Peptidome Profiling of Bubalus bubalis Urine and Assessment of Its Antimicrobial Activity against Mastitis-Causing Pathogens

Round 1

Reviewer 1 Report

The antimicrobial peptides (AMPs), have raised great interest and many investigations are focused on these natural compounds as potential innovative drug candidates. Therefore, the antibiotic resistance issue has made it urgent to search for alternatives to conventional antibiotic in human and veterinary medicine. AMPs are small, natural bioactive proteins, produced by living organisms, and representing the first line of defense against microorganisms.

AMPs display immunomodulatory, antioxidant, antimicrobial and anti-inflammatory activities. There are many articles which clearly showed bactericidal activity on Gram-positive and Gram-negative bacteria, so the findings of the authors for antimicrobial properties just confirmed the already known information.

 The authors use the same research design as in their previous article “Peptide profiling in cow urine reveals molecular signature of physiology-driven pathways and in-silico predicted bioactive properties”, concerning peptide profile in cow urine. Figure 1 is almost the same as figure 1 used in the article mentioned above. The difference is that in the first article the authors used urine from cows instead buffalo, but the most findings are the same.

Line 160-162 Is it true for all three test bacteria. Please check for S. agalacticae and revise.

Line 165-168 Kill kinetics assay - please provide more details.

It is not clear what is the final concentration of SPE in MIC determination. What is the concentration in 100 µl. Please clarify.

Line 501-502 - 0,5 McFarland is 1.5x10⁸cfu/ml for bacteria, when the suspension was diluted 20 times it is not 5x10⁵ cfu/ml. After that 5 µl from that suspension was added to 50 µl - what is the final concentration. Please revised the whole scheme of procedure and clarify the final concentration of bacterial test strains.

Why did you decided to use resazurin dye for determination of MIC. What is the reproducibility and repeatability of the method?

Line 523 - It’s not appropriate the way of blood collecting. Who is the donor? There is no information about the health status etc.

 Line 565-577 Conclusion - Most of the findings were already demonstrated in privious article “Peptide profiling in cow urine reveals molecular signature of physiology-driven pathways and in-silico predicted bioactive properties” of the team. Please revise.

Author Response

The authors use the same research design as in their previous article “Peptide profiling in cow urine reveals molecular signature of physiology-driven pathways and in-silico predicted bioactive properties”, concerning peptide profile in cow urine. Figure 1 is almost the same as figure 1 used in the article mentioned above. The difference is that in the first article the authors used urine from cows instead buffalo, but most findings are the same.

We agree that we’ve used the same design experiment as stated in “Peptide profiling in cow urine reveals molecular signature of physiology-driven pathways and in-silico predicted bioactive properties” but :

• The former article was more focussed on the physiological variation and briefly discusses the antimicrobial activity of the urinary peptide while the present article discusses the antimicrobial activity of the total urinary peptide in detail.
• The aim of the present study was to focus on the urinary peptides and draw a parallel between two dairy species
• The study also shows that biological fluid contains thousands of peptide sequences generated from precursor proteins by the action of endogenous proteases. These sequences by the virtue of their amino acid composition possess a variety of bioactivities. A qualitative analysis shows that buffalo urinary peptides possess more antimicrobial activity than cows.
• The method detailed in figure 1 has been perfected over time with a large sample volume and sample size and is the core of a urinary peptide-based study.

Line 160-162 Is it true for all three test bacteria. Please check for S. agalacticae and revise.

• Correction introduced in line no. 186

Line 165-168 Kill kinetics assay - please provide more details.

• Details added in the mentioned section line 196-202

Line 501-502 - 0,5 McFarland is 1.5x10⁸cfu/ml for bacteria, when the suspension was diluted 20 times it is not 5x10⁵ cfu/ml. After that 5 µl from that suspension was added to 50 µl - what is the final concentration. Please revised the whole scheme of procedure and clarify the final concentration of bacterial test strains.

