Author response:
The following is the authors’ response to the original reviews.
eLife assessment
In this useful study, Wang and colleagues investigate the potential probiotic effects of Bacillus velezensis to prevent colitis in a mouse model. They provide solid evidence that B. velezensis limits the growth of Salmonella typhimurium in lab culture and in mice, together with beneficial effects on the microbiota. The work will be of interest to infectious disease researchers and those studying the microbiome.
Response: Thanks for the constructive comments and the positive reception of the manuscript.
Public Reviews:
Reviewer #1 (Public Review):
Summary:
Wang and colleagues presented an investigation of pig-origin bacteria Bacillus velezensis HBXN2020, for its released genome sequence, in vivo safety issue, probiotic effects in vitro, and protection against Salmonella infection in a murine model. Various techniques and assays are performed.
Response: Thanks for the constructive comments and the positive reception of the manuscript.
Strengths:
An extensive study on the probiotic properties of the Bacillus velezensis strain HBXN2020.
Response: Thank you very much for your reading and comments our manuscript.
Weaknesses:
- The main results are all descriptive, without new insight advancing the field or a mechanistic understanding of the observed protection.
Response: Thank you for your comments and suggestions on our manuscript. In later work, we will focus on exploring the antibacterial substances and bactericidal mechanisms of B. velezensis. We appreciate your review and feedback.
- Most of the results and analysis parts are separated without a link or any story-telling to deliver a concise message.
Response: Thank you for your comments and suggestions on our manuscript. The comments improve the quality and depth of manuscript. Based on your suggestions, we have revised modifications to the entire manuscript.
The updated contents were presented in the revised manuscript.
- For the Salmonella Typhimurium-induced mouse model of colitis, it is not clear how an oral infection of C57BL/6 would lead to colitis. Streptomycin is always pretreated (https://link.springer.com/protocol/10.1007/978-1-0716-1971-1_17).
Response: Thank you very much for your reading and comments our manuscript. The S. Typhimurium ATCC14028 (STm) used in this study is a highly virulent strain. The findings of the predimed trial indicated that mice infected with 107 CFU STm exhibited notable symptoms in the absence of streptomycin pretreatment. Hence, streptomycin was not utilized as a pretreatment for mice in this study. We appreciate your review and feedback and hope that our response adequately addresses your concerns.
Reviewer #2 (Public Review):
Summary:
In this study, Wang and colleagues study the potential probiotic effects of Bacillus velezensis. Bacillus species have the potential benefit of serving as probiotics due to their ability to form endospores and synthesize secondary metabolites. B. velezensis has been shown to have probiotic effects in plants and animals but data for human use are scarce, particularly with respect to salmonella-induced colitis. In this work, the authors identify a strain of B. velezensis and test it for its ability to control colitis in mice.
Response: Thanks for the constructive comments and the positive reception of the manuscript.
Key findings:
(1) The authors sequence an isolate for B. velezensis - HBXN2020 and describe its genome (roughly 4 mb, 46% GC-content etc).
Response: Thanks for the constructive comments and the positive reception of the manuscript.
(2) The authors next describe the growth of this strain in broth culture and survival under acid and temperature stress. The susceptibility of HBXN2020 was tested against various antibiotics and against various pathogenic bacteria. In the case of the latter, the authors set out to determine if HBXN2020 could directly inhibit the growth of pathogenic bacteria. Convincing data, indicating that this is indeed the case, are presented.
Response: Thanks for the constructive comments and the positive reception of the manuscript.
(3) To determine the safety profile of BHXN2020 (for possible use as a probiotic), the authors infected the strain in mice and monitored weight, together with cytokine profiles. Infected mice displayed no significant weight loss and expression of inflammatory cytokines remained unchanged. Blood cell profiles of infected mice were consistent with that of uninfected mice. No significant differences in tissues, including the colon were observed.
Response: Thanks for the constructive comments and the positive reception of the manuscript.
