Genomic Characterization of Salmonella Isolates Causing Infections in Children with Sickle Cell Disease in Dakar, Senegal

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Diop et al. is important having addressed an underexplored topic which is the genomic traits of Salmonella from children with sickle-cell disease in sub-Saharan Africa. However, the following comments should be addressed for the manuscript to be publishable:

TITLE

I think the title is overly broad and blurred transparency because the word “Senegalese” suggests a comprehensive national-level study whereas the content showed that the isolates originated from a single hospital in Dakar. Although the words “Phenotypic and Genomic Profiling” are often used by authors, I think they are generic because they did not highlight what is novel (e.g., diversity, plasmids, SPIs, SCD context) in the current study. However, authors may consider rephrasing to “Genomic Characterization of Salmonella Isolates Causing Infections in Children with Sickle-Cell Disease in Dakar, Senegal”

ABSTRACT

31-33: The aim appears vague and non-hypothesis driven because this statement “We wanted to conduct a detailed characterization” is informal and weak. There is no need to give overemphasis on technical details by listing specific SPIs and genes because this overwhelms the abstract without clear clinical relevance. The conclusions in line 41-42 are generic because it is expected and not specific to findings: “underscore the importance of genomic surveillance”. Authors should clearly state the knowledge gap, objective, and key contribution, then emphasize diversity, rarity of resistance, and clinical context, and tamper the conclusion by mentioning key limitations such as small sample size and retrospective design

KEYWORDS

Using “Invasive” as a keyword is vague, rather “Invasive nontyphoidal Salmonella” would be more appropriate. Why not reflect key findings of the study such as plasmids, SPIs, or whole-genome sequencing, virulence determinants?

INTRODUCTION

47-47: Too lengthy background on textbook-level Salmonella biology that is not directly relevant. General Salmonella description should be shortened.

Notably, there is weak linkage between SCD and genomics. Authors should clearly discuss the rationale for genomic analysis in SCD patients. I expected to see why genomic data in SCD patients matter (e.g., virulence adaptation, invasive potential)

73-83: Epidemiological statements are redundant. The study rationale appeared only at the very end in lines 103–107. Authors should make a concise study aim paragraph earlier

MATERIALS AND METHODS

109–123: A reader may want to know why the isolates were stored for a long period of time (2007–2019)? The phrase “Occasionally stored strains” is vague. What culture medium and conditions were the isolates stored to ensure viability for long duration? Is there potential bias that may ensue due to temporal spread? What inclusion criteria were used to select isolates for characterization? The incubation conditions and name of companies of materials used in bracket should be mentioned. What’s the justification for selecting the antimicrobials used for AST? Which quality control strain was used for the AST? I understand EUCAST employs only Susceptible and Intermediate categorization but using 2021 version whereas 2025 version is freely available online is not acceptable

Genome Sequencing and Assembly

126–146: Although the reference-guided assembly followed by plasmid de novo assembly is described, why was this approach was chosen? What is the coverage depth, and number of contigs? Justification of the assembly strategy might be important for reproducibility

Bioinformatics Analyses

159-160: SPI detection thresholds are arbitrary without justification for 60% coverage cutoff. Any justification or reference?

161-162: Why was phylogenetic comparison limited to Enteritidis excluding other serovars?

Sequencing Data Availability

169–177: EnteroBase accessions appears not clearly traceable/clickable, and some listed BioProject numbers appear unrelated or fragmented. A supplementary table linking each isolate to accession numbers might be useful

RESULTS

187–202: Result cannot all be descriptive, there should be element of statistical or comparative analysis

192-199: Suits better in Discussion section as reference must not be in the Result section. However, the comparison to prior studies is weak because cited study involves diarrheal cases, not SCD. I think the emphasis should be on clinical relevance (e.g., invasive sources). And over-interpreting comparisons across different populations should be avoided

203–213: There is overemphasis of a single MDR isolate. Authors should note that one MDR Salmonella isolate limits epidemiological inference. It is better to state that MDR findings are rare in this dataset. Moreover, the methodology did not show how the multidrug resistance was assessed

211-212: It is confusing to mention natural resistance. Authors should clarify intrinsic vs acquired resistance, and if Salmonella is naturally resistant to aminoglycosides, why was gentamicin used for the AST?

