Avian and Human Turicibacter Isolates Possess Bile Salt Hydrolases with Activity Against Tauro-Conjugated Bile Acids

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a straightforward and well-executed paper that explores the bile salt hydrolase activity of two newly identified strains, with a particular preference for chicken bile acids. I find the work solid overall.  The significance is not groundbreaking, I think it adds some valuable insights to the field. The article is well-written, and the findings are presented clearly.

 

Minor Concern:

 

In Figures 1 and 2, I think it would be helpful to clarify the limit of detection. This would give readers a better understanding of how truly zero your zero is.

 

Major Concern 1:

 

In both the introduction and the discussion, I believe it’s important to explain why these two specific chicken strains were chosen for this study. What are the implications for agriculture, human health, or other areas? Why focus on these two strains specifically? Providing more context here would really help strengthen the relevance of the study.

 

Major Concern 2:

 

In the discussion, it would be valuable to address how likely the identified genes are to be the only bsh genes in the genome. Are there other bsh genes present, and if so, how can the authors be certain that the genes they identified are the only ones responsible for the observed activity?  What would the Bile analysis data look like if multiple enzymes were at work?

 

Minor Concern:

 

I think it would be useful to discuss how genetically tractable these organisms are and whether deletions could be used to confirm the identity of the identified genes as BSH. This is a point I would suggest addressing in the discussion, though I don’t think it needs to be incorporated just yet.

Author Response

Comment 1: In Figures 1 and 2, I think it would be helpful to clarify the limit of detection. This would give readers a better understanding of how truly zero your zero is.

Response 1: Thank you for the comment. We agree. The limit of detection has been added to the figure legend for both figures 1 and 2 (lines 251 and 281). Lines 251-252 (page 6, figure 1 caption) and 281-283 (page 7, figure 2 caption) read, “Limit of detection (LOD) for taurochenodeoxycholic acid/chenodeoxycholic acid = 0.0001% (w/v). LOD for taurocholic acid/cholic acid = 0.0003% (w/v).”

Comment 2: In both the introduction and the discussion, I believe it’s important to explain why these two specific chicken strains were chosen for this study. What are the implications for agriculture, human health, or other areas? Why focus on these two strains specifically? Providing more context here would really help strengthen the relevance of the study.

Response 2: Thank you for the comment. These specific strains of Turicibacter bilis were chosen as they were the only chicken Turicibacter isolates we had at the time. They are also, to our knowledge, the only Turicibacter strains that have been isolated from chickens to date. If we had more chicken isolates available to us, we would have included them in this work. As for the question on relevance, Turicibacter represent a ubiquitous organism in the gastrointestinal tract of chickens (and other livestock species) and their ability to modify bile acids has important implications for chicken nutrient absorption, making this study of interest to the agricultural industry. Bile acids can also act as important chemical signals for foodborne pathogens like Campylobacter jejuni and Escherichia coli, making bile acid-modifying bacteria, such as Turicibacter, an important are of study as these organisms may enhance or inhibit colonization of potential pathogens. We have added a sentence at line 40 to reflect why the strains were chosen. Line 40 (page 1, paragraph 3) reads, “These two strains represent the only known isolates of T. bilis from poultry”. We also added a section (lines 75-83) to address the relevance of the work for the agricultural industry and human health. Lines 75-83 (page 2, paragraph 3) now read, “Characterizing the range and extent of BA modification by Turicibacter spp. would be of interest to the poultry industry, as BA modifications may impact nutrient absorption in chickens (26, 28, 29). Additionally, modified BAs also serve as chemical signals that modulate the virulence and colonization capabilities of poultry pathogens, such as Clostridium perfringens, and food safety pathogens such as Campylobacter jejuni (42, 43, 44). Understanding BA-modifying organisms, like Turicibacter spp., and the BA preferences of their BSH could allow for the development of targeted interventions to reduce disease in poultry and make poultry products safer for consumers.”

Comment 3: In the discussion, it would be valuable to address how likely the identified genes are to be the only bsh genes in the genome. Are there other bsh genes present, and if so, how can the authors be certain that the genes they identified are the only ones responsible for the observed activity?  What would the Bile analysis data look like if multiple enzymes were at work?

Response 3: Thank you for the comment. We are confident in the annotation of the bsh genes across the different Turicibacter strains, though we are only able to operate off the genome annotations generated in this work and how those results align with our previous work and the work of others (Lynch et al., 2023). We did not undertake any cloning or knockout mutant generation in this work, so the possibility that we are "missing" genes capable of deconjugating bile acids is a possibility. We do note that this is a limitation and further work into the individual bsh genes would help to clarify the activity of BSH in the poultry gut (lines 518-522), but more text has been added to the discussion (line 508) to explicitly mention the need for knockout mutants generation of individual bsh genes would help to clarify if these bsh genes are the only ones responsible for bile acid deconjugation and to assess the deconjugation activity of these BSH individually and in combination. Lines 508-511 (page 13, paragraph 1) read, “Further studies into the deconjugative capabilities of bsh knockout mutants would provide greater understanding of the spectrum of activity and rate of deconjugation for Turicibacter BSH from different clades and sub-clades, both individually and in combination.”

