Synergistic Antibiofilm Effects of Exopolymers Produced by the Marine, Thermotolerant Bacillus licheniformis B3-15 and Their Potential Medical Applications

Round 1

Reviewer 1 Report

The exopolysaccharide (EPS B3-15) and biosurfactant (BS B3-15) were recently reported to possess different antibiofilm activities. In this study, the synergistic effects of the two exopolymers on the bacterial adhesion and biofilm disruption of P. aeruginosa and S. aureus were evaluated on different materials, and the result showed that the mixture exhibited synergistic antibiofilm effects, indicating their potential medical applications in the future. However, there are some advices as following:

(1)  Since BPS B3-15 did not affect the growth of P. aeruginosa or S. aureus up to 1000 µg mL-1, it is better to show the growth curves at the concentration up 1000 µg mL-1 in figure 1.

(2)  Even at the concentration of 300 µg mL-1, BPS B3-15 still could not completely inhibit biofilm formation. Why not try higher concentration? Such as 1000 µg mL-1.

(3)  In line 269, is EPS B3-15 correct? According to your result, it looks like BPS B3-15.

(4)  In figure 2, why detected the biofilm formation of P. aeruginosa at 48 h, and detected the biofilm formation of S. aureus at 24 h, please explain it. It is better to detect the biofilm formation of them both at 24 and 48 h.

(5)  On polystyrene microplates and PVC medical device, the biofilm detection was done with BPS B3-15 at different concentration, but on Contact Lenses (CL) and CL-care solution, why only detect the biofilm formation at 300 µg mL-1? To compare the antibiofilm activity on different device, it is better to keep consistent.

The manuscript is easy to understand, but it can be improved by moderate editing.

Author Response

Dear Reviwer and Editor, 

thank you for your valuable suggestions

The exopolysaccharide (EPS B3-15) and biosurfactant (BS B3-15) were recently reported to possess different antibiofilm activities. In this study, the synergistic effects of the two exopolymers on the bacterial adhesion and biofilm disruption of P. aeruginosa and S. aureus were evaluated on different materials, and the result showed that the mixture exhibited synergistic antibiofilm effects, indicating their potential medical applications in the future. However, there are some advice as following:

• Since BPS B3-15 did not affect the growth of  aeruginosa or S. aureus up to 1000 µg mL-1, it is better to show the growth curves at the concentration up 1000 µg mL-1 in figure 1.

R- Although the BPS B3-15 did not affect the bacterial growth up to 1000 ug/ml, its components EPS and BS have been previously reported to show low toxicity above 300 ug ml to human cells. Zammuto et al (2023) [23,24]. Therefore, in this study, we did not exceed this dose. Consequently, the bacterial growth curve was presented in the presence of BPS B3-15 at 300 ug mL

• Even at the concentration of 300 µg mL^-1, BPS B3-15 still could not completely inhibit biofilm formation. Why not try higher concentration? Such as 1000 µg mL-1.

R- As reported above, the EPS B3-15 and BS B3-15 were not cytotoxic at 300 ug mL-1, therefore all the experiments were performed using this biocompatible concentration.

• In line 269, is EPS B3-15 correct? According to your result, it looks like BPS B3-15.

It was corrected.

(4)  In figure 2, why detected the biofilm formation of P. aeruginosa at 48 h and detected the biofilm formation of S. aureus at 24 h, please explain it. It is better to detect the biofilm formation of them both at 24 and 48 h.

R- As previously reported in Zammuto et al (2023), and according with the literature (Pericolini et al 2018 and Oliveira et al. 2007), P. aeruginosa forms mature biofilm after 48h, and S. aures after 24h.

(5)  On polystyrene microplates and PVC medical device, the biofilm detection was done with BPS B3-15 at different concentration, but on Contact Lenses (CL) and CL-care solution, why only detect the biofilm formation at 300 µg mL-1? To compare the antibiofilm activity on different device, it is better to keep consistent.

R- The effects of exopolymers at different concentrations on the biofilm formation of P. aeruginosa and S. aureus on contact lenses are showed in the novel Figure 5.

