Assessment of the Possibility of Using Bacterial Strains and Bacteriophages for Epidemiological Studies in the Bioaerosol Environment

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1.       In comparing Tables 1, 2, 5, and 6, it is inconsistent that the detection time for the survival rate in physiological saline is 4 hours, while in air it is 30 hours."

 2.       In Table 4, the inconsistency of the various concentrations of bacteriophages, as well as their nebulization and homogenization times, is noticable"

 3.       Considering the configuration of these fans, it cannot be ascertained if the indoor microbial concentration is uniform. To verify this, multiple detectors should be placed in various locations."

 4.        The indicators labeled as 'good', 'very good', etc. in Table 8 lack explicit concentrations."

 5.       The aim of this study is to investigate a model virus and bacteria for aerosol transmission research. We only looked at how well they survived under constant humidity, temperature, and in a saline solution. However, the control group was limited, so we need to compare them in different environments too. Also, using bacteriophage as a model virus might not be the best choice because coronaviruses like COVID-19 have different characteristics. It would be better to use similar weakly pathogenic coronaviruses or pseudo-viral particles for this research.

 

Comments on the Quality of English Language

Some sentences need to be revised to make them more concise and readable.

Author Response

Thank you very much for taking the time to review this manuscript and for your valuable comments, which in the future will allow more accurate rephrasing of the research results. All comments have been corrected in the text of the article. Below are the answers to each question.

1. In comparing Tables 1, 2, 5, and 6, it is inconsistent that the detection time for the survival rate in physiological saline is 4 hours, while in air it is 30 hours."

 

There was an error in the results, which has already been corrected in the article. Survival in saline fluid was tested over a longer period of time, as this was what the test required. The procedure itself involved preparation of the suspension, preparation of the nebulization station, nebulization and post-test control analysis. Such a procedure took between 1.5 and 2h, so survival in physiological fluid had to be no less than 2 hours. Survival in air, on the other hand, had to be longer than the test performed on the nebulization bench, i.e. no less than 5 minutes. The different survival analysis times are thus due to the different survival needs in a given medium (physiological fluid and air).

 

2. In Table 4, the inconsistency of the various concentrations of bacteriophages, as well as their nebulization and homogenization times, is noticable"

 

This inconsistency is due to the search for appropriate parameters, for each bacteriophage, based on the characteristics of the bacteriophage in question and the experience of those performing the tests. Table 4 lists the preliminary tests that were performed to select the appropriate bacteriophage. Detailed selection of nebulization parameters was performed in the next stage of the study. The authors realized that bacteriophage culture on host medium and storage may not be reflected in the nebulization analysis, as confirmed by the results in Table 6.

 It can be seen from Table 4 that both bacteriophage phi6 and phiX174 are countable using the nebulization method described, but phiX174 would require the use of a very large amount of bacteriophage (nebulization of the suspension without dilution) and a long nebulization time.  It turned out that the duration of the other elements of the procedure would be longer than the assumed time of passage through the airlock (as indicated by the project assumptions). This means that although a given phage may be countable after nebulization, the parameters of its nebulization may not meet the assumptions of the experiment (e.g., procedure time). It can be seen from Table 6 that only phage phi6 is countable and meets the assumptions of the experiment (passage time of 5 minutes), while phages phiX174 and T4 do not meet the assumptions of the experiment (very poor survival in air or the need to use a large amount of phage and increase the nebulization time).

 

3. Considering the configuration of these fans, it cannot be ascertained if the indoor microbial concentration is uniform. To verify this, multiple detectors should be placed in various locations."

This is a very valuable observation. In the future, we plan to check the uniformity of microbial concentration in the tested rooms. In this test, the conditions were reproducible, and from this we determined the effect on survival rates.

4. The indicators labeled as 'good', 'very good', etc. in Table 8 lack explicit concentrations."

The authors found this to be the most illustrative description of the results. The indicators are qualitative and were used to compare the suitability of bacteria and phages in the study.

5. The aim of this study is to investigate a model virus and bacteria for aerosol transmission research. We only looked at how well they survived under constant humidity, temperature, and in a saline solution. However, the control group was limited, so we need to compare them in different environments too. Also, using bacteriophage as a model virus might not be the best choice because coronaviruses like COVID-19 have different characteristics. It would be better to use similar weakly pathogenic coronaviruses or pseudo-viral particles for this research.