The dilutions were prepared as following:

The saline suspension was prepared of the isolated colony and the turbidity was adjusted to 0.5 Mc Farland equivalent which usually give bacterial suspension with 1-2 x 10⁸ CFUs. The suspension was further diluted by 1:20 for broth microdilution assay and 5 µL of this dilution was later added into 50 µL of test peptide containing broth.

For 1:20 dilution (for 2 ml volume):

C₁V₁=C₂V₂

1x10⁸ X 100 = ? X 2000

? = 1x10⁸ X 100 / 2000

? = 5x10⁶

For last dilution (5 µl for 50 µl)

C₁V₁=C₂V₂

5x10⁶ X 5 = ? X 50

? = 5x10⁵

Why did you decided to use resazurin dye for the determination of MIC. What is the reproducibility and repeatability of the method?

• Given that wells appear transparent in some cases, it is sometimes very difficult to differentiate between the wells where bacteria is alive and where bacteria is dead from the turbidity. Additionally, resazurin dye helps to discern the wells where bacteria are metabolically active. The turbid appearance on the bottom of the well, in some cases, is due to cell debris of dead bacteria. The method is reproducible and its efficiency has been discussed in several studies.

It’s not appropriate the way of blood collecting. Who is the donor? There is no information about the health status etc.

• The blood for the estimation of the haemolytic activity of the peptides was provided by the first author (Rohit Kumar). The blood was collected by venous puncture using a butterfly needle. Roughly 10 ml of the blood was drawn with the help of a vacutainer. The donor was healthy at the time of blood collection and volunteered for the study.

Line 565-577 Conclusion - Most of the findings were already demonstrated in privious article “Peptide profiling in cow urine reveals molecular signature of physiology-driven pathways and in-silico predicted bioactive properties” of the team. Please revise.

Though the study was conducted with a hypothesis that we will observe differences in different aspects of urinary peptides but we obtained similar trend in both cow and buffalo. We’ve tried to revise the conclusion statement by stating that these two are closely related groups (line 610-615). However rest of our statement are observation and core to this study and cannot be changed entirely. By the permission of reviewers and editors we’d like to introduce the following figures that will explain the correlation between two species. The data shown below is the amalgamation of our previous and present study and clearly explain the discussed similarity between the two datasets.

Author Response File: Author Response.docx

Reviewer 2 Report

The article submitted for possible publication in Antibiotics discusses and determines the peptidome profiling of Bubalus bubalis urine and its antimicrobial property against mastitis-causing pathogens. The manuscript provides sufficient results. However, the article needs to be improved, especially the abstract and introduction sections. The revisions required to ameliorate the article quality:

v  Abstract section:

-          Authors are invited to add a background at the beginning of the first paragraph of the abstract section

-          The abstract section should contain the most important results obtained in the study

-          Add a conclusion at the end of the abstract section

v  Introduction section:

-          Line 49: Authors are invited to add a section explaining the mastitis disease as one of the most infections of the urinary tract and mention its treatment with available antibiotics and their use limitations.

-          Bacterial strain names should be in italic

-          Enrich the introduction part with recent advances on AMPs and their biological properties

-          At the end of the introduction part, authors are invited to cite the originality and novelty of their manuscript

v  Results and discussion

-          This part is well presented and discussed

v  References

-          Reference number 17 should be rectified 

Author Response

-          Authors are invited to add a background at the beginning of the first paragraph of the abstract section

-          The abstract section should contain the most important results obtained in the study

-          Add a conclusion at the end of the abstract section

• The abstract was modified to introduce the above mentioned correction. Kindly refer line10-33

v  Introduction section:

-          Line 49: Authors are invited to add a section explaining the mastitis disease as one of the most infections of the urinary tract and mention its treatment with available antibiotics and their use limitations.

• The reviewer might have mistaken the term mastitis for metritis. Mastitis is the inflammatory disease of the mammary gland of the udder while the latter is the inflammation of uterus. The idea behind the study was to explore the biological fluid to find a novel approach (AMPs) to treating infectious disease, in this case, mastitis-causing prevalent pathogens. Our current study focuses on working with a single synthesized peptide to functionally validate its antimicrobial activity and we’ve obtained good results in this direction. Our aim is to generate a list of such functionally validated peptide sequences with good antimicrobial parameters. Such sequences will be of relevance in near future considering the rise of MDR pathogens cases.

