Epidemiology, Temporal Trends and Resistance Patterns of ESBL-Producing Non-Typhoidal Salmonella Isolated from Blood Cultures in Kisantu, DRC (2019–2022)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The well-written manuscript describes the rapid increase in prevalence of ESBL-producing suspected non-typhoidal Salmonella involved in septicemia in Kisantu area (Republic of Congo) in a period of about 3 years from about 14 to about 95%. It provides possible reasons for such a strong increase and measures to manage AMR issues.

a few comments:

1. perhaps modify the title slightly. "temporal Evolution" is the part of the title that doesn't describe the issue.
2. Line 73:  "Klebiellapneumoniae" should be  Klebiella pneumoniae
3. A general remark related to the curve fitting Figure 2: it seems that the logistic regression did not predict the enormous increase in ESBL producing NTS in septicemia to 95% in three years. Perhaps there is a formula predicting the increase in a better way
4. Line 172 - 173: insert here a reference to Figure 1 for clarity reasons
5. Line 288: in relation to mentioning the age group 24 months in the manuscript, please substitute in line 288 "2 years"by 24 months

Author Response

1. perhaps modify the title slightly. "temporal Evolution" is the part of the title that doesn't describe the issue.

Answer/Comment: We sincerely thank you for this valuable comment. We modified the title as follow:

’’Epidemiology, Temporal Trends and Resistance Patterns of ESBL-Producing Non-Typhoidal Salmonella Isolated from Blood Cultures in Kisantu, DRC (2019–2022).’’

2. Line 73:  "Klebiellapneumoniae" should be  Klebiella pneumonia

Answer/comment: Thank you for this observation. We have corrected the name of the bacteria as follow ‘‘Klebsiella pneumonia’’ (see line 74).

3. A general remark related to the curve fitting Figure 2: it seems that the logistic regression did not predict the enormous increase in ESBL producing NTS in septicemia to 95% in three years. Perhaps there is a formula predicting the increase in a better way

Answer/comment: Thank you for this pertinent comment, we changed our approach by working with data grouped by years (using 2019 data as a reference). The Odd ratios were calculated for each year 2020, 2021 and 022, allowing us to observe the upward trend over time in terms of NTS BLSE (see lines 146 to 156).

4. Line 172 - 173: insert here a reference to Figure 1 for clarity reasons

Answer/comment: Thank you for this suggestion, we have inserted the reference to figure 1 into the text (see line 177).

5. Line 288: in relation to mentioning the age group 24 months in the manuscript, please substitute in line 288 "2 years"by 24 months

Answer/comment: Thank you for this comment. However, in order to standardize the terminology for patient age, we decided to express ages in years throughout the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, I believe the article is interesting because there are not many studies like this in your country. In the current version, we have several areas for improvement that, when corrected, will enhance the quality of the article for its subsequent reconsideration for publication in the journal.

1. The first observation is that, according to iThenticate, the similarity percentage is 26%. I consider this to be somewhat high, so I suggest you reduce it to less than 10%, preferably, as this will increase the quality of the article.
2. I recommend that you verify that the font, font size, and line spacing are consistent throughout the entire document, so that it looks as professional as possible.
3. The "Abstract" is good since it gives a complete overview of the research, but I suggest that you join all the paragraphs into a single paragraph, this will improve this section.
4. In section "1. Introduction" it is necessary to explain in more detail the impact of the presence of bacteria in blood and ESBL on human health. Also investigate further on the prevalence of bacteria, specifically ESBL-producing NTS in the world, in Africa and in DRC, this will allow readers to adequately understand the information necessary for the research.
5. In section "2.1. Blood culture Outcomes and NTS isolation" I believe the explanation of the results shown in Figure 1 could be improved. It would be helpful to include an image that visualizes the results supporting the identification of the bacteria as ESBL producers. This would allow non-specialist readers to better interpret the results of the techniques used in the research.
6. In section "2.2. Epidemiologic characteristics of patients with ESBL-NTS Bacteremia" I recommend that the age ranges of the patients be in years, not months, since this could generate confusion among the readers, correcting it will improve the quality of this section. Additionally, I believe that the name of Table 1 "Epidemiologic characteristics..." is too long; it should be specifically delimited to ESBL-NTS cases, so I suggest correcting the name of said table.
7. In section "2.3. Temporal Trends in ESBL production (2019 to 2022)," I believe the information presented in Table 2 would be much better presented graphically, for example, as a bar chart. This would allow readers to more clearly visualize the quarterly distribution of ESBL-producing NTS isolates. Additionally, the results of the logistic regression in Figure 2 should be explained in more detail, which would significantly improve the quality of this section.
8. In section "2.4. Resistance Profile of ESBL-NTS (n=1,184)" it is not necessary to put the n in the title, in addition, it is necessary to explain why the azithromycin sensitivity tests were only performed between September 2021 and December 2022, personally since these results are not comparable to the other antibiotics I would remove them from the article.
9. In section "3. Discussion," I have several observations:

• It is necessary to explain why the 113 isolates were not analyzed and what the consequences would be for public health, given that it had an impact on determining the proportion of ESBL-producing NTS isolates.
• It is necessary to discuss more deeply why most ESBL isolates recovered in children under 5 years of age, particularly those under 2 years of age, and to conduct in-depth research into the determinants that produce greater vulnerability to invasive infections and how this is associated with the results obtained in the research.
• It is mentioned that the quarterly analysis revealed a significant upward trend in the prevalence of ESBL-NTS and may be influenced by other unmeasured factors. I recommend that these factors be discussed appropriately, along with their impact on this increase in prevalence and its repercussions on public health.
• It is mentioned that high percentages of XDR and PDR isolates were detected, and the importance of ESBL genes associated with resistance determinants is discussed. I believe it would be appropriate to investigate and discuss which genes these are and what role they play in human and public health.
• It is unclear how azithromycin is related to Stevens-Johnson syndrome, nor the role that ESBL-NTS plays in this syndrome, and as I mentioned earlier, I believe that the proportion of azithromycin-resistant isolates is comparable to other antibiotics, because it was not monitored throughout the study period.
• Finally, it would be appropriate to discuss in depth the impact of ESBL-NTS on human and public health, as well as mention treatment alternatives for patients infected with these bacteria; this will improve the quality of this section.

10. In section "4. Materials and methods," I recommend adding numbers to the subtopics (which should not contain punctuation marks such as colons (:) - please remove them). Regarding section "4.1 Study Design:" it needs improvement. I recommend explaining the study population, how it was accessed, and the inclusion/exclusion criteria more clearly. This will allow readers to properly understand this important section of the article. The colon (:) in section "4.2 Study site:" must also be removed.
11. In the "4.3 Sample collection" section, it is necessary to explain in detail the conditions of blood sample collection, storage (if carried out), transport and processing performed with the samples; this will allow non-specialized readers to adequately understand the importance of the pre-analytical phase of the research.
12. In section "4.4 Laboratory analysis," the biochemical tests used need to be explained in more detail (or were only Simmons citrate and ODC tests performed?). I recommend a brief explanation of the Kirby-Bauer disk diffusion method and the combined disk diffusion method. It is necessary to explain how fluoroquinolone resistance was assessed based on observed pefloxacin resistance; this will allow non-specialist readers to fully understand all the methodologies used in the research.
13. In section "4.5 Data Collection and Statistical Analysis," you must explain the criteria used to categorize patients into the four groups mentioned. Additionally, you should provide a more detailed description of the data, statistical analysis, and processing used in the research. This will improve the quality of section "4. Materials and Methods" and make it as comprehensive as possible.
14. Section "5. Conclusion" could be improved; I recommend explaining a little better the impact that their results have on human health and whether they adequately meet the research objectives. This will further emphasize their results and their impact on public health.

Author Response

Comment 1: The first observation is that, according to iThenticate, the similarity percentage is 26%. I consider this to be somewhat high, so I suggest you reduce it to less than 10%, preferably, as this will increase the quality of the article.

Answer/comment: We thank you for this observation. In order to reduce similarity percentage, we managed to make some edits/modifications in several sections of the article. After that, we assessed once again the article for similarity using Plagiarism checker and we found that the highest percentage of similarity was related to the preprints version this article which was available online since November 19^th, 2025.

Comment 2. I recommend that you verify that the font, font size, and line spacing are consistent throughout the entire document, so that it looks as professional as possible.

Answer/comment: Thank you very much for this comment. After your comment we scrutinized the article and managed to harmonize front size of the text in accordance to the journal guidelines for titles, subtitles and core text.