 

This is a very valuable comment. We are aware of the limitations of our research. Studies of this type are limited by access to specialized BSL-3 class laboratories, and there are no known laboratories of this type that would ensure the safety of personnel and the environment with a large enough room in which to place a nebulization station.

Reviewer 2 Report

Comments and Suggestions for Authors

-I am very glad to review the paper in greater depth because the topic was interesting. And the authors chose three strains of bacteria and three bacteriophages similar to the COVID-19 and SARS_CoV-2 viruses, respectively, in order to study precautions for the spread of infectious microorganisms in aerosol environments and stated that the research process does not pose undue risks to researchers. For the benefit of the reader, however, a number of points need clarifying and certain statements require further justification. There are given below.

 

- This may be an interesting study, but I feel that it does not articulate what is new, what is important, and what is the value of the study. When I read the title of the article, I was hoping that the manuscript would describe a new specific method or device to effectively address the issue of microbial transmission in aerosols, and what contribution this new method might make in epidemiology, and how the study is an improvement over previously reported studies. In addition to this, aerosol-borne epidemiological studies have been reported quite a few times before this, so the novelty of this study is debatable.

 

Specific comments:

Abstract

-Lines 18-20, Lacks a description of the findings of the study and does not express the scientific questions and hypotheses that the study was intended to address.

 

Introduce

-Lines 42-45, There has been a lot of research reported on aerosol-borne epidemics, so why is there no description of the relevant control measures that have been taken?

-Lines 47-48, The authors mentioned that these bacteria and bacteriophages were chosen because of their similarity to the COVID-19 and SARS_CoV-2 viruses, but they did not explain the specific similarities, I think we need to describe in detail the pathogenicity and physicochemical properties of these bacteria and bacteriophages as well as their relevance to the COVID-19 and SARS_CoV-2 viruses.

-Lines 58-61, The authors mention several times that the study process does not pose unnecessary risks to researchers, but the manuscript does not explain why.

 

Materials and methods

-Lines 127-132. This section has clearly described the components of the device, can you be specific about how the device works?

-What was the starting point for studying the survival of strains and phages in saline and air.

-The experimental methodology does not mention that biological replicates were set up, and without replicated experiments and results, the results obtained are incomplete.

 

Discussion

-Figure 3-Figure 4. It is recommended to change the presentation of horizontal and vertical coordinates, the chart format is not standardized.

-Table 8. There is some overlap in content with Figure 3-Figure 4, suggested changes.

 

Conclusion

Lines -314-322. The conclusion simply summarizes the results of the experiment without a complete analysis, lacks a statement of the author's opinion, and does not excite the reader about the prospects of the study.

Lines -323-326. The authors state that the most effective strains in the method were Micrococcus garciniae strain ATCC 7468 and phage phi6, please analyze why in detail. What were the most effective evaluation criteria? What is the significance of the effective strain and phage evaluated?

-The results of the study were not analyzed in comparison with previous studies.

-The limitations of the study were not elaborated.

Comments on the Quality of English Language

Moderate editing of English language required

FIG 1 in English

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L232 38% within 30 minutes (Table 3) 

Author Response

Thank you very much for taking the time to review this manuscript and for your valuable comments, which in the future will allow more accurate rephrasing of the research results. All comments have been corrected in the text of the article. Below are the answers to each question. The results of the presented research actually served in further stages to develop a method and equipment, serving as mobile buffer rooms. The research procedure used made it possible to determine the effectiveness of the different variants of buffer rooms used to isolate patients.

Specific comments:

Abstract

1. Lines 18-20, Lacks a description of the findings of the study and does not express the scientific questions and hypotheses that the study was intended to address.

 In the article, this was completed.

Introduce

2. Lines 42-45, There has been a lot of research reported on aerosol-borne epidemics, so why is there no description of the relevant control measures that have been taken?

 

In the article, this was completed.

