Antimicrobial Resistance Patterns and Serological Diversity of Shigella Species from Patient Isolates at University Teaching Hospital in Lusaka, Zambia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study is well designed however there are some comments should be corrected:

- The samples are collected in 2021, and the study didnt mention if any changes in resistance patterns happened after then. given the dynamic nature of antibiotic resistance epidemiological studies should be longitudinal or update.

- in a group sample demographic informations are not enough specialy age is very wide group from 1 to 75 years old, also in statistical approach it is not considered.

- importantly within antibiotic resistance testing control is not considered

 

Author Response

Point-to-point responses to Reviewer # 1

Comments and Suggestions for Authors

The study is well designed however there are some comments should be corrected:

Responses:

1. i) The samples are collected in 2021, and the study didn’t mention if any changes in resistance patterns happened after then. given the dynamic nature of antibiotic resistance epidemiological studies should be longitudinal or update.

Thank you for the observation. Our study looked at archived samples after confirming them biochemically. The quantities of isolates per year were not adequate to analyze the resistance trends. However, our future studies will make reference to this study. We have included this as one of our limitations.

 

1. ii) In a group sample demographic informations are not enough specialy age is very wide group from 1 to 75 years old, also in statistical approach it is not considered.

Thank you for this observation, however, Shigella infections in Zambia are seasonal, and at the time of the study, only forty-eight suspected Shigella archived isolates were available, representing an exhaustible list of Shigella samples. This could have contributed to the wide range of age observed in the sample demographics. Looking at the small sample size and the convenient sampling method, we could not do inferential analysis.

 

iii) importantly within antibiotic resistance testing control is not considered

Thank you for the comment.

The antibiotic resistance testing control was considered and this was mentioned in the material and methods section subheading 2.2. We have however revised it from “The laboratory strain Escherichia coli (ATCC 25922) was used for quality control. This organism is a whole-genome sequenced quality control strain recommended by CLSI for antimicrobial susceptibility testing” to “For quality control, a whole-genome sequenced laboratory strain of Escherichia coli (ATCC 25922), recommended by CLSI for antimicrobial susceptibility testing, was utilized.” See lines 121-123 of the revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled "Antimicrobial Susceptibility Patterns and Sero-Diversity of Shigella Species from Patients’ Isolates at the University Teaching Hospitals in Lusaka, Zambia" (bacteria-3385228) was authored by Mike Nundwe et al. The authors investigated the occurrence of Shigella species in patients diagnosed with diarrhea across various locations in Zambia. The researchers also evaluated the antibiotic resistance profiles of Shigella species and their serotypes.

This manuscript would make a significant contribution to the Journal of Bacteria by examining the prevalence of Shigella species across different age groups, their antibiotic resistance, and serotypes.

The authors may wish to consider making some revisions in certain sections to further strengthen the presentation and impact of their work. These could include:

Title of the manuscript: Alternatively, the topic can be written as: "Antimicrobial Resistance Patterns and Serological Diversity of Shigella Species from Patient Isolates at University Teaching Hospitals in Lusaka, Zambia." However, the title is not sufficiently reflected in the main text of the document, especially in the introduction, which lacks information on the serotypes of Shigella species. Additionally, the title suggests that more than one hospital was involved in the study.

Authors’ information: Authors should consider providing the full address for all.

Abstract: The authors should consider revising the background information to focus on the relevant issue, followed by a clear statement of the study's aim before introducing the methodology. The results may also indicate the serotypes of the Shigella species identified in the study.

Line 33: Write the name of the organism in full, follow the rules of Taxonomy.

Line 39: “WHO” the term is being introduced for the first time, authors should consider writing it out in full.

 

Introduction:

Line 48-49: Authors can consider to revising the sentence to: In Zambia, more than 30% of children experiencing diarrhea are infected with Shigella.

Line 54-55: Sentence can be revised to: Shigella has a notable resistance to stomach acid, and an infectious dose can be as low as 10 bacteria.

The first paragraph doesn't transition well into the second. The authors could move the third sentence of the first paragraph to the end of that paragraph to create a better connection to the second.

The authors should revise the study's aim, as they are not only examining phenotypic characteristics but also conducting molecular analysis on beta-lactamase enzymes. Additionally, the antibiotic resistance profiles should be included.

