Molecular Characterization of Bacillus anthracis from Selected Districts of Bangladesh

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments to the Authors:

This study aims to molecularly identify and characterize Bacillus anthracis from soil, blood, and meat samples collected in three districts of Bangladesh. PCR-based detection confirmed the presence of B. anthracis in 21 soil samples and all blood and meat samples, with the phylogenetic analysis showing that the 16S rRNA gene of the Sirajganj-1 strain was identical to other known strains, highlighting the need for effective control measures to address zoonotic diseases in the region. However, the article requires revisions to enhance its clarity and scientific rigor before it can be considered for publication. My detailed comments are as follows:

1. Is the methodology for sample collection and PCR analysis clearly described, and do you think it is adequate for identifying B. anthracis?
2. Do the results provide sufficient evidence to support the conclusions regarding the presence of B. anthracis in the studied areas?
3. Do you think the study addresses the significance of B. anthracis in relation to the Sustainable Development Goals and the control of zoonotic diseases in Bangladesh?
4. Are the use of MEGA5 software and the phylogenetic analysis appropriate for this type of study?
5. Do you have any recommendations on how the study can be expanded to further explore the virulence factors of B. anthracis?

Author Response

Comment 1: Is the methodology for sample collection and PCR analysis clearly described, and do you think it is adequate for identifying B. anthracis?

Response 1: Yes, the methodology that we used is described in lines 92-98.

Comment 2: Do the results provide sufficient evidence to support the conclusions regarding the presence of B. anthracis in the studied areas?

Response: Thanks for the comment. PCR is more reliable and accurate due to testing the specific genetic material of the organism. Additionally, we also used positive control. 

Comment 3: Do you think the study addresses the significance of B. anthracis in relation to the Sustainable Development Goals and the control of zoonotic diseases in Bangladesh?

Response: A single livestock is a small source of income for a micro family here in the village of Bangladesh. Healthy livestock not only helps to lower the poverty but also meets the protein requirement for the society.

Comment 4: Are the use of MEGA5 software and the phylogenetic analysis appropriate for this type of study?

Response: The foundation of genomic studies is DNA and protein sequences, which MEGA5 specifically designed to analyze. It helps researchers understand the evolutionary history and relationships of genes and species, which is crucial in genomic research. MEGA5 offers various statistical methods for phylogenetic analysis, including maximum likelihood (ML), maximum parsimony, and distance-based methods.

Comment 5: Do you have any recommendations on how the study can be expanded to further explore the virulence factors of B. anthracis?

Response: To expand the exploration of Bacillus anthracis virulence factors, researchers can leverage whole genome sequencing (WGS), single nucleotide polymorphism (SNP) analysis, and pan-genome analysis to identify novel virulence genes, track plasmid evolution, and differentiate B. anthracis from closely related species.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript provides a valuable look into the molecular surveillance of Bacillus anthracis in a high-risk region of Bangladesh. By focusing on three districts with a known history of anthrax outbreaks, the authors aim to detect and characterize B. anthracis from both environmental and clinical samples, using PCR and sequencing of the 16S rRNA gene. Their work is relevant, particularly in the context of endemic zoonotic disease control in resource-limited settings.

Here are my observations for the Authors:

First, while the study includes PCR confirmation of virulence plasmids (pX01 and pX02), only one isolate was subjected to 16S rRNA sequencing and phylogenetic analysis. Considering that 21 isolates were confirmed as B. anthracis, including additional sequences would provide a more robust phylogenetic perspective. This is particularly important to assess intra-regional strain variation and to monitor for the possible emergence of new or divergent strains.

Second, the decision to only sequence a single isolate also limits the potential insights into gene-level variability or plasmid carriage patterns. While the authors do report plasmid presence across isolates in different locations, whole-genome sequencing or even multilocus sequence typing (MLST) could enhance understanding of the molecular epidemiology of the strains circulating in these districts.

Another limitation lies in the statistical treatment of the data. While the manuscript presents prevalence percentages, it does not report confidence intervals or use inferential statistics to explore associations between sampling location and detection rates. A simple chi-square analysis, for instance, could support the interpretation of differences in positivity rates across the districts surveyed.