(4) Next, the authors tested the ability of HBXN2020 to inhibit the growth of Salmonella typhimurium (STm) and demonstrate that HBXN2020 inhibits STm in a dose-dependent manner. Following this, the authors infect mice with STm to induce colitis and measure the ability of HBXN2020 to control colitis. The first outcome measure was a reduction in STm in faeces. Consistent with this, HBXN2020 reduced STm loads in the ileum, cecum, and colon. Colon length was also affected by HBXN2020 treatment. In addition, treatment with HBXN2020 reduced the appearance of colon pathological features associated with colitis, together with a reduction in inflammatory cytokines.
Response: Thanks for the constructive comments and the positive reception of the manuscript.
(5) After noting the beneficial (and anti-inflammatory effects) of HBXN2020, the authors set out to investigate the effects on microbiota during treatment. Using a variety of algorithms, the authors demonstrate that upon HXBN2020 treatment, microbiota composition is restored to levels akin to that seen in healthy mice.
Response: Thanks for the constructive comments and the positive reception of the manuscript.
(6) Finally, the authors assessed the effect of using HBXN2020 as prophylactic treatment for colitis by first treating mice with the spores and then infecting them with STm. Their data indicate that treatment with HBXN2020 reduced colitis. A similar beneficial impact was seen with the gut microbiota.
Response: Thanks for the constructive comments and the positive reception of the manuscript.
Strengths:
(1) Good use of in vitro and animal models to demonstrate a beneficial probiotic effect.
Response: Thank you very much for your reading and comments our manuscript.
(2) Most observations are supported using multiple approaches.
Response: Thanks for the comments and the positive reception of the manuscript.
(3) The mouse experiments are very convincing.
Response: Thanks for the comments and the positive reception of the manuscript.
Weaknesses:
(1) Whilst a beneficial effect is observed, there is no investigation of the mechanism that underpins this.
Response: Thank you for pointing this out. We apologize for any inconvenience caused by the lack of mechanism research of the manuscript. In later work, we will focus on exploring the antibacterial substances and bactericidal mechanisms of B. velezensis. Thank you for your suggestions, and we hope our response has addressed your concerns.
(2) The mouse experiments would have benefited from the use of standard anti-inflammatory therapies to control colitis. That way the authors could compare their approach of using bacillus spores with the current gold standard for treatment.
Response: We gratefully appreciate for your valuable comments. The objective of this study is to investigate the potential of B. velezensis spores in mitigating bacterial-induced colitis. In this experiment, animal experimental design referred to the method described in previous studies with slight modifications (10.1038/s41467-019-13727-9, 10.1126/scitranslmed.abf4692). We appreciate your review and feedback. We hope that our response adequately addresses your concerns.
Reviewer #3 (Public Review):
Summary:
The manuscript by Wang et al. investigates the effects of B. velezensis HBXN2020 in alleviating S. Typhimurium-induced mouse colitis. The results showed that B. velezensis HBXN2020 could alleviate bacterial colitis by enhancing intestinal homeostasis (decreasing harmful bacteria and enhancing the abundance of Lactobacillus and Akkermansia) and gut barrier integrity and reducing inflammation. Overall, the manuscript is of potential interest to readers.
Response: Thanks for the comments and the positive reception of the manuscript.
Strengths:
B. velezensis HBXN2020 is a novel species of Bacillus that can produce a great variety of secondary metabolites and exhibit high antibacterial activity against several pathogens. B. velezensis HBXN2020 is able to form endospores and has strong anti-stress capabilities. B. velezensis HBXN2020 has a synergistic effect with other beneficial microorganisms, which can improve intestinal homeostasis.
Response: Thanks for the comments and the positive reception of the manuscript.
Weaknesses:
There are few studies about the clinical application of Bacillus velezensis. Thus, more studies are still needed to explore the effectiveness of Bacillus velezensis before clinical application.
