Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Resistome and virulence factor gene profiles during anaerobic  digestion of poultry litter: effects of micro-aeration and sound treatments

In this study, the inactivation of resistome and virulence factor genes in poultry litter (PL) using anaerobic digestion was investigated. The topic is of significant interest, and the study design is well structured with results that are clearly presented. However, several revisions are required to improve the manuscript at the scientific level.

 Following requests are necessary to be addressed to improve quality of the study.

 Title:

• In my opinion, the presented results suggest that the study focuses on evaluating anaerobic digestion for the inactivation or removal of antibiotic resistance genes (ARGs) in poultry litter. A revision of the title is recommended to improve alignment with the study aim.

Abstract:

• Line 11-12: The phrase “that also produce biogas” is unclear and the sentence should be rewritten for clarity.
• Line 12: How does your previous work in anaerobic digestion relate to this project, and how would you evaluate the link between increasing biogas production and the resulting impact on the resistome?
• Line 13 The study should first present a clearly defined aim, and then describe the procedures undertaken to address this aim.
• Line 14 The type of control mentioned is unclear and should be clearly explained.
• Line 17: It is necessary to clarify what optimization was performed; for clarity, the phrase “after optimization” should be deleted.
• Line 17: On what criteria did you base the selection of weeks 6, 23, and 42?
• Line 18: It should be clarified whether the statement 'Over 80% of the resistome was composed of four antimicrobial classes' applies to all four types of digesters
• Line 21: It is necessary to clearly define the control digester to properly interpret the results presented in this section.
• Line 21 -23: This observed result where ARG levels in the control digester declined while increasing over time in the other three digesters needs to be clearly interpreted. It should also be indicated whether this pattern is expected or unexpected, and whether the observed decline or increase is statistically significant.
• Line 31 : it would be interesting if the conclusion included results that recommend a specific type of digesters
• Line 32: Limitations of the current study and suggestions for future research should be included at the end of this section.

Introduction:

- Line 61: Adding a diagram, cartoon, or figure illustrating the components and processes of anaerobic digestion (AD) or the digesters would be helpful and make it easier for readers to understand

 

 

Results

• Line 91, As the total sample size was 21, the weight of each sample should be provided, along with clarification on whether the samples were collected from a single poultry farm or multiple farms.
• Line 104: The current subtitle does not correspond to the presented results and should be revised to clearly reflect the impact of various anaerobic digestion (AD) treatments on resistance genes
• Line 112: A title and descriptive legend for the figure are missing and should be included.
• If the efficiency of anaerobic digestion (AD) in inactivating or removing genes is influenced by gene type, function, or other characteristics, it should be explained why this was not tested. Conversely, if it is not influenced, the rationale for focusing on gene profiling despite the study’s primary aim being to evaluate AD efficiency should be clarified.

Discussion

 

• In need to explain if the result gotten is logical or some of them is unexpected
• It is necessary to cite recent studies that have profiled antibiotic resistance genes in the manure of other farm animals ( e.g DOI: 10.22207/JPAM.17.3.35)
• Clearly explain the limitations of the study and recommend further research
• Include a section that highlights and explains the clear contributions and implications of this study.

 

Method :

• Line 407: Details of the farm, including location, size, management practices, and the quantity of poultry litter used, should be included in this section

 

 

Author Response

Reviewer 1

Title: old: Resistome and virulence factor gene profiles during anaerobic digestion of poultry litter: effects of micro-aeration and sound treatments

 

In this study, the inactivation of resistome and virulence factor genes in poultry litter (PL) using anaerobic digestion was investigated. The topic is of significant interest, and the study design is well structured with results that are clearly presented. However, several revisions are required to improve the manuscript at the scientific level.

Response: We sincerely thank you for the positive feedback and constructive comments. We have revised the manuscript to address your concerns.

 Following requests are necessary to be addressed to improve quality of the study.

 

 Title: New: Evaluation of anaerobic digestion amended with micro-aeration and/or sound treatment on the resistome and virulence factor gene profiles in poultry litter

In my opinion, the presented results suggest that the study focuses on evaluating anaerobic digestion for the inactivation or removal of antibiotic resistance genes (ARGs) in poultry litter. A revision of the title is recommended to improve alignment with the study aim.

Response: Modified

 

Abstract:

Line 11-12: The phrase “that also produce biogas” is unclear and the sentence should be rewritten for clarity.

Response: revised for clarity.

Line 12: How does your previous work in anaerobic digestion relate to this project, and how would you evaluate the link between increasing biogas production and the resulting impact on the resistome?

Response: We removed the statement to avoid ambiguity.

Line 13 The study should first present a clearly defined aim, and then describe the procedures undertaken to address this aim.