 216–234:  Yes, plasmids were profiled, but I expected to see whether isolates from invasive sites were more likely to carry virulence plasmids

236: “Salmonella pathogenicity islands (SPIs) are important virulence factors that contribute to various steps of bacterial pathogenesis” should be in the Introduction or Discussion section

244-245: This statement “Surprisingly, SPI-12 and SPI-20, reported 244 as always present in Salmonella [16], were detected in 6/23 and none” contradicts with published literature. It should therefore be critically discussed in the discussion section, so remove the discussion statement and reference from the result

I observed limited Heatmap interpretation. State what the findings reflect (e.g. assembly issues, strain diversity, or biological variation)

259–283:  Limiting analysis to Enteritidis limits manuscript cohesion. And MGT discussion is highly technical with limited clinical interpretation. Authors should reduce technical detail while emphasizing what phylogeny added to understanding local versus global transmission

DISCUSSION

334-349: Too lengthy and speculative vaccine discussion which is disproportionate to the data. Similarly, 250-266 on mouse model. Authors should shorten these paragraphs, add a clear limitations paragraph that would address sample size, retrospective design, single-center sampling and long collection period

CONCLUSIONS

Discussion points were repeated. Authors made claims about pathogenesis which are not directly supported by data. I suggest they focus conclusions on what was demonstrated such high serotype diversity, low resistance prevalence, and presence of virulence plasmids

Italicize names of organisms like Salmonella throughout the manuscript

Author Response

Responses to Reviewer 1

The manuscript by Diop et al. is important having addressed an underexplored topic which is the genomic traits of Salmonella from children with sickle-cell disease in sub-Saharan Africa. However, the following comments should be addressed for the manuscript to be publishable:

TITLE

I think the title is overly broad and blurred transparency because the word “Senegalese” suggests a comprehensive national-level study whereas the content showed that the isolates originated from a single hospital in Dakar. Although the words “Phenotypic and Genomic Profiling” are often used by authors, I think they are generic because they did not highlight what is novel (e.g., diversity, plasmids, SPIs, SCD context) in the current study. However, authors may consider rephrasing to “Genomic Characterization of Salmonella Isolates Causing Infections in Children with Sickle-Cell Disease in Dakar, Senegal”.

We have changed the title of the manuscript as suggested by the reviewer.

ABSTRACT

31-33: The aim appears vague and non-hypothesis driven because this statement “We wanted to conduct a detailed characterization” is informal and weak. There is no need to give overemphasis on technical details by listing specific SPIs and genes because this overwhelms the abstract without clear clinical relevance. The conclusions in line 41-42 are generic because it is expected and not specific to findings: “underscore the importance of genomic surveillance”. Authors should clearly state the knowledge gap, objective, and key contribution, then emphasize diversity, rarity of resistance, and clinical context, and tamper the conclusion by mentioning key limitations such as small sample size and retrospective design

We have rewritten the abstract taking into account the reviewer’s remarks.

KEYWORDS

Using “Invasive” as a keyword is vague, rather “Invasive nontyphoidal Salmonella” would be more appropriate. Why not reflect key findings of the study such as plasmids, SPIs, or whole-genome sequencing, virulence determinants?

We added the keywords suggested by the reviewer.

INTRODUCTION

47-47: Too lengthy background on textbook-level Salmonella biology that is not directly relevant. General Salmonelladescription should be shortened. Notably, there is weak linkage between SCD and genomics. Authors should clearly discuss the rationale for genomic analysis in SCD patients. I expected to see why genomic data in SCD patients matter (e.g., virulence adaptation, invasive potential). Epidemiological statements are redundant. The study rationale appeared only at the very end in lines 103–107. Authors should make a concise study aim paragraph earlier.