Furthermore, while the study touches on implications for vaccination and early detection, it stops short of making specific recommendations based on its findings. For example, the observation that all clinical samples (blood and meat) were PCR-positive suggests these sample types might be prioritized in future outbreak investigations. This point could be emphasized more strongly as a practical takeaway.

The conclusion would benefit from a more specific discussion on how molecular detection could be integrated into existing surveillance frameworks or what barriers exist to doing so in rural Bangladesh.

Line 11 – delete (B)

Line 80 – more information about soil samples must be included; add a map with sample locations and pictures from sampling;

After line 218 must be added comprehensive discussion and comparation with literature; why did you do this research? What is important? What is novel?

Comments on the Quality of English Language

The manuscript could benefit from tighter language editing in places, especially in the results and discussion sections, where clarity sometimes suffers due to grammar and phrasing issues.

Author Response

Firstly, I appreciate the reviewer for the valuable comments. It was a small MS study, and there was no funding, so we couldn't afford to do more sequencing. Also, there is a sequential work published with big data, and we cite that work. In lines 181-183, we described that blood and meat samples might be prioritized in future outbreak investigations. 

In line 11 we used the first time Bacillus and then a short abbreviation of Bacillus, so we used "B" there. We also add a citation in the sample collection part for more info and visibility of this work.

Not sure what the reviewer meant after line 218. Some comprehensive discussion and comparison with literature is present in the discussion part.

Thanks

Reviewer 3 Report

Comments and Suggestions for Authors

Review of the article “Molecular Characterization of Bacillus Anthracis From Selected Districts Of Bangladesh”

Anthrax is a zoonotic disease caused by the bacterium Bacillus anthracis, which is a non-hemolytic, non-motile, Gram-positive, spore-forming rod. Bacillus anthracis is a facultative anaerobe. Virulence depends on two plasmids, one of which carries the protein capsule gene and the other the exotoxin gene. The exotoxin consists of three proteins: a protective antigen, an edema factor, and a lethal factor. Anthrax disease is caused by its spores, which germinate in bacilli inside the macrophages of an infected host. Once inside the host, the bacilli begin to cause disease by releasing toxins that cause cell death, lymphadenopathy, and edema. Spores can remain dormant and viable in the environment for decades. Anthrax is classified as a Category A priority pathogen by the Centers for Disease Control and Prevention because it has the potential to be used as a biological weapon. There are other urgent concerns about anthrax in wildlife, livestock, and humans. Animal anthrax typically occurs during the summer and fall seasons and is most common in grazing mammals, including domestic goats, sheep, and cattle, as well as wild deer and antelope. Transmission to humans occurs through contact with infected animals during butchering and skinning, or by eating raw or undercooked meat. Populations at risk for anthrax include those who eat undercooked meat contaminated with spores and those living in rural and agricultural areas. Exposure to livestock, contaminated meat, and contaminated soil increases the risk of developing the disease.

The manuscript presented provides a significant contribution to the field of medicine, addressing a topic that is both original and highly relevant. The authors have effectively communicated the stages of their research, allowing for a clear understanding of their methodology and findings. Each section of the study is meticulously crafted, presenting information in a clear and detailed manner. The evidence and conclusions drawn are robust and credible, reflecting a well-founded argument that is supported by thorough analysis. The references cited throughout the manuscript are adhere to the required formatting standards.

Minimal remarks:

1) According to the International Code of International Classification, the species name must always be written in lower case, even in the title of an article.

2) It is not good to start a sentence with an abbreviation of a generic name - “B. anthracis is a Gram-positive..........”. Must be full name - Bacillus anthracis.

Please correct!

Author Response

Comment 1: According to the International Code of International Classification, the species name must always be written in lower case, even in the title of an article.

Response: Thanks for the critical comment. We just edited that one. 

Comment 2: It is not good to start a sentence with an abbreviation of a generic name - “B. anthracis is a Gram-positive..........”. Must be full name - Bacillus anthracis.

Response: Thanks for the comment. We added your suggestion. 

