Metabacillus schmidteae sp. nov., Cultivated from Planarian Schmidtea mediterranea Microbiota

Round 1

Reviewer 1 Report

The authors performed the description of a novel Bacillus species with proposed name as Bacillus schmidteae sp. nov., isolated from the microbiota of planarian Schmidtea mediterranea.

The manuscript is well written, the experiments have been well designed and performed, also the results and methodology are clearly and well presented to the reader.

However, I have a minor comment for the authors that in my opinion, need to be addressed before manuscript publication.

Minor comment:

The discovery and description of novel bacterial species is not a minor finding. Therefore, I think that the introduction deserves to be a little bit more developed and improved, since in this state is very poor and does not correlate to the important finding presented in this manuscript.

 

Author Response

R1

The authors performed the description of a novel Bacillus species with proposed name as Bacillus schmidteae sp. nov., isolated from the microbiota of planarian Schmidtea mediterranea.

The manuscript is well written, the experiments have been well designed and performed, also the results and methodology are clearly and well presented to the reader.

However, I have a minor comment for the authors that in my opinion, need to be addressed before manuscript publication.

Minor comment:

The discovery and description of novel bacterial species is not a minor finding. Therefore, I think that the introduction deserves to be a little bit more developed and improved, since in this state is very poor and does not correlate to the important finding presented in this manuscript.

Authors response: the authors thank the reviewer’s remark and we have added details in the manuscript introduction from lines 45 to 60. “The birth of genomics, followed by the development of Next Generation Sequencing (NGS) methods has allowed the characterization, classification and nomenclature of a large number of prokaryotic species; using taxonogenomics strategy that combines phenotypic assays and genome sequencing [1]–[3]. The phenotype of an organism is typically taken to parameters such as morphological, physiological, chemical and biochemical characteristics [4]. Whereas genotypic characterization uses the genetic material that is essential of species description, as genetic information sheds light on the evolutionary relationships between various lineages. First, the16S rRNA gene sequences were used to determine of similarity between sequences and phylogenetic analysis [68]; then DNA–DNA hybridization, which evaluates the degree of genetic similarity between two genomes, has been used for bacterial species demarcation by providing a constant numerical threshold (DDH value >70 %) for the species boundary [69]. Planarian Schmidtea mediterranea, is an invertebrate living in freshwater and an excellent models to investigate the host-pathogen relationship in the context of human pathogens [5]–[7]. To understand the role of the microbiota in the immune response of S. mediterranea, we have investigating the composition of its microbiota. From the S. mediterranea microbiota we have  isolated by culturomics [70] a bacterial strain (Marseille-P9898).”

Reviewer 2 Report

The paper is well written, clear and for easy reading. The description of the new species is interestingly done. Improvement could be the description of the aspect of the colonies on more solid agar media and not only blood agar, because is not easy to identify colonies only on non selective agar media.

The study of the genome could also give to the microbiologist the selection of sequences to put in place PCR for fast identification of Bacillus schmidteae.

 

General consideration

May be is not of paramount importance for the characterization but you presented results of AMR, then why not to report data on AMR in silico?

Requests for checking and amendments

40          double space after described

52          the “latter” for clarity better to report water

54          please explain how the sterility was checked

58          “the worm was sprayed”? Please explain clearly

69          repetition of sequencing, please check the sentence

75          it is not clear the usage of “two” sequencing …. May be it is better to use “the”?

81          nr database is Blast nr database please clarify in the text

89          Comparison COG functional categories are carried out using BLASTP (E-value 10-3, 89 coverage 0.7 and identity percent 30%) against the clusters of orthologous groups (COG) 90 database.

Please report the “clusters of orthologous groups” after Comparison and delete after 30%)

93          “sought” is not a proper word for streaking or spreading or pour-on, the meaning of the sentence is not clear. Does the strain was streaked and then grown at different temperatures?