Comment 3. The "Abstract" is good since it gives a complete overview of the research, but I suggest that you join all the paragraphs into a single paragraph, this will improve this section.

Answer/comment: Thank you for this suggestion, we modified the abstract and we joined all the paragraphs in one paragraph (see lines 41 to 61).

Comment 4. In section "1. Introduction" it is necessary to explain in more detail the impact of the presence of bacteria in blood and ESBL on human health. Also investigate further on the prevalence of bacteria, specifically ESBL-producing NTS in the world, in Africa and in DRC, this will allow readers to adequately understand the information necessary for the research.

Answer/comment: Thank you for your pertinent comment. We added some sentences to explain the impact of NTS bacteremia and ESBL-producing NTS on human health (lines 82 to 102).

Comment 5. In section "2.1. Blood culture Outcomes and NTS isolation" I believe the explanation of the results shown in Figure 1 could be improved. It would be helpful to include an image that visualizes the results supporting the identification of the bacteria as ESBL producers. This would allow non-specialist readers to better interpret the results of the techniques used in the research.

Answer/comment: Thank you for comment. We added biochemical reactions use to identify NTS at the lab and a photo of ESBL screening using the double disk method. (see lines 205 to 206).

Comment 6. In section "2.2. Epidemiologic characteristics of patients with ESBL-NTS Bacteremia" I recommend that the age ranges of the patients be in years, not months, since this could generate confusion among the readers, correcting it will improve the quality of this section. Additionally, I believe that the name of Table 1 "Epidemiologic characteristics..." is too long; it should be specifically delimited to ESBL-NTS cases, so I suggest correcting the name of said table.

Answer/comment: Thank you for your suggestion, we harmonized the ages ranges in patients in year. Further, we modified name of table as follow: Age and gender distribution of ESBL-producing NTS isolates

Comment 7. In section "2.3. Temporal Trends in ESBL production (2019 to 2022)," I believe the information presented in Table 2 would be much better presented graphically, for example, as a bar chart. This would allow readers to more clearly visualize the quarterly distribution of ESBL-producing NTS isolates. Additionally, the results of the logistic regression in Figure 2 should be explained in more detail, which would significantly improve the quality of this section.

Answer/comment: Thank you for this pertinent comment, we modified the name of the section 2.3. We modified the presentation of the temporal trends of ESBL-producing NTS isolates. We regrouped data by years which clearly display the temporal increase of ESBL-producing nts from 2019 to 2022. However, the details on quarterly distribution of ESBL producing NTS by years are displayed in the supplemental information using a bar chart (supplemental figure 1). Regarding the current figure 2, we have provided additional details highlighting the increase in resistance over the years.

Comment 8. In section "2.4. Resistance Profile of ESBL-NTS (n=1,184)" it is not necessary to put the n in the title, in addition, it is necessary to explain why the azithromycin sensitivity tests were only performed between September 2021 and December 2022, personally since these results are not comparable to the other antibiotics I would remove them from the article.

Answer/comment: Thank you very much for these comments. We removed the "n" in the title. Regarding the azithromycin susceptibility testing, this only started in 2021 due to the limited therapeutic options resulting from the surge in resistance, but also following the encouraging results obtained from the study by Tack et al., as described in the Methods section. We understand your suggestion to remove the azithromycin results from the article; however, this antibiotic is one of the antibiotics of choice that is available and affordable in the DRC. We think it is important to display these results (see line 308 to 316).

Comment 9. In section "3. Discussion," I have several observations:

- It is necessary to explain why the 113 isolates were not analyzed and what the consequences would be for public health, given that it had an impact on determining the proportion of ESBL-producing NTS isolates.

Answer/comment: Thank you for rising this observation. Regarding this comment, the major problem of the country's permanent supply of laboratory inputs was mentioned in the manuscript as having led to this situation (see lines 174 to 177).

 - It is necessary to discuss more deeply why most ESBL isolates recovered in children under 5 years of age, particularly those under 2 years of age, and to conduct in-depth research into the determinants that produce greater vulnerability to invasive infections and how this is associated with the results obtained in the research.

Answer/comment: Thank you for raising this observation. Children in these age groups are generally already vulnerable to malaria, malnutrition, and anemia, which are prevalent in an endemic manner in this region. Third-generation cephalosporins have been primarily used for the management of infections in this age group, reflecting the high antibiotic pressure within this population. We have added these details to the Discussion section (see lines 180-183).