Response: Thanks for your suggestion. This study serves as an exploratory investigation before the application of Bacillus velezensis. The main purpose of this study is to explore the potential of Bacillus velezensis in application. We appreciate your review and feedback and hope that our response adequately addresses your concerns.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
Abstract:
It is quite wordy, without a clear emphasis on the major point of the study. It is obvious how the host-probiotic-microbiota behaves and why it works out well, which is the key part.
Response: Thank you for your valuable suggestion. The comments improve the quality of manuscript. We have modified this in the revised manuscript as suggested.
The updated contents were presented in line 30-32, 34-39 and 41-46 in abstract section of the revised manuscript.
Please remove "novel", Many previous works have already documented the probiotic Bacillus velezensis. It is also NOT novel species...
Response: Thank you for your suggestion. We have corrected it as suggested. Please see line 26 in abstract section of the revised manuscript.
Lines 44-46. The way this conclusion is delivered is inappropriate; it should be clarified exactly according to the supported results.
Response: Thank you for your valuable suggestion. The comments improve the quality of manuscript. We have corrected this in the revised manuscript as suggested.
The updated contents were presented in line 44-46 in abstract section of the revised manuscript.
Introduction:
Lines 71-71, Lines 75-77, Line 92 "the homeostasis of", please remove.
Response: Thank you for pointing this out. We have corrected this in the revised manuscript as suggested.
The updated contents were presented in line 96 in introduction section of the revised manuscript.
Are the Salmonella loads the key indicator for this model?
Response: We gratefully appreciate for your valuable comments. In this study, we aimed to evaluate whether B. velezensis can alleviate S. Typhimurium-induced colitis in mice. It has been reported that S. Typhimurium enters the intestine, colonizes and proliferates in the intestinal epithelium, and then breaks through the intestinal barrier to reach the whole body with the blood circulation system, leading to systemic infection. Thereby, the load of Salmonella in the intestine and tissue organs is also one of the key indicators reflecting Salmonella infection. We appreciate your review and feedback and hope that our response adequately addresses your concerns.
The introduction should really focus on the knowledge gap in general and in a specific field, which is not available in the current version.
Response: Thank you for your valuable suggestion. The comments improve the depth of the manuscript. We have corrected it as suggested.
The updated contents were presented in line 53-57, 61-64, 69-75, 85-88 and 97-100 in introduction section of the revised manuscript.
Results:
"Genomic Characteristics" of B. velezensis HBXN2020 are separated. There are no links between this work for safety and probiotic effects.
Response: Thank you for your suggestion. Based on your suggestion, we have revised modifications to the "genomic characteristics" in the results section. Please see line 104-110 and Supplementary Table 2 in revised manuscript and supplemental material.
Are the AMR and virulent genes available on the chromosome? Is there any gene cluster that codes useful stuff that is linked to probiotic efficacy in vitro and in vivo?
Response: Thanks for your suggestion. The comments improve the quality and depth of manuscript. In this study, the HBXN2020 genome contains fragments of AMR and virulence genes. However, the results of antibiotic sensitivity test and safety test showed that HBXN2020 did not exhibit resistance and toxicity. Furthermore, the HBXN2020 genome contains 13 different clusters of secondary metabolic synthesis genes. such as surfactin (genomic position: 323,509), macrolactin H (genomic position: 1,384,185), bacillaene (genomic position: 1,691,549), fengycin (genomic position: 1,865,856), difficidin (genomic position: 2,270,091), bacillibactin (genomic position: 3,000,977) and Bacilysin (genomic position: 3,589,078) (Table S2). These secondary metabolites have been shown to have varying degrees of inhibition on fungi (10.3390/foods11020140), Gram-positive pathogens (10.1371/journal.pone.0251514) and Gram-negative pathogens (10.1007/s00253-017-8095-x). We appreciate your review and feedback and hope that our response adequately addresses your concerns. We have marked the updated contents in the revised manuscript.
The updated contents were presented in line 108-110 in results section of the revised manuscript and supplementary Table 2 in the revised supplemental material.
Finally, the raw data (Illumina, Pacbio) should also be provided.