Materials and Methods:

The authors referenced a supplementary file, Table S1; they should consider including a title for the supplementary table.

Line 103: The authors should be prompted to mention that the bacterial isolates identified as Shigella species underwent antimicrobial susceptibility testing using the Kirby Bauer method, as detailed by ..........

Line 108-110: Authors should consider specifying the volume plated on MHA plates.

Line 113-115: Authors may want to consolidate the information into a single summarized sentence.

Line 119: Instead of using the term "single primer PCR," it would be better to say "conventional PCR."

Line 117-127: Authors should ensure consistency in time units by using either all abbreviations or full terms. For instance, "seconds" can be shortened to "sec."

Authors should make sure that the names of microorganisms are italicized consistently throughout the manuscript.

Results:

Figure 1: The materials and methods section mention the collection of samples from different regions of the country, but this is not discussed in the results section. Furthermore, the ages of the patients are introduced here for the first time.

Table 1: The serogroups for the Shigella species are not indicated. For example, Shigella flexneri serotypes 2a, 1b, 3a, 4a, and 6 are responsible for the majority of shigellosis cases in developing countries.

Line 158: Since TET was defined earlier, the abbreviation can be used.

Line 162: Remove brackets on AMC

In Figure 2, authors may want to label the Y-axis with "antibiotics" instead of "antimicrobials."

In section 3.4, authors should specify whether the PCR products were sequenced to confirm the genes of interest.

Figure 4: What is the supplier for the molecular weight marker? Authors might also consider relocating the image of the PCR products to the supplementary file.

Line 200-201: Authors should consider rephrasing the sentence to: This study, along with others conducted in different locations [32,33], also identified S. sonnei as the most predominant species among children.

Line 229 to 234: Authors may want to consider the availability of sanitation services in the mentioned area, including access to clean water and sanitation facilities as part of the discussion of their results.

The conclusion still does not include the serotype data or the relationship to sanitation services in the areas from which the samples were collected.

Comments on the Quality of English Language

The quality of English used in the manuscript is good, but the problem on linking some sections as indicated in the comments for authors. 

Author Response

Point-to-point responses to Reviewer # 2

 Comments and Suggestions for Authors

The manuscript titled "Antimicrobial Susceptibility Patterns and Sero-Diversity of Shigella Species from Patients’ Isolates at the University Teaching Hospitals in Lusaka, Zambia" (bacteria-3385228) was authored by Mike Nundwe et al. The authors investigated the occurrence of Shigella species in patients diagnosed with diarrhea across various locations in Zambia. The researchers also evaluated the antibiotic resistance profiles of Shigella species and their serotypes. This manuscript would make a significant contribution to the Journal of Bacteria by examining the prevalence of Shigella species across different age groups, their antibiotic resistance, and serotypes. The authors may wish to consider making some revisions in certain sections to further strengthen the presentation and impact of their work. These could include:

Responses:

Thank you for finding our piece of work interesting and for your recommendations. We would like to provide more information to the comments as follows:

• Title of the manuscript: Alternatively, the topic can be written as: "Antimicrobial Resistance Patterns and Serological Diversity of Shigella Species from Patient Isolates at University Teaching Hospitals in Lusaka, Zambia." However, the title is not sufficiently reflected in the main text of the document, especially in the introduction, which lacks information on the serotypes of Shigella Additionally, the title suggests that more than one hospital was involved in the study.

 

This is well noted. The authors have revised the title of the manuscript to read “Antimicrobial Resistance Patterns and Serological Diversity of Shigella Species from Patient Isolates at University Teaching Hospitals in Lusaka, Zambia”. 

The study was conducted at the largest reference hospital but it is called “hospitals” because it has different specialties though using one microbiology laboratory. The information on serotypes of Shigella species has been included in the introduction and it reads “The genus Shigella consists of four species—Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei—all of which are pathogenic to humans and cause shigellosis, a severe form of bacillary dysentery characterized by diarrhea, fever, and abdominal pain [1,2]. The four Shigella species are classified into serotypes and subserotypes based on the O-antigen of their cell envelope lipopolysaccharides [3].  S. dysenteriae comprises 15 serotypes, with S. dysenteriae type 1 being the most pathogenic, S. flexneri (19 serotypes and subserotypes), S. boydii (19 serotypes), and S. sonnei (one serotype).” See lines 46-53 of the revised manuscript.