140 – 147  Could you please explain why you cited B. malikii as closely related strain but you did not report the strain in Table 1.

164 please check the tense of “present” and amend if needed

169 please check the usage of “enough” and amend if needed

185 please explain the meaning of “aerobic atmosphere and strictly aerobic” and amend if needed

188 please check the term “Tableau 2” and amend

190 please check the meaning of “with yellowish white” and explain

228 – 229 “please check the sentence “were not inhibited the growth of strain Marseille-P9898” and amend

Figure 1 please add the last 4 rows of the caption to M&M, starting from “Sequences where aligned … divergence”

Figure 2 is reported without any connection to the textIn the Section 1.1

Author Response

R2

The paper is well written, clear and for easy reading. The description of the new species is interestingly done. Improvement could be the description of the aspect of the colonies on more solid agar media and not only blood agar, because is not easy to identify colonies only on non selective agar media.

Authors response: We agree, however the aims of the manuscript is to characterize by genomics the nature of the colonies selected. The colors of the colonies is just an indication but cannot be a way to identify this microbes

 

The study of the genome could also give to the microbiologist the selection of sequences to put in place PCR for fast identification of Bacillus schmidteae.

Authors response: We agree, but pangenome is required. PCR Primers need to be define in function of the PCR methods used and it is important to check cross reaction with other species.

 

General consideration

May be is not of paramount importance for the characterization but you presented results of AMR, then why not to report data on AMR in silico?

Authors response: we are not sure to understand what the reviewer mean by AMR in silico. Anyway instead of to report data a from the literature, we have preferred to measure again the MIC from the different bacterial strain used in this study

 

Requests for checking and amendments.

40  double space after described

Authors response: corrected accordingly

 

52 the “latter” for clarity better to report water.

Authors response: the authors thanks the reviewer’s remark, in lines 52 we replace latter to “filtered water” (now line 76).

 

54 please explain how the sterility was checked

Authors response: the authors thanks the reviewer’s remark and the method details now provided in lines 78 to 83 “The sterility of the filtered water has been checked before use for S. mediterranea. Microbiological analysis of the filtered water was performed by inoculation of the filtered water (25, 50, 75 and 100 µL) in 5 % sheep blood-enriched Columbia agar plate (bioMérieux, Marcy l’étoile, France) and incubated at various temperatures (5, 10, 19, 28, 37 and 45 °C). After  4 days the absence of bacterial colonies was checked.”

 

58 “the worm was sprayed”? Please explain clearly

Authors response: sentence was clarified (now line 86 to 88). “The worms were starved for 2 weeks, washed in filter-sterilized water, one worm was smashed and inoculated in 5% sheep blood-enriched Columbia agar (bioMérieux, Marcy l’étoile, France), and incubated at 28°C.”

 

69 repetition of sequencing, please check the sentence

Authors response: the authors thanks the reviewer’s remark, we delete “of sequencing” now lines 99 to 100. “We used two technologies for the sequencing of the strain Marseille-P9898.”

 

75  it is not clear the usage of “two” sequencing …. May be it is better to use “the”?

Authors response: the authors thanks the reviewer’s remark, now lines 97 to 98. “We used two technologies for the sequencing of the strain Marseille-P9898.”

 

81 nr database is Blast nr database please clarify in the text

Authors response: the authors thanks the reviewer’s remark, now line 111 “For taxonomic assignation, we use nr database (Standard databases) for BLASTn search”

 

89 Comparison COG functional categories are carried out using BLASTP (E-value 10-3, 89 coverage 0.7 and identity percent 30%) against the clusters of orthologous groups (COG) 90 database.

Please report the “clusters of orthologous groups” after Comparison and delete after 30%)

Authors response: the authors thanks the reviewer’s remark, now lines 125 to 127 Comparison “COG functional categories are carried out using BLASTP (E-value 10^-3, coverage 0.7 and identity percent 30%) against the clusters of Bacillus orthologous groups (COG) database”.