 

3. Lines 47-48, The authors mentioned that these bacteria and bacteriophages were chosen because of their similarity to the COVID-19 and SARS_CoV-2 viruses, but they did not explain the specific similarities, I think we need to describe in detail the pathogenicity and physicochemical properties of these bacteria and bacteriophages as well as their relevance to the COVID-19 and SARS_CoV-2 viruses.

 

This was completed in the introduction.

 

4. Lines 58-61, The authors mention several times that the study process does not pose unnecessary risks to researchers, but the manuscript does not explain why.

 

This was completed in the article. In the work, microorganisms that do not threaten human life were used and all safety measures were observed. The test room was additionally equipped with an outdoor station for testing the survival of bioaerosol components in the air. The samplers used made it possible to run them at any time, according to the test procedure. It allowed sampling while being outside the test room at any time interval. The station has its own "airlock", which allows placing the sampler / plates with the medium inside in a way that does not interfere with the homogenization of the bioaerosol inside. After the analysis is complete, disinfection of the chamber (ozonation) is also triggered remotely, from outside the test room.

 

Materials and methods

5. Lines 127-132. This section has clearly described the components of the device, can you be specific about how the device works?

 

To spray the prepared suspension with the test microorganism, a 6 Jet Collison nebulizer, (CH Technologies, USA), made of stainless steel and glass, was used, which produces an aerosol with parameters - MMAD 2.5 μm and GSD 1.8. A compressed air bottle was connected to the nebulizer, a PTFE tube was used for connection, with a 0.2 µm pore diameter filter. The nebulizer was sterilized in an autoclave (steam sterilizer) for 15 minutes at 121°C. The nebulizer at 20 psi sprays 0.0254 ml / min. Suspension (8,262 - 1010 particles). During nebulization of bacterial and phage suspension, bacterial cells (single, aggregates, packets or cells in the process of division) or bacteriophage virions are sprayed from droplets of solution (saline fluid or broth / broth diluted with saline fluid).

 

6. What was the starting point for studying the survival of strains and phages in saline and air.

 

The survival study was designed to determine the relationship of microorganism survival over time under different conditions. For each case, the conditions were different and the comparison followed between microorganisms under the same test conditions.

 

7. The experimental methodology does not mention that biological replicates were set up, and without replicated experiments and results, the results obtained are incomplete.

 

As for independent repetitions of the entire experiments - they were not performed - the research served to select a suitable bacterial strain and bacteriophage for further bioaerosol studies. This will be taken into account in the future.

 Discussion

8. Figure 3-Figure 4. It is recommended to change the presentation of horizontal and vertical coordinates, the chart format is not standardized.

 

The charts have been rearranged according to the reviewer's instructions

 

9. Table 8. There is some overlap in content with Figure 3-Figure 4, suggested changes.

 

In the paper, detailed results are presented in order to further describe the results obtained. The graphs show quantitative data, while the table presents qualitative - comparative results. This was necessary to compare the obtained results as qualitative "parameters" for further research.

Conclusion

10. Lines -314-322. The conclusion simply summarizes the results of the experiment without a complete analysis, lacks a statement of the author's opinion, and does not excite the reader about the prospects of the study.

 

In the article, this part has been completed. The use of non-hazardous bacteriophages, with the accepted time limitation, will much better reflect the way pathogenic viruses propagate through the air (similar size). Having at one's disposal the characteristics presented in the publication, it is possible to correct the concentration values for the effect of survival, isolating the influence of the tested protection agents. The results of the study will be used in the future to develop a universal procedure and test bed dedicated to devices for reducing the concentration of microorganisms in the air when exiting a room. Then, the effectiveness of the devices will be tested in different variants using selected bacteriophage and bacteria.

 

11. Lines -323-326. The authors state that the most effective strains in the method were Micrococcus garciniae strain ATCC 7468 and phage phi6, please analyze why in detail. What were the most effective evaluation criteria? What is the significance of the effective strain and phage evaluated?

 

In terms of the usefulness of bioaerosol testing, survival rate (time not less than that required to perform a nebulization experiment on the test bench) and countability of the phage in the air (which is closely related to its survival rate) are important. Practical issues are also important - reproducibility of the culture and readout (large, clear colonies, distinguishable from the background).