-          Bacterial strain names should be in italic

Bacterial strain names corrected

-          Enrich the introduction part with recent advances on AMPs and their biological properties

• A detailed section describing the recent advance on AMPs and their biological properties has been added in the Introduction section. Kindly refer line 59-70

-          At the end of the introduction part, authors are invited to cite the originality and novelty of their manuscript

• We’ve conducted a similar study in Sahiwal cow (India milch breed). However, the present study provides a more in-depth investigation approach and tries to draw a parallel between the previous and the present study. Identification of urinary peptides belonging to different repertoire of bioactivities. These sequences by the virtue of compositional biases have been shown to possess different bioactivities. Till now, this is the second study focussing on the antimicrobial activity of the urinary peptides.

v  References

-          Reference number 17 should be rectified 

• The reference is corrected (reference 17 is now reference 28)

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript by Kumar et al studied the peptidome profile from the urine of Murrah Buffaloes and measured the antimicrobial properties of the urinary aqueous phase. The LC-MS/MS analysis identified 8165 peptides originating from 6041 parent proteins. They identified 76 Proteases responsible for the release of these sequences from precursor proteins. 

Further, the identified peptide sequences were analyzed to identify bioactive peptides and tried to classify them into anti-cancerous, anti-hypertensive, anti-microbial, and anti-inflammatory groups particular antimicrobial agents. 

I think a few issues need clarification and corrections to be fixed, as follows:

1.    The authors identified alanine, glycine lysine, leucine, and serine as the abundant amino acid residues in buffalo urinary peptides: any specific explanation for it?

2.    The reference to figures in the text is incorrect at multiple places.

3.    It is not entirely clear from the manuscript how the anti-microbial activity is determined. Did author determine the antimicrobial activity of the mixture of peptides? What is the control used?

4.    It would be useful to provide details about the quantification of different peptides observed.

Author Response

1. The authors identified alanine, glycine lysine, leucine, and serine as the abundant amino acid residues in buffalo urinary peptides: any specific explanation for it?

• The amino acid distribution trend observed in our data shows similarity with the other studies as well as previous studies. We don’t exactly know the answer but our speculation is that the amino acid composition of proteins follows specific pattern, mostly depending upon the taxa of the organism. Here’s one study that supports it.

Bogatyreva, N.S., Finkelstein, A.V. and Galzitskaya, O.V., 2006. Trend of amino acid composition of proteins of different taxa. Journal of bioinformatics and computational biology, 4(02), pp.597-608.

2. The reference to figures in the text is incorrect in multiple places.

• We only found one figure (fig .2) that wasn’t cited in the main text and corrected it.

3. It is not entirely clear from the manuscript how the anti-microbial activity is determined. Did author determine the antimicrobial activity of the mixture of peptides? What is the control used?

• As pointed out by the reviewer, we’ve determined the antimicrobial activity of pooled crude urinary peptides extracted from ten different Murrah buffaloes. For the antimicrobial activity, we used BSA protein digest as a negative control just to rule out the possibility of the generation of antimicrobial peptides from any kind of proteolytic degradation. This negative control was used in the disc diffusion assay. In the case of broth microdilution assay, we used two controls: sterility control and growth control as suggested in CLSI guidelines.

4. It would be useful to provide details about the quantification of different peptides observed.

• The general practice is to inject a control substance with a known amount, once its retention time and peak area are obtained it can be used to calculate the quantity of the test substance. However, in our case, the data is qualitative. We have mentioned the peak area and peak intensity of peptides detected in the supplementary data. But reporting an exact quantity will be difficult considering that it is not relative quantification data (label or label-free). However, we did find a way to report the abundance of peptides in terms of the peak area they occupy by using following formula:

Percent peak area occupied by a peptide= (Peak area of a peptide/∑ Peak area of all the reported peptides) X 100

Please check supplementary table no.1

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

I am satisfied with the revised version.