Control of Multidrug-Resistant Pathogenic Staphylococci Associated with Vaginal Infection Using Biosurfactants Derived from Potential Probiotic Bacillus Strain
, Karima Ncib 2, Wael Bahia 3, Mouna Ghorbel 1, Nadia Leban 4, Nouha Bouali 1, Olfa Bechambi 1, Ridha Mzoughi 2 and Abdelkarim Mahdhi 2,*,†Round 1
Reviewer 1 Report
Please find the review in attachment.
Comments for author File: 
Comments.pdf
Author Response
Manuscript ID: fermentation-1511414
Control of multidrug-resistant pathogenic Staphylococci associated with vaginal infection using biosurfactants derived from potential probiotic Bacillus strain (N Haddaji et al.)
Response to Reviewer’s Comments
Reviewer #1:
Reviewer: “The manuscript contains valuable information on the antimicrobial effect of a biosurfactant isolated from a potentially probiotic strain of Bacillus sp. However, the scope of research is relatively small and the work requires significant improvement, especially in terms of methodology description. There are also factual errors in the work (drug susceptibility test). The research would gain significantly in quality if the authors determined the chemical composition of the obtained biosurfactant.”
Authors: This is an accurate summary, and we appreciate the positive assessment.
Changes: Appropriate modifications were made in the revised manuscript. Please see below for a full point-by-point response to the reviewer’s comments. Also, please note that the rectifications were done with blue color.
Reviewer: “Various microbes can be involved in vaginitis. Why did the authors decide to evaluate the antimicrobial activity of the biosurfactant only in relation to staphylococci?”
Authors: Thank you for pointing this out.
During this study we focus our interest on Staphylococcus sp because according our obtained results using sequencing of 16sRNA gene ported on a large number of women at the Neonatal and maternity center of Monastir we found that Staphyloccocus is among the predominant pathogens of the microflora (data under publication). Also, according litterature Staphylococci is considered as one among the most important pathogen associated with vaginal infection. On the other hand, Staphylococcus is conseidered among the leading pathogens responsible for chronic biofilm infections. Also, infections associated to biofilm are difficult because of the resistance to antibiotics. It is becoming increasingly difficult to treat these infections due to drug resistance, especially since biofilms created by S. aureus can limit the effectiveness of antibiotics, causing severe morbidity and mortality
Reviewer: “The potentially probiotic strain of Bacillus sp. HM117834 should be identified to the species level”
Authors: This identification is based on partial 16 sRNA gene. The strain was used in previously published study and their probiotic properties were investigated.
Reviewer: “L30 and L36 - The abstract should contain more precise information, i.e. please provide the species and strain number of Bacillus sp. and list the pathogenic strains (number, ie 4, and strain species) against which the biosurfactant was tested.”
Authors: We thank the reviewer for the constructive comment. We have addressed such concern in the abstract section.
Changes: Appropriate changes were made in the text, as recommended by the reviewer
Reviewer: “Biosurfactant, methicillin-resistant, nutrient broth, antibiotic names and haemophilus should be lowercase – L30, L99, L115, L213, L228?”
Authors: Will do.
Changes: Corrected in the main text.
Reviewer: “L60 – staphylococci – remove italics, L67 – add surname of author before [9]”
Authors: We acknowledge this obvious error
Changes: We corrected this error in the revised manuscript
Reviewer: “ L119-127 - The description of the biosurfactant production procedure is unclear and should be much more precise.”
Authors: We appreciate this comment.
Changes: Changes are made in the revised manuscript
Reviewer: “L120 - in what medium was the probiotic strain of Bacillus sp propagated? what was the volume of the medium?. L121 - the sentence "Initially, flasks were used .... is unclear”
Authors: More details are given in production BS part, and reformulation was done.
Reviewer: “ L122 - what kind of bacteria? In the description of the method, it should be clearly stated which strain was used for the production of the biosurfactant.”
Authors: Bacillus sp "HM117834 was used for production of the biosurfactant.
Reviewer: “L125 - equal parts - how many ml exactly?”
Authors: Extractions were performed three times using ethyl acetate in equal parts 500ml each
Reviewer: “L125 - what was the mass of Na2SO4 and the volume of the extract?”
Authors: Anhydrous sodium sulfate (Na2SO4) was applied at a concentration of 10 g / 100 ml.
Reviewer: “L127- Was BioSa3 ultimately a dry powder? grams per liter of what?”
