Mutations associated with tetracycline resistance detected in Treponema spp.- an analysis of 4,355 Spirochaetales genomes
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Background The resurgence of syphilis has necessitated novel prophylactic strategies, such as the use of doxycycline post-exposure prophylaxis (doxy-PEP). However, the potential for increased doxycycline use to select for tetracycline resistance represents significant challenges in managing this sexually transmitted infection. This study aims to identify chromosomal mutations associated with tetracycline resistance in Spirochaetales to inform molecular surveillance tools. Methods Whole genome sequences (WGS) from the Spirochaetales order, including 4,355 genomes, were analyzed for the presence of mutations in 16S rRNA and non-synonymous mutations in the rpsC and rpsJ genes. The study utilized WGS from GenBank® and sequence data from the PubMLST Treponema pallidum isolate collection. Genetic resistance to tetracycline was detected using a combination of BLASTN searches and gene-gene analysis. Results A transition mutation TGA to TGG at positions 965-967 in the 16S rRNA gene was detected in 5.6% of Treponema spp. and 4.0% of Spirochaeta spp. genomes. The rpsJ gene exhibited a V57G amino acid substitution across a significant subset of Treponema spp. (n=14) and Spirochaeta spp. (n=1). Notably, the V57K substitution was present in Spirochaeta spp. (n=17) and Treponema spp. (n=15). The rpsC gene had the H178Q mutation and was found to be present in the Spirochaetales bacterium (n=4). Conclusion The identification of mutations associated with tetracycline resistance in Spirochaetales provides a foundation for the development of rapid molecular tests. This study underscores the complexity of antibiotic resistance mechanisms and the critical importance of surveillance of genetic resistance determinants in the era of antibiotic prophylaxis for STI management.
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Yor manuscript has been assessed by two independent reviewers. Although both of them expressed interest in the manuscript, the extent of new data analysis and further development viewed as necessary to substantiate the conclusions indicates to me that the manuscript is currently too preliminary for further consideration as a Research Article. I believe this manuscript would be suitable for publication as a Short Communication. Therefore, I invite you to change the article type to Short Communication and submit it for further consideration. I stress, however, that you will still be required to provide a ponti-by-point response to the reviewers' comments and to provide tracked and clean versions of the correctted manuscript. I will look forward to receiving a corrected version of the manuscript. Best regards, Gustavo
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Comments to Author
Considering the initial focus of the study, only 417 genomes belonged to Treponema pallidum and 614 to Treponema spp. from the total dataset, the work shows that there is a low frequency of mutations to the tetracycline resistance profile for the T. pallidum species. Given that some core genes were not detected, it would have been useful to perform a CheckM analysis to assess genome completeness and contamination. Additionally, a taxonomic classification using Kraken could have helped improve species identification and increase the number of T. pallidum genomes for analysis. Regarding the detection of genetic resistance to tetracycline, the gene-by-gene analysis approach is not clearly explained. Is this analysis referring to the strategy by chewBBACA tool, which identifies core genes and allele …
Comments to Author
Considering the initial focus of the study, only 417 genomes belonged to Treponema pallidum and 614 to Treponema spp. from the total dataset, the work shows that there is a low frequency of mutations to the tetracycline resistance profile for the T. pallidum species. Given that some core genes were not detected, it would have been useful to perform a CheckM analysis to assess genome completeness and contamination. Additionally, a taxonomic classification using Kraken could have helped improve species identification and increase the number of T. pallidum genomes for analysis. Regarding the detection of genetic resistance to tetracycline, the gene-by-gene analysis approach is not clearly explained. Is this analysis referring to the strategy by chewBBACA tool, which identifies core genes and allele profiles? Clarifying this point would improve the reader's understanding of the methodology. Variant prediction was performed using BLASTN, but why was the allele profile analysis of genes target from chewBBACA not considered for this purpose? Furthermore, why was strain typing based on three genes instead of using a cgMLST schema from chewBBACA ? This resulted in the typig of only 178 genomes, limiting the resolution of diversity. Considering the challenges associated with T. pallidum, I would suggest for next work, an alternative approach based on the collection data from read sequences analyses and metagenomic data, and a variant calling for discover new and know variants in resistance-associated genes. The strain typing of T. pallidum was not discussed in detail—what were the resistance profiles identified by typing? Additionally, the mutation profiles previously described in the literature in association with tetracycline resistance could have been better contextualized, specifying which mutations were not found in this dataset. Were any novel resistance-associated mutations discovered and potencial impact discuss? Since the mutation frequency target genes in T. pallidum, was relatively low, and there was a lack of metadata to establish phenotypic correlations of mutant identified, the study considered highlight results at other pathogenic genus (Treponema spp.) and order (Spirochaetales) levels but lack discuss its relevance clinical.
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?
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?