Reviewer 2 Report

I have read the manuscript entitled “Synergistic antibiofilm effects of exopolymers produced by the marine, thermotolerant Bacillus licheniformis B3-15 and their potential medical applications” with great interest and I think it is in principle suited for a publication in the JMSE, Special Issue “Marine Microbes and Their Application in Biotechnology”. This study presents a significant advancement in understanding the antibiofilm activities of exopolysaccharide (EPS B3-15) and biosurfactant (BS B3-15) produced by the marine Bacillus licheniformis B3-15. Beyond individual exopolymer performance, the research provides valuable insights into the synergistic potentials when used in tandem. The ability of the BPS combination to disrupt mature biofilms, especially its pronounced effects against S. aureus, is a significant highlight. However, I also have minor comments.

 

Comments:

 

Line 31. The term "oxopolymers" is introduced without previous mention or explanation. It seems to be a typo. The correct term should probably be "exopolymers" based on the context.

Line 37. "self-synthetized" should be "self-synthesized".

Line 38. "Differently to the free-living style" is unclear. Consider rephrasing to "Unlike free-living bacteria".

Line 88. "expolymers" should be "exopolymers".

Lines124, 139. “Muller Hinton broth” should be “Mueller Hinton broth”; “Muller Hilton Agar” should be “Mueller Hinton Agar”. Please check.

Line 174. The dimensions provided for the segments ("1 cm × 0.5 cm") seem to imply a 2D surface. If these segments are 3D (which they likely are), then the third dimension should be provided for clarity.

Line 189. If you are describing the cleaning process of the contact lenses, ensure that the "Zeiss Dayfilcon A 42% and water 58%" describes the material composition of the contact lens, and not the washing solution. If it does describe the washing solution, the sentence should be clearer about it.

Lines 192-193. If the same method was applied for both bacterial strains, there should be consistency in the incubation time. Ensure that the incubation times for both P. aeruginosa and S. aureus are the same unless the difference is intentional and justified.

Lines 194-195. The concentration of the crystal violet solution ("0.001%") seems unusually low. Please check.

Line 220. Ensure that referencing the method as "previously reported [31,32]" aligns with the specific method described. If this method deviates in any significant manner from the sources [31,32], then the use of "previously reported" may be misleading.

Line 224. Please change "descried" to "described".

Line 340. “S. aures” should be “S. aureus”.

Line 379. “chloroforms” should be “chloroform”. 

Author Response

Deat Reviewer and Editor, 

thank you for your valuable suggestions

Line 31. The term "oxopolymers" is introduced without previous mention or explanation. It seems to be a typo. The correct term should probably be "exopolymers" based on the context.

R: It has been corrected

Line 37. "self-synthetized" should be "self-synthesized".

R: It has been corrected

.Line 38. "Differently to the free-living style" is unclear. Consider rephrasing to "Unlike free-living bacteria".

R: The sentence has been modified.

Line 88. "expolymers" should be "exopolymers".

 R: It has been corrected

Lines124, 139. “Muller Hinton broth” should be “Mueller Hinton broth”; “Muller Hilton Agar” should be “Mueller Hinton Agar”. Please check.

R: It has been corrected

Line 174. The dimensions provided for the segments ("1 cm × 0.5 cm") seem to imply a 2D surface. If these segments are 3D (which they likely are), then the third dimension should be provided for clarity.

R: The third dimension has been added.

Line 189. If you are describing the cleaning process of the contact lenses, ensure that the "Zeiss Dayfilcon A 42% and water 58%" describes the material composition of the contact lens, and not the washing solution. If it does describe the washing solution, the sentence should be clearer about it.

R:The sentence has been rewritten more clearly

Lines 192-193. If the same method was applied for both bacterial strains, there should be consistency in the incubation time. Ensure that the incubation times for both P. aeruginosa and S. aureus are the same unless the difference is intentional and justified.

R: As previously reported in Zammuto et al (2023) [23,24], the time to form mature biofilm by P. aeruginosa and S. aures are different, and determined in 48h and 24h, respectively.

Lines 194-195. The concentration of the crystal violet solution ("0.001%") seems unusually low. Please check.

R: Since polymers of contact lenses seem to have a high affinity to the stain, the concentration of crystal violet was optimized at this concentration, which allows us to estimate the biofilm formed.