Authors: The BioSa3 isn’t as a dry powder. Grams per liter of centrifuged and filtered culture
Changes: Mentioned in the text.
Reviewer: “L134 - solution containin the BioSa3 - what was the concentration of BioSa3 in mg/ml?”
Authors: An equal volume of the solution containing the crude BioSa3 was deposited.
Changes: Mentioned in the text.
Reviewer: “L138-143 & L212-221 - The EUCAST 2021 Guideline for Staphylococcus does not include cut-off points for kanamycin and methicillin - his is a factual error.”
Authors: We are aware that this is one of the most controversial points. Therefore, we made a control investigation ported on the sensible strain of Staphylococcus against kanamycin, and methicillin. Results show an inhibitory zone around the disc with the charge recommended by the EUCAST guideline (data not shown in the manuscript). For the evaluated strains in the current study, there is no inhibitory zone. This means that these strains aren't sensible towards the tested ATB.
Reviewer: “L218 – antibacterial activity of what?”
Authors: antibacterial activity of BioSa3
Changes: Rectified in the title and the table.
Reviewer: “Table 2 – first line – change “Antibiotics” into “ Antibiotics susceptibility” and “Antibacterial activity” into “Antibacterial activity of BioSa3”
L2018-219 - Table 2 - The test to assess the antimicrobial activity of the biosurfactanate was performed in one repetition, which significantly reduces the reliability of the obtained results. Each experiment should be performed at least twice to make sure that the results are reproducible.
Last column(MIC) in Table 2 – lack of value b S3 and S4”
Authors: Will do.
Changes: Corrected in the revised manuscript.
Reviewer: “L139 - what isolates?”
Authors: The four pathogenic strains
Changes: corrected in the text.
Reviewer: “L141-142 - the concentration of the antibiotic in the discs is expressed in μg or U, not in μg/ml; ampicillin 10 mg or 10 ug?”
Changes: Corrected in the Text and the table.
Reviewer: “L145-151 - the description of the methodology is unclear”
Authors: A point well taken and appreciated
Changes: Appropriate modifications were made in the Materials and Methods.
Reviewer: “L146- Staphylococcus - italics”
Authors: Done
Reviewer: “L147 - nutrient agar or nutrient broth?”
Authors: Done
Reviewer: “L148 - 10 ml from colonies ??! what was the density of the bacteria suspension in saline?”
Authors: Corrected in the text.
Reviewer: “L149 - 10 ul of BioSa3 was applied to the well or dotted on the agar ?? what was the concentration of BioSa3? What was BioSa3 dissolved in?”
Authors: Details are given in the text.
Reviewer: “L150 - what well? did the authors cut out agar wells? if so, what was their diameter?”
Authors: We thank the reviewer for this comment. A diffusion agar assay, using direct contact between the biosurfactant and pathogens, was used to test the antibacterial activity of the extracted BioSa3 against multidrug-resistant Staphylococcus strains.
Changes: Added in the revised manuscript.
Reviewer: “L173 – UFC? 106 change into 10(6) or 106”
Authors: 106 UFC/ml
Reviewer: “L174 – what was the volume of BioSa3?”
Authors: 100 µl of BioSa3
Reviewer: “L181-189 - The description of the method does not include information on testing the effect of BioSa3 on the hydrophobicity of staphylococci; there is no consistency with the description of the results - L241”
Authors: Corrected in the part of Hydrophobicity essay
Reviewer: “193 – what samples? – unclear”
Authors: Sample of Biosa3
Changes: Added in the text.
Reviewer: “L195 – what was the concentration of ascorbic acid?”
Authors: 250 µg / ml
Changes: Added in the text.
Reviewer: “L204 – put dot after “production”
Authors: Will do
Changes: Corrected in the revised manuscript
Reviewer: “L204& L256 – table and figure names should be capitalized”
Authors: Will do
Changes: We revised the name of figures and tables.
Reviewer: “L213 – what does mean “complete resistance”? there is no such term in the EUCAST guidelines”
Authors: Rectified in the text as resistance
Reviewer: “L230 – membrane or cell wall?”
Authors: membrane
Reviewer: “L238-239 - move this information to the M&M department”
Authors: Will do
Changes: Reformulated in the manuscript to be more informative
Reviewer: “L255 – in vitro – italics”
Authors: Will do
Changes: Corrected in the revised manuscript
Reviewer: “L259 – 1mg/ml – add space”
Authors: Will do
Changes: Added in the text
Reviewer: “L263 – [37-38]”
Authors: Done
Reviewer: “L273 – predominantly dominated – improve style o evaluate the antimicrobial activity of the biosurfactant only in relation to staphylococci?”