- It is mentioned that the quarterly analysis revealed a significant upward trend in the prevalence of ESBL-NTS and may be influenced by other unmeasured factors. I recommend that these factors be discussed appropriately, along with their impact on this increase in prevalence and its repercussions on public health.

Answer/comment: Regarding this comment, we have updated the discussion. A paragraph has been added briefly mentioning these factors, taking into account the country and local context (see lines 212 to 221).

- It is mentioned that high percentages of XDR and PDR isolates were detected, and the importance of ESBL genes associated with resistance determinants is discussed. I believe it would be appropriate to investigate and discuss which genes these are and what role they play in human and public health.

Answer/comment: Thank you for your comment. Although genetic analysis has not yet been performed as part of the present study, previous studies have reported the presence of Salmonella Typhimurium ST313, which is characterized by high levels of antibiotic resistance. This represents a limitation of our study. We also plan to carry out genetic analyses in the coming months.

- It is unclear how azithromycin is related to Stevens-Johnson syndrome, nor the role that ESBL-NTS plays in this syndrome, and as I mentioned earlier, I believe that the proportion of azithromycin-resistant isolates is comparable to other antibiotics, because it was not monitored throughout the study period.

Answer/comment: Thank you for this comment. The present study used retrospective data from the surveillance network which is laboratory based. We did not make a follow up of patient treatment. Further, we did not assess patients for the occurrence for the Stevens-Johnson syndrome. egarding the resistance of ESBL-producing NTS to azithromycin, we believe this is a very important finding, as it is one of the only two drugs that still retain full activity against these bacteria.

- Finally, it would be appropriate to discuss in depth the impact of ESBL-NTS on human and public health, as well as mention treatment alternatives for patients infected with these bacteria; this will improve the quality of this section.

Answer/comment: Thank you for thie comment. We take this into account in the current version of the article.

Comment 10. In section "4. Materials and methods," I recommend adding numbers to the subtopics (which should not contain punctuation marks such as colons (:) - please remove them). Regarding section "4.1 Study Design:" it needs improvement. I recommend explaining the study population, how it was accessed, and the inclusion/exclusion criteria more clearly. This will allow readers to properly understand this important section of the article. The colon (:) in section "4.2 Study site:" must also be removed.

Answer/comment: Thank you for this observation. We have specifically created a section dedicated to describing the study population. This helps to improve the understanding of the Methods section (see line 240-251)

Comment 11. In the "4.3 Sample collection" section, it is necessary to explain in detail the conditions of blood sample collection, storage (if carried out), transport and processing performed with the samples; this will allow non-specialized readers to adequately understand the importance of the pre-analytical phase of the research.

Answer/comment: Thank you for While we thank you for this pertinent observation, we have taken it into account by briefly explaining the procedure for collecting and transferring blood cultures directly to the laboratory (see lines 269 to 275).

Comment 12. In section "4.4 Laboratory analysis," the biochemical tests used need to be explained in more detail (or were only Simmons citrate and ODC tests performed?). I recommend a brief explanation of the Kirby-Bauer disk diffusion method and the combined disk diffusion method. It is necessary to explain how fluoroquinolone resistance was assessed based on observed pefloxacin resistance; this will allow non-specialist readers to fully understand all the methodologies used in the research.

Answer/comment: We thank you for this observation, we have also taken it into account by briefly explaining the method used in the antibiogram that were performed (see lines 280 to 290).

Comment 13. In section "4.5 Data Collection and Statistical Analysis," you must explain the criteria used to categorize patients into the four groups mentioned. Additionally, you should provide a more detailed description of the data, statistical analysis, and processing used in the research. This will improve the quality of section "4. Materials and Methods" and make it as comprehensive as possible.

Answer/comment: Thank you for this insightful comment. We have taken it into account (see lines 307 to 322).

Comment 14. Section "5. Conclusion" could be improved; I recommend explaining a little better the impact that their results have on human health and whether they adequately meet the research objectives. This will further emphasize their results and their impact on public health.

Answer/comment: Thank you this observation. We have improved the conclusion based on your latest observations.