 

Authors’ information: Authors should consider providing the full address for all.

The full addresses of all the authors have been provided in the revised manuscript.

 

 

• Abstract:

The authors should consider revising the background information to focus on the relevant issue, followed by a clear statement of the study's aim before introducing the methodology.

Thank you for the comment.

The background has been revised to “Shigella species, are the leading cause of human shigellosis. In Zambia, more than 30% of children experiencing diarrhea are infected with Shigella species. The increasing resistance of Shigella spe-cies to the recommended therapy is of great concern.”

The results may also indicate the serotypes of the Shigella species identified in the study.

Speciation of the Shigella isolates was done using the slide agglutination method using the antisera and manufacturer’s manual. We could only identify Shigella species at the genus level due to limited resources. Therefore, we hope to explore more on the Shigella serotypes in future studies.

However, we have revised our introduction section as below ; (revised manuscript, lines 46-53).

“The genus Shigella consists of four species—Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei—all of which are pathogenic to humans and cause shigellosis, a severe form of bacillary dysentery characterized by diarrhea, fever, and abdominal pain. [1,2]. The four Shigella species are classified into serotypes and subserotypes based on the O-antigen of their cell envelope lipopolysaccharides [3].  S. dysenteriae comprises 15 serotypes, with Shigella dysenteriae type 1 being the most pathogenic, S. flexneri (19 serotypes and subserotypes), S. boydii (19 serotypes), and S. sonnei (one serotype).”

 

Line 33: Write the name of the organism in full, follow the rules of Taxonomy.

This has been worked on, it now reads “Shigella flexneri” in lines 34 and 35 of the revised manuscript.

Line 39: “WHO” the term is being introduced for the first time, authors should consider writing it out in full.

This has been written in full “World Health Organization”, line 39 of the revised manuscript.

 

• Introduction:

Line 48-49: Authors can consider to revising the sentence to: In Zambia, more than 30% of children experiencing diarrhea are infected with Shigella.

This has been revised as “In Zambia, more than 30% of children experiencing diarrhea are infected with Shigella species”, lines 54-55 of the revised manuscript.

Line 54-55: Sentence can be revised to: Shigella has a notable resistance to stomach acid, and an infectious dose can be as low as 10 bacteria.

This has been worked on, as indicated in lines 60-62 of the revised manuscript.

The first paragraph doesn't transition well into the second. The authors could move the third sentence of the first paragraph to the end of that paragraph to create a better connection to the second.

The authors have worked on this based on the recommendation, see lines 66-68 of the revised manuscript.

The authors should revise the study's aim, as they are not only examining phenotypic characteristics but also conducting molecular analysis on beta-lactamase enzymes. Additionally, the antibiotic resistance profiles should be included.

The authors have worked on this based on the recommendation it now reads; “This study, therefore, investigated the antibiotic resistance profiles, phenotypic and genotypic characteristics of Shigella isolates at the University Teaching Hospitals, the highest referral hospital in Zambia.” See the revised manuscript lines 94-96.

• Materials and Methods:

The authors referenced a supplementary file, Table S1; they should consider including a title for the supplementary table.

Thank you.

The title of the supplementary table has been included and it now reads as “Metadata and susceptibility patterns for the studied isolates”. see the attached revised supplementary table.

Line 103: The authors should be prompted to mention that the bacterial isolates identified as Shigella species underwent antimicrobial susceptibility testing using the Kirby Bauer method, as detailed by ..........

Thank you.

This has been worked on and it reads as “The bacterial isolates identified as Shigella species underwent antimicrobial susceptibility testing using the Kirby Bauer method, as detailed by ....” Kindly see the revised manuscript lines 113-114.

 

Line 108-110: Authors should consider specifying the volume plated on MHA plates.

The Kirby-Bauer disk diffusion method was carried out as described in the protocol by Hudzicki (2009). However, the actual volume of the inoculum could not be quantified, because a dry sterile swab was used for inoculation of the prepared bacterial suspension.  

