Identification of pandemic clade-specific genetic marker with genomic insight into Vibrio parahaemolyticus
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Vibrio parahaemolyticus is a foodborne pathogen commonly present in seafood. Of the various V. parahaemolyticus serotypes reported, O3:K6, O1:K25, O1:KUT and O4:K68 represent the major serotypes among pandemic clones that emerged from 1995 onward. However, new molecular markers of pandemic clones remain unidentified, and limited genomic sequence data are available for non-pandemic strains. Therefore, we aimed to identify novel genetic markers specific to pandemic V. parahaemolyticus strains by comparing non-pandemic and pandemic strains using whole-genome sequencing. Phylogenetic analysis of 163 V . parahaemolyticus strains revealed high genomic diversity within the species. The analysis also revealed a pandemic clade consisting of serotypes O3:K6, O1:K25, O1:KUT and O4:K68 strains isolated after 1995. We identified the genomic island GI-110 (VPaI-5) as a potential marker exclusive to the pandemic clade. Multiplex PCR detection of VPaI-5 demonstrated high specificity for pandemic strains, outperforming the detection of existing markers. The capacity of multiplex PCR for VPaI5 in distinguishing between pandemic and non-pandemic strains was confirmed using clinical isolates from Thailand. Our findings provide valuable insights into the genetic diversity of V. parahaemolyticus and establish a reliable method for monitoring pandemic strains.
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This is a study that would be of interest to the field and community.
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Thank you for addressing the reviewer comments and submitting your revised manuscript. I have reviewed the amendments and am satisfied that they address the reviewers’ suggestions. However, I have identified additional edits that require your attention before publication. Please see these below, and I look forward to receiving your amended manuscript. Lines 91-93 – Please include the length of the sequencing reads. Lines 94-95 – Please indicate the read lengths and if this was run as paired-ed sequencing. Lines 96-97. Please state the public database(s) used. Please add accession numbers to the strains in the table S4. Line 102 – Please include the reference accession number (and assembly version if applicable) used in this study. Line 211 – “and no alternative methods have been replaced” should instead read “and no alternative …
Thank you for addressing the reviewer comments and submitting your revised manuscript. I have reviewed the amendments and am satisfied that they address the reviewers’ suggestions. However, I have identified additional edits that require your attention before publication. Please see these below, and I look forward to receiving your amended manuscript. Lines 91-93 – Please include the length of the sequencing reads. Lines 94-95 – Please indicate the read lengths and if this was run as paired-ed sequencing. Lines 96-97. Please state the public database(s) used. Please add accession numbers to the strains in the table S4. Line 102 – Please include the reference accession number (and assembly version if applicable) used in this study. Line 211 – “and no alternative methods have been replaced” should instead read “and no alternative methods have been proposed.” Line 214-215 – it is not clear what this sentence means, please reword this sentence to improve clarity “This suggests that a positive GS-PCR result is not sufficient condition” Lines 230-232 – please add the strain ID’s in-text for these isolates to enable the reader to understand which strains these refer to.
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Dear Authors, thank you for submitting your manuscript to Access Microbiology. The manuscript has now been evaluated by two experts in the field, and their comments are attached at the end of this email. Both reviewers agree that the presented research would be of interest to the field. It was also highlighted that there could be a grater emphasis on the context of the study, especially in the introduction and discussion sections. I welcome the authors to address these concerns as well as all other reviewer comments that aim to strengthen this manuscript towards publication.
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Comments to Author
The manuscript is written in fluent and concise manner. Abstract seems to have the sufficient details. Introduction probably needs more background information to provide some comparisons and findings related to this project. I think it will be helpful if the authors provide their overall goal for the project in their introduction. Authors need to check their sampling numbers, was not sure how many samples they analysed. They also indicate 167 V. parahaemolyticus strains in the abstract but it is over 170 in another section. Their method can use some graphical representation to assist the reviewers about the process. At least some sort of diagram showing the steps. Are there any statistical assessments in this project? It does not seem to be clear. I recommend the authors to provide some details on …
Comments to Author
The manuscript is written in fluent and concise manner. Abstract seems to have the sufficient details. Introduction probably needs more background information to provide some comparisons and findings related to this project. I think it will be helpful if the authors provide their overall goal for the project in their introduction. Authors need to check their sampling numbers, was not sure how many samples they analysed. They also indicate 167 V. parahaemolyticus strains in the abstract but it is over 170 in another section. Their method can use some graphical representation to assist the reviewers about the process. At least some sort of diagram showing the steps. Are there any statistical assessments in this project? It does not seem to be clear. I recommend the authors to provide some details on their quantitative assessments of their data at the end of their methods. Results provide sufficient details and explanation. Their discussion should provide a pig picture, impact. This section can also use some discussion on the recent findings from other research perhaps with slightly different bacterial population. Perhaps, they may provide some conclusion after their discussion or add additional sentences. Overall, this manuscript provides novel findings that will interest to the readers of this journal. I recommend the authors to format their manuscript further. I am not sure if it is possible to provide some sort of numbering or alphabet coding for the titles and subtitles. Some of the figures may use larger text font sizes and perhaps better resolution.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Not at all
