Inter-Species Competition of Mono- or Dual Species Biofilms- of MDR-Staphylococcus aureus and Pseudomonas aeruginosa Promotes the Killing Efficacy of Phage or Phage Cocktail

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors and editor

Concerning the MS Inter-species competition of mono or dual species biofilms of MDR-Staphylococcus aureus and Pseudomonas aeruginosa promotes the killing efficacy of phage or phage cocktail", there is some preliminary positive data concerning the effect of phage or phage cocktail on mixed bacterial infection, more experimental work need to be performed to confirm the provided data  and the mechanism of phage on biofilm.

 

Methods

-        In the step of Determination of Biofilm biomass of single or dual species, Is the incubation of bacterial for 4hr is enough to study the effect of  page on biofilm.

-        Using 100 micro of culture will not be enough to coat the well of 24 well plate, also using 200 micro of CV is not enough to stain the cells, please revise the quantities as they are for 96 not 24 well plates

-        What is the starting bacterial conc used for the step of biofilm formation

The authors studied the effect of phage on the mature biofilm, the authors need to add more experimental work such as study the effect on bacterial adhesion, study the effect on bacteria viability inside the biofilm using TTC assay, study the effect of phage on polysaccharides.

-        What was the effective phage conc, how the authors determined it and what is the effect of various phage concentrations on the biofilm?

-        The effect of phage and its adhesion to biofilm matrix can be detected by transmission electron microscopy

-         

Results

-Table 1 and fig 1 represents the same data please use one of them

-What is the data added by fig 4 (A)

Discussion

-There is No data discussion concerning confocal electron microscopy

 

Minor

·        All the names of MO need to be italic

·        The tab at the begin of each paragraph is not adjusted

·        Gram Not gram

·        Line 136, we do not start the sentence with number

 

 

Comments on the Quality of English Language

Dear authors and editor

Concerning the MS Inter-species competition of mono or dual species biofilms of MDR-Staphylococcus aureus and Pseudomonas aeruginosa promotes the killing efficacy of phage or phage cocktail", there is some preliminary positive data concerning the effect of phage or phage cocktail on mixed bacterial infection, more experimental work need to be performed to confirm the provided data  and the mechanism of phage on biofilm.

 

Methods

-        In the step of Determination of Biofilm biomass of single or dual species, Is the incubation of bacterial for 4hr is enough to study the effect of  page on biofilm.

-        Using 100 micro of culture will not be enough to coat the well of 24 well plate, also using 200 micro of CV is not enough to stain the cells, please revise the quantities as they are for 96 not 24 well plates

-        What is the starting bacterial conc used for the step of biofilm formation

The authors studied the effect of phage on the mature biofilm, the authors need to add more experimental work such as study the effect on bacterial adhesion, study the effect on bacteria viability inside the biofilm using TTC assay, study the effect of phage on polysaccharides.

-        What was the effective phage conc, how the authors determined it and what is the effect of various phage concentrations on the biofilm?

-        The effect of phage and its adhesion to biofilm matrix can be detected by transmission electron microscopy

-         

Results

-Table 1 and fig 1 represents the same data please use one of them

-What is the data added by fig 4 (A)

Discussion

-There is No data discussion concerning confocal electron microscopy

 

Minor

·        All the names of MO need to be italic

·        The tab at the begin of each paragraph is not adjusted

·        Gram Not gram

·        Line 136, we do not start the sentence with number

 

 

Author Response

Comments 1: In the step of Determination of Biofilm biomass of single or dual species, Is the incubation of bacterial for 4hr is enough to study the effect of phage on biofilm.

 

Response: 1 Thank you for pointing this out. Yes, 4 hrs of phage incubation is enough to showcase noticeable effect on the biofilms. We optimized the phage-bacterial biofilm interactions at various concentrations of phages, multiplicity of infection and time up to 16 hrs. Accidentally we observed bacteria got phage resistance after 16 hrs of incubation. Through our experimentation we find 4 h incubation is optimal.

Comments 2:        Using 100 micro of culture will not be enough to coat the well of 24 well plate, also using 200 micro of CV is not enough to stain the cells, please revise the quantities as they are for 96 not 24 well plates.

Response: 2. Thank you for your keen observations. Sorry for the mistake. For SEM and CLSM we employed 24 well plates because of coverslip added into it to grow the biofilm. Yes, I employed 94 well micro titer plate for the estimation of biofilm biomass of bacteria and phage. Corrected as suggested.  

 

Comments 3:  What is the starting bacterial conc used for the step of biofilm formation

Response: 3. We observed the MOI (Multiplicity of infection) of phages with respective host bacteria is 1. All over the experiment we employed 1:1 ratio of bacteria and phage initial concentration I,e, Bacteria 10⁹ CFU/µL and phage 10⁹ PFU/µL.  

 

Comments 4:  The authors studied the effect of phage on the mature biofilm, the authors need to add more experimental work such as study the effect on bacterial adhesion, study the effect on bacteria viability inside the biofilm using TTC assay, study the effect of phage on polysaccharides.