No: not contain
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Comments to Author
The manuscript „Mutations associated with tetracycline resistance detected in Treponema spp. - an analysis of 4,355 Spirochaetales genomes" by Manoharan-Basil et al., describes an attempt to find previously described mutations associated with tetracycline resistance within published treponemal or other spirochaetal genomes. This attempt is a very important step as the use of doxycycline for post-exposure prophylaxis increases antibiotic pressure and could lead to development of resistance as already happened with macrolides. Identification of existing mutations could help in molecular surveillance. Even though this reviewer appreciates the work of authors, the manuscript itself looks unfinished and preliminary, and needs more work (e.g., broadening and clarifying the Results section) to bring more …
Comments to Author
The manuscript „Mutations associated with tetracycline resistance detected in Treponema spp. - an analysis of 4,355 Spirochaetales genomes" by Manoharan-Basil et al., describes an attempt to find previously described mutations associated with tetracycline resistance within published treponemal or other spirochaetal genomes. This attempt is a very important step as the use of doxycycline for post-exposure prophylaxis increases antibiotic pressure and could lead to development of resistance as already happened with macrolides. Identification of existing mutations could help in molecular surveillance. Even though this reviewer appreciates the work of authors, the manuscript itself looks unfinished and preliminary, and needs more work (e.g., broadening and clarifying the Results section) to bring more valuable message. More details are in comments below. Material and Methods Dataset - The authors should add a table, which would summarize analyzed genomes regarding the species and genome numbers, including description of bigger groups if authors create some (e.g., Other Treponema spp. - should be described what all species is in here and how many analyzed strains) - The authors should mention how many genes were available and analyzed (possible even for individual groups; it is not clear if there were genomes in supplementary table 1 which were not analyzed at all for not containing sequence of selected genes (see comments to Supplementary table). How many of analyzed genomes had available sequence for all 3 genes, etc. - The authors should try to better denote species and subspecies, i.e., Treponema pallidum group contains genomes which belong to group T. pallidum ssp. pallidum (and other subspecies as well). This imprecision is undermining the value of authors' findings. - How were solved overlaps between PubMLST and GenBank results? Were there any? - How authors resolved additional copies of 16S rRNA genes in genomes? Were all of them having the same sequence? Were all of them checked? Detection - line 130: why authors selected as a reference sequence Nichols strain AE000520, and not CP004010, which is more precise and more frequently used? - line 137-138: how were these mutations chose? - line 139-142: which sites were analyzed for mutation presence? All of 16S rRNA gene sequence or just selected positions? According to Table 1, only positions 965-968 were analyzed, why not others which was also described to be connected with changed susceptibility to tetracycline? What about other positions containing substitution(s)? (e.g., there is a substitution in authors' alignment in Treponema pallidum subsp. endemicum strains on position 1371). Supplementary material Table 1_V2 - abbreviation used is not described - why some numbers of MLST gene are in red? - missing information about PubMLST isolates - Authors should include information about which loci were available for each genome. In this moment is not possible to verify changes claimed by authors because of missing information. - Authors should try to define genomes belonging to individual subspecies of T. pallidum, if not in Table S1, then in data analysis and results as problem of increased resistance is expected in those genomes. File - alignment - line 160: should be stated that alignment consist only of part of the data. According to number of genomes, I assume it is PubMLST genomes? - line 192—193: alignment does not support data (does not show more than 1000 mutations) - authors could present also alignment for other 2 analyzed genes Results - in general, this section should be broaden to describe more details, e.g., pinpointing Treponema pallidum ssp. pallidum (pertenue, and endemicum, respectively) as it is the main concern of authors according to Introduction. - it would be useful to show the alignments of all analyzed genes, and show information which genomes were analyzed (and what were the results). - as supplementary file does not contain enough information it is impossible to verify the stated findings - Why not all substitutions in the 16S rRNA genes were evaluated? - line 170: What is Spirochaetales bacterium? Table 1 - total no of genomes does not match the number of genomes in the Supplementary table 1 (e.g., T. parvum only 3 there, T. brennaborense only 2, etc.) - What belongs to "Other Treponema spp.? - No of analyzed genomes from PubMLST is in here 544; does it mean that not all 613 16S rRNA sequences were analyzed? - Did authors find mutations in all copies of 16S rRNA present in genomes? - Spirochaeta spp. contains all non-treponemes? - It would be useful to more specify T. pallidum subspecies. Introduction - line 94: there are two copies of 16S and 23S rRNA genes in Treponema pallidum ssp. pallidum. Discussion - line 186-190: this should be partially described in Results section as well. - line 186: "Among the five genomes analyzed…" should be rephrased to "Among the five genomes from PubMLST containing mutations…" as authors analyzed more than 5 genomes. - line 192-193: supplementary material does not show more than 1000 mutations… more comments in the Results section. Other comments - unite the use of latin names (e.g., line 104, 109) - line 144: heading should be in bolt - unite the use of italics for names of species - data summary posted online does not contain adjusted version of Supplementary table 1. - keyword syphilis in this moment is not really matching the content of article
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
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?
No: There were no human or animal work
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