Safety Assessment of Five Candidate Probiotic Lactibacilli using Comparative Genome Analysis
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Microorganisms belonging to the Lactobacillus genus complex (LGC) are often used for oral consumption and are generally considered safe but can exhibit pathogenicity in rare and specific cases. Therefore, screening and understanding genetic factors that may contribute to pathogenicity can yield valuable insights regarding probiotic safety. Limosilactobacillus mucosae LM1, Lactiplantibacillus plantarum SK151, Lactiplantibacillus plantarum BS25, Limosilactobacillus fermentum SK152, and Lactobacillus johnsonii PF01 are current probiotics of interest; however, their safety profiles have not been explored. The genome sequences of LM1, SK151, SK152, and PF01 were downloaded from the NCBI GenBank, while that of L. plantarum BS25 was newly sequenced. These genomes were then annotated using the Rapid Annotation using Subsystem Technology (RAST) tk pipeline. Subsequently, a command line BLAST was performed against the Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD) to identify potential virulence factors and antibiotic resistance (AR) genes. Furthermore, ResFinder was used to detect acquired AR genes. The query against the VFDB identified genes that have a role in bacterial survivability, platelet aggregation, surface adhesion, biofilm formation and immunoregulation; and no acquired AR genes were detected using CARD and ResFinder. The study shows that the query strains exhibit genes identical to those present in pathogenic bacteria with the genes matched primarily having roles related to survival and surface adherence. Our results contribute to the overall strategies that can be employed in pre-clinical safety assessments of potential probiotics. Gene mining using whole genome data, coupled with experimental validation, can be implemented in future probiotic safety assessment strategies.
Article activity feed
-
You have addressed the raised concerns.
-
-
Overall the manuscript has improved, however there is still a number of things that need to be adressed: 1.5 Repositories – add the name of the repository here as well not just accession number Reference 3 is not an actual reference- please correct this and add an appropiate reference- this looks like some automatically generated suggestions l. 30 pathogenesis is wrongly used here, I think you mean pathogenecity since the bacteria do not develop a disease but rather cause it Abstract is missing a final summary sentence of what your findings contribute or how they can be used for policies, what should happen next? l. 76 Lactobacillus should be in italics l. 83 please add the reference to this documentation? l. 85-86 Reference Binda et al 2020 is in a different format than the rest of the references l 96-97 „strains have the ability to” …
Overall the manuscript has improved, however there is still a number of things that need to be adressed: 1.5 Repositories – add the name of the repository here as well not just accession number Reference 3 is not an actual reference- please correct this and add an appropiate reference- this looks like some automatically generated suggestions l. 30 pathogenesis is wrongly used here, I think you mean pathogenecity since the bacteria do not develop a disease but rather cause it Abstract is missing a final summary sentence of what your findings contribute or how they can be used for policies, what should happen next? l. 76 Lactobacillus should be in italics l. 83 please add the reference to this documentation? l. 85-86 Reference Binda et al 2020 is in a different format than the rest of the references l 96-97 „strains have the ability to” replace by “strains can” l 101 „of disease“ is redundant when using pathogenesis is used prior l 172-175 species names need to be in italics l 180 change „ may cause a disease process“ to „cause disease“ L 185 RAST version needs to be stated L193 be more specific which sequences are you referring to here? It is assumed the 5 genomes?! Ll 209-211 references for the tools are missing L 225 would add „encoding“ before genes, bsh should be in capital letter as you are referring to an enzyme L 226 bsh should be Bsh as I presume you are referring to the enzyme and not the gene L 230 suddenly it is BSH here, it should be consistent throughout. Genes need to be small letters and in italics, proteins first letter in capital and no italics L233 bsh genes – this needs to be in italics L 239 I think you mean here „a priori“ L 252 – 253 give a bit more detail on the genes here, what do they encode and how are they involved in pathogenecity? Or refer that each will be in detail discussed next. L 267 in vitro in italics, testing not in italics L 281 reference in different format (Granato et al) – no number reference at end of sentence? L 290/294/297 species name should be small letter (T -> t) L 306 species names in italics L 308/309 reference not in number at end of sentence L333-335 please put a reference for this statement L 335 „Hsa expressed in Challis“ Challis is a subspecies? Please use the full term. L 382 genes not in italics? L395 pathology should this not rather be pathogenecity? L 428 reference Allard et al 2002 is not at the end of the sentence in number format L429 italics Lactobacillus L 444 Campedelli reference is not at the end of the sentence in number reference format l 445 italics Lactobacillus l 456-7 gene name spelling incorrect Table 1 Mycobacterium needs to be in italics Table 2 genes need to be in italics Editor Review Questions
-
-
-
The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. Please provide more detail in the Methods section and ensure that software is consistently cited and its version and parameters included. In addition to the points raised by the reviewers, please also consider the following For the purpose of clarity I suggest you put a line which repositry you have used under the data statement. Under data summary please also mention the accession number of the strain you have sequenced as part of your study. It would help clarity if you introduced bacteriocins in the introduction as refer later to why analysing this is of importance and include this in your discussion as well. Please state the version and settings for each pipeline that was used (missing for RAST and also under 6.5)