Line 113-115: Authors may want to consolidate the information into a single summarized sentence.

Thank you.

This has been worked on, from “The laboratory strain Escherichia coli (ATCC 25922) was used for quality control. This organism is a whole-genome sequenced quality control strain recommended by CLSI for antimicrobial susceptibility testing” to “For quality control, a whole-genome sequenced laboratory strain of Escherichia coli (ATCC 25922), recommended by CLSI for antimicrobial susceptibility testing, was utilized.” See lines 123-125 of the revised manuscript.

Line 119: Instead of using the term "single primer PCR," it would be better to say "conventional PCR."

Thank you.

This has been changed to read “Conventional.” See the revised manuscript line 129.

Line 117-127: Authors should ensure consistency in time units by using either all abbreviations or full terms. For instance, "seconds" can be shortened to "sec."

Thank you for your feedback. This is well noted, and authors have opted to go for a full term “seconds”. See the revised manuscript lines 135 and 136.

Authors should make sure that the names of microorganisms are italicized consistently throughout the manuscript.

Thank you for the feedback.

This has been worked on, as all the names of microorganisms have been italicized in the revised manuscript.

 

Results:

Figure 1: The materials and methods section mention the collection of samples from different regions of the country, but this is not discussed in the results section. Furthermore, the ages of the patients are introduced here for the first time.

Thank you very much for the comment.

We agree with the observation, however, the study used archived clinical isolates that were collected from different regions of the country and were referred to the largest referral hospital (The University Teaching Hospitals) where the study was conducted. Therefore, at the time of the study, the authors used the information collected from the laboratory registers as shown in the attached Supplementary table. We have however included a sentence on metadata collection “The information on the archived isolates such as age, sex, and residence was collected from the laboratory registers” in the methodology section, lines 109-111.

 

Table 1: The serogroups for the Shigella species are not indicated. For example, Shigella flexneri serotypes 2a, 1b, 3a, 4a, and 6 are responsible for the majority of shigellosis cases in developing countries.

Thank you very much for this observation. However, at the time of the study, we could only identify Shigella species at the genus level due to limited resources. Therefore, we hope to explore more on the Shigella serotypes in future studies.

Line 158: Since TET was defined earlier, the abbreviation can be used.

Thank you.

This has been worked on, it's now “TET”. Line 177 of the revised manuscript.

Line 162: Remove brackets on AMC

Thank you.

This has been worked on, it's now “AMC”.  Line 181 of the revised manuscript.

In Figure 2, authors may want to label the Y-axis with "antibiotics" instead of "antimicrobials."

Thank you.

This has been worked on.

 

In section 3.4, authors should specify whether the PCR products were sequenced to confirm the genes of interest.

Thank you for the feedback.

Sequencing was not done to confirm the resistance gene of interest. Only PCR was performed to confirm the presence of the bla_TEM gene. However, we intend to perform WGS in future studies, which would possibly include the samples from this current study.

Figure 4: What is the supplier for the molecular weight marker? Authors might also consider relocating the image of the PCR products to the supplementary file.

Thank you very much. The supplier of the molecular weight marker has been included as “(HiGenoMB-Himedia, Mumbai, India)” and Figure 4 has been relocated to the Supplementary file as “(Figure S1)”. See the revised manuscript line 211-212 and the attached file named Figure S1.

Line 200-201: Authors should consider rephrasing the sentence to: This study, along with others conducted in different locations [32,33], also identified S. sonnei as the most predominant species among children.

Thank you very much for the recommendation. The sentence has been rephrased accordingly and it now reads as “This study, along with others conducted in different locations [36,37] also identified S. sonnei as the most predominant species among children.” See the revised manuscript lines 219-220.

 

Line 229 to 234: Authors may want to consider the availability of sanitation services in the mentioned area, including access to clean water and sanitation facilities as part of the discussion of their results.

Thank you for the feedback.

In the manuscript, we have added, "Furthermore, these facilities are located in densely populated urban areas with inadequate sanitation and hygiene services.” Lines 250-251 of the revised manuscript. The demographic data captured from the registers was limited which affected our discussion on specific sanitation services per residence area. Therefore, we aim to capture more epidemiological details in future studies to explain better the impact of sanitation services in the mentioned areas.

