Characterization of MultidrugResistant serogroup 19 Streptococcus pneumoniae isolated from healthy children below 5 years of age in Indonesia
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We investigated the resistance genes, pilus islets, biofilm formation ability and sequence types of multidrug-resistant Streptococcus pneumoniae (MDRSP) isolated from healthy children below 5 years of age in Indonesia. In all, 104 archived MDRSP isolates from previous carriage studies in Indonesia in 2016–2019 were screened for the presence of antibiotic resistance genes and the rrgC (pilus islet 1) and pitB (pilus islet 2) genes. Multilocus sequence typing and biofilm formation were determined by PCR sequencing and the ability of cells to adhere to the walls, respectively. Results have shown that the mefA , ermB and tetM genes were found in 93, 52 and 100 % of MDRSP isolates, respectively. Insertions of arginine, proline and Ile-100–Leu were the most common mutations in the folA and folP genes. Pilus islets 1 and 2 were discovered in 93 and 82 % of MDRSP isolates, respectively. The MDRSP isolates showed no biofilm formation ability (64 %), and 5 out of 10 strains of MDRSP strains were ST1464. This finding can be used to provide further considerations in implementing and monitoring pneumococcal vaccination in Indonesia.
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This study would be a valuable contribution to the existing literature.
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Thank you for your response. I'm happy with the correction and find the address reviewers concerns. I've selected minor corrections however as it appears that at least figure 1 is missing from the manuscript and is not available in my Editor's file inventory. Can you please confirm that this is included or include it.
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This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
Salsabila et al report on the antimicrobial resistance profiles of serogroup 19 isolates of Streptococcus pneumoniae sampled from Indonensian children. They use robust methods to detect the presence of genes conferring resistance to a range of different antibiotics. They also test for the presence of other virulence factors among these isolates, such as the pilus genes, and the isolates' ability to form biofilms. The authors also type a subset of their collection to place them in a global context. They find widespread resistance in this subset of Indonesian isolates, linking this spread to a known pneumococcal MDR clone. This is an important and interesting finding. The manuscript would benefit from a review of the grammar and spelling, just to ensure the clarity of its points. I have highlighted a …
Comments to Author
Salsabila et al report on the antimicrobial resistance profiles of serogroup 19 isolates of Streptococcus pneumoniae sampled from Indonensian children. They use robust methods to detect the presence of genes conferring resistance to a range of different antibiotics. They also test for the presence of other virulence factors among these isolates, such as the pilus genes, and the isolates' ability to form biofilms. The authors also type a subset of their collection to place them in a global context. They find widespread resistance in this subset of Indonesian isolates, linking this spread to a known pneumococcal MDR clone. This is an important and interesting finding. The manuscript would benefit from a review of the grammar and spelling, just to ensure the clarity of its points. I have highlighted a few smaller errors below, in the minor review section. The referencing in the paper should also be revised, currently the format seems to differ, with some references not containing names of the journal or source, i.e references 10, 11, 12, 16, 18, 19, 21, 22, 24, 25, 34 and 36. The authors should choose a style accepted by the journal and standardise all references to this. The methods were in-depth and clearly explained. However it was not clear to me where the MIC values for isolates were calculated. Was this done during this study? If so these techniques should also be described in the methods section. If not the study which performed this work should be cited. Additionally, it was unclear to me how the 104 isolates in the study were sampled. Was this from a carriage study? If so the authors should also briefly explain their methodology in the S. pneumoniae isolate collection section of the methods. The authors should also explain how they chose the 10 isolates which were selected for MLST. The results section was clear and concise. The authors should though, also report both the raw number of isolates that have the phenotype in question as well as the percentage through-out the results section. In the section detailing the results of the flouroquinolone resistance mutations, it would be helpful to state the number of isolates which were predicted to be resistant. The figures presented in the text would benefit from grid lines, at present it is hard to tell the exact values for the individual columns. An eBURST figure of the 10 MLST profiles in the manuscript would also be helpful. This allows the reader to see the relationship between the isolates more clearly. It can be created using the BURST analysis on the pubMLST website, where the authors have checked these profiles. The discussion is broad and takes in a wide range of the current literature on the topic. The discussion of biofilm formation and serotype is slightly confusing however. On line 395 for instance the authors state "Biofilm formation ability is independent of serotypes", the following lines then links certain serotypes as being good or bad biofilm producers. This should be cleared up. The paragraph starting on line 431 also appears to imply the authors have tested the causality of AMR, pilus and biofilm formation on the invasiveness of strains. This hypothesis has not been tested in this study, it is an observational study of the current pneumococcal population in Indonesia. The authors should be careful about implying this from their work. Some minor reviews of the paper are below: Line 37: Suggest changing "most of the sequence type was ST1464" to "the most frequent sequence type was ST1464" Line 54: Remove "fastidious" Line 58: Need to state that IPD has not been monitored in Indonesia, before mentioning "the country" Line 67: Need a reference for the 59.3% prevalence figure. Line 71: Need a reference for