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
This study reassesses the previously established genomic markers for pandemic V. parahaemolyticus, a food-borne pathogen which requires rapid detection of pandemic clones to prevent outbreaks. The authors propose VPaI-5-PCR, a primer focused on the genomic island VPaI-5, as a novel marker due to its high specificity in pandemic strains, compared to existing markers. The analysis was based on 162 initial V. parahaemolyticus strains to identify pandemic clusters and appropriate genomic islands, and then tested on 71 isolates from Thailand and 118 from public collections. It is concisely communicated with strong figures, but to provide a more convincing argument that VPaI-5 could be a novel pandemic marker, a few results and comparisons need greater reporting quantitatively in the text, and some greater …
Comments to Author
This study reassesses the previously established genomic markers for pandemic V. parahaemolyticus, a food-borne pathogen which requires rapid detection of pandemic clones to prevent outbreaks. The authors propose VPaI-5-PCR, a primer focused on the genomic island VPaI-5, as a novel marker due to its high specificity in pandemic strains, compared to existing markers. The analysis was based on 162 initial V. parahaemolyticus strains to identify pandemic clusters and appropriate genomic islands, and then tested on 71 isolates from Thailand and 118 from public collections. It is concisely communicated with strong figures, but to provide a more convincing argument that VPaI-5 could be a novel pandemic marker, a few results and comparisons need greater reporting quantitatively in the text, and some greater contextual issues could be addressed (such as in the context of both global expansion)- see comments below. General - If VPaI-5 was identified as a genomic island associated with pandemic V. parahaemolyticus in Hurley et al., 2006, why has it not yet been suggested as a marker? A discussion on whether this is due to previous markers being well-established with no reassessment (which is alluded to in the introduction) would be beneficial. - Greater discussion on the one pandemic isolate that didn't test positive for VPaI-5 e.g. comparison to other studies where not always present (e.g. Guerrero et al., 2021, BMC Genom Data 10.1186/s12863-021-00985-0), potential drivers of this and implications for use of VPaI-5 as a primer - If possible, list locations (e.g. country) where the initial 162 isolates and 118 publicly available isolates were collected (Table S1 and S4). For VPaI-5 to be adopted as a pandemic marker, it would need to be shown that it is uniquely present in pandemic isolates obtained across geographic locations, as Vp undergoes global expansion leading to evolutionary divergence of pandemic isolates in different regions. Currently, we only know the primer was tested on Thailand isolates, so if the original strain collection and the public collection was more global, this would add more support towards the use of VPaI-5 as a validated marker. - Additionally- it is unclear if the other pandemic markers tested on the Thailand and public samples for a comparison? If so, this needs to be quantified more clearly- this analysis is important to report for comparison, to show if VPaI-5 is performing better than existing markers here. - The exploration on the PGS-PCR positive in a non-pandemic strain as a consequence of horizontal gene transfer affecting chromosome 2 is interesting. If I'm correct, I believe orf8-PCR is also based on genomic regions in chromosome 2, which would be interesting to include to (alongside supporting the use of VPaI-5 as a pandemic marker) when highlighting limitations of the previous pandemic markers. Potentially a short discussion could be added on the trade-off between marker stability (focusing on chromosome 1 regions), and overlooking the rapid adaptation that might be associated with pandemic strains in the future using chromosome 2. Specific - In the abstract, it is claimed that VPaI-5 demonstrates high specificity for pandemic strains. Both specific and sensitivity could be quantified, but are not currently explicitly reported in the results. This should be added in sections 7.2 and 7.3. - Line 73: Specify and cite when the last studies that set the current standard biomarkers were undertaken - Lines 90-92: Add in a sentence about the 118 public isolates used - The phylogenetic and Bayesian analysis of population structure (BAPS) analysis is not currently described in the methodology. Line 110-111 mentions it briefly (without describing the acronym), but the implementation and algorithm needs to be described in more detail. - Lines 112 and 158: Full use of 'genomic islands' in subtitles would be preferential - Lines 147-149: unclear if this is one of the study's results or referring to previous studies' use of pandemic markers. In the next sentences the authors show how this results was found in their analyses, which suggests to me this should be presented as a result summarising the results of all pandemic markers before individual results are specified for each marker, and then this similarity to previous results can be discussed in the discussion. - Line 149: 'Positive results' is vague. Specify how many of the strains in subcluster 6.1 had positive results for GS-PCR marker- was it 100% of them? - Line 220-222: 'Further studies characterize' Is this a recommendation for further studies to characterize VPaI-5 functionality? In which case, add 'should'. Or a statement that they are? In which case they should be cited. - Figure 1 caption should explain how the clusters were generated or at least expand the acronym - Further discussion on the subcluster that yielded positive results for GS-PCR could be included- were these pre-pandemic?
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?
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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