Response: Thanks for pointing this out. According to your suggestion, we have submitted the raw data of the HBXN2020 genome to the GenBank database, GenBank accession number CP119399.1. We appreciate your review and feedback and hope that our response adequately addresses your concerns.
The updated contents were presented in line 770-773 in data availability section of the revised manuscript.
Lines 100-108, please replace this part for a more meaningful investigation that could be possibly supported by the following experimental assays.
Response: We gratefully appreciate for your valuable comments. The comments improve the quality and depth of manuscript. Based on your suggestion, we try our best to remove some minor results and supplement more meaningful research findings. We appreciate your review and feedback, and have marked the updated contents in the revised manuscript. Please see line 104-110 and Supplementary Table 2 in revised manuscript and supplemental material.
Lines 119-126, which are not important, did you further check what or which parts make the bacteriostasis?
Response: Thanks for pointing this out. According to your suggestion, we try our best to remove some minor results by removing unnecessary words and sentences. Furthermore, in the following research, we will focus on exploring the antibacterial substances and bactericidal mechanisms of B. velezensis. We appreciate your review and feedback and hope that our response adequately addresses your concerns. We have marked the updated contents in the revised manuscript.
The updated contents were presented in line 122-124 in results section of the revised manuscript.
"Biosafety"? Is there a standard way to conduct this investigation? please clarify.
Response: Thank you for pointing out this problem in manuscript. In this experiment, Biosafety assessment of B. velezensis HBXN2020 referred to the method described by Zhou et al. with slight modifications (10.1038/s41467-022-31171-0). We appreciate your review and feedback and hope that our response adequately addresses your concerns.
The updated contents were presented in line 651-652 in results section of the revised manuscript.
Why are spores used, not whole bacteria? Please clarify.
Response: Thanks for pointing this out. We apologize for any incomprehension caused by the use of B. velezensis HBXN2020 spores in manuscript. In this study, mice were treated with B. velezensis by oral gavage, while gastric acid will drastically reduce the activity of B. velezensis. However, spores tolerated strong acidic environments well. Additionally, previous studies have also precedents of using spores (10.1126/scitranslmed.abf4692). Thank you for your comments and feedback and hope that our response adequately addresses your concerns.
Line 196, line 287, repeated assays were conducted, but the logical link is missing.
Response: We gratefully appreciate for your valuable comments. We apologize for any inconvenience caused by the organization and coherence of our results section. According to your suggestion, we try our best to improve the manuscript's layout by removing unnecessary words and revising sentences. We would like to express our apologies once again and hope that the revised manuscript meets your expectations. We have marked the updated contents in the revised manuscript.
The updated contents were presented in line 195-198, 246-248, 256-257 and 285-287 in results section of the revised manuscript.
Discussion:
Please shorten it; it is wordy but without focus.
Response: We gratefully appreciate for your valuable comments. The comments improve the quality and depth of manuscript. According to your suggestion, we try our best to shorten the discussion length by removing unnecessary words and revising sentences. We would like to express our apologies once again and hope that the revised manuscript meets your expectations. We have marked the updated contents in the revised manuscript.
The updated contents were presented in line 353-355, 358-360, 366-371, 381-385, 395-401, 417-419, 430-438, 459-466, 478-481 and 484-485 in discussion section of the revised manuscript.
Conclusion:
Please clarify and rework it.
Response: Thanks for your suggestion. The comments improve the quality and depth of manuscript. Based on your suggestion, we have now rewritten the conclusion.
The updated contents were presented in line 492-496 in conclusion section of the revised manuscript.
Materials and Methods:
Much more detailed information should be provided.
Response: Thank you for your suggestion. The comments improve the quality and depth of manuscript. Based on your suggestion, we have revised detailed modifications to the experimental method. We appreciate your review and feedback, and have marked the updated contents in the revised manuscript. Please see line 513-515, 530-533 and Supplementary Table 5 in revised manuscript and supplemental material.
All previous bacterial sampling and a list of results should be provided as the supplemental document.