 

12. The results of the study were not analyzed in comparison with previous studies.

 

Due to the highly elaborate form and unique conditions of the study, such an analysis was difficult. The authors found no analogous published studies in which this parameter was determined under similar conditions. The analysis of the results in the article were enriched by comparison with other articles on the selected microorganisms.

 

13. The limitations of the study were not elaborated.

 

The limitation is the testing time (the concentration decreases over time) and the need to choose such a concentration so that the amount is quantifiable. Consequently, when using aerosolized microorganisms, the initial concentration must be limited (to be measurable) and thus the concentration after using a given solution to limit it may be too small to measure at some point.  In addition, the ability to determine the concentration over time is a limitation. Due to the nature of the samplers, which contain a certain number of holes in the head - there can be no more than 216 colonies. In order to determine the effectiveness of the solution based on such a bioaerosol, it is also necessary to achieve a concentration higher than 0 - which can be achieved by manipulating the parameters of nebulization or suspension parameters (abundance of bacteria/phages in the nebulization fluid, dilution factor).

Moderate editing of English language required done

FIG 1 in English- done

L166  2-3? done

L194  m3 superscript done

L232 38% within 30 minutes (Table 3) done

Reviewer 3 Report

Comments and Suggestions for Authors

During the COVID-19 pandemic, there was a neglect of microbiological controls regarding the spread of viruses through the air. Recognizing the need for research to identify and implement control measures against airborne transmission, the study focuses on using analogous microorganisms, which pose minimal risk to research staff, to validate or challenge methods for preventing the spread of infectious microorganisms. The work outlines a selection process for bacteria and viruses, particularly bacteriophages, with potential for experimental studies on airborne-droplet transmission indoors, specifically in hospital facilities.

 

The summary acknowledges the neglect of microbiological controls during the pandemic, highlighting the importance of addressing airborne transmission. This is a valid and critical observation, as airborne transmission played a significant role in the spread of COVID-19.

 

Some comments:

 

1.     The study suggests using analogous microorganisms to simulate the behavior of infectious agents without the high risk associated with COVID-19 and SARS-CoV-2. This approach is reasonable, as it allows for experimental validation or challenge of control methods. However, the success of this strategy depends on the chosen microorganisms accurately representing the behavior of the targeted viruses.

 

2.     The emphasis on studying airborne-droplet transmission in hospital facilities is relevant, given the higher vulnerability of such environments to the rapid spread of infectious diseases. This focus aligns with the practical need to enhance infection control measures in healthcare settings.

 

3.     The concern for the high risk involved in research on COVID-19 and SARS-CoV-2 viruses is appropriately addressed by proposing the use of safer microorganisms for experimentation. This reflects a responsible approach to scientific research, prioritizing the safety of research staff.

 

4.     The text suggests that additional research is required to identify and implement control measures. While this is acknowledged, it would be helpful to specify the specific areas of research that need further exploration for a more targeted approach.

 

5.     The summary provides a general overview, but more detail on the specific methods of microorganism selection, experimental design, and potential control measures would enhance the clarity and depth of understanding. In conclusion, the work recognizes a critical gap in microbiological controls related to airborne transmission and proposes a responsible approach to address this gap through the use of analogous microorganisms. However, further details on the methodology and specific areas requiring additional research would strengthen the study's overall impact and applicability.

 

6.     Adding statistical information in abstract and conclusion would improve the quality of work.

 

7.     Elaborate all figure captions.

 

 

Comments on the Quality of English Language

none

Author Response

Thank you very much for taking the time to review this manuscript and for your valuable comments, which in the future will allow more accurate rephrasing of the research results. All comments have been corrected in the text of the article. Below are the answers to each question.

1. 
   The study suggests using analogous microorganisms to simulate the behavior of infectious agents without the high risk associated with COVID-19 and SARS-CoV-2. This approach is reasonable, as it allows for experimental validation or challenge of control methods. However, the success of this strategy depends on the chosen microorganisms accurately representing the behavior of the targeted viruses.