Response: These are separated. Thanks!

Line 14 The type of control mentioned is unclear and should be clearly explained.

Response: clarified.

Line 17: It is necessary to clarify what optimization was performed; for clarity, the phrase “after optimization” should be deleted.

Response: deleted

Line 17: On what criteria did you base the selection of weeks 6, 23, and 42?

Response: these were clarified in the materials and methods. In the abstract, we modified them for clarity. Week 6 was when micro-aeration and sound treatments started. Weeks 23 and 42 were chosen based on the increasing levels of poultry litter addition schemes.

Line 18: It should be clarified whether the statement 'Over 80% of the resistome was composed of four antimicrobial classes' applies to all four types of digesters

Response: This is clarified by adding more information to the abstract. It is across all sequence reads and all 21 samples including experimental components (digestate used to initially seed the digesters, raw poultry litter samples, initial mix, and wood discs as a bedding material).

Line 21: It is necessary to clearly define the control digester to properly interpret the results presented in this section.

Response: This is now clearly defined above. Thanks.

Line 21 -23: This observed result where ARG levels in the control digester declined while increasing over time in the other three digesters needs to be clearly interpreted. It should also be indicated whether this pattern is expected or unexpected, and whether the observed decline or increase is statistically significant.

Response: A statement was added. Obviously, since the study was novel by applying sound and little air to a fully anaerobic system, we did not know what to expect. It is a new finding. Statistical comparison was not possible since only one sample was processed per digestion at each time point. The inference was merely empirical based on observation of the bar plots.

Line 31 : it would be interesting if the conclusion included results that recommend a specific type of digesters

Response: This was added. Sound treated AD seems promising for reducing the abundance of ARGs and VFGs. But more research is needed.

Line 32: Limitations of the current study and suggestions for future research should be included at the end of this section.

Response: included in the abstract briefly due to word limitation. The points raised by the Reviewer in this section were elaborated under Discussion and Conclusion section of the paper.

 

Introduction:

- Line 61: Adding a diagram, cartoon, or figure illustrating the components and processes of anaerobic digestion (AD) or the digesters would be helpful and make it easier for readers to understand

Response: Added.

 

Results

Line 91, As the total sample size was 21, the weight of each sample should be provided, along with clarification on whether the samples were collected from a single poultry farm or multiple farms.

Response:

The weight of samples used for DNA extraction was in the commercial kit manual (100 mg for solids and 250 µl for liquids) which was already referenced in the Material and Methods. We note that DNA extraction, library preps, sequencing, and bioinformatics were done by a commercial sequencing service. So, we followed their typical report for publications. Also, as stated in Materials and Methods,” poultry litter [was] obtained from a commercial broiler chicken farm”.

 

Line 104: The current subtitle does not correspond to the presented results and should be revised to clearly reflect the impact of various anaerobic digestion (AD) treatments on resistance genes

Response: modified. Thanks!

 

Line 112: A title and descriptive legend for the figure are missing and should be included.

Response: sorry, if the Reviewer missed it but these were presented below the Figure.

 

If the efficiency of anaerobic digestion (AD) in inactivating or removing genes is influenced by gene type, function, or other characteristics, it should be explained why this was not tested. Conversely, if it is not influenced, the rationale for focusing on gene profiling despite the study’s primary aim being to evaluate AD efficiency should be clarified.

Response: Although these were presented in Table 1, the Figures, and Supplementary Tables, we did not sufficiently describe subtle details. Now, we highlighted both when differences were not found, and when differences were evident.

 

Discussion

In need to explain if the result gotten is logical or some of them is unexpected

Response: This is added to the discussion part here and there.

 

It is necessary to cite recent studies that have profiled antibiotic resistance genes in the manure of other farm animals ( e.g DOI: 10.22207/JPAM.17.3.35)

Response: a reference to a comprehensive review of field studies that evaluated anaerobic digestion of animal manure on tetracycline resistance genes (as an example) is added. Most studies focused on dairy cattle manure, and scarce for poultry litter which was the focus of the present study. Studies were based on PCR (including qPCR) based approaches that could only analyze a handful of resistance genes.

 

Clearly explain the limitations of the study and recommend further research

Response: This was done already: example evaluating different temperatures since the present study was done at ambient temperature. We also mentioned the need for on-farm studies to clearly understand the dynamics of ARGs in poultry production. Further assessment of on-farm AD systems for the removal of ARGs and bacteria using the shotgun metagenomics will be helpful. Limitations with areas for future research are now included in more detail.

 

Include a section that highlights and explains the clear contributions and implications of this study.

