Review Reports - Environmental Surveillance of ESKAPE Bacteria in Wastewater and Rivers in the Vhembe District, South Africa: Public Health Risks from a One Health Perspective

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The abstract repeats “health risks to humans” and “One Health risks” (Lines 8–12, 22–23), which could be streamlined for clarity and conciseness.

Several sentences are long and contain grammatical errors that reduce readability. For example, Lines 76–85 contain a 10-line sentence that mixes justification, regional context, and study aims without proper breaks. Use Shorter, focused sentences

The claim that “approximately two million people will die worldwide because of antibiotic-resistant pathogens in the next two decades” (Lines 64–66) is written without specific recent reference

Lines 102–106) Only 500 mL of water was collected per sample, was this volume sufficient for detecting low pathogens.

(Lines 123–157) PCR controls are described, there is no mention of how contamination during DNA extraction or amplification was prevented (e.g., use of negative extraction controls).

(Lines 161–250) Table 2 is overly dense and difficult to interpret. The plus/minus symbols (+/–) for detection could be accompanied by quantitative counts or PCR Ct values to better reflect pathogen load.

(Lines 181–246) Claims of treatment effectiveness are descriptive rather than supported by evidence. The authors note seasonal differences in detection (e.g., August vs. December), but there is no statistical analysis of these trends (e.g., chi-square or ANOVA).

Large sections of the discussion repeat literature findings (Lines 170–198) analyse critically how the current results compare with or differ from these studies with latest references

(Lines 251–347) there is no information on the specific genes used for alignment, nor is there a discussion of bootstrap support values beyond a general statement (Lines 344–346).

Lines 329–333 the paper frequently mentions the One Health perspective, there is no analysis of animal or agricultural sources beyond wastewater.

(Lines 350–364) The statement that “environmental strains closely aligned with globally significant clinical reference sequences” (Lines 350–353) implies direct transmission but does not provide evidence of genetic exchange or directionality.

Check and correct frequent typographical issues such as “catapulted by” (Line 48), “antimicrobial resistance (AR) priority list” instead of AMR (Line 49), and tense inconsistencies (e.g., “were not detected” vs. “was detected”)

Author Response

Reviewers' comments and rebuttals

Reviewer one

The abstract repeats “health risks to humans” and “One Health risks” (Lines 8–12, 22–23), which could be streamlined for clarity and conciseness.

Response

Thank you for your comment

This has been amended in the manuscript

Several sentences are long and contain grammatical errors that reduce readability. For example, Lines 76–85 contain a 10-line sentence that mixes justification, regional context, and study aims without proper breaks. Use Shorter, focused sentences

Response

Thank you for your comment

This has been amended in the manuscript

The claim that “approximately two million people will die worldwide because of antibiotic-resistant pathogens in the next two decades” (Lines 64–66) is written without a specific recent reference

Response

Thank you for your comment

This has been amended in the manuscript

Lines 102–106) Only 500 mL of water was collected per sample. Was this volume sufficient for detecting low pathogens?

Response

Thank you for your comment

Yes, this volume was more than enough because it was sampled in duplicates

(Lines 123–157) PCR controls are described, there is no mention of how contamination during DNA extraction or amplification was prevented (e.g., use of negative extraction controls).

Response

Thank you for your comment

This has been amended in the manuscript

(Lines 161–250) Table 2 is overly dense and difficult to interpret. The plus/minus symbols (+/–) for detection could be accompanied by quantitative counts or PCR Ct values to better reflect pathogen load.

Response

Thank you for your comment

In the current study, we used conventional PCR as described in lines 120-148

(Lines 181–246) Claims of treatment effectiveness are descriptive rather than supported by evidence. The authors note seasonal differences in detection (e.g., August vs. December), but there is no statistical analysis of these trends (e.g., chi-square or ANOVA).