Authors: Rectified.
Reviewer: “L274 – Lactobacilli, which act as protective surfactants? This surfactants cause an acidic pH? The environment is acidified by the lactic acid produced by the LAB.”
Authors: Corrected.
Reviewer: “L284 – antioxidant activity of what?”
Authors: Corrected.
Reviewer: “L285 - sentence without a verb”
Authors: Will do
Changes: Corrected in the main text
Reviewer: “L288 – quantity of what?”
Authors: Quantity of the used biosurfactant
Reviewer: “L293 – albicans[45] – add space. L297 – improve style”
Authors: Will do
Changes: Corrected in the revised manuscript.
Reviewer: “L337 – lichenysin biofilms?”
Authors: Corrected.
Reviewer: “L340 – properties[68] – spce. L341 – [69]. – add space”
Authors: Will do
Changes: Corrected in the revised manuscript.
Reviewer: “L346 – demonstrated by…- add surname of author”
Authors: Will do
Changes: Added in the text
Reviewer: “L354 – Bacillus sp….- add strain number as it is not known if all Bacillus strains produce the same biosurfactant”
Authors: Will do
Changes: reference added in the conclusion section.
Reviewer: “L355-356 – clinical multidrug-resistant strains”
Authors: Corrected in the text.
Reviewer: “References – improve whole chapter – names of journals should be capitalized”
Authors: Will do
Changes: References adjusted as recommended by the ACS style guide.
Reviewer 2 Report
The submitted article deals with the production of a biosurfactant by a Bacillus strain and its use as an antibacterial agent. Below are some comments that I feel will improve the manuscript.
1) Line 89: rather "liquid interfaces" then just "surfaces".
2) Lines 109, 111 and 112: the numbers given are, I assume, the sequence numbers in the Gen Bank database, but this is not explicitly written.
3) Table 2: it is not explicitly stated which part of the table refers to the biosurfactant properties.
4) Figure 1: The description of the "formation" and "inhibition" series is inappropriate because it suggests the effect of the activity to the reader unnecessarily. It would be more appropriate to describe the series as "without biosurfactant" and "with biosurfactant".
5) Discussion: there is no reference of specific values in the measurements obtained by the authors to the results of analogous studies with other strains or other biosurfactants.
General comments:
6) A major shortcoming is the failure to perform analyses of the chemical composition of the biosurfactant, or even to indicate to which group of biosurfactants it belongs. This makes it difficult to compare the results with other publications.
7) There is no indication on what basis biosurfactant concentrations were chosen to the experiments.
8) The text contains many editorial errors, including unnecessary capital letters in the middle of the sentence (e.g. in the case of names of antibiotics), lack of spaces in the names of strains, etc.
Author Response
Manuscript ID: fermentation-1511414
Control of multidrug-resistant pathogenic Staphylococci associated with vaginal infection using biosurfactants derived from potential probiotic Bacillus strain (N Haddaji et al.)
Response to Reviewer’s Comments
Reviewer #2:
Dear reviewer
First of all, thank you for your interesting and constructive comments on the manuscript. Kindly find here the response to your remarks. Also, some modifications were done with red color in the manuscript in response to your comments. The quality of the manuscript was improved through the text. The modifications were done with red color.
The submitted article deals with the production of a biosurfactant by a Bacillus strain and its use as an antibacterial agent. Below are some comments that I feel will improve the manuscript.
1) Line 89: rather "liquid interfaces" then just "surfaces".
* Corrected in the manuscript.
2) Lines 109, 111 and 112: the numbers given are, I assume, the sequence numbers in the Gen Bank database, but this is not explicitly written.
* We reformulate this part in order to be clearer.
3) Table 2: it is not explicitly stated which part of the table refers to the biosurfactant properties.
* Corrected in the manuscript.
4) Figure 1: The description of the "formation" and "inhibition" series is inappropriate because it suggests the effect of the activity to the reader unnecessarily. It would be more appropriate to describe the series as "without biosurfactant" and "with biosurfactant".
* Reformulated according your recommendation.
5) Discussion: there is no reference of specific values in the measurements obtained by the authors to the results of analogous studies with other strains or other biosurfactants.