Comment 4: I think it would be useful to discuss how genetically tractable these organisms are and whether deletions could be used to confirm the identity of the identified genes as BSH. This is a point I would suggest addressing in the discussion, though I don’t think it needs to be incorporated just yet.

Response 4: Thank you for the comment. To our knowledge, no group has tried to generate knockout mutants of any genes in Turicibacter so it is unknown how genetically tractable the organism is. We have noted the need for further work into the individual Turicibacter bsh genes in the discussion (Line 518) but we also added a section to the discussion (line 508) to address the need for bsh knockout mutants in Turicibacter to get a true sense of the spectrum of activity. Lines 508-511 (page 13, paragraph 1) read, “Further studies into the deconjugative capabilities of bsh knockout mutants would provide greater understanding of the spectrum of activity and rate of deconjugation for Turicibacter BSH from different clades and sub-clades, both individually and in combination.”

Reviewer 2 Report

Comments and Suggestions for Authors

The present manuscript aims to compare the bile salt hydrolase (BSH) activity and genome of Turicibacter strains from chickens, pigs, and humans. By analyzing Turicibacter strains from different hosts, the study reveals variations in bile acid metabolism and explores how these differences influence host adaptation. However, several issues need to be addressed before publication.

Major Comments:

1. Bacterial growth should be monitored throughout the experiment (e.g., through bacterial counting or optical density measurements). Changes in bile acids could result from either bacterial metabolic activity or population changes. Determining whether bile acids and related compounds inhibit bacterial growth would significantly strengthen the study's logical rigor.
2. The clarity of figures requires improvement, as several are currently difficult to interpret.
3. The metabolic experiment uses only a single 8-hour time point for bile acid measurements. A time-course experiment would be ideal, and at minimum, this limitation should be explicitly acknowledged.
4. Please clarify the methodology for identifying shared CDS among species.

Minor Comments:

1. Some species names are not italicized as required.
2. Lines 278-280 should be relocated to the Methods section."

 

Author Response

Comment 1: Bacterial growth should be monitored throughout the experiment (e.g., through bacterial counting or optical density measurements). Changes in bile acids could result from either bacterial metabolic activity or population changes. Determining whether bile acids and related compounds inhibit bacterial growth would significantly strengthen the study's logical rigor.

Response 1: Thank you for the comment. We previously conducted growth curve analyses with Turicibacter bilis in another publication (Maki et al., 2023), so we can say these strains were likely in late-log phase to early stationary phase when the spent media samples were collected for metabolomic analysis. The reviewer brings up a valid criticism about potential bile acid inhibition of the Turicibacter strains. A section has been added to the discussion (line 475) to acknowledge the need for growth inhibition assays for different Turicibacter strains under different bile acids. Lines 473-477 (page 12, paragraph 2) now read, “Further work needs to be conducted in Turicibacter and other bsh-encoding organisms to characterize this potential link between BSH spectrum of activity and the BA profile of its preferred host species as well as the potential inhibitory effects of bile and BAs from different hosts and how the transformation of these BAs impacts Turicibacter growth dynamics and survival.”

Comment 2: The clarity of figures requires improvement, as several are currently difficult to interpret.

Response 2: Thank you for the comment. Figures 3 (page 8) and 4 (page 9) have been revised to enhance clarity and increase font size.

Comment 3: The metabolic experiment uses only a single 8-hour time point for bile acid measurements. A time-course experiment would be ideal, and at minimum, this limitation should be explicitly acknowledged.

Response 3: Thank you for the comment. The reviewer is correct that a time course trial would be warranted to further determine the impacts of growth phase and incubation times on bile acid deconjugation. A sentence has been added to the discussion (line 477) acknowledging the need for follow-up studies to assess impacts of growth phase on Turicibacter BSH activity. Lines 477-481 (page 12, paragraph 2) read, “In this study, spend media samples were collected after 8 hours of incubation, which correspond to the late-log to early stationary phase of growth for Turicibacter bilis (37). Follow-up studies should be conducted to further assess the impacts of growth phase and incubation times on the extent of BA deconjugation for these and other Turicibacter strains”

Comment 4: Please clarify the methodology for identifying shared CDS among species.

Response 4: Thank you for the comment. CDS where considered "shared" between strains if they possessed the same "seed_eggNOG_ortholog" annotation. We have updated the methods (Line 148) to provide additional information on how shared CDSs were determined. Line 148-150 (page 4, paragraph 2) read, “CDSs were considered “shared” between strains if they possessed the same “seed_eggNOG_ortholog” ID from the eggNOG-mapper annotation.”

Comment 5: Some species names are not italicized as required.

Response 5: Thank you for the comment. We have scanned the manuscript and italicized species names in the main body of the text as required.

Comment 6: Lines 278-280 should be relocated to the Methods section.

Response 6: Thank you for the comment. We have removed this sentence from the results as it was redundant with what was already present in the methods (lines 131-138).