 

93 “sought” is not a proper word for streaking or spreading or pour-on, the meaning of the sentence is not clear. Does the strain was streaked and then grown at different temperatures?

Authors response: the authors thanks the reviewer’s remark, now lines 130 to 132 “Strain Marseille-P9898 was attempted at different growth temperatures (4, 19, 28, 30, 37 and 45°C) in 5% sheep blood-enriched Columbia agar (bioMérieux) under anaerobic and aerobic atmospheres using GasPak™ EZ generators (Becton- Dickinson, Maryland, USA).”.

 

140 – 147 Could you please explain why you cited B. malikii as closely related strain but you did not report the strain in Table 1.

Authors response: the authors thanks the reviewer’s remark. We do not report this strain in the table 1 because the genomics information are not available

 

164 please check the tense of “present” and amend if needed

Authors response: corrected accordingly

 

169 please check the usage of “enough” and amend if needed

Authors response: the authors thanks the reviewer’s remark, we replace enough to sufficient

 

185 please explain the meaning of “aerobic atmosphere and strictly aerobic” and amend if needed

Authors response: the authors thanks the reviewer’s remark, we delete “and strictly aerobic”

 

188 please check the term “Tableau 2” and amend

Authors response: corrected accordingly

 

190 please check the meaning of “with yellowish white” and explain

Authors response: corrected accordingly

 

228 – 229 “please check the sentence “were not inhibited the growth of strain Marseille-P9898” and amend

Authors response: corrected accordingly

 

Figure 1 please add the last 4 rows of the caption to M&M, starting from “Sequences where aligned … divergence”

Authors response: corrected accordingly. Now lines 115 to 120 “DNA sequences were downloaded in fasta format to NCBI. Methods for estimating phylogenetic trees is Maximum Likelihood. The sequences were aligned using the MUSCLE algorithm with default parameters. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 1000 times to generate a majority consensus tree. Only bootstrap values ≥50% were retained. The scale bare indicates a 0.01% sequence divergence”

 

Figure 2 is reported without any connection to the textIn the Section 1.1

Authors response: corrected accordingly. Now lines 215 we add “(Figure 2)”.

Reviewer 3 Report

The article “Bacillus schmidteae sp. nov., cultivated from microbiota planarian Schmidtea mediterranea” concerns the description of a Marseille-P9898 strain isolated from the planarian Schmidtea mediterranea. The authors identified and characterized bacterium upon (1) phenotypic and biochemical properties, (2) sequence of 16S rRNA, (3) functional annotation of proteins encoded by sequenced genomes, (5) DNA-DNA hybridization, (6) average nucleotide identity, (7) antibiotic susceptibility, (8) content of fatty acids, and (9) phylogenetic relationships. According to the authors, the results of their work show that the strain Marseille-P9898 is a new bacterial species of the genus Bacillus and they propose to name it Bacillus schmidteae sp. nov. Nevertheless, some issues raised in the article are questionable and require clarification. The manuscript describes interesting research results but in a very uninteresting and messy way. Moreover, it contains unacceptable shortcomings and errors that indicate either a rush in the preparation of the manuscript or the authors' ignorance of microbiology. Additionally, the manuscript contains a number of inaccuracies and some parts in the manuscript need to be improved. I suggest accepting the manuscript, but it needs changes.

 

Major remarks:

1. The bacteria cultured from Schmidtea mediterranea were identified by MALDI-TOF-MS (page 2, lines 59-64), but the authors did not mention about the results of this identification in the manuscript. The authors are asked to present the results and discuss them in the manuscript, especially for the strain Marseille-P9898.

2. Currently, two strains are considered as belonging to distinct species if they share 16S rRNA gene sequence identity lower than 98.7% (Yarza et al., 2014, Nature Reviews Microbiology 12, 635–645; Rossi-Tamisier et al., 2015, International Journal of Systematic and Evolutionary Microbiology 65,1929–1934).