* A reference describing the effect of Biosurfactant on Staphylococcus and other pathogens was added in this part.
General comments:
6) A major shortcoming is the failure to perform analyses of the chemical composition of the biosurfactant, or even to indicate to which group of biosurfactants it belongs. This makes it difficult to compare the results with other publications.
* You have reason; the chemical determination is important. For us the study of composition is under study. Optimization will take time, for that reason we used the crude biosurfactant during the current investigation.
7) There is no indication on what basis biosurfactant concentrations were chosen to the experiments.
* More details are given in the section of diffusion agar assay. A reference also was added.
The antibacterial activity at the concentration used in the agar assay method was important. For this reason, we used during a microdilution assay a lower concentration.
8) The text contains many editorial errors, including unnecessary capital letters in the middle of the sentence (e.g. in the case of names of antibiotics), lack of spaces in the names of strains, etc.
* A general revision for grammatical, English improvement and reformulations were done through the text. Please you find them with red color
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors corrected the text, but they did it carelessly - it still contains many errors.
The authors do not know the spelling rules for the names of microorganisms. Systematic names like Staphylococcus should be written with a capital letter in italics, but staphylococci (not a Latin name) should be written with a lower case and without italics.
The authors did not follow the previous remark regarding the capitalization of table names.
L116-118 Pathogenic multi- 5 117 resistant belong to the genus of Staphylococci and submitted to Gen Bank under the following 118 accession numbers: […] – unclear - GenBank is not a collection of micoorganism, but database of DNA/RNA sequences. Have the authors sequenced the entire genomes of these four strains and put them in GenBank? Or is it just some selected genes? If yes – what genes exactly did you sequenced?
L118-119 and L231 - Is the ID (numer of strains) and GenBank Acc. Numbers the same? It is imposible
L117 – GenBank (www.ncbi.nlm.nih.gov/genbank/)
L120 – had had
L133 – v/v
L133-134 – 10 g/100 ml
L151 – 10 ug – double space
L159 - 25mg/mg lack of space
L161 – remove dot […] was determined. [30].
L191 & 223 & 228– Staphylococci shoud be lowercase
L191 - before and after treatment with BioSa3 – unclear, How did you treat the bacteria with biosurfactate? - it should be described in detail
L2016 - 250 µg / ml remove space
L216 - µg / ml space
L2015 & 224 – names of tables should be capilatized
L224 – remove „(Antibiotics)”
L148-150 & 224-228- I do not accept the authors' responses to the comment regarding the criteria for assessing drug susceptibility to kanamycin and methicillin. If authors have used their own criteria for classifying strains as resistant and susceptible, they should describe it in detail in M&M.
Which specific reference strain was used as control in this test? (susceptible to metycylin)
Table 2, first line - Antibiotics susceptibility – should be Antibiotic
L235 – Staphylococci – change to lowrecase
L240-241 – move this senetnce into Discussion and and expand this thought; explain, please, how the biosurfacant changes the cell membrane and how it affects the cell wall in G + bacteria, and thus their hydrophobicity
L285 – In my opinion the statement „Lactobacilli, which act as protective surfactants? These surfactants inhibit the adhesion and growth of pathogens” is not correct; Bacteria are not a surfactant. There are several mechanisms by which Lactobacillaceae bacteria inhibit adhesion (to the epithelium) and the growth of unfavorable microflora.
Author Response
Manuscript ID: fermentation-1511414 R2
Control of multidrug-resistant pathogenic Staphylococci associated with vaginal infection using biosurfactants derived from potential probiotic Bacillus strain (N Haddajiet al.)
Response to Reviewer’s Comments
Reviewer #1:
Reviewer: The authors corrected the text, but they did it carelessly - it still contains many errors.
The authors do not know the spelling rules for the names of microorganisms. Systematic names like Staphylococcus should be written with a capital letter in italics, but staphylococci (not a Latin name) should be written with a lower case and without italics.
Authors: We corrected this error in the revised manuscript
Reviewer: The authors did not follow the previous remark regarding the capitalization of table names.
Authors: we corrected the mentioned names.
Reviewer: L116-118 Pathogenic multi- 5 117 resistant belong to the genus of Staphylococci and submitted to Gen Bank under the following 118 accession numbers: […] – unclear - GenBank is not a collection of microorganism, but database of DNA/RNA sequences. Have the authors sequenced the entire genomes of these four strains and put them in GenBank? Or is it just some selected genes? If yes – what genes exactly did you sequenced?