We removed part of the first sentence on Salmonella description. The remaining of the introduction comprises three parts corresponding to (i) Salmonella pathogenesis and infection of different organs, (ii) serotypes associated with invasive Salmonella in sub-Saharan Africa, (iii) SCD and susceptibility to bacterial infections especially to Salmonella, we think that it is relevant to the work and suggest to keep it.

We mentioned the absence of reports linking Salmonella virulence factors and pathogenesis in individuals with SCD (lines 106-107 of the revised manuscript). Additionally, we expanded on the objectives of this work in the last paragraph of the introduction section lines 112-114 of the revised manuscript.

MATERIALS AND METHODS

109–123: A reader may want to know why the isolates were stored for a long period of time (2007–2019)? The phrase “Occasionally stored strains” is vague. What culture medium and conditions were the isolates stored to ensure viability for long duration? Is there potential bias that may ensue due to temporal spread? What inclusion criteria were used to select isolates for characterization? The incubation conditions and name of companies of materials used in bracket should be mentioned. What’s the justification for selecting the antimicrobials used for AST? Which quality control strain was used for the AST? I understand EUCAST employs only Susceptible and Intermediate categorization but using 2021 version whereas 2025 version is freely available online is not acceptable.

We thank the reviewer for pointing out the absence of these relevant details. We answered to the questions raised by the reviewer lines 122-141 of the revised manuscript. Regarding the AST, it was performed in 2022 using 2021 EUCAST guidelines.

Genome Sequencing and Assembly

126–146: Although the reference-guided assembly followed by plasmid de novo assembly is described, why was this approach was chosen? What is the coverage depth, and number of contigs? Justification of the assembly strategy might be important for reproducibility.

There are not universally agreed procedures for plasmid detection and assembly from WGS data. We described the procedure we routinely use to assemble genomes, identify plasmid reads and assemble plasmids. We added a supplementary table showing sequencing depth, genome coverage, and additional assembly data.

Bioinformatics Analyses

159-160: SPI detection thresholds are arbitrary without justification for 60% coverage cutoff. Any justification or reference?

We agree with the reviewer: we arbitrarily set the threshold for SPI detection to 60% coverage and 90% nucleotide identity following in this known web platform for genome analysis (e.g. ResFinder). We acknowledged that this might introduce a limit. We formally recognized this limit in the last paragraph of the Discussion section, lines 395-402 of the revised manuscript.

161-162: Why was phylogenetic comparison limited to Enteritidis excluding other serovars?

We conducted phylogenetic analysis with Enteritidis isolates because this serovar (with Typhimurium) is the most frequently associated with iNTS in sub-Saharan Africa and it was predominant in the isolates we analyzed. Our goal was to see whether our isolates corresponded to African or international lineages.

Sequencing Data Availability

169–177: EnteroBase accessions appears not clearly traceable/clickable, and some listed BioProject numbers appear unrelated or fragmented. A supplementary table linking each isolate to accession numbers might be useful.

We added the information requested by the reviewer in a supplementary table.

RESULTS

187–202: Result cannot all be descriptive, there should be element of statistical or comparative analysis.

Given the small sample size, we did not conduct any statistical analysis, which could not enable any inference.

192-199: Suits better in Discussion section as reference must not be in the Result section. However, the comparison to prior studies is weak because cited study involves diarrheal cases, not SCD. I think the emphasis should be on clinical relevance (e.g., invasive sources). And over-interpreting comparisons across different populations should be avoided.

We thank the reviewer for giving us the opportunity to clarify the reference to diversity of isolates in stool samples. Actually, there are a few laboratories that perform Salmonella serotyping in Senegal and, to our knowledge, none of these do it systematically. The same applies to the majority of the other sub-Saharan African countries. Therefore, data on the serovars causing clinical infections are scarce. What our results suggest is that serovars that are likely associated with gastroenteritis in healthy individuals are able to cause invasive disease in SCD. We added this information in the Discussion section, lines 336-339 of the revised manuscript.