Reviewer 4 Report

Comments and Suggestions for Authors

This study aims to identify and characterize Bacillus anthracis at the molecular level in soil, meat, and blood samples collected from three districts in Bangladesh using 16S rRNA gene sequencing. By analyzing the prevalence and genetic composition of anthrax-causing bacteria, the research contributes to controlling zoonotic diseases in developing nations. The relevance of this study lies in addressing a critical gap in anthrax detection and characterization in Bangladesh, where frequent outbreaks occur but remain inadequately studied. The application of molecular techniques such as PCR and phylogenetic analysis for detecting B. anthracis is particularly novel in the context of Bangladesh’s unique environmental and socioeconomic conditions. This approach offers a rapid and sensitive method for identifying anthrax spores, facilitating early diagnosis and informing vaccine development strategies.

Unlike conventional studies, which often rely on traditional diagnostic methods, this research highlights the significance of molecular characterization in a resource-limited setting, where large-scale depopulation programs may not be economically viable. Furthermore, it provides valuable genetic data, comparing local B. anthracis strains with global variants, which could enhance vaccine formulation and improve outbreak management strategies.

Methodological Considerations

• Sample Diversity: Although 128 soil samples and three clinical specimens were analyzed, increasing the number of clinical samples, particularly from blood and meat, would enhance statistical reliability.

• Experimental Controls: Including negative controls during PCR amplification would help ensure specificity and accuracy.

• Sequencing Depth: Expanding sequencing efforts beyond a single positive sample would offer a more comprehensive view of genetic variability.

• Environmental Influences: Incorporating data on soil properties such as pH, temperature, and moisture content could provide insights into spore viability under diverse ecological conditions.

The conclusions drawn from the study align with the presented evidence. The successful identification of B. anthracis via PCR underscores the significance of molecular techniques in pathogen detection. Moreover, by linking findings to sustainable development goals, the study advocates for vaccination initiatives and awareness programs as crucial measures for anthrax prevention. However, while the phylogenetic analysis presents promising insights, the limited sequencing sample restricts broader applicability. Nonetheless, key research questions were effectively addressed through PCR-based identification and phylogenetic comparison.

Suggestions for Enhancement

• References: The citations are appropriate, covering anthrax outbreaks, molecular methodologies, and environmental factors influencing spore persistence. However, incorporating more recent studies on anthrax management strategies could further strengthen the discussion.

• Tables and Figures:

  • The tables clearly outline primer sequences and sample results but could be supplemented with graphical representations to illustrate prevalence across different districts.

  • The phylogenetic tree is informative but would benefit from annotations that highlight significant clades and genetic relationships.

• Data Presentation: While the PCR results are well-documented, electrophoresis images should be improved for better clarity and interpretation.

By addressing these aspects, the study could further refine its impact, providing a more robust framework for understanding anthrax epidemiology and informing future control measures in Bangladesh and similar regions.

Comments on the Quality of English Language

The quality of English in the manuscript is generally acceptable, but there are areas where the language could be improved for clarity, grammar, and flow. Below is a detailed assessment:

Strengths in Language Use

1. The manuscript effectively conveys the scientific content and methodology in a structured manner.
2. Technical terms and scientific jargon are used appropriately, indicating familiarity with the subject matter.
3. The overall message of the study is clear, and the conclusions are understandable.

Areas for Improvement

1. Grammar and Sentence Structure:
• Several sentences are overly long and complex, making them difficult to follow. For example:
• "Neutral to slightly alkaline soil, calcareous, vegetative, or humic rich soil, drought followed by heavy rainfall, high mean ambient temperature..." (lines 31–33).
  → This sentence can be simplified for better readability.
• Incorrect verb usage: "These spores have the potential to live in soil for decades" (line 63) should be revised to "These spores can remain viable in soil for decades."
2. Repetition:
• Some ideas are repeated unnecessarily, such as the persistence of spores in soil (lines 30–40 and lines 62–65). Consolidating these sections would improve conciseness.
3. Word Choice:
• Words like "approace" (line 141) and "ca�le" (line 60) seem to be typographical errors. These should be corrected to "approach" and "cattle."
• Phrases like "depopulation program doesn’t support" (line 145) should be revised to "depopulation programs are not feasible."
4. Flow and Transitions:
• Some paragraphs lack smooth transitions between ideas, making the text feel disjointed. For instance, the discussion about anthrax outbreaks globally (lines 46–50) could be better connected to the specific context of Bangladesh.
5. Punctuation:
• Missing or misplaced commas make some sentences harder to read. For example:
• "When it comes to the management and control of anthrax risk having a technique that is both quick and sensitive..." (line 146).
  → Should be revised to: "When it comes to managing and controlling anthrax risk, having a technique that is both quick and sensitive..."
6. Clarity in Results Section:
• The description of results could benefit from more precise language. For example:
• "21 of the 128 samples were believed to be B. anthracis" (line 153) could be revised to: "21 out of 128 samples were identified as presumptive positive for B. anthracis based on colony morphology."
7. Consistency in Terminology:
• The manuscript alternates between "Bacillus anthracis" and "B. anthracis." While this is acceptable in scientific writing, ensure consistency within a section.
8. Use of Passive Voice:
• Excessive use of passive voice can make sentences less direct. For example:
• "The genomic DNA of every B. anthracis isolate was achieved by the use of the boiling process" (line 102).
  → Revise to: "Genomic DNA was extracted from each B. anthracis isolate using the boiling method."