 

The authors realize that bacteriophages are not coronaviruses, but their choice was dictated by the need to use a microorganism similar in size and structure to the SARS-CoV2 coronavirus as a complement to bioaerosol studies using bacteria. Bacteriophages pose no risk to personnel and allow multiple replicates to be performed without the need to work with a BSL-3 grade laboratory. bacteriophage phi6 and SARS-CoV-2 virus are two different categories of viruses, but there are some similarities in their structure and function, although they are two completely different classes of pathogens. Both bacteriophage phi6 and SARS-CoV-2 are viruses. This means that they are microscopic organisms that do not have their own metabolism or ability to multiply on their own. Instead, they must infect host cells to multiply. Both phi6 and SARS-CoV-2 have a capsid, which is an outer protective layer. In the case of phi6, it is the capsid that surrounds its RNA genome, while in the case of SARS-CoV-2, it is the protein envelope that surrounds its RNA genome. Both phi6 and SARS-CoV2 contain an RNA nucleic acid-based genome. The phi6 bacteriophage has a three-segmented RNA genome, while SARS-CoV-2 is a single-stranded RNA virus. Both phi6 and SARS-CoV-2 need to infect host cells to multiply. Phi6 infects bacteria, while SARS-CoV-2 infects human cells. Both viruses bind to receptors on the surface of host cells, allowing them to enter the cell and begin the multiplication process.

The phi6 virus is highly specific to its bacterial hosts, specifically bacteria of the Pseudomonas genus. It has no ability to infect or replicate in human cells or other animal organisms. Phi6 does not have the ability to attach to receptors on the surface of human cells, meaning it is unable to penetrate or infect human cells. Viruses that pose a threat to humans often have pathogenic genes or toxins that can cause disease. Since phi6 is a bacterial virus, there are no mechanisms for it to replicate inside the human body. In this study, two other bacteriophages were additionally selected to compare their survival rates under the same conditions.

 

2. The emphasis on studying airborne-droplet transmission in hospital facilities is relevant, given the higher vulnerability of such environments to the rapid spread of infectious diseases. This focus aligns with the practical need to enhance infection control measures in healthcare settings.

 

The authors fully agree with the reviewer.

 

3. The concern for the high risk involved in research on COVID-19 and SARS-CoV-2 viruses is appropriately addressed by proposing the use of safer microorganisms for experimentation. This reflects a responsible approach to scientific research, prioritizing the safety of research staff.

 

The authors agree with the reviewer. Safety was a key theme in the reposted study.

 

4. The text suggests that additional research is required to identify and implement control measures. While this is acknowledged, it would be helpful to specify the specific areas of research that need further exploration for a more targeted approach.

 

The research conducted and presented in the article was the beginning to develop a complete method and devices to limit the spread of dangerous microorganisms via the air-droplet route. It is planned to present these results in subsequent articles.

 

5. The summary provides a general overview, but more detail on the specific methods of microorganism selection, experimental design, and potential control measures would enhance the clarity and depth of understanding. In conclusion, the work recognizes a critical gap in microbiological controls related to airborne transmission and proposes a responsible approach to address this gap through the use of analogous microorganisms. However, further details on the methodology and specific areas requiring additional research would strengthen the study's overall impact and applicability.

 

In order to select test strains for further study, the following characteristics were compared with the test bioaerosol:

For bacteria:

- ability to obtain a quantifiable bioaerosol from a suspension with turbidity in the McFarland scale range;

- ease of colony reading (color, shape, colony size);

- survival in physiological fluid;

- survival in artificial saliva;

- survival in air.

 In the case of bacteriophages:

- ability to obtain a quantifiable bioaerosol;

- number of phages in suspension (phage titer);

- ease of reading baldness (magnitude of translucency);

- survival in physiological fluid;

- survival in air

 

6. Adding statistical information in abstract and conclusion would improve the quality of work.

 

Statistical data was compiled and entered.

 

7. Elaborate all figure captions.

 

In the article, all signatures have been checked and corrected.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The time formats in Table 2 and Table 3 are not uniform, so the format of the entire manuscript should be carefully checked and unified.

Author Response

Thank you very much for your careful analysis of the article. The times in Tables 2 and 3 depend on the testing environment, in the saliva environment the time was measured in hours while in air the time was measured in minutes. At these times, changes in the given conditions of microorganism survival were recorded.

Reviewer 2 Report

Comments and Suggestions for Authors The manuscript has been sufficiently improved to warrant publication in Microbiology Research.

Author Response

thank you for this feedback