single ESBL E. coli strain. Curiously, the improved efficacy does not seem to result from insertions into the modified region of the vector, as might be expected. These findings are potentially mechanistically very interesting, but are not expanded upon within the paper. The paper is generally well-written with good use of English language throughout and mostly accurate, well-produced images (see below for specific comments). The methodology is relatively clear and most of the relevant sequences generated during the study have been …
Comments to Author
In this manuscript, Tansirichaiya et al. present a series of pBACpAK-derived entrapment vectors for improved transposon detection and analysis. They demonstrate enhanced function of their vectors over the original vector for the capture of IS26, IS1 and IS186B from a single ESBL E. coli strain. Curiously, the improved efficacy does not seem to result from insertions into the modified region of the vector, as might be expected. These findings are potentially mechanistically very interesting, but are not expanded upon within the paper. The paper is generally well-written with good use of English language throughout and mostly accurate, well-produced images (see below for specific comments). The methodology is relatively clear and most of the relevant sequences generated during the study have been deposited. There are some key omissions from the text that would improve the manuscript if redressed. Preferably, extra experimental controls should be conducted to justify some of the claims made in the Discussion, but failing that, extra citations from existing literature are needed. Points for consideration/improvement: * Line 22: "experimentally proven insertion sites of transposable elements" is over-reaching. If these insertion sites have been experimentally proven, please cite the published data (i.e. not just a GenBank search). * The authors state only that "the extended-spectrum β-lactamase (ESBL) E. coli ESI123 clinical isolate was obtained from the routine bacteriology laboratory at Siriraj Hospital, Bangkok, Thailand" (line 93). Beyond its AMR profile, no further information about the strain is presented. E. coli is more than just a collection of AMR genes and this is highly relevant to MGE studies. The authors should at least present the serotype and putative pathovar, retrievable bioinformatically from the WGS they've performed. If possible, they should also note the disease that the strain was associated with in the patient. * Lines 168-172: Whilst the authors do present primers for their constructs and supply a supplementary weblogo figure from which the information can be extracted, the authors could include the inserted sequences unambiguously in this section of text for clarity. * Figure 1: The legend states "dashed purple lines" (no such dashed lines exist on the figure; presumably they mean the purple box) and "at the end of the cI repressor gene" (the purple box is at the start of the gene, not the end). * Figure 2: Again, the insertion is not at the end of the cI gene as stated in the legend. * Table 2: Did the authors analyse any of the tetracycline-resistant colonies that did not show evidence of transposon capture or chromosomal integration? An explanation of what has occurred here would be useful to bolster the study's rigour and conclusions, in particular since more colonies were isolated when using the parent vector than its derivatives. * Line 263: "Data not shown" should be avoided. It is my understanding that the journal's policy is for authors to provide all relevant data. * Line 265: The authors based their inserted sequences on the supposed consensuses derived from IS26 and IS1 insertions, but these sequences appear quite inconsistent/random in the weblogo comparison. Instead of being sequence-specific, the results may be due to increased spacing between the promoter and cI gene (as the authors state in the Discussion). The authors could therefore construct additional controls containing different sequences of the same lengths, or other lengths, which would presumably answer this question. Without these experiments, citations are needed in the Discussion at this point in order to back up their arguments regarding polymerase vs transposase activity. Is there existing evidence from the literature to support the claims being made? * Since this paper primarily focuses on vector modification and validation, it would be appropriate for the authors to make available the full, annotated sequences of the modified pBACpAK plasmids (separate from the WGS sequence data).
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Good
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
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