the 46% of children in Lombok carrying S. pneumoniae and 19F being the second most common serotype. Line 76: Replace "dan" with "and" Line 78: Replace "pilus and biofilm" with "pilus and biofilm formation" Line 104-107: The resistance rates are more suited to results - move this information to that section. Line 230: Authors should remove the adjective significantly here, it implies a statistical test has been performed on this difference. Line 239: Suggest rephrasing the title to "Mutations in folA and folP". Line 246-248: This final sentence, detailing the upload of sequence data, should be in the methods section. Line 259: Quote the raw number of isolates not able to form biofilms instead of "most of the isolates" Line 265: For these MLST results don't quote percentages, as a rule don't use percentages for numbers where the total is less than 100. This should be observed through-out the manuscript. Line 300: Start of the sentence should be "The majority of the MDRSP…" Line 314: The 916 in Tn916 should be italicized. Line 317: Rephrase to "This study has shown that macroide-resistant S. pneumoniae can also be tetracycline-resistant" Lines 329-330: No need to spell out the acronyms for DHFR and DHPS here again, just use the acronyms. Line 354: Make sure et al is italicized. Line 371: Refer again to table 2 to make the reader aware of the overall distribution of resistance mutations. Line 392: What do the authors mean by "greater advantage"? It should be stated more directly if they believe this would be a fitness benefit or not. Lines 399-400: This sentence, which starts with "Meanwhile, MDRSP" is slightly confusing and shoule be rephrased. How did the isolates show invasiveness? Line 417: Need a reference for this claim of interspecies recombination due to penicillin use. Line 442-445: This study hasn't investigated invasiveness, it has described the virulence factors that are present in the genome. Line 662: Should be ribosome not "ribosom"
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
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
1. Methodological rigour, reproducibility and availability of underlying data The authors do not adequately describe their data source and how the data was generated. The authors also need to better acknowledge the limitations of their study, particularly in relation to the MLST results. The sample size is too small to support the conclusions that are made in the paper. 2. Presentation of results The figures need to be adjusted to better convey the intended message. I would advise changing the current figures to tables to make the values easier to read and suggest adding figures that display the range of MICs and compare these to the genotyping results. Some of the figures could benefit from being converted into tables. I also think the paper could benefit from presentation of the MIC data. This …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data The authors do not adequately describe their data source and how the data was generated. The authors also need to better acknowledge the limitations of their study, particularly in relation to the MLST results. The sample size is too small to support the conclusions that are made in the paper. 2. Presentation of results The figures need to be adjusted to better convey the intended message. I would advise changing the current figures to tables to make the values easier to read and suggest adding figures that display the range of MICs and compare these to the genotyping results. Some of the figures could benefit from being converted into tables. I also think the paper could benefit from presentation of the MIC data. This may have been presented elsewhere but it is used enough in this study to warrant presentation here. There also need to be figures that display a comparison between the genotypic and phenotypic data. This is mentioned heavily in the text but there is no figure displaying this. 3. How the style and organization of the paper communicates and represents key findings The results section would benefit from a more direct comparison of AMR genotype and phenotype. It is mentioned for some gene/antibiotics but should be calculated for all. Please be consistent in use of the results headings, some are statements that summarise the results sections while others are overall topics. 4. Literature analysis or discussion The introduction needs to provide more information on the resistance genes being investigated and which antibiotics they confer resistance to. Providing this information in the discussion will aid in interpreting the results sections. There is discussion on the dominant genotypic mechanisms leading to phenotype but no results have been presented on the concordance that was observed between these two data sources. In general, the discussion section needs more references and the study data needs to be more directly compared to these references rather than stating what other studies found. 5. Any other relevant comments Line 29: Please include the time frame in which the isolates were collected. Line 37: Change to the most common sequence type. Line 58: Specify the country. Line 76: and spelt incorrectly. PCR methods: There is large amounts of repetition in these sections. The authors should consider writing a generic PCR protocol and then a table that describes any of the primers, timings or temperatures that were changed for each gene. Line 171: Please include references describing the biofilm forming abilities of these strains. Line 204: Please describe how and why these isolates were chosen for MLST. Line 221: Please reference the PubMLST database. Line 232: Unclear which isolates you are referring to as "These isolates" and please give MIC interpretation values to aid in interpreting the >2 MIC. Line 239: Please be consistent when refering to folA and folP, I think it is easier to use these gene names rather than DHFR and DHPS. Line 259: It is not needed to refer to the study isolates as "MDR S. pneumoniae 260 serotypes 19F and 19A" each time, they can be referred to as the "study isolates" after they have been described once. Line 270 - 275: I question how much of this is results and how much is merely a description of the different MLSTs available on PubMLST. Line 359: This statement should have a reference.
Please rate the manuscript for methodological rigour
Satisfactory
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?
Yes
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