Antimicrobial resistance and molecular characteristics of Neisseria gonorrhoea isolates in Ghana
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Introduction. Gonorrhoea is a disease associated with humans and caused by Neisseria gonorrhoea. N. gonorrhoea’ s ability to evolve and evade various treatment regimens can lead to untreatable gonorrhoea. In the absence of a viable vaccine and a national database on the antimicrobial resistance (AMR) and molecular characteristics of N. gonorrhoea, and with reliance on a syndromic management regime, continuous national antimicrobial resistance surveillance and molecular characterization of N. gonorrhoea remain imperative. Only two gonococcal studies have described N. gonorrhoea’s molecular characteristics linked to AMR in Ghana.
Methods. Secondary N. gonorrhoea isolates ( n =4) were collected from two metropolises in Ghana: Tamale in the northern sector ( n =1) and Accra in the southern sector ( n =3). The isolates were confirmed and characterized using polymerase chain reaction (PCR) targeting the por B and tbp B genes, and the disc diffusion method was used to evaluate AMR. N. gonorrhoea multi-antigen sequence typing (NG-MAST) and porin B ( por B) gene sequence analyses were employed to reveal the molecular epidemiology and evolutionary trajectory, respectively.
Results. All four isolates showed resistance to at least four of the tested antibiotics. One isolate showed resistance to all seven antibiotics, i.e. ceftriaxone, azithromycin, ciprofloxacin, tetracycline, erythromycin, togamycin and penicillin. NG-MAST typing revealed isolate S3 (MZ313864) as ST211. The locus of S2 (MZ313863) (transferrin-binding protein B; tbp B) was identified as tbp B1844, and its por B locus, as por B6412, with only 4 closely related variants but with 15 nucleotide differences. However, its sequence type does not exist. The por B analysis identified isolate S3 (MZ313864) to be found globally, while S2 (MZ313863) is unique to this study.
Discussion. Despite the small number of isolates tested, this study recorded multidrug resistance and previously unknown gonococcal variants based on por B gene. Additionally, the molecular typing schemes revealed a disparity between NG-MAST and the National Center for Biotechnology Information (NCBI) platforms. There is a need for continuous gonococcal AMR and molecular surveillance in Ghana to contribute to the global efforts to describe circulating strains and support proper application of the syndromic management regime to gonorrhoea.
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I am satisfied that you have responded thoroughly to all comments raised by the reviewers.
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This work, whilst only describing a small number of isolates, provides a useful addition to the understanding of multidrug resistant Neisseria gonorrhoeae in Ghana. The reviewers make several suggestions for improvement that I would like you to address in a revision. Please pay particular attention to the request for more detailed methodology and to the suggestions that will improve the clarity of the results. Whilst I think a study of this size is appropriate for publication in Access Microbiology, please address the comment of Reviewer 2 and mention this in the Discussion.
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Comments to Author
Thank you to the authors for this piece of work. It will bring good value to understanding MDR Gonococcal infection in Ghana. Overall I found the paper relatively easy to follow and of an appropriate length in most places. The results section could do with a little more framework and detail, but the introduction and discussion are well-written, with a strong impact statement at the end. Therefore, they only need some amendments. One comment throughout would be that the author's should try to use more approachable language. There were some instances where words did not really fit what they were trying to say, such as the use of microcosm (line 69) and buttresses (line 258), and that more simplified English would make a greater impact. This would also help the paper be more reader friendly for those …
Comments to Author
Thank you to the authors for this piece of work. It will bring good value to understanding MDR Gonococcal infection in Ghana. Overall I found the paper relatively easy to follow and of an appropriate length in most places. The results section could do with a little more framework and detail, but the introduction and discussion are well-written, with a strong impact statement at the end. Therefore, they only need some amendments. One comment throughout would be that the author's should try to use more approachable language. There were some instances where words did not really fit what they were trying to say, such as the use of microcosm (line 69) and buttresses (line 258), and that more simplified English would make a greater impact. This would also help the paper be more reader friendly for those where English is not a first language. Please also ensure that all in-text references are correctly formatted and consistent. There are some instances where over all names are listed in the reference structure and others where et al is used. There are also some instances where references are missing in the text, so make sure to add these in. This was noted for pubMLST which is mentioned as a URL but missing the associated reference (PMID: 30345391). In terms of grammar, there are a few times where capitals have/have not been used incorrectly. For example, there is a capital A for 'Antimicrobial' on line 74 which is not needed. However, names of antibiotics mentioned throughout need to be changed to have capital letters. Having numbers in brackets after the word is also not required and makes more sense to be removed (e.g. (7) on line 106) as it would be less confusing. Abstract: 1. Please define what secondary Neisseria gonorrhoea isolates are as this is not clear (line 23) 2. Make sure to specify what the acronym means in all first instances, including in the abstract (example on line 26) 3. Instead of just listing three antibiotics, please list all 7 to make it clear (lines 28-29) 4. Lines 31-32 could be changed to make it a little clearer to read. It appears that the author's have found a unique PorB allele in isolate S3 and this is why they are unable to give it a Sequence Type (ST). This is a good finding and useful to pinpoint. 