Response: 4. We investigate the phage lytic action on respective host bacterial biofilms by employing FilmTracer™ LIVE/DEAD® Biofilm viability kit (Molecular Probes, Life Technologies Ltd) under confocal laser scanning electron microscopic studies.  We maintained the test (Phage with respective bacteria) and control (Salts of Magnesium buffer SM with respective bacteria, is represented in Figure 2. Live biofilms were represented in Green colour due to Syto®9 and dead cells in Red colour because of propidium iodide intake of bacteria.

Comments 5:  What was the effective phage conc, how the authors determined it and what is the effect of various phage concentrations on the biofilm?

Response: 5. Initially we tried with various concentration such as 10⁶, 10⁸ and 10⁹ PFU/µL, but the results showed 10⁹ PFU/µL is the optimized one to combat with 24 h old biofilm under static conditions.

Comments 6:   The effect of phage and its adhesion to biofilm matrix can be detected by transmission electron microscopy

Response: 6.  No, we employed TEM images is only for the external morphology of the bacteriophages mainly to know the tail morphology.  We employed SEM and CLSM images for showcasing phage-biofilm interactions.

-         

Results

Comments 7-Table 1 and fig 1 represents the same data please use one of them

Response: 7. Removed Figure1 and kept the table in the manuscript as suggested.

Comments 8-What is the data added by fig 4 (A)

Response: 8.  Now is Figure 3, it’s about TEM images of bacteriophages which showed lytic action on their respective host bacteria. Up to now naming of phages were done based on their tail morphology through Transmission electron microscopic images. A   vB_PAnP_PADP4: phage isolated and characterized against to P. aeruginosa, B. vB_SAnS_SADP1: phage specific to S. aureus. (vB_PAnP_PADP4: vB: Virus of bacteria; PAnP: P. aeruginosa phage belonging to Podoviridae family (No tail or short tail); PADP4: P. aeruginosa, Durbaka Pallavali series number 4)

 

Discussion

Comments 9 -There is No data discussion concerning confocal electron microscopy

Response: 9: Thank you pointing out, we the more data related to biofilm-phage analysis by employing CLSM as suggested.

Minor

Comments 10:   All the names of MO need to be italic

Response: 10 Corrected as suggested.

Comments 11:        The tab at the begin of each paragraph is not adjusted

Response: 11 Corrected as suggested.

Comments 12:     Gram Not gram

Response: 12 Corrected as suggested.

Comments 13 Line 136, we do not start the sentence with number

Response: 13 Corrected as suggested.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The complexity of biofilm-associated infections is now a worldwide concern. The authors have addressed an interesting topic. However, the editorial quality is poor (some comments below) and some issues need clarification. Therefore, I include the comments below:

- please make superscript next to authors' names

- line 15 - please provide the purpose and a brief methodology in the abstract

- line 17 - if species names appear again, please use abbreviated species names

- e.g. line 31- species names in italics - please correct

- e.g. line 56, 57 - if species names appear again, please use abbreviated species names

-  in the introduction, please supplement with epidemiological data - what percentage of the mentioned species is responsible for wound infections. Are there any factors / location conducive to infection with a particular microbial species. What treatment is introduced for MDR strains ?

- Have more strains been considered ? After one strain from a species does not give authoritative results - this could be a pilot study or a short message
- what was the control in this study ? were reference strains used ?

- line 91 - please state the abbreviation MDR in the appropriate place in the introduction, that is, where it first appears

- line 94 - specify the time and conditions of the incubation atmosphere

- line 93 - The study should have approval from the Bioethics Committee - please provide the number

- line 97 - please write the formulas of chemical compounds correctly

- line 105 - please elaborate on the abbreviation PFU

- line 116 - please specify the manufacturer of all reagents, e.g., missing with methanol and ethanol

- line 114 - please provide full name of PBS (appears for the first time) and manufacturer

- line 140 - in how many repetitions was the experiment conducted ?

- line 144 - was a statistical analysis performed ?

-  line 151 - incorrect numbering

- e.g. line 156 - please correct the degree notation

-  line 169 - table description should be above the table - please correct

- figure 1 - please put species names in italics

- line 189 - please correct the degree notation

 

Author Response

Reviewer:2

The complexity of biofilm-associated infections is now a worldwide concern. The authors have addressed an interesting topic. However, the editorial quality is poor (some comments below) and some issues need clarification. Therefore, I include the comments below:

Thank you reviewer for your

Comments 1- please make superscript next to authors' names

Response: 1 Done affiliation numbers in superscript as suggested.

Comments 2 line 15 - please provide the purpose and a brief methodology in the abstract

Response: 2.  As suggested, I included the purpose and methodology of the study and submitted both tracked and accepted tracked manuscript files.

Comments 3- line 17 - if species names appear again, please use abbreviated species names

Response: As suggested, we utilized the abbreviated species names after the first representation in abstract.

Comments 4- e.g. line 31- species names in italics - please correct

- e.g. line 56, 57 - if species names appear again, please use abbreviated species names

Response 4:  Updated the suggestions.