The authors are asked to explain in the manuscript on what basis they propose establishing a new bacterium species, since the identity of 16S rDNA sequence of the strain Marseille-P9898 and Bacillus niabensis is above 98.7%. It should be discussed.

 

Minor remarks:

1. The authors are asked to rethink the content of the Abstract. Why are some results presented in detail and others not at all? Maybe it is worth emphasizing the essence of the work and summarizing the results instead of listing them in detail?

2. There is insufficient information in the Introduction section. It should be rewritten. The authors are asked to clearly describe the criteris for establishing a new bacterial species. Additionally, the authors should consider including in Introduction characteristics of Bacillus species showing high similarity to the strain Marseille-P9898. The characteristics of the host Schmidtea mediterranea would also be desirable in the Introduction section.

3. The characterization of the Bacillus genus presented in the Introduction (page 2, lines 42-45) in not complete and the data is based on incorrect references. The authors are asked to start reading about Bacillus with Bergey's Manual of Systematic Bacteriology, and then find publications concerns Bacillus spp.

4. The authors used API test kits (bioMérieux, USA) for biochemical characterization of bacterial strain Marseille-P9898. The API kits are a basic tool for identifying bacteria to the species level in any microbiological laboratory. Maybe the authors should consider using APIWEB software (bioMérieux, USA) for a reliable automated interpretation of their API strip results. Maybe the results should be provided in the manuscript and discussed.

5. Materials and Methods. What model of sequence evolution was chosen in the phylogenetic analysis using the MEGAX 10.1 software? What software was applied for model selecting?

6. The authors are asked to consider the change in the order of methods and results presented. Maybe first the methods (in the Materials and Methods section) and results (in the Results and Discussion section) of bacterial identification should be presented (MALDI-TOF-MS, API, analysis of 16S rRNA, DNA-DNA hybridization), and then, the methods allowing detailed the characterization of the bacterium (antibiotic susceptibility, phylogeny). The authors are asked to rethink the structure of the manuscript with regards to the weight and purpose of the methods used.

7. There is erroneous information in the manuscript. For example in Table 4, Bacillus litoralis was indicated as Gram-negative and the reference Yoon & Oh (2005, Int. J. Syst. Evol. Microbiol. 55, 1945–1948) was provided. But in the publication mention above, Bacillus litoralis is decribed as a Gram-variable bacterium. The authors are asked to carefully check the credibility of the presented content and to cite them correct.

8. Table 4. Why there are so many NA in the table?

9. Only results are presented in the Results and Discussion section. There is no discussion.

 

The manuscript is written messily and contains numerous inaccuracies. For example:

- Title. “microbiota planarian Schmidtea mediterranea”. Maybe it should be: “microbiota of…”,

- Title page, abbreviation. The authors are asked to add the English translation of the abbreviation expansion and to complete the list of abbreviations with the others mentioned in the manuscript: NCCP, DMS, GGDC and ect.

- Keywords. Latin species names should be italicized.

- Page 2, line 42 and page 4, line 145. The names of bacterial family and phylum are written in italics. Table 2 and page 15, line 246. These names are not written in italics. The authors are asked to unify this.

- The correction of the numbering of sections and subsections in the manuscript is needed.- Materials and Methods. Results and Discusson. Why the description of phylogenetic analysis and genomic comparison are not divided? These are two completely different analyzes, the results of which lead to completely different conclusions. Similarly, the description of antibiotic susceptibility and the content of fatty acids.

- “Hexadecanoic acid”, „12-methyl-Tridecanoic acid”, “Maximum likelihood Phylogenetic”, and etc. Maybe it should be: “hexadecanoic acid”, “12-methyl-tridecanoic acid”, “Maximum likelihood phylogenetic” or “Maximum Likelihood Phylogenetic”?