Authors: Pathogenic multi- 5 117 resistant belong to the genus of staphylococci and submitted to Gen Bank under the following 118 accession numbers:
Reviewer: L118-119 and L231 - Is the ID (numer of strains) and GenBank Acc. Numbers the same? It is impossible
Authors:
Sincerely your comments aren’t clear. But we will try to response in order to clear things up.
First of all, we know well that Gen Bank is dedicated to submit sequences or partial sequences of some genes like 16sRNA used in our work and not collection of organisms. In our case, MZ475010 for example, is a Gen Bank accession number which corresponds to the identity of the identified strain (in our case: S. aureus). When you will search for the identity you will don’t find results because the submitted sequences will be available on 1 July 2022 as indicated in the received message from GenBank Direct Submission Staff (see attached file below). In fact, the used strains are among a large number of isolates identified in the context of a study about the diversity and pathogenic properties of pathogens implicated in the phenomena of Recurrent Pregnancy Loss and we have delayed the publication, to protect our results until their publication.
Reviewer: L117 – GenBank (www.ncbi.nlm.nih.gov/genbank/)
Authors: Done.
Reviewer: L120 – had had. L133 – v/v. L133-134 – 10 g/100 ml. L151 – 10 ug – double space. L159 - 25mg/mg lack of space. L161 – remove dot […] was determined. [30].
Authors: Revised in the manuscript
Reviewer: L191 & 223 & 228– Staphylococci shoud be lowercase
Authors: rectified in the text
Reviewer: L191 - before and after treatment with BioSa3 – unclear, How did you treat the bacteria with biosurfactate? - it should be described in detail
Authors: Added in the text
Reviewer: L2016 - 250 µg / ml remove space. L216 - µg / ml space. L2015 & 224 – names of tables should be capilatized. L224 – remove „(Antibiotics)”
Authors: Corrected in the text.
Reviewer: L148-150 & 224-228- I do not accept the authors' responses to the comment regarding the criteria for assessing drug susceptibility to kanamycin and methicillin. If authors have used their own criteria for classifying strains as resistant and susceptible, they should describe it in detail in M&M.
Authors: The CA-SFM/EUCAST guidelines clearly indicate for the Kanamycin; If the 30 µg kanamycin disc is unavailable, strains with a diameter <12 mm around a 30 IU kanamycin disc (24 µg) be characterized by a diameter of <12 mm. But in the case when the 30 µg kanamycin disc is available, strains with a diameter <18 mm the strain is Resistance and ≥18 is Sensible for S. aureus and <22 mm the strain is Resistance and ≥22 is sensible for S. non-aureus.
According to the recommendations by CA-SFM/EUCAST, the diameter of the inhibition zone is proportional to the sensitivity of the tested microorganism, and in its lack, the clinical categorization of this microorganism will be interpreted as resistant for the evaluated ATB concentration. (The case of Achromobacter xylosoxidans as an example). During the present study, we did not find an inhibitory zone around the disc, nor the culture in contact with the disc for the strains which was indicated as resistant. Whereas, for the strain considered as sensible, we used, as indicated in our first response and for a comparative purpose, an isolate known by its sensibility to methicillin. Data will be published soon, because this a part from a general study in which we characterized vaginal and cervicovaginal pathogenic bacteria, implicated in the Recurrent Pregnancy Loss complication, in term of their antimicrobial susceptibility.
Reviewer: Table 2, first line - Antibiotics susceptibility – should be Antibiotic. L235 – Staphylococci – change to lowrecase
Authors: Corrected.
Reviewer: L240-241 – move this senetnce into Discussion and and expand this thought; explain, please, how the biosurfacant changes the cell membrane and how it affects the cell wall in G + bacteria, and thus their hydrophobicity
Authors: Explication about the mechanism of action is given in the discussion L350 -353.
Reviewer: L285 – In my opinion the statement „Lactobacilli, which act as protective surfactants? These surfactants inhibit the adhesion and growth of pathogens” is not correct; Bacteria are not a surfactant. There are several mechanisms by which Lactobacillaceae bacteria inhibit adhesion (to the epithelium) and the growth of unfavorable microflora.
Authors: Modified in the manuscript
Author Response File: Author Response.pdf
Reviewer 2 Report
Thank you for your kind response.
Author Response
We sincerely express our deepest gratitude for the positive responses to our manuscript.
