COVID-19 Pandemic Modulates the Environmental Contamination Level of Enteric Bacteria from WWTPs

Round 1

Reviewer 1 Report

1. The paper provides a comprehensive analysis of the modulation of environmental contamination levels with enteric bacteria through WWTPs during the COVID-19 pandemic. It is recommended to further discuss the diversity of enteric bacteria species and their specific impacts on the environment and public health.

2. The methods section details the sample collection, microbiological analysis, and statistical analysis methods. However, a more thorough discussion on the representativeness and limitations of the sample selection is recommended to enhance the universality of the research findings. 

3. The results section clearly shows the variations in the load of enteric bacteria in the influent and effluent of WWTPs in different regions during the pandemic. A comparison with similar studies in other parts of the world is recommended to demonstrate the universality or specificity of the situation in Romania. 

4. The discussion offers an in-depth analysis of the results and explores the impact of the COVID-19 pandemic on the efficiency of WWTPs. Discussions on improving WWTP treatment strategies to cope with similar public health crises in the future are recommended. Several paper related to wastewater treatment suggested to be read. (e.g. ECOL CHEM ENG S. 2014, 21(1):89-99) 

5. Overall, the study provides valuable insights into the impact of the COVID-19 pandemic on environmental microbial pollution. It is suggested to emphasize the policy and practical implications of the research in the conclusion, especially recommendations for enhancing WWTP treatment efficiency and environmental monitoring. 

Moderate editing of English language required. 

Author Response

Thanks to your observations, we improved the English quality and scientific aspects of the manuscript taking into account your recommendations as following:

• ESKAPE enteric bacteria impact on human and environmental health were discussed on lines 102-110
• the sampling protocol was detailed with the number of samples (replicates and average samples) and collection time at the 2.1 “sampling” section
• results from the WWTP were compared with literature studies at line 230
• we added some research options for enhancing WWTP treatment efficiency

Reviewer 2 Report

The research presented in the manuscript provides an interesting look at how the pharmaceuticals used COVID-19 pandemic on WWTP processes. However, to be suitable for publication, some further refinement is required. 

Introduction

The introduction's flow is difficult to follow, and the information, while interesting, is not relevant to the topic at hand.  For example, the first paragraph does not introduce the research topic well, and much of the information is tangential to the research topic. The research topic and relevance are not provided until line 52. 

Line 45-47: statements like the one made should have supporting references. 

Line 153-158: This information is perhaps more suitable for the methods section

Materials and Methods

The sampling information is not clear on the number of samples taken and the dates of sampling

Results and Discussion

It is unclear  what the authors mean by Covid-19 incidence average on lines 204 and 205

Lines 220-221: the statement made requires referencing due to the importance of the following statements 

Lines 237-238: What is included in the bacteriological loading discussed?

Lines 250 - 253: what is the supporting basis for the statement made?

The results linking pathogen loading and pharmaceuticals are not well supported. While the literature supports the hypothesis, how are the authors sure of the assumed interactions between antibiotics, pathogen loads, and antimicrobial resistance? 

Some information in the results and discussion is perhaps better suited to the introduction (e.g., lines 209-218) or edited to better relate to the interpretation of the results presented. 

The statistical analysis is very superficial; why have the authors limited the analysis to ANOVA only? There is no detailed information provided on the statistical analysis or the results observed. There needs to be more detailed analysis than noteing p values in the figure text. 

The English needs proofreading, focusing on clear, concise sentences and the overall flow of information. 

Author Response

Thank you for your comments which helped us improve the English quality and scientific aspects of the manuscript taking into account your recommendations as following.

The manuscript highlighted the impact of the Covid-19 pandemic on microbial populational structural as well as on microbial adaptation capacity induced by pharmaceutical compounds used in Covid-19 treatments. Microbial adaptation was mainly based on antibiotic resistant genes which were disseminated from wastewaters to aquatic environment through WWTP.

The introduction section was rearranged for an easier understanding and more supplemental information were provided to enhance the information flow of this section.

The sampling protocol was detailed with the number of samples (replicates and average samples) and collection time at the 2.1 “sampling” section. In addition, the Covid-19 incidence information was explained on line 205.

The “results and discussion” section was improved by adding references as a support basis for our statements. We also clarified in the text the significant differences obtained among various sets of results through the statistical analysis. The statistical technique used (Anova) provided the certainty of the results so that p value < 0.005 supports the significant differences obtained.

Round 2

Reviewer 1 Report

It can be accept. 

Author Response

Thank you for your kind words. 

Reviewer 2 Report

The authors have made a number of edits based on the review provided. Due to the lack of marked changes, it is difficult to assess all the changes made. 

There is still concern that the adequacy of a single 24-hour sample period for each of the three sampling events is adequate to account for the expected temporal variation in wastewater. Likewise, for surface water sampling. The authors may want to consider referring to comparable sampling approaches. 

The manuscript should consider the limitations of the study and further research to overcome limitations. 

Efforts have been made to improve the English; however, the paper could do with a thorough review. 

Author Response

We understand the concerns about the sampling campaigns, but we would like to draw the attention on the fact that these sampling campaigns were taken during Covid-19 peak periods with the same incidences values such as 5 for Timisoara and 3 for Valcea and Iasi. This link between sampling campaign period dates and incidences gave a robust results, backed up by p values, as mentioned in text (please the second row, first paragraph, Results and Discussion Section.

We hope that with the above explanations of the sampling campaigns correlated with the information about microbiological modulation environment by the Covid-19, especially enterobacteria from hospitals to environment, highlighted this manuscript value. In addition, we observed a high interests for this manuscript downloaded on preprint site

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

While the authors have made efforts to improve the manuscript, the conclusions are superficial and fail to highlight limitations and further research adequately.  Consideration should be given to these aspects by the authors prior to publication.