Reviewer 3 Report

Comments and Suggestions for Authors

This study investigates the prevalence, and resistance patterns of ESBL-producing non-typhoidal Salmonella in Kisantu, DRC. The manuscript is overall very good, but would benefit from appropriate revisions to improve readability and interpretation.

1. The term “temporal evolution” in the manuscript title may be misleading because “evolution” can suggest biological evolution (e.g., selection or adaptation), whereas the analyses primarily describe epidemiologic changes over time in prevalence and resistance. Consider replacing it with clearer wording such as “temporal trends” or “temporal dynamics.”
2. The Results section is appropriately concise; however, it would benefit from slightly more narrative detail to better highlight the major findings. Please explicitly highlight the key patterns and important differencesbetween comparison groups to improve readability and interpretation.
3. The manuscript compares outcomes across groups (for example, by sex and by age), but no statistical analyses are provided to support these comparisons. Please perform and report appropriate statistical testing (e.g., Fisher’s s exact test) and ensure that the text distinguishes between descriptive differences and statistically supported differences.
4. Table 3. Consider stratifying the dataset by year/quarter and, for each antibiotic category, reporting the resistance counts/denominators (n/N) and corresponding percentages for each time period. In addition, please perform appropriate trend analyses to evaluate how resistance changes over time and whether temporal trends differ across antibiotic categories.
5. Lines 160-162: Consider providing the data in the supplement.

Author Response

Comment 1. The term “temporal evolution” in the manuscript title may be misleading because “evolution” can suggest biological evolution (e.g., selection or adaptation), whereas the analyses primarily describe epidemiologic changes over time in prevalence and resistance. Consider replacing it with clearer wording such as “temporal trends” or “temporal dynamics.”

Answer/comment: We would like to thank you for your comment and suggestions. We have opted for the title Epidemiology, temporal trends and Resistance Patterns of ESBL-producing non-typhoidal Salmonella isolated from Blood cultures in Kisantu, DRC (2019–2022).

Comment 2. The Results section is appropriately concise; however, it would benefit from slightly more narrative detail to better highlight the major findings. Please explicitly highlight the key patterns and important differences between comparison groups to improve readability and interpretation.

Answer/comment: Thank you for this comment. We have proceeded to the updated of this section (see lines 140 to 165).

Comment 3. The manuscript compares outcomes across groups (for example, by sex and by age), but no statistical analyses are provided to support these comparisons. Please perform and report appropriate statistical testing (e.g., Fisher’s s exact test) and ensure that the text distinguishes between descriptive differences and statistically supported differences.

Answer/comment: Thank you for this pertinent remark. We performed Pearson's chi-square test to analyze the association between the production of extended-spectrum β-lactamases (ESBL) by non-typhoidal Salmonella (NTS) (dependent variable) and explanatory variables, in particular the sex and age of patients (see table between lines 141 and 145).

Comment 4. Table 3. Consider stratifying the dataset by year/quarter and, for each antibiotic category, reporting the resistance counts/denominators (n/N) and corresponding percentages for each time period. In addition, please perform appropriate trend analyses to evaluate how resistance changes over time and whether temporal trends differ across antibiotic categories.

Answer/comment: Thank you for this observation. Regarding this pertinent observation, we have stratified the data by year for greater clarity in data analysis. In this study, we specifically focused on studying the dynamics of ESBLs, taking into account the local context, which is marked by intensive use of third-generation cephalosporins. With regard to assessing the evolution of resistance to other antibiotics, we plan to use this data in the context of studies on resistance prediction that we intend to conduct in the future.

Comment 5. Lines 160-162: Consider providing the data in the supplement.

Answer/comment: Regarding the azithromycin susceptibility testing, this only started in 2021 due to the limited therapeutic options resulting from the surge in resistance, but also following the encouraging results obtained from the study by Tack et al., as described in the Methods section. We understand your suggestion to remove the azithromycin results from the article; however, this antibiotic is one of the antibiotics of choice that is available and affordable in the DRC. We think it is important to display these results (see line 308 to 316).

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, I believe that this new version satisfactorily addresses all the observations I shared with you previously, which has improved the quality of the article. Congratulations.

Author Response

Dear Reviewer,
We sincerely thank you for your insightful comments, which have greatly contributed to the improvement of this manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

My comments have been fully addressed.

Author Response

Dear Reviewer,
We sincerely thank you for your insightful comments, which have greatly contributed to the improvement of this manuscript.