203–213: There is overemphasis of a single MDR isolate. Authors should note that one MDR Salmonella isolate limits epidemiological inference. It is better to state that MDR findings are rare in this dataset. Moreover, the methodology did not show how the multidrug resistance was assessed

The text mentioned by the reviewer, which list the antibiotic resistance genes found in the MDR isolate, only aimed to show that the resistance phenotype was consistent with the genomic data.

We added the definition of MDR phenotype in the Materials and Methods section lines 139-141 of the revised manuscript.

211-212: It is confusing to mention natural resistance. Authors should clarify intrinsic vs acquired resistance, and if Salmonella is naturally resistant to aminoglycosides, why was gentamicin used for the AST?

We thank the reviewer for requesting this clarification. We replaced «natural» with «intrinsic» line 235 of the revised manuscript. Actually, almost all Salmonella isolates harbor aminoglycoside acetyl transferase (e.g. aac(6’)-Ia) and or aminoglycoside phosphatidyl transferase (e.g. aph(3’)-) that presumably confer resistance to aminoglycosides. This has led to preventing the use of this antibiotic class for treatment of Salmonella infection. However, it is known that the presence of these genes is not associated with resistance in vitro. We test aminoglycosides when we characterize Salmonella isolates in order to possibly identify genes that confer resistance in vitro and for epidemiological value since aminoglycoside resistance genes can be encoded by plasmid and be disseminated by horizontal transfer.

216–234:  Yes, plasmids were profiled, but I expected to see whether isolates from invasive sites were more likely to carry virulence plasmids

We thank the reviewer for suggesting a comparative analysis of plasmid harbored by invasive vs gastrointestinal isolates. There were not differences between gastrointestinal and invasive isolates. Rather, isolates belonging to the same serovars tend to harbor the same plasmids as shown for isolates belonging to Enteritidis and Chester serovars.

236: “Salmonella pathogenicity islands (SPIs) are important virulence factors that contribute to various steps of bacterial pathogenesis” should be in the Introduction or Discussion section.

We removed the mentioned sentence from the results and added the description of SPIs in the Introduction section lines 103-107 of the revised manuscript.

244-245: This statement “Surprisingly, SPI-12 and SPI-20, reported as always present in Salmonella [16], were detected in 6/23 and none” contradicts with published literature. It should therefore be critically discussed in the discussion section, so remove the discussion statement and reference from the result

We thank the reviewer for catching this erroneous statement. The sentence has been removed from the revised manuscript.

I observed limited Heatmap interpretation. State what the findings reflect (e.g. assembly issues, strain diversity, or biological variation)

We have tightened the description of SPI distribution and clarified that SPI profiles were conserved within serovars (lines 265-280). Additionally, the limitations of the detection approach (threshold of 60% coverage and 90% identity) are now addressed in the final paragraph of the Discussion section (lines 395-402).

259–283:  Limiting analysis to Enteritidis limits manuscript cohesion. And MGT discussion is highly technical with limited clinical interpretation. Authors should reduce technical detail while emphasizing what phylogeny added to understanding local versus global transmission

To date, MGT analysis has been developed for only five bacterial species/serotypes: Salmonella Enteritidis, SalmonellaTyphimurium, Vibrio cholerae, Bordetella pertussis, and Staphylococcus aureus. Enteritidis, together with Typhimurium, represents the most extensively studied nontyphoidal Salmonella serovar worldwide. We therefore performed MGT analysis on our Enteritidis isolates, as (i) they were predominant in this study, and (ii) this serotype is frequently associated with outbreaks in Senegal (unpublished data). The objective of this phylogenetic analysis was to assess whether the isolates belonged to geographically restricted or globally disseminated lineages.

DISCUSSION

334-349: Too lengthy and speculative vaccine discussion which is disproportionate to the data. Similarly, on mouse model. Authors should shorten these paragraphs, add a clear limitations paragraph that would address sample size, retrospective design, single-center sampling and long collection period.