We also explained the sanitation situation in Zambia in the lines Lines 81-89 of the revised Manuscript.

“In Zambia, access to clean water and sanitation infrastructure is limited; the majority of the population has inadequate sanitary facilities, with only 64% using basic drinking water services and 33% basic sanitation services, while 10% practice open defecation [22]. Additionally, only 24% of the population can access basic hygiene services such as hand washing facilities with soap and water [22]. Therefore, contaminated water sources, inadequate sewage disposal, and limited access to healthcare facilities increase the frequency and spread of Shigella. Public health efforts in Zambia involve initiatives to improve water and sanitation infrastructure, promote hygiene practices, and enhance healthcare services to diagnose and treat Shigella infections promptly [23,24]”. Lines 81-89 of the revised Manuscript.

The conclusion still does not include the serotype data or the relationship to sanitation services in the areas from which the samples were collected.

Thank you for the comment

We could only identify Shigella species at the genus level due to limited resources. Therefore, we hope to explore more on the Shigella serotypes in future studies. However, a sentence has been added and it reads “Speciation of the isolates revealed a serological diversity of the four Shigella species – S. flexneri, S. sonnei, S. boydii, and S. dysenteriae.” Lines 278-280 of the revised manuscript.

A statement has been added on the sanitation services in the conclusion. It reads “Additionally, we recommend improved sanitation services as key to infection prevention and control, particularly in endemic areas with a high prevalence of shigellosis." Lines 286-288 of the revised manuscript.

 

Comments on the Quality of English Language

The quality of English used in the manuscript is good, but the problem on linking some sections as indicated in the comments for authors.

Thank you very much for the comment. We have read through again to correct the grammatical errors using Grammarly.

Reviewer 3 Report

Comments and Suggestions for Authors

This is a retrospective and descriptive study that reports on the antimicrobial susceptibility profile and serodiversity of Shigella species isolated from patients admitted to Lusaka University Hospital, a referral hospital in Zambia. Although the number of isolates analyzed was small (a limitation that authors must present in the manuscript), a high prevalence of multidrug-resistant Shigella species was identified (importance of the study for the region).

The manuscript is technically sound and generally well written. A few minor issues need to be clarified.

 

As Shigella corresponds to the genus, whenever referring to the bacterium include spp, sp or species (whichever is most appropriate). Please also check that all scientific names are in italics.

Lines 83-84: “While other studies have reported AMR in children among Shigella species in Zambia”... The bacterium that presents AMR, not the child. I suggest changing it to: ”While other studies have reported AMR in Shigella species isolated from children in Zambia”...

In section 3.2. the authors describe the general antimicrobial susceptibility profile and then present it for S. flexneri and S. sonnei. In line 158, referring to fluoroquinolones, is the result general or only for S. flexneri? If it is general, the data has already been presented for CIP. Please clarify.

Line 162: please remove the brackets from (AMC)

Table 1: As in the discussion the authors state that as in other studies, the present study identified S. sonnei (lines 200-201) as the predominant species in children, I suggest adding a column in Table 1 indicating the age group in which the bacterium was isolated.

Table 2: Does the “Facility of origin” column correspond to the sectors in the same hospital? In the discussion it was mentioned that the isolates were recovered in the reference hospital (line197). The methodology states that the bacteria were isolated from 48 patients in 10 health facilities in three districts (lines 97-98). Please clarify.

Please add limitations of the study.

Author Response

Point-to-point responses to Reviewer # 3

 

Comments and Suggestions for Authors

This is a retrospective and descriptive study that reports on the antimicrobial susceptibility profile and serodiversity of Shigella species isolated from patients admitted to Lusaka University Hospital, a referral hospital in Zambia. Although the number of isolates analyzed was small (a limitation that authors must present in the manuscript), a high prevalence of multidrug-resistant Shigella species was identified (importance of the study for the region).

The manuscript is technically sound and generally well written. A few minor issues need to be clarified.

 Responses:

Thank you for finding our piece of work technically sound and generally well-written, and for your recommendations. We would like to provide more information to the comments as follows:

As Shigella corresponds to the genus, whenever referring to the bacterium include spp, sp or species (whichever is most appropriate). Please also check that all scientific names are in italics.

Thank you for the feedback.