Response: the use of shotgun to assess the novel method of aeration and sound treatment was presented under discussion. It gives a comprehensive understanding of total resistome. Analysis of the components provides information as to the source of ARGs into AD system. We added this information in the revised manuscript, limitations and recommendations for future research under the Conclusion section.

 

Method :

Line 407: Details of the farm, including location, size, management practices, and the quantity of poultry litter used, should be included in this section

Response: we already provided the location of the farm, included its size typical of U.S. broiler production as far as management goes. Sorry, unfortunately, we can’t provide more than what is provided in the manuscript for the privacy of the farm. Farm description is not important for this manuscript since we conducted experimental study and not farm survey. We could have used poultry litter from any other farm. The amounts of poultry litter added to the digesters were already clearly presented in the material and methods.

Reviewer 2 Report

Comments and Suggestions for Authors

Poultry litter contains organic matter that is degradable and can generate biogas under anaerobic conditions. This research holds great importance in terms of biogas generation, and the authors studied the antibiotic and virulence factors associated with genes in PL anaerobic digestion.

Material/method:

Line 410: The authors used a single digester for each condition, which is a limitation of this study. Did the authors further use triplicate PL for DNA extraction and metagenomics from each digester, and how can the authors justify the statistical significance of the study using 4 digesters, one for each treatment?

Did the authors measure physicochemical parameters such as pH and ammonia during the digestion of PL?

Line 415: Author used 1KHz frequency? What was the reason behind this specific frequency?

Line 426: The authors used week 6 as a baseline, but why was time 0 not used?

Was the author normalizing the sample across the treatment and control groups before the bioinformatic analysis?

Author Response

Reviewer 2

Comments and Suggestions for Authors

Poultry litter contains organic matter that is degradable and can generate biogas under anaerobic conditions. This research holds great importance in terms of biogas generation, and the authors studied the antibiotic and virulence factors associated with genes in PL anaerobic digestion.

Response: We thank the Reviewer for their feedback and constructive comments and suggestions. We presented biogas production data in Loughrin et al 2020 (doi: 10.3390/environments7020011). Both the sound and micro-aeration treatments were found to enhance biogas production whereas the combination of the two did not significantly increase biogas production. This was presented in the Introduction with the newly added reference with respect to sound.

 

Material/method:

Line 410: The authors used a single digester for each condition, which is a limitation of this study. Did the authors further use triplicate PL for DNA extraction and metagenomics from each digester, and how can the authors justify the statistical significance of the study using 4 digesters, one for each treatment?

Response: We admit that this is a limitation of the study, but practical limitations were imposed by the scale of the digesters and the amount of poultry litter, and time, that would have been required to feed multiple replicates of the digesters. Metagenomic sequencing and bioinformatics is also cost prohibitive. We did not use triplicates of PL for DNA extraction and metagenomics. I believe having more biological replicates, i.e., increasing the sample size with more digesters per treatment group is more powerful to make rigorous statistical and biological inferences than technical replicates. However, due to practical limitations (as stated above), this is impossible for small scale studies. This study may be used to generate hypothesis that can be tested in future large-scale studies or using multi-center studies to pool the results to have more statistical power. We did not compare digesters on each sampling week: thus, any inference was made based on the bar-graphs. We conducted statistical comparison of the treatment groups by aggregating the results of the three weeks observation into the AD treatment group (in which case we had three observation/treatment). We agree this is another limitation of the study. We added these limitations under conclusions.

 

Did the authors measure physicochemical parameters such as pH and ammonia during the digestion of PL?

Response: Yes, we did and the results are presented in Loughrin et al. 2020, and Loughrin et al. 2023 (doi: 10.3390/microorganisms11092349). This information is included in the manuscript.

 

Line 415: Author used 1KHz frequency? What was the reason behind this specific frequency?

Response: We added the rationale for using the 1 kHz frequency in the text. Thank you!

 

Line 426: The authors used week 6 as a baseline, but why was time 0 not used?

Response: Up until week 6 all four digesters were managed the same, without exposing any of them to micro-aeration or sound. Since micro-aeration and sound treatments started on week 6, it was considered the baseline for this experiment. The first 6 weeks of the experiment was the startup phase to ensure digesters became fully active. Nevertheless, we also presented the analysis of the resistome in the initial digestate, mix and poultry litter components. These were clearly described in the Materials and Methods.

 

Was the author normalizing the sample across the treatment and control groups before the bioinformatic analysis?

Response: As described on the ZYMOS sequencing service manual, the pipeline was made simple by auto normalization of the DNA inputs. What is needed is to pool libraries at equal amounts. Furthermore, microbial community standards ensure normalization of processes among samples.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors’ revisions satisfactorily address the requested corrections.