The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. Please provide more detail in the Methods section and ensure that software is consistently cited and its version and parameters included. In addition to the points raised by the reviewers, please also consider the following For the purpose of clarity I suggest you put a line which repositry you have used under the data statement. Under data summary please also mention the accession number of the strain you have sequenced as part of your study. It would help clarity if you introduced bacteriocins in the introduction as refer later to why analysing this is of importance and include this in your discussion as well. Please state the version and settings for each pipeline that was used (missing for RAST and also under 6.5) The results appear very much as a list of different findings (and there seems to be a high similarity phrases from references used- you need to address this), I highly recommend to connect/relate these findings in the text and establish more of a narrative for the reader (please also see point 4 from reviewer 1) I also strongly agree with the suggestion to rephrase your title. This would also aid establishment of a narrative. Individual Reviewer Comments to Author (Editor's Copy)
-
Comments to Author
Please address areas of high similarity concern highlighted on the iThenticate report. The article deals with comparative genomics of Lactobacillus The author failed to incorporate reference for "Probiotics…. Gras status". The safety and toxicity investigations of probiotic microorganisms are the main area of interest for the author. Its hemolytic nature is one of the primary determinants of safety and toxicity. Although the author makes no mention of it, bioinformatics technologies may also be used to anticipate this. Bile salt hydrolases, the adhesion genes, must be taken into account for the investigation. The author should demarcate survival and adaptation factors of the control, and various microbes considered for the study. Subsequently, they may discuss in comparison with the control so that …
Comments to Author
Please address areas of high similarity concern highlighted on the iThenticate report. The article deals with comparative genomics of Lactobacillus The author failed to incorporate reference for "Probiotics…. Gras status". The safety and toxicity investigations of probiotic microorganisms are the main area of interest for the author. Its hemolytic nature is one of the primary determinants of safety and toxicity. Although the author makes no mention of it, bioinformatics technologies may also be used to anticipate this. Bile salt hydrolases, the adhesion genes, must be taken into account for the investigation. The author should demarcate survival and adaptation factors of the control, and various microbes considered for the study. Subsequently, they may discuss in comparison with the control so that the study will be much clearer. A small write-up of these factors and their link in the said functions may enhance the credibility. The table may give good clarity. At present the MS looks just WGS analysis data has been exhibited it does not give clear picture of the objectives. The following hypothesis / report needs a reference "Pathogenic bacteria may acquire antibiotic resistance genes from the probiotics via horizontal gene transfer. When the probiotic has an intrinsic resistance, it means that all AR genes are typical of all strains of that species" "In healthy individuals, opportunistic pathogens are not able to produce infections as they lack the necessary mechanisms of toxicity and invasiveness to overcome the immune system". Here the author may need to discuss dysbiosis and eubiosis phenomenon, followed by immune response.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data The methods and description of methods appear appropriate. 2. Presentation of results Results are generally well presented. 3. How the style and organization of the paper communicates and represents key findings I think the current title of the manuscript should be reconsidered. Aspects of the title could be more concise, i.e. is it necessary to list each candidate probiotic? Furthermore, the element of investigating genetic similarities between pathogenic bacteria and LAB should be more clearly reflected in the manuscript title. 4. Literature analysis or discussion In the Results and Discussion, I suggest that sections 7.2 and 7.5 are combined. Currently, section 7.2 focuses on discussing the identified virulence genes, …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data The methods and description of methods appear appropriate. 2. Presentation of results Results are generally well presented. 3. How the style and organization of the paper communicates and represents key findings I think the current title of the manuscript should be reconsidered. Aspects of the title could be more concise, i.e. is it necessary to list each candidate probiotic? Furthermore, the element of investigating genetic similarities between pathogenic bacteria and LAB should be more clearly reflected in the manuscript title. 4. Literature analysis or discussion In the Results and Discussion, I suggest that sections 7.2 and 7.5 are combined. Currently, section 7.2 focuses on discussing the identified virulence genes, but in the context of pathogens. I would argue that these should be discussed in relation to the candidate probiotic strains / lactic acid bacteria, which is somewhat done in section 7.5. By integrating sections 7.2 and 7.5, the main focus of the manuscript (i.e. probiotics/LAB) would be maintained, and the narrative easier to follow. It is not clear what the bacteriocin mining aspect adds to the manuscript. Furthermore, in section 7.3, there is no discussion of the bacteriocin mining results. I suggest that the authors should reconsider the purpose of bacteriocin mining in this manuscript, or provide justification for its inclusion in the manuscript. 5. Any other relevant comments Line 31-32: I think this should state "used for oral consumption" Line 32: I think this should read "but can exhibit pathogenesis" Line 39: Please check the phrasing/grammar Line 77: I think this should read "in some instances" Line 107: check spelling of "status" Line 134: The references cited should be listed in ascending order Line 173: Provide some appraisal of the potential implications of this statement i.e. multiple genes related to beta-lactamase resistance Line 175-176: Provide a citation for this statement Line 300: Correct spelling should be "Campedelli" Line 302: Check phrasing "while other reports...." Details of Reference number [7] are incomplete/incorrect
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-