5. The first sentence on line 33 does not really make sense and could be omitted as is not necessarily needed. Otherwise, please can the author's expand on what they did to record the results if they wish to keep it in the abstract. Introduction: 6. Great impact statement used at the end of page 3. It shows why the research has been done and how it will contribute to vaccine research. Very clear. 7. Line 69 reads as if there is currently no other work on the molecular characteristics of N. gonnorhoaea, but then is contradicted using two references. This could be re-worded to frame it more that there is limited work in this area for Ghana/Africa, stating the two references at the start instead. There is a similar incidence on line 71 regarding no existing antimicrobial resistance database but there are publications. It would be good if the author's could also expand on this as to why it would be advantageous to have a Gonococcal antibiotic resistance database, with scope for future work later on (which may be more of a discussion point). Materials and Methods: 8. Please could there be a little more detail here. For instance, how were the isolates stored (e.g. it -80 oC in Cryovials containing TSB with 8% DMSO (v/v)) on line 81. Similar for the GC agar supplemented with Vitox, extra information on the quantity is required with appropriate units. 9. In terms of media preparation, it is important to state which manufacturer's instructions the agar was made to. It appears that it would be Oxoid, but it would be good to make this clearer in the sentence (line 84). 10. On line 87, could the author's please add more detail on how long the isolates were incubated for. There is temperature, but no time. 11. Could the author's give more indication as to why reference strain ATCC 49226 was used in the methodology? Has it been used as a positive control elsewhere? If so, please cite. 12. Author's have stated the concentration units of antibiotics and versions of bioinformatics software used which is great and makes the work more easily reproducible. They just need to make sure all methods and software packages are referenced too as there are a few missing. A reference for the disk diffusion method is required on line 109, BLAST reference needed on line 133 (along with the parameters used), PopArt reference needed on line 136/137, MEGA X reference needed on line 138, and a ClustalW reference needed on line 139. Results: 13. The results section of the work could do with some more detail. It had a good foundation, but more interpretation and links to the literature could be made. I would suggest the author adds a few sentences to the start of each results section which explains the introduction of that result to the work followed by brief sentence on how they did it (which links back to the methods). Also make sure the number of replicates for each of each result is clear throughout, as this is not immediately visible in the manuscript. 14. How many times was the PCR performed? Could this be made more clear in the results section, with reason as to why it was performed X number of times. Make sure to add in the sensitivity and specificity of the assay where possible. 15. For Figure 1, please add the full set of numbers for the ladder to make each band clearer? There is a good start by showing the PorB and tbpB band numbers. In the legend, to make it clearer and link to the text, please link the isolate numbers to the full isolate name. 16. On line 155, could you please expand on what an antibiotic resistance index is, and what one with > 0.5 means (with references, if applicable), as it is not immediately clear. 17. For the explanation Figure 2, on line 158, could the authors please expand the explanation on what was used to illustrate percentage resistance. Were the isolates tested multiple times and the average was taken? Or does the Figure only contain one replicate/all values taken for each isolate? 18. Table 1 is helpful, with resistant, intermediate, and sensitive isolates against each antibiotic very clear. To improve this even more, a suggestion would be to also add the raw disk diffusion values (average) into the table with the respective units. 19. On line 173 a chromatogram is mentioned and that only two of the isolates were readable. It would be good to get a Figure of this in the supplementary data, with addition of an explanation as to why there may have been issues as to why the data was unreadable. Further explanation for what is meant by 'trimmed appropriately', including the techniques used and the result of this, for isolates S2 and S3 would be good. 20. The NG-MAST results state that the ST for S2 could not be determined due to the lack of a defined PorB allele number. This indicates that there may be a novel PorB allele in this sequence, which is a great find. To expand on this, the authors could submit the PorB allele in this sequence, plus the genome data, for curation on pubMLST. A curator will be able to assign a novel PorB allele number and in turn novel ST number for this sequence, if the finding is rendered unique overall. This would be a fantastic addition to the findings of the paper if this is the case. 21. Please could further explanation be given as to how the alignment was performed for the N. gonorrhoeae porB gene. The use of BLAST and GENTle are mentioned in the methods, so it would be good to make links to these so it is clearer. Likewise with the haplotype network analysis, it would be good to link it back to the use of PopArt mentioned in the methods for clarity. 22. On line 197, please mention the N number for the other isolates included in the analysis, and make it clear why and how you chose these. It could be good to include a table in the results section of all the isolates used in the network analysis, with associated metadata (such as country, sequence type, year of isolation, etc.). Use of colours or symbols would help the reader make links between your isolates and the others included in the analysis. 23. A table would be good for the phylogenetic analysis (line 208), as well as why you chose these 22. The table could include the accession number of the sequences alongside any references. 