Response

Thank you for your comment

The effectiveness of the treatment was demonstrated in Table 2, where the presence of ESKAPE in the influent was detectable in comparison to the treated final effluent during the sampling period, as opposed to the seasonality. This was further supported by the fact that it was a surveillance study for different sampling periods, not in different seasons, which would require ANOVA or Chi-square to show any significant variation.

Large sections of the discussion repeat literature findings (Lines 170–198) analyse critically how the current results compare with or differ from these studies with latest references

Response

Thank you for your comment

This has been amended in the manuscript

(Lines 251–347) there is no information on the specific genes used for alignment, nor is there a discussion of bootstrap support values beyond a general statement (Lines 344–346).

Response

Thank you for your comment

This has been amended in the manuscript

Lines 329–333, the paper frequently mentions the One Health perspective; there is no analysis of animal or agricultural sources beyond wastewater.

Response

Thank you for your comment

The frequent reference to “One Health” is relevant to this study because activities such as agricultural work, bricklaying, fishing, swimming, and animals grazing are prominent along the adjacent rivers and around WWTPs in the setting of this study. This is why the One Health approach was relevant and still is.

(Lines 350–364) The statement that “environmental strains closely aligned with globally significant clinical reference sequences” (Lines 350–353) implies direct transmission but does not provide evidence of genetic exchange or directionality.

Response

Thank you for your comment

This has been amended in the manuscript

Check and correct frequent typographical issues such as “catapulted by” (Line 48), “antimicrobial resistance (AR) priority list” instead of AMR (Line 49), and tense inconsistencies (e.g., “were not detected” vs. “was detected”)

Response

Thank you for your comment

This has been amended in the manuscript

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

10-11. «In this approach, Wastewater treatment plants (WWTPs) play an important role in the dissemination of bacteria and antibiotic-resistant genes (ARGs) in the environment».

This is not quite correctly written: not in dissemination, but in hindering.

34-37. The authors write only and exclusively about surveillance, it may be worth asking a question (or even giving an answer to it) 1). Is it possible to do something with the purification system and the installation of sewage treatment plants in hospitals? 2). Adopt (and comply with) rules for the disposal of antibiotics in medical institutions and pharmaceutical companies.

43-44. «….ESKAPE pathogens have become a global pandemic …»

That's too big a statement. Better a problem, not a pandemic

64-66. «The authors also reported that, in the next two decades, approximately two million people will die worldwide because of antibiotic-resistant pathogens if no mitigation action is in place».

The link?

82-83. «....linking the inefficiency wastewater treatment…»

That's too big a statement. Cleansing statements are really ineffective and do not delay antibiotics at all. That wastewater treatment plants should directly detain an antibiotic or a bacterium resistant to it?

Was sampling carried out directly in the equipment complexes in the sewage treatment plant system at different stages of purification?

201-202. «…This was due to less runoff that was experienced in that month…»

How much wastewater was exactly discharged monthly?

You write about «….effective therapeutic treatment by dosing enough chlorine into the final effluent before…..»

To what extent does it quantitatively/concentrationally affect the contents of ESKAPE bacteria in wastewater?

When you write that « was not detected either in the upper or lower reaches of the river…» how many km is it? What is the flow rate, is it possible to propose a mathematical model of wastewater dilution by the volume of flowing river waters?

I would like to read in the Discussion or Conclusion about the solutions to the problem posed in the title of the article. You write that surveillance is needed, we know that surveillance in European countries has been conducted very carefully and meticulously for 20 years. How did he help in solving this problem? Might it be worth paying more attention to sewage treatment plants? What laws in Vhembe District, South Africa regulate the discharge of antibiotics into wastewater?

Author Response

Reviewer Two and rebuttals

10-11. «In this approach, Wastewater treatment plants (WWTPs) play an important role in the dissemination of bacteria and antibiotic-resistant genes (ARGs) in the environment». This is not quite correctly written: not in dissemination, but in hindering.