We thank the reviewer for this comment. The discussion of vaccines was included to emphasize the need for Salmonellacountermeasures in sub-Saharan Africa, where a significant proportion of individuals have SCD or are otherwise highly susceptible to invasive Salmonella infection (children, the elderly, and the immunocompromised). We believe this contextual discussion adds value to our study. Additionally, we have now added a dedicated paragraph on the limitations of our study, as requested (lines 395-402 of the revised manuscript).

CONCLUSIONS

Discussion points were repeated. Authors made claims about pathogenesis which are not directly supported by data. I suggest they focus conclusions on what was demonstrated such high serotype diversity, low resistance prevalence, and presence of virulence plasmids.

We have rewritten the conclusion as suggested by the reviewer.

Italicize names of organisms like Salmonella throughout the manuscript.

This correction has been done.

We are sincerely grateful to the reviewer for the insightful comments, corrections, and suggestions, all of which have helped us strengthen our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Diop et al. titled "Phenotypic and Genomic Profiling of Salmonella Isolates from Senegalese Children with Sickle Cell Disease" presents a well-structured genomic study of Salmonella isolates from a clinically vulnerable population—children with sickle-cell disease in Senegal. The research addresses an important gap in the literature, particularly in the context of low- and middle-income countries where SCD is prevalent and invasive bacterial infections pose significant risks. The study is methodologically sound, employing whole-genome sequencing, antimicrobial susceptibility testing, and comprehensive bioinformatic analyses to characterize serovar diversity, virulence determinants, plasmid profiles, and pathogenicity islands. The manuscript is generally well-written, though there are some areas where language clarity and grammatical precision could be improved.

Minor editing by a native English speaker or professional language service is recommended to polish the final version. While generally clear, there are occasional grammatical errors and awkward phrasings that could be refined, for example:
"In this study, we wanted to conduct…" → "In this study, we aimed to conduct…"
"All but one isolate was susceptible…" → "All but one isolate were susceptible…"
Some sentences are overly long and could be split for clarity.

Minor issues identified in the manuscript:
Line 38: "harboring the spvRABCD gene cluster, a plasmid-encoded fimbriae (pef), and the complement resistance gene (rck)" could be removed, as it is redundant when referring to the virulence plasmid.
Line 44: It is recommended to replace "Invasive" with "iNTS".
Line 48: For consistency, I strongly recommend using either serovar or serotype throughout the manuscript.
Line 71: Please add a reference for the statement "Typhimurium and Enteritidis are the most frequently isolated iNTS serovars."
Line 74: What does “sSA” stand for?
Table 1: It seems highly unlikely that all 23 isolates contained only one replicon/plasmid. The possible presence of small cryptic plasmids should be discussed even if they were not detected.
Table 1: The IncI1_1_alpha (67.9 kb) plasmid appears to be a hybrid plasmid fusing both virulence and antibiotic resistance genes. This should be discussed and compared with similar plasmids from databases for this serovar.
Table 1: For the source "Lung" please clarify whether this refers to a lung biopsy or sputum.
Table 1: "Peritoneal" is listed in the text here but not in the table - "Ascite" appears instead. Please correct this inconsistency.
Table 1: For strain M257 the last column should read "N/A" instead of "None" as no plasmids were detected.
Line 129: "TM" should be written as a superscript.
Line 186: "(see materials and methods)" should be removed.
Lines 208–211: Gene names should be italicized.
Lines 213, 224: Salmonella should be italicized.
Line 233: Table 1 indicates that the Liverpool serovar has no plasmids/replicons. Please clarify or correct.
Line 277: Specify clearly which database was used: Warwick University’s EnteroBase or South Wales University’s MGTdb?
Line 284: Salmonella should be italicized.
Line 286: Please include reference to the Multilevel Genome Typing database platform (MGTdb, https://mgtdb.unsw.edu.au/enteritidis/) in the Materials and Methods section.
Line 290: Were the new allelic profiles ("X") added to MGTdb? If so, it would be preferable to list their actual numbers rather than "X".
Throughout Introduction and Conclusion: Latin names such as Salmonella and Streptococcus pneumoniae should be italicized.
Line 307: The text above states that the strain was resistant to tetracyclines; this should be checked for consistency.
Line 310: Please add "(LMICs)" in parentheses.
Discussion: It would strengthen the manuscript to briefly compare the SPI profiles observed here with those from other African or global studies.
Discussion: A short paragraph on study limitations (e.g., sample size, retrospective design) is recommended. Additionally, it would be useful to clarify whether samples were collected from living patients or postmortem, and how many of the 23 patients survived.
Line 382: Please specify who received the grant and who participated in it (initials).