The authors have addressed this and “Shigella” is now accompanied by species as “Shigella species” in the revised manuscript.

 

Lines 83-84: “While other studies have reported AMR in children among Shigella species in Zambia”... The bacterium that presents AMR, not the child. I suggest changing it to: ”While other studies have reported AMR in Shigella species isolated from children in Zambia”...

Thank you for this recommendation. We have rephrased the sentence accordingly. It now reads as “While other studies have reported AMR in Shigella species isolated from children in Zambia” See lines 91-92 of the revised manuscript.

 

In section 3.2. the authors describe the general antimicrobial susceptibility profile and then present it for S. flexneri and S. sonnei. In line 158, referring to fluoroquinolones, is the result general or only for S. flexneri? If it is general, the data has already been presented for CIP. Please clarify.

Thank you very much for this observation. The author would like to clarify that the result is general and they have since deleted the repeating statement. See the revised manuscript lines 177-178.

 

Line 162: please remove the brackets from (AMC)

Thank you.

This has been worked on, it's now “AMC”. See the revised manuscript line 181

 

Table 1: As in the discussion the authors state that as in other studies, the present study identified S. sonnei (lines 200-201) as the predominant species in children, I suggest adding a column in Table 1 indicating the age group in which the bacterium was isolated.

Thank you very much for the feedback.

This has been worked on, see Table 1 of the revised manuscript.

 

Table 2: Does the “Facility of origin” column correspond to the sectors in the same hospital? In the discussion it was mentioned that the isolates were recovered in the reference hospital (line197). The methodology states that the bacteria were isolated from 48 patients in 10 health facilities in three districts (lines 97-98). Please clarify.

Thank you very much for the comment, the authors would like to clarify as follows:

Yes, the samples were collected from 10 facilities in three districts. Of the 10 health facilities, two are sectors of the study center. While 8/10 are facilities that refer patients to the University Teaching Hospital. These samples were referred to the University Teaching Hospitals which is also the center for all clinical microbiological investigations. This means that microbiological investigations on these samples were conducted at the referral hospital and not the health facilities where these samples came from.

 

Please add limitations of the study.

Thank you very much for the recommendation and this has been added to the study.  

The limitation reads “The small sample size affected the generalization of the findings and to identify significant differences or relationships. Furthermore, the absence of comprehensive epidemiological data hindered the contextualization of the results, making it challenging to account for confounding variables such as socioeconomic factors and underlying clinical conditions. Consequently, future studies should involve larger sample sizes and robust epidemiological data to make meaningful statistical inferences”. See lines 290-296 of the revised manuscript.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled "Antimicrobial Susceptibility Patterns and Sero-Diversity of Shigella Species from Patients’ Isolates at the University Teaching Hospitals in Lusaka, Zambia" (bacteria-3385228) was authored by Mike Nundwe et al. The authors have addressed some of the concerns raised during the initial review process. However, a few comments remain unaddressed; please see the comments below:

Introduction:

The section has improved, author should consider sticking to the format, with full stop before reference (line 50).

Line 97-99: The sentence could be taken out and this can be used in the conclusion of the findings.

Materials and Methods:

Authors should consider specifying the volume plated on MHA plates.

Section 2.2: Authors should clarify on the antibiotic susceptibility tests if they were conducted in duplicate, triplicate or not?

Section 2.3: The authors did not specify whether the PCR products were sequenced, which is crucial information for confirming the analyzed genes. Additionally, what positive controls were employed in the PCR if sequencing was not performed?

Line 136: Authors should ensure consistency in time units by using full word. For instance, "min" can be written as "minutes"

Line 137-138: Authors should include the voltage used in electrophoresis.

Results:

Table 1: The authors should clarify what the numbers in brackets represent in the "most prevalent age category" column.

Table 2: The percentages should be expressed as the exact value of 12.5%, as the current figures have been rounded and total 102%.

The formatting of the references is inconsistent; for instance, reference 30 is in all caps, while reference 35 has the authors' names in bold. Additionally, the names of microorganisms are not italicized in references 4, 6, 33, 42, 53, etc.