- Subtitles. “Antibiotic susceptibility and chemotaxonomic Analysis”. Maybe it should be “Antibiotic susceptibility and chemotaxonomic analysis” or „Antibiotic Susceptibility and Chemotaxonomic Analysis”?

-Page 9, line 188. “Tableau”?

-Page 13, line 231. No full stop at the end of the sequence.

- Page 14, line 233. The explanation of the unit abbreviation μg/ml is unnecessary.

- Page 15, line 242. The strain “was first isolated from microbiota of planarian and abundant”. What does it mean “abundant” in this sentence? The authors are ask to explain this issue in the Result section.

- Figure 1. Some names of bacterial species are italicized and others not. The authors are asked to unify this.

- The Figure 2 is poorly visible. Names of bacterial species should be in italics.

 

This is only a brief summary of inaccuracies in the manuscript. Similar mistakes should be found and corrected throughout the manuscript.

Comments for author File: Comments.pdf

Author Response

R3

The article “Bacillus schmidteae sp. nov., cultivated from microbiota planarian Schmidtea mediterranea” concerns the description of a Marseille-P9898 strain isolated from the planarian Schmidtea mediterranea. The authors identified and characterized bacterium upon (1) phenotypic and biochemical properties, (2) sequence of 16S rRNA, (3) functional annotation of proteins encoded by sequenced genomes, (5) DNA-DNA hybridization, (6) average nucleotide identity, (7) antibiotic susceptibility, (8) content of fatty acids, and (9) phylogenetic relationships. According to the authors, the results of their work show that the strain Marseille-P9898 is a new bacterial species of the genus Bacillus and they propose to name it Bacillus schmidteae sp. nov. Nevertheless, some issues raised in the article are questionable and require clarification. The manuscript describes interesting research results but in a very uninteresting and messy way. Moreover, it contains unacceptable shortcomings and errors that indicate either a rush in the preparation of the manuscript or the authors' ignorance of microbiology. Additionally, the manuscript contains a number of inaccuracies and some parts in the manuscript need to be improved. I suggest accepting the manuscript, but it needs changes.

 

 

Major remarks:

1. The bacteria cultured from Schmidtea mediterranea were identified by MALDI-TOF-MS (page 2, lines 59-64), but the authors did not mention about the results of this identification in the manuscript. The authors are asked to present the results and discuss them in the manuscript, especially for the strain Marseille-P9898.

 

Authors response: the authors thank the reviewer’s remark. Now line 177 to 181 “Otherwise, the iidentification by MALDI-TOF-MS of the strain Marseille-P9898 showed a score of 1.77 matching for Bacillus niabensis. This value is less than 2 and does not allow the strain Marseille-P9898 to be identified as Bacillus niabensis; but this score shows the strain Marseille-P9898 belongs to genus Bacillus and related species is not identified. Hence the interest of doing a genomic study”

 

2. Currently, two strains are considered as belonging to distinct species if they share 16S rRNA gene sequence identity lower than 98.7% (Yarza et al., 2014, Nature Reviews Microbiology 12, 635–645; Rossi-Tamisier et al., 2015, International Journal of Systematic and Evolutionary Microbiology 65,1929–1934).

The authors are asked to explain in the manuscript on what basis they propose establishing a new bacterium species, since the identity of 16S rDNA sequence of the strain Marseille-P9898 and Bacillus niabensis is above 98.7%. It should be discussed.