Response

Thank you for your comment

This has been amended in the manuscript

34-37. The authors write only and exclusively about surveillance, it may be worth asking a question (or even giving an answer to it) 1). Is it possible to do something with the purification system and the installation of sewage treatment plants in hospitals? 2). Adopt (and comply with) rules for the disposal of antibiotics in medical institutions and pharmaceutical companies.

Response

Thank you for your comment

Yes indeed.

This is a brilliant question, which we have also made recommendations to the Water Service Boards and the Department of Water and Sanitation based on the current and previous findings of our research

43-44. «….ESKAPE pathogens have become a global pandemic …»

That's too big a statement. Better a problem, not a pandemic

Response

Thank you for your comment

This has been amended in the manuscript

64-66. «The authors also reported that, in the next two decades, approximately two million people will die worldwide because of antibiotic-resistant pathogens if no mitigation action is in place». The link?

Response

Thank you for your comment

This has been amended as it was also raised by the other reviewer in the manuscript

82-83. «....linking the inefficiency wastewater treatment…»

Response

Thank you for your comment

This has been amended as suggested in the manuscript.

Was sampling carried out directly in the equipment complexes in the sewage treatment plant system at different stages of purification?

Response

Thank you for your comment

The sampling question was carried out as indicated in lines 99-101 of the manuscript.

201-202. «…This was due to less runoff that was experienced in that month…»

How much wastewater was exactly discharged monthly?

Response

Thank you for your comment

This has been amended in the manuscript

You write about «….effective therapeutic treatment by dosing enough chlorine into the final effluent before…..»

To what extent does it quantitatively/concentrationally affect the contents of ESKAPE bacteria in wastewater?

Response

Thank you for your comment

The recommended quantity of chloring dosage is between 5 to 20 mg/L, while the plants were dosing approximately 10 mg/L, which was considered enough.

When you write that « was not detected either in the upper or lower reaches of the river…» how many km is it? What is the flow rate, is it possible to propose a mathematical model of wastewater dilution by the volume of flowing river waters?

Response

Thank you for your comment

The distance of the sampling point from both upstream and downstream in relation to the discharge point of the WWTP was between 100 to 200 metres.

I would like to read in the Discussion or Conclusion about the solutions to the problem posed in the title of the article. You write that surveillance is needed, we know that surveillance in European countries has been conducted very carefully and meticulously for 20 years. How did he help in solving this problem? Might it be worth paying more attention to sewage treatment plants? What laws in Vhembe District, South Africa regulate the discharge of antibiotics into wastewater?

Response

Thank you for your comment

You are right with various groundbreaking research on the surveillance in the Northern hemisphere (Europe and the USA) and the far east of the Asian continent (China, Australia, etc.), yet in the southern hemisphere, particularly Sub-Saharan Africa (South Africa), little of this surveillance work is just in their initial phases. This study is even first of its kind in this part of the country (Vhembe) of South Africa which is triggered with what has been done across the globe hence that is why we are recommending expanded genomic surveillance and integrated AMR monitoring strategies, particularly in peri-urban and rural settings where sanitation infrastructure is limited as part of our conclusions.

Again, it was worth paying attention to sewage treatment and laws that govern the discharge of antibiotics. This is because there are no laws governing the discharge of antibiotics, which is strictly limited to the Vhembe district, but rather governing the whole country in terms of treatment of any hospital waste, agricultural, pharmaceuticals and industrial waste before being discharged into the main sewer line into the WWTP. There is a lack of commitment or illegal discharge of this waste from the facilities mentioned above into the main sewer line, which then enters the WWTP before treatment. This is the reason why we recommend an extensive review and continuation of policy/infrastructure development and further enforcement of laws regarding wastewater treatment. Further enforcement of both green (Wastewater treatment) and blue (treatment of drinking water) reports that are in place by water authority services and the Department of Water and Sanitation, together with the Department of Environmental Affairs.