24. The phylogenetic analysis results are very brief, so more detail/analysis would be better. As part of this, could the authors make any links to published literature as to why their PorB phylogeny grouped this way? Please also state the software (MEGA X) used to build the phylogenetic tree in the Figure legend. Can the authors infer any further evolutionary traits from the tree, and possibly link this to other PorB phylogenies within the literature? Further analysis could be performed by adding both sequence type (ST) and phenotype data onto the tree. If there is any data available for the isolates on NCBI and pubMLST, links to antimicrobial resistance patterns could be inferred. Discussion: 25. The discussion is the strongest part of this manuscript. It is clear and written well. There are only a few minor improvements suggested: 26. On line 228, it was mentioned that multi drug resistance of N. gonorrhoaea to the tested antibiotics was also found elsewhere. Whilst references have been given, could the author please also note the places it was found in the text. Could the authors also make a statement on how this links to/affects those found in Ghana. 27. Line 230 has an impactful statement about the fact that the antibiotics can still be resisted years after therapeutic withdrawal. To make this even stronger, the authors may like to add in the exact years in which these were withdrawn. This could then give an exact amount of time and less vague than years. 28. On line 234, can the authors please expand, with references, why there are some parts of Ghana which still use Ciprofloxacin despite its removal as a first-line antibiotic? 29. It is interesting that isolate S2 has a relation to TbpB allele ST-10251. The high number on this suggests that it is a more recently discovered allele and this is why it is not as widespread. The authors make a good point in the discussion about the discrepancies in the NG-MAST database compared to NCBI, where similarities of PorB to the query are vastly different. This is because NG-MAST is held within pubMLST which is a database that relies on manual submissions by researchers, and therefore often has a smaller dataset than NCBI. The authors make a good comment that there is need for consolidation of these. Could they suggest how this could be done and what would be the best approach? 30. On line 282, the authors mention that they need more isolates from Tamale Metropolis for the research. Please state how you will collect these samples in future. Will it be random or routine, for example, or if you already have access to further samples. 31. The authors have identified another N. gonorrhoeae study in Ghana, but the sequences are not publicly available. If possible, it would be interesting if the authors could collaborate with Attram et al. with their isolates to enhance the phylogenetic and genomic analysis in the publication. 32. Please can the authors clarify what they mean by 'singleton sites' on line 287. Does this mean single nucleotide polymorphisms? 33. In the conclusion, please specify which one of the Accra isolates was resistant to all seven antibiotics by using the strain name.
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?
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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Comments to Author
Although the laboratory methods seem appropriate in line with diagnostic limitations in the reported country, the sample size is incredibly small to conclude anything other than a brief description of the two isolates. My sincere recommendation is to test more isolates before publishing this work. Specific comments: Abstract Throughout: Organism should be Neisseria gonorrhoeae or N. gonorrhoeae Abstract has an 'Introduction' heading but no 'methods', 'results' or 'discussion'. Please streamline. Line 18: gonorrhoea should be non capitalised Line 21-22: Last sentence on that page needs re-phrasing (only two gonococcal data - do they mean studies?) Lines 29-32 need re-phrasing. Introduction Line 42: Change the word maladies to infection or something more scientific. Line 43: N. gonorrhoeae is not …
Comments to Author
Although the laboratory methods seem appropriate in line with diagnostic limitations in the reported country, the sample size is incredibly small to conclude anything other than a brief description of the two isolates. My sincere recommendation is to test more isolates before publishing this work. Specific comments: Abstract Throughout: Organism should be Neisseria gonorrhoeae or N. gonorrhoeae Abstract has an 'Introduction' heading but no 'methods', 'results' or 'discussion'. Please streamline. Line 18: gonorrhoea should be non capitalised Line 21-22: Last sentence on that page needs re-phrasing (only two gonococcal data - do they mean studies?) Lines 29-32 need re-phrasing. Introduction Line 42: Change the word maladies to infection or something more scientific. Line 43: N. gonorrhoeae is not known to cause meningitis. Lines 67-68: Unclear how molecular typing will provide information for vaccine development Please add clear aims and objectives for the study. Would like to see some of the data from previous studies referenced. Materials and Methods: Line 78: Should have the sub-heading 'Gonococcal Isolates' Disk diffusion method should go before PCR Amplification so that amplification and sequencing follow on from each other. Other than that, methods seem appropriate Results: I don't think Figure 1 is necessary in the main body of the manuscript. NG-MAST is a well-accepted and established typing method so the gel image is not adding any value to the manuscript. Line 154: explain in the methods how the AMR index is calculated Figure 2: Please put axis number on y axis and remove percentage from inside the bars. The data for NG-MAST is extremely limited - only 50% of isolates yielded any results and for the remaining some were incomplete. It will not be possible to apply data from 2 isolates to the entire gonococcal population of a country. Discussion I can't see anywhere outlined what the first line treatment for gonorrhoea is in Ghana, would be helpful to state. Line 272-273: I don't think the authors can imply that the STs found in this study have not been previously reported, if molecular typing isn't routinely done. It's possible that these strains are widespread but undetected due to lack of testing. Line 274: Should be 1A allele or IA allele? From the referenced study it should be P.IA. Line 283: corroborates There isn't a discussion of the limitations of the study; namely the extremely small sample size. Please add a section in the discussion to outline the limitations.
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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