Authors response: the authors thank the reviewer’s remark, we explain the details of this information in introduction. Lines 45 to 60 “The birth of genomics, followed by the development of Next Generation Sequencing (NGS) methods has allowed the characterization, classification and nomenclature of a large number of prokaryotic species; using taxonogenomics strategy that combines phenotypic assays and genome sequencing [1]–[3]. The phenotype of an organism is typically taken to parameters such as morphological, physiological, chemical and biochemical characteristics [4]. Whereas genotypic characterization uses the genetic material that is essential of species description, as genetic information sheds light on the evolutionary relationships between various lineages. First, the16S rRNA gene sequences were used to determine of similarity between sequences and phylogenetic analysis [68]; then DNA–DNA hybridization, which evaluates the degree of genetic similarity between two genomes, has been used for bacterial species demarcation by providing a constant numerical threshold (DDH value >70 %) for the species boundary [69]. Planarian Schmidtea mediterranea, is an invertebrate living in freshwater and an excellent models to investigate the host-pathogen relationship in the context of human pathogens [5]–[7]. To understand the role of the microbiota in the immune response of S. mediterranea, we have investigating the composition of its microbiota. From the S. mediterranea microbiota we have  isolated by culturomics [70] a bacterial strain (Marseille-P9898).”

 

 

Minor remarks:

1. The authors are asked to rethink the content of the Abstract. Why are some results presented in detail and others not at all? Maybe it is worth emphasizing the essence of the work and summarizing the results instead of listing them in detail?

Authors response: corrected accordingly

 

2. There is insufficient information in the Introduction section. It should be rewritten. The authors are asked to clearly describe the criteris for establishing a new bacterial species. Additionally, the authors should consider including in Introduction characteristics of Bacillus species showing high similarity to the strain Marseille-P9898. The characteristics of the host Schmidtea mediterranea would also be desirable in the Introduction section.

Authors response: corrected accordingly

 

3. The characterization of the Bacillus genus presented in the Introduction (page 2, lines 42-45) in not complete and the data is based on incorrect references. The authors are asked to start reading about Bacillus with Bergey's Manual of Systematic Bacteriology, and then find publications concerns Bacillus spp.

Authors response: corrected accordingly

 

4. The authors used API test kits (bioMérieux, USA) for biochemical characterization of bacterial strain Marseille-P9898. The API kits are a basic tool for identifying bacteria to the species level in any microbiological laboratory. Maybe the authors should consider using APIWEB software (bioMérieux, USA) for a reliable automated interpretation of their API strip results. Maybe the results should be provided in the manuscript and discussed.

Authors response: we agree, but to date we proceed as described in manuscript. The combination of this different methods describe in manuscript lead us to go from species identification to the subspecies and thus the characterization of a new species

 

5. Materials and Methods. What model of sequence evolution was chosen in the phylogenetic analysis using the MEGAX 10.1 software? What software was applied for model selecting?

Authors response: the authors thanks the reviewer’s remark. Now lines 115-117“DNA sequences were downloaded in fasta format to NCBI. Methods for estimating phylogenetic trees is Maximum Likelihood.”

 

6. The authors are asked to consider the change in the order of methods and results presented. Maybe first the methods (in the Materials and Methods section) and results (in the Results and Discussion section) of bacterial identification should be presented (MALDI-TOF-MS, API, analysis of 16S rRNA, DNA-DNA hybridization), and then, the methods allowing detailed the characterization of the bacterium (antibiotic susceptibility, phylogeny). The authors are asked to rethink the structure of the manuscript with regards to the weight and purpose of the methods used.

Authors response: manuscript has reorganized

 

7. There is erroneous information in the manuscript. For example in Table 4, Bacillus litoralis was indicated as Gram-negative and the reference Yoon & Oh (2005, Int. J. Syst. Evol. Microbiol. 55, 1945–1948) was provided. But in the publication mention above, Bacillus litoralis is decribed as a Gram-variable bacterium. The authors are asked to carefully check the credibility of the presented content and to cite them correct.

Authors response: corrected accordingly

 

8. Table 4. Why there are so many NA in the table?

Authors response: the authors thanks the reviewer’s remark, many NA because this data are not available in the literature used to design the table 4.