Response: Thank you for your valuable suggestion. The comments improve the quality and depth of manuscript. In this study, we conducted preliminary biological activity testing on 362 isolates of Bacillus against pathogenic bacteria, which included S. Typhimurium ATCC14028, E. coli ATCC35150, S. aureus ATCC43300 and ATCC29213. We found that the antagonistic activity of four strains of Bacillus （B. subtilis H1, B. velezensis HBXN2020, B. amyloliquefaciens 6-1 and B. licheniformis BSK14）against these pathogenic bacteria, while the rest have no significant activity. So we chose these four strains to further evaluate their antibacterial activity against Gram-negative and Gram-positive pathogens (Supplementary Table 5). Based on the antibacterial test results, we found that B. velezensis HBXN2020 strain had the best antibacterial activity. so we chose B. velezensis HBXN2020 for subsequent experiments.
The updated contents were presented in Supplementary Table 5 in supplemental material.
Minor points:
All bacterial genera and species should be italicized.
Response: Thank you for pointing this out. We have corrected this in the revised manuscript as suggested.
The updated contents were presented in line 26 in abstract section and line 67, 69 in introduction section and line 111 in results section of the revised manuscript.
Line 39, remove repeated "importantly"
Response: Thanks for your useful suggestion. We have corrected this in the revised manuscript as suggested.
The updated contents were presented in line 39 in abstract section of the revised manuscript.
Lines 55-56, please rewrite.
Response: Thanks for your suggestion. We have now rephrased the sentence.
The updated contents were presented in line 56-57 in introduction section of the revised manuscript.
The relevant references should be updated, in the right format.
Response: Thanks for your suggestion. Based on your suggestion, we have revised modifications according to the literature format of eLife magazine.
The updated contents were presented in reference section of the revised manuscript.
Reviewer #2 (Recommendations For The Authors):
Major concerns:
(1) In Figure 2, the authors make the argument that the increased survival of Bacillus spores at high temperatures and low pH renders the strain useful as a probiotic as it would survive in the gut. However, the gut temperature is not significantly higher than the rest of the body (certainly not 95 degrees). One assumes the pH argument applies to surviving in stomach acid so that spores can travel to the gut. These conclusions should be clarified/revised. The survival in bile salts gastric fluid etc makes more sense.
Response: Thank you for your suggestion. The comments improve the quality and depth of manuscript. Based on your suggestion, we have revised these conclusions. We would like to express our apologies once again and hope that the revised manuscript meets your expectations. We have marked the updated contents in the revised manuscript.
The updated contents were presented in line 129-132 in results section of the revised manuscript.
(2) The overall differences in the microbiota on the stacked bar graphs are difficult to determine. In many cases, it looks like the HBXN2020 does not have a significant effect. The subsequent scattergrams are more convincing. Perhaps the authors can think of a better way to compare composite populations. If not, I suggest moving these stacked graphs to the supplementary information.
Response: We gratefully appreciate for your valuable comments. The comments improve the quality and depth of manuscript. Based on your suggestion, we have moved stacked graphs to the supplemental material. In addition, we replaced bar graphs with heatmaps, the differences of microbial community composition among different experimental groups were evaluated using the depth of color. We appreciate your review and feedback, and have marked the updated figures in the revised manuscript. Please see Figure 7and 10 in revised manuscript and supplemental material.
Minor editorial:
(1) Line 55 - "....antibiotic therapy is...".
Response: Thank you for your suggestion. We have corrected it as suggested.
The updated contents were presented in line 56-57 in introduction section of the revised manuscript.
(2) Line 60 - replace "emergent search" - poor syntax.
Response: Thank you for your suggestion. The comments improve the quality of manuscript. We have corrected this in the revised manuscript as suggested.
The updated contents were presented in line 61-62 in introduction section of the revised manuscript.
(3) Line 63 - "...play an important...".
Response: Thanks for pointing this out. We have now rephrased the sentence.
The updated contents were presented in line 63-64 in introduction section of the revised manuscript.