Author Response

RESPONSE TO THE REVIEWER’S COMMENTS ROUND 2

The manuscript titled "Antimicrobial Susceptibility Patterns and Sero-Diversity of Shigella Species from Patients’ Isolates at the University Teaching Hospitals in Lusaka, Zambia" (bacteria-3385228) was authored by Mike Nundwe et al. The authors have addressed some of the concerns raised during the initial review process. However, a few comments remain unaddressed; please see the comments below:

 

Thank you very much for your feedback and comments. The authors have highlighted in yellow all the changes made to the manuscript. The authors would like to respond as follows:

Introduction:

The section has improved, author should consider sticking to the format, with full stop before reference (line 50).

Thank you for your observation. The authors have adhered to the format with a period at the end of the reference. See the revised manuscript (line 50)

 

Line 97-99: The sentence could be taken out and this can be used in the conclusion of the findings.

Thank you for the feedback. The authors have moved out the sentence. See lines 96-98 of the revised manuscript.

 

Materials and Methods:

Authors should consider specifying the volume plated on MHA plates.

Thank you for your feedback.

The Kirby-Bauer disk diffusion method was carried out as described in the protocol by Hudzicki (2009). This protocol involves dipping a sterile swab in a 0.5 McFarland-standardized bacterial suspension and pressing against the tube to remove excess inoculum but does not accurately measure the inoculum on the swab, thus we could not determine the amount plated.

We have since included the following statement in the revised manuscript, which now reads “According to the Kirby-Bauer disk diffusion method described by [29], inoculation involves dipping a sterile swab in a 0.5 McFarland-standardized bacterial suspension and pressing against the tube to remove excess inoculum but does not accurately measure the inoculum on the swab, thus we could not determine the amount plated.”  See lines 120-124

Section 2.2: Authors should clarify on the antibiotic susceptibility tests if they were conducted in duplicate, triplicate or not?

Thank you for your comment. The authors want to clarify that antibiotic susceptibility tests were not conducted in duplicate or triplicate.

Section 2.3: The authors did not specify whether the PCR products were sequenced, which is crucial information for confirming the analyzed genes. Additionally, what positive controls were employed in the PCR if sequencing was not performed?

Thank you for your comment. In the previous review we responded to this comment “In section 3.4, authors should specify whether the PCR products were sequenced to confirm the genes of interest” as follows:

“Thank you for the feedback.

Sequencing was not done to confirm the resistance gene of interest. Only PCR was performed to confirm the presence of the bla_TEM gene. However, we intend to perform targeted or WGS in future studies, which would possibly include the samples from this current study”

For the PCR positive control, a previously characterized strain of Escherichia coli using whole genome sequencing with Strain-ID, Zam_UTH_03, was employed (Shawa et al., 2021).  

We have included this statement in the methods section 2.3 of the revised manuscript, which now reads “For the PCR positive control, a previously characterized strain of Escherichia coli using whole genome sequencing with Strain-ID, Zam_UTH_03 was employed [35].” Lines 141-143)

Line 136: Authors should ensure consistency in time units by using full word. For instance, "min" can be written as "minutes"

Thank you for your observation. We have written the time unit in full, which now reads “minutes" instead of "min." See the revised manuscript line 139.

 

Line 137-138: Authors should include the voltage used in electrophoresis.

Thank you for your observation. The authors have included the voltage used in electrophoresis. Please refer to the revised manuscript line 141.

Results:

Table 1: The authors should clarify what the numbers in brackets represent in the "most prevalent age category" column.

Thank you for your comment. The authors would like to clarify that the numbers in brackets represent proportions. We have added the percentages; they now appear as “(6/16; 37.5%)” etc.

Additionally, an explanation has been given below the table “*In brackets are the proportions and percentages for the most prevalent age category”

Table 2: The percentages should be expressed as the exact value of 12.5%, as the current figures have been rounded and total 102%.

Thank you for your feedback. We have addressed this as suggested; please refer to Table 2 in the revised manuscript line 201.

The formatting of the references is inconsistent; for instance, reference 30 is in all caps, while reference 35 has the authors' names in bold. Additionally, the names of microorganisms are not italicized in references 4, 6, 33, 42, 53, etc.

Thank you for the feedback. This has been addressed. Please refer to the revised manuscript for references 30, 35, 4, 6, 33, 42, 53, etc.