 

Comments 5- In the introduction, please supplement with epidemiological data - what percentage of the mentioned species is responsible for wound infections. Are there any factors / location conducive to infection with a particular microbial species. What treatment is introduced for MDR strains ?

Response:5.  Included the epidemiological data of predominant bacterial isolates isolated from the various wound infections in introduction of the manuscript Reference [2,3,4 and 5]. I included my own research inputs about the Isolation and Characterization of Bacteriophages against MDR-Bacterial infections (P. aeruginosa, S. aureus, K. pneumoniae, and E. coli) from Septic wounds and the possibility of their application in (Biofilms) biological control”. We employed bacteriophages as controlling agents against multidrug resistant bacterial isolates and studies proved the phages as permissive agents even on Biofilms irrespective of their drug-resistant nature. We included the reference as well to support the study. The employed bacteria for this study were isolated from RIMS, hospital, Kadapa, Andhra Pradesh, India.

2. Pallavali, R. R., Degati, V. L., Lomada, D., Reddy, M. C., & Durbaka, V. R. P. (2017). Isolation and in vitro evaluation of bacteriophages against MDR-bacterial isolates from septic wound infections. PloS one, 12(7), e0179245.
3. Pallavali, R. R., Avula, S., Degati, V. L., Penubala, M., Damu, A. G., & Durbaka, V. R. P. (2019). Data of antibacterial activity of plant leaves crude extract on bacterial isolates of wound infections. Data in brief, 24, 103896.
4. Pallavali, R., Shin, D., & Choi, J. (2023). Phage-based Biocontrol of antibiotic-resistant bacterium isolated from Livestock Wastewater Treatment Plant. Water, 15(8), 1616.

 

 

 

Comments:6. Have more strains been considered? After one strain from a species does not give authoritative results - this could be a pilot study or a short message

Response:6.  From our research study we isolated the 115 bacterial isolates from 130 septic wound subject, among 115 bacterial isolates P. aeruginosa was the most predominant 26 (22.6%), followed by S. aureus in 22 (19.1%), K. pneumoniae in 20 (17.3%), E. coli (E. coli) in 19 (16.5%), S. pyogenes in 9 (7.8%), Coagulase-negative Staphylococci spp. 8 (6.9%), Enterococcus spp. 6 (5.2%), Enterobacter spp. 3 (2.6%) and Proteus spp. 2 (1.7%). The employed P. aeruginosa and S. aureus is a multidrug resistant and detailed study were published in PLOS one journal (Pallavali et al,.2017).

Comments: 7. what was the control in this study ? were reference strains used ?

 Response:7. The following strains were used for the study

1. Pseudomonas aeruginosa strain DR4 16S ribosomal RNA gene, partial sequence GenBank: KY018605.1
2. Staphylococcus aureus subsp. aureus strain yvu2 16S ribosomal RNA gene, partial sequence GenBank: KY496615.1

Comments:8. - line 91 - please state the abbreviation MDR in the appropriate place in the introduction, that is, where it first appears

Response: Updated in the manuscript as suggested.

Comments:9. line 94 - specify the time and conditions of the incubation atmosphere

Response: 9.  Included the time and conditions as suggested I,e, At 37°C for 24 hrs for bacteria and Bacteriophages were stored in Salts of Magnesium (SM) buffer at 4°C

Comments:10.  line 93 - The study should have approval from the Bioethics Committee - please provide the number

Response:10.  Resolution Number: 002/2013-2014/ii/b/IEC/YVU/DVRPdT11/10/2014 included in the manuscript as suggested.

 

Comments:11.  line 97 - please write the formulas of chemical compounds correctly

Response:11.  Corrected as suggested

Comments:12 line 105 - please elaborate on the abbreviation PFU

Response:12  Elaborated plaque forming units at first cite as suggested

Comments:13 line 116 - please specify the manufacturer of all reagents, e.g., missing with methanol and ethanol

Response:13 Updated as suggested.

Comments:14 line 114 - please provide full name of PBS (appears for the first time) and manufacturer

Response:14 Phosphate buffer saline is prepared in our laboratory and Updated as suggested.

Comments:15.  line 140 - in how many repetitions was the experiment conducted ?

Response:15 Triplicates were maintained, mentioned in 131 line.

Comments:16 line 144 - was a statistical analysis performed ?

Response:16 Yes, statistical analysis is done by employing Graph Pad Prism software. The difference between the before and after phage treatments are statistically significant at the P < 0.05. ANOVA and Bonferroni's selective comparison tests were done by using Graph Pad Prism software.

 

Comments:17.  line 151 - incorrect numbering

Response:17. Corrected as suggested.

Comments:18- e.g. line 156 - please correct the degree notation

Response:18. Corrected as suggested.

Comments:19  line 169 - table description should be above the table - please correct

Response:19. Corrected as suggested.

Comments:20 figure 1 - please put species names in italics

Response:20. As suggested by another reviewer to remove either Table 1 or Figure 1due to showcasing the same data, I removed the Figure 1 from the manuscript.

Comments:21 line 189 - please correct the degree notation

Response:21. Corrected as suggested.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for introducing my suggestions. In my opinion, the article is suitable for publication.