Author Response

Responses to Reviewer 2

The manuscript by Diop et al. titled "Phenotypic and Genomic Profiling of Salmonella Isolates from Senegalese Children with Sickle Cell Disease" presents a well-structured genomic study of Salmonella isolates from a clinically vulnerable population—children with sickle-cell disease in Senegal. The research addresses an important gap in the literature, particularly in the context of low- and middle-income countries where SCD is prevalent and invasive bacterial infections pose significant risks. The study is methodologically sound, employing whole-genome sequencing, antimicrobial susceptibility testing, and comprehensive bioinformatic analyses to characterize serovar diversity, virulence determinants, plasmid profiles, and pathogenicity islands. The manuscript is generally well-written, though there are some areas where language clarity and grammatical precision could be improved.

Minor editing by a native English speaker or professional language service is recommended to polish the final version. While generally clear, there are occasional grammatical errors and awkward phrasings that could be refined, for example:
"In this study, we wanted to conduct…" → "In this study, we aimed to conduct…"
"All but one isolate was susceptible…" → "All but one isolate were susceptible…"
Some sentences are overly long and could be split for clarity.

We thank the reviewer for drawing our attention to these important issues. The manuscript has been carefully revised to correct all grammatical errors and improve the overall phrasing and clarity.

Minor issues identified in the manuscript:
Line 38: "harboring the spvRABCD gene cluster, a plasmid-encoded fimbriae (pef), and the complement resistance gene (rck)" could be removed, as it is redundant when referring to the virulence plasmid.

We remove details on the virulence plasmids from the Abstract of the revised manuscript.

Line 44: It is recommended to replace "Invasive" with "iNTS".

“Invasive” was replaced with “Invasive nontyphoidal Salmonella” in the list of keywords of the revised manuscript.

Line 48: For consistency, I strongly recommend using either serovar or serotype throughout the manuscript.

Serovar is now used in the entire text of the revised manuscript.

Line 71: Please add a reference for the statement "Typhimurium and Enteritidis are the most frequently isolated iNTS serovars."

The requested references were added, lines 74 of the revised manuscript.

Line 74: What does “sSA” stand for?

“sSA” stands for “sub-saharan African”. The revised manuscript was corrected accordingly (line 76).

Table 1: It seems highly unlikely that all 23 isolates contained only one replicon/plasmid. The possible presence of small cryptic plasmids should be discussed even if they were not detected.

We mentioned the possibility of missing cryptic plasmids lines 241-242 of the revised manuscript.

Table 1: The IncI1_1_alpha (67.9 kb) plasmid appears to be a hybrid plasmid fusing both virulence and antibiotic resistance genes. This should be discussed and compared with similar plasmids from databases for this serovar.

We thank the reviewer for suggesting this important addition. We added a description of the IncI1-1-alpha plasmid, lines 253-259 of the revised manuscript.

Table 1: For the source "Lung" please clarify whether this refers to a lung biopsy or sputum.

Lung aspirate is now shown in Table 1 and line 205 of the revised manuscript.

Table 1: "Peritoneal" is listed in the text here but not in the table - "Ascite" appears instead. Please correct this inconsistency.

Ascites is now in both the text (line 205) and Table 1 of the revised manuscript.

Table 1: For strain M257 the last column should read "N/A" instead of "None" as no plasmids were detected.

Actually, M257 harbors a plasmid that does not encode antibiotic resistance. Table 1 was corrected accordingly.

Line 129: "TM" should be written as a superscript.