Recommendations

1. Proofread the entire manuscript carefully or use professional editing services to catch grammatical errors and improve readability.
2. Simplify complex sentences while retaining scientific accuracy.
3. Ensure proper use of punctuation marks to enhance sentence clarity.
4. Avoid redundancy by consolidating repetitive information.
5. Use active voice where appropriate for more engaging writing.

Assessment

• Overall Quality: The English is adequate but could benefit from improvements for clarity and fluency.
• Recommendation: The English language should be improved before publication.

Author Response

Comment 1: Add more samples from meat and blood.

Response: Thanks for the comment. Hopefully next time, if we do more analysis, we would like to add a larger number of samples.

Comment 2: Add the negative control in PCR.

Response: We are also thinking of adding this in our next experiment. 

About the reference, we tried to add the more recent citation; maybe more than 75% of the citations are from the last 10 years.

Comment 3. Incorrect verb usage: "These spores have the potential to live in soil for decades" (line 63)

Response: Edited. Thanks.

Comment 4: Some ideas are repeated unnecessarily, such as the persistence of spores in soil (lines 30–40 and lines 62–65). Consolidating these sections would improve conciseness.

Response: Thanks for the observation. You're correct, but sometimes it's necessary to reiterate certain points to ensure the reader's understanding. Thanks for understanding. 

Comment 5: Words like "approace" (line 141) and "ca�le" (line 60) seem to be typographical errors. These should be corrected to "approach" and "cattle."

Response: Edited.

Comment 6: Phrases like "depopulation program doesn’t support" (line 145) should be revised to "depopulation programs are not feasible."

Response: Edited. 

Comment 7: Missing or misplaced commas make some sentences harder to read. For example:

"When it comes to the management and control of anthrax risk having a technique that is both quick and sensitive..." (line 146).

Response: Thanks and edited. 

Comment 8: The description of results could benefit from more precise language. For example:

"21 of the 128 samples were believed to be B. anthracis" (line 153) could be revised to: "21 out of 128 samples were identified as presumptive positive for B. anthracis based on colony morphology."

Response: Thanks , edited. 

Comment 9: Excessive use of passive voice can make sentences less direct. For example:

"The genomic DNA of every B. anthracis isolate was achieved by the use of the boiling process" (line 102).
→ Revise to: "Genomic DNA was extracted from each B. anthracis isolate using the boiling method."

Response: Thanks and edited.

Comment 10: The manuscript alternates between "Bacillus anthracis" and "B. anthracis." While this is acceptable in scientific writing, ensure consistency within a section.

Response: Thanks for the observation. Edited.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The paper is improved a lot and I recommend the acceptance.

Author Response

Thanks!

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Despite you brief explanation of limitations you had for this study; I think that you need to improve quality of the paper according to recommendations and contribute with point-to-point responses for each observation.

Author Response

Here is an attached file where all the responses are present. 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have corrected all comments on this article. I have no more comments on the material. I believe that the article can be accepted for publication.

Author Response

Thanks!

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have incorporated the suggestions, and I believe this manuscript is now ready for publication as an article.

Author Response

Thanks for reviewing our manuscript!!!

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

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