 

9. Only results are presented in the Results and Discussion section. There is no discussion.

Authors response:  we agree, modified the manuscript however it is important to note that discussion is difficult to establish based only on the identification of a new species. Now lines 242 to 257 “Description of the bacterial new species requires several approaches, most recurrent is the approach taxonogenomics [1]–[3]. In this approach, we describe the main phenotypic and genotipic characteristic from the strain Marseille-P9898. Use of the MALDI-TOF-MS are showed a protein profile of the stain Marseille-P9898, but incapable to discriminate Marseille-P9898 with other species of genus Bacillus. As far as that goes,  the sequence 16S rRNA gene show a value than more to 98.65% [25], a cut to demarcate the new species. But we know that the conventional low divergence between two 16S rRNA genes from two organisms, results in a slight and limited bacterial description [71]–[72]. However, the use of the genome gives access to complete genetic information, allows to evaluate the degrees of genomic similarity using the tools as Genome-to-Genome Distance Calculator (GGDC) [28] and Orthologous Average Nucleotide Identity (Ortho-ANI) [29]. Genomic data shows that Marseille-P9898 is a bacterial new species of genus Bacillus. Otherwise, phenotypic, biochemical and chemotaxonomic analysis are showed Marseille-P9898 are obligate aerobic, aquatic habitats, moderately halophilic or halotolerant, and 12-methyl-tetradecanoic acid which are the signature of the Bacillus genus. [8], [13]–[17]. “

 

The manuscript is written messily and contains numerous inaccuracies. For example:

- Title. “microbiota planarian Schmidtea mediterranea”. Maybe it should be: “microbiota of…”,

Authors response: corrected accordingly

 

- Title page, abbreviation. The authors are asked to add the English translation of the abbreviation expansion and to complete the list of abbreviations with the others mentioned in the manuscript: NCCP, DMS, GGDC and ect.

Authors response: corrected accordingly

 

- Keywords. Latin species names should be italicized.

Authors response: corrected accordingly

 

- Page 2, line 42 and page 4, line 145. The names of bacterial family and phylum are written in italics. Table 2 and page 15, line 246. These names are not written in italics. The authors are asked to unify this.

Authors response: corrected accordingly

 

- The correction of the numbering of sections and subsections in the manuscript is needed.- Materials and Methods. Results and Discusson. Why the description of phylogenetic analysis and genomic comparison are not divided? These are two completely different analyzes, the results of which lead to completely different conclusions. Similarly, the description of antibiotic susceptibility and the content of fatty acids.

Authors response: corrected accordingly

 

 

- “Hexadecanoic acid”, „12-methyl-Tridecanoic acid”, “Maximum likelihood Phylogenetic”, and etc. Maybe it should be: “hexadecanoic acid”, “12-methyl-tridecanoic acid”, “Maximum likelihood phylogenetic” or “Maximum Likelihood Phylogenetic”?

Authors response: corrected accordingly

 

- Subtitles. “Antibiotic susceptibility and chemotaxonomic Analysis”. Maybe it should be “Antibiotic susceptibility and chemotaxonomic analysis” or „Antibiotic Susceptibility and Chemotaxonomic Analysis”?

Authors response: corrected accordingly

 

-Page 9, line 188. “Tableau”?

Authors response: corrected accordingly

 

 

-Page 13, line 231. No full stop at the end of the sequence.

Authors response: corrected accordingly

 

- Page 14, line 233. The explanation of the unit abbreviation μg/ml is unnecessary.

Authors response: corrected accordingly

 

- Page 15, line 242. The strain “was first isolated from microbiota of planarian and abundant”. What does it mean “abundant” in this sentence? The authors are ask to explain this issue in the Result section.

Authors response: corrected accordingly

 

- Figure 1. Some names of bacterial species are italicized and others not. The authors are asked to unify this.

Authors response: corrected accordingly

 

- The Figure 2 is poorly visible. Names of bacterial species should be in italics.

Authors response: corrected accordingly

 

Supplementary information:

We provide the supplementary information in the manuscript:

-In line 34, we delete space

-In line 41, we delete space

-In line 51, we delete space