(4) Figure 1C is not very useful, simply reinforces the data from 1A and 1B - this can be moved to the supplementary information.
Response: Thank you for your valuable suggestion. The comments improve the quality and depth of manuscript.
Based on your suggestion, we have moved figure 1C to the supplemental material. We appreciate your review and feedback, and have marked the updated figures in the revised manuscript. Please see figures in revised manuscript and supplemental material.
(5) Line 126, "...that the growth of B. velezensis HBXN2020 was relatively stable." What do the authors mean by this? "Stable" implies no increase in biomass, but the growth curve does not indicate this, there was an increase in biomass after which, the culture appeared to reach a stationary phase. This should be clarified.
Response: Thanks for pointing this out. The comments improve the quality of manuscript. We have corrected this in the revised manuscript as suggested.
The updated contents were presented in line 122-124 in results section of the revised manuscript.
(6) In Figure 5 - all the graphs in panel A can be amalgamated into one figure using different colours/symbols.
Response: Thank you for your suggestion. The comments improve the quality and depth of manuscript. Based on your suggestion, we have merged all the graphics in panel A in Figure 5 into one figure.
The updated contents were presented in Figure 5 in the revised manuscript.
(7) The overall cohesiveness of the manuscript could be improved.
Response: Thank you for your valuable comments. The comments improve the quality and depth of manuscript. We have revised the entire manuscript based on your suggestions. The updated contents were presented in the revised manuscript.
Reviewer #3 (Recommendations For The Authors):
There are some issues that following issues require clarification to improve the quality of the manuscript further.
(1) L.55: Replace "antibiotic therapies" with "antibiotic therapy".
Response: Thank you for your suggestion. We have corrected it as suggested.
The updated contents were presented in line 56-57 in introduction section of the revised manuscript.
(2) "Bacillus" should be modified to italics in the manuscript (see e.g., L. 26, 65, 68, 109).
Response: Thank you for your suggestion. The comments improve the quality of manuscript. We have corrected this in the revised manuscript as suggested.
The updated contents were presented in line 26 in abstract section and line 67, 69 in introduction section and line 111 in results section of the revised manuscript.
(3) The first appearance of bacterial names in the manuscript requires the full English name (see e.g., L. 158, 159, 160).
Response: Thank you for pointing out this problem in manuscript. We have corrected this in the revised manuscript as suggested.
The updated contents were presented in line 153-156 in results section of the revised manuscript.
(4) L.166 and 167: "we evaluated its biological safety in a mouse model" suggest modifying to "we evaluated the biological safety of HBXN2020 in a mouse model".
Response: Thanks for your suggestion. We have corrected this as suggested.
The updated contents were presented in line 163-164 in results section of the revised manuscript.
(5) L.229: Replace "suggest" with "suggested".
Response: Thanks for your suggestion. We have corrected this as suggested.
The updated contents were presented in line 226 in results section of the revised manuscript.
(6) L.367: The tense of "can" should be consistent with "demonstrated".
Response: Thanks for pointing this out. We have corrected this as suggested.
(7) L.368 and L. 369: Replace "Gram positive and Gram negative" with "Gram-positive and Gram-negative".
Response: Thanks for your suggestion. We have corrected this as suggested.
(8) L.372: Replace "and" with "as well as".
Response: Thanks for your useful suggestion. We have corrected this in the revised manuscript as suggested.
The updated contents were presented in line 365 in discussion section of the revised manuscript.
(9) NCBI accession number of supplementing 16SrRNA sequencing raw data.
Response: Thank you for your suggestion. We have added it in the revised manuscript.
The updated contents were presented in line 770-773 in data availability section of the revised manuscript.
(10) L. 1020 and L. 1073: It's recommended to reduce the word count in the annotations of Figures 5 and 8.
Response: Thank you for your valuable suggestion. We have corrected it as suggested.
The updated contents were presented in the annotations of Figure 5 and Figure 8 in figure legends section of the revised manuscript.