This correction was made.

Line 186: "(see materials and methods)" should be removed.

The expression "(see materials and methods)" was removed from the revised manuscript.

Lines 208–211: Gene names should be italicized.

Gene names are italicized in the revised manuscript (lines 230-233).

Lines 213, 224: Salmonella should be italicized.

This change was made.

Line 233: Table 1 indicates that the Liverpool serovar has no plasmids/replicons. Please clarify or correct.

We thank the reviewer for catching this error. Isolate M257 harbors a small plasmid of the Col family. We corrected the details in Table 1 of the revised manuscript.

Line 277: Specify clearly which database was used: Warwick University’s EnteroBase or South Wales University’s MGTdb?

We thank the reviewer for requesting this clarification. The location of the MGTdb was mentioned in both the Results (line 302) and Materials and Methods (lines 180-181) sections.

Line 284: Salmonella should be italicized.

Done.

Line 286: Please include reference to the Multilevel Genome Typing database platform (MGTdb, https://mgtdb.unsw.edu.au/enteritidis/) in the Materials and Methods section.

This change was made lines 180-181 of the revised manuscript.

Line 290: Were the new allelic profiles ("X") added to MGTdb? If so, it would be preferable to list their actual numbers rather than "X".

We thank the reviewer for this suggestion. We have requested MGTdb to assign new allele numbers for our isolates.

Throughout Introduction and Conclusion: Latin names such as Salmonella and Streptococcus pneumoniae should be italicized.

This change was made.

Line 307: The text above states that the strain was resistant to tetracyclines; this should be checked for consistency.

The strain was resistant to tetracycline but susceptible to quinolones. The error was corrected lines 229 of the revised manuscript.

Line 310: Please add "(LMICs)" in parentheses.

The full expression and abbreviation are now added in the Results section, line 207 of the revised manuscript, as well as in the abbreviation list.

Discussion: It would strengthen the manuscript to briefly compare the SPI profiles observed here with those from other African or global studies.

There are only a few articles performing SPI profiling using WGS data. The findings align with our results showing that SPI profiles are generally conserved within serovars, and do not vary according to the clinical presentation of the disease. We added this observation in the revised manuscript, lines 278-280.

Discussion: A short paragraph on study limitations (e.g., sample size, retrospective design) is recommended. Additionally, it would be useful to clarify whether samples were collected from living patients or postmortem, and how many of the 23 patients survived.

We added a paragraph detailing the limit of this study, lines 395-402 of the revised manuscript. Regarding the outcome of the diseases, these data are available. The microbiology laboratory receives and processes samples from all departments of the children’s hospital, including the SCD Center. Clinicians from the SCD Center submit laboratory request forms indicating the clinical diagnosis prompting the microbiological analysis; however, no additional patient-specific information is routinely provided to the microbiology laboratory.

Line 382: Please specify who received the grant and who participated in it (initials).

The requested information was added.

We would like to thank the reviewer for his comments, corrections, and suggestions, all of which have contributed to improving our manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors made significant effort to address all my comments.

However, for reproducibility, they should add the number of cells (in cfu/mL) in specific McFarland's turbidity standard used to inoculate the Mueller-Hinton agar plate in AST.

Since AST is aimed for effective treatment, the latest breakpoints recommended by CLSI or EUCAST should be used for interpreting AST results irrespective of date at which AST was conducted 

Check to ascertain that the Accession numbers are clickable

Author Response

However, for reproducibility, they should add the number of cells (in cfu/mL) in specific McFarland's turbidity standard used to inoculate the Mueller-Hinton agar plate in AST.

The requested details were added lines 132-134 of the revised manuscript.

Since AST is aimed for effective treatment, the latest breakpoints recommended by CLSI or EUCAST should be used for interpreting AST results irrespective of date at which AST was conducted.

We corrected the used guidelines as requested, line 140 of the revised manuscript.

Check to ascertain that the Accession numbers are clickable.

All the accession numbers in Supplementary Table 1 are now clickable directing to their corresponding pages at NCBI.