Line 220. Ensure that referencing the method as "previously reported [31,32]" aligns with the specific method described. If this method deviates in any significant manner from the sources [31,32], then the use of "previously reported" may be misleading.

R: The sentence has been modified.

Line 224. Please change "descried" to "described".

R: Done

Line 340. “S. aures” should be “S. aureus”.

R: Done

Line 379. “chloroforms” should be “chloroform”.

R: Done

Reviewer 3 Report

The article "Synergistic antibiofilm effects of exopolymers produced by the marine, thermotolerant Bacillus licheniformis B3-15 and their potential medical applications" is interesting and well written. In this article, two exopolymers were evaluated for the prevention of biofilm formation and their subsequent use in biomedical applications. The introduction to this article was well written by reviewing the relevant literature and the methods were also well described. The results were also presented in a good format through the visualisation of the data, and there was a good discussion of the results. However, there are some suggestions for improvement of the article, so I am recommending the article for publication after a minor revision.

 

1.   Line 80-81: „The search for new strategies capable of counteracting the formation of biofilms on different surfaces and devices is important in medical and non-medical fields.“ - Please explain why this is important and provide some references.

2. Lines 83-84: Please also explain why you chose Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 for this study.

3.    Figure 2a: This looks good, but it is a little bit confusing. Please do a Tukey HSD test to compare them better.

4.      Figure 2b: Please label the figure clearly, it took me a minute to work out that the one on the right is ATCC 29213.

5.  Figure 2b: Is there a significant difference between the hours of treatment that are treated with BPS B3-15? Please do a Tukey HSD test or ANVA test.

6.    Figure 3: Use different letters on the bars (e.g. a, b, c...) to represent the significance (ANOVA results) instead of stars. It would be easier.

7.      Line 337: " The combination of EPS B3-15, BS B3-15 and the BPS B3-15 with CS reduced the biofilm of P. aeruginosa on CLs (64, 72, and 88%, respectively) more efficiently than the CS (55%) or each exopolymer alone (Figure 5b)". In the figure, they are only compared with the control by ANOVA, but you have not compared them one by one, as mentioned above, a Tukey HSD test would work well here.

8.   Line 339: " Furthermore, each exo-polymer in combination with CS moderately inhibited the biofilm formation of S. aures (< 40%), being BPS B3-15 the most active (39%)." - There seems to be no difference between EPS B3-15, BS B3-15 and BPS B3-15, as shown in the graph, but how is that more active?

The English language of the article is fine, a little spell check and some sentence changes are needed.

Author Response

Dear Reviewer and Editor

thank you for your suggestions here following our responses:

The article "Synergistic antibiofilm effects of exopolymers produced by the marine, thermotolerant Bacillus licheniformis B3-15 and their potential medical applications" is interesting and well written. In this article, two exopolymers were evaluated for the prevention of biofilm formation and their subsequent use in biomedical applications. The introduction to this article was well written by reviewing the relevant literature and the methods were also well described. The results were also presented in a good format through the visualisation of the data, and there was a good discussion of the results. However, there are some suggestions for improvement of the article, so I am recommending the article for publication after a minor revision.

1. Line 80-81: „The search for new strategies capable of counteracting the formation of biofilms on different surfaces and devices is important in medical and non-medical fields.“ - Please explain why this is important and provide some references.

R: The sentence has been modified

2. Lines 83-84: Please also explain why you chose Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 for this study.

R: The sentence has been modified.

2. Figure 2a: This looks good, but it is a little bit confusing. Please do a Tukey HSD test to compare them better.

R: Done

3. Figure 2b: Please label the figure clearly, it took me a minute to work out that the one on the right is ATCC 29213.

R: the figure 2 has been replaced

4. Figure 2b: Is there a significant difference between the hours of treatment that are treated with BPS B3-15? Please do a Tukey HSD test or ANVA test.

R: Done

5. Figure 3: Use different letters on the bars (e.g. a, b, c...) to represent the significance (ANOVA results) instead of stars. It would be easier.

R: Done

6. Line 337: " The combination of EPS B3-15, BS B3-15 and the BPS B3-15 with CS reduced the biofilm of P. aeruginosa on CLs (64, 72, and 88%, respectively) more efficiently than the CS (55%) or each exopolymer alone (Figure 5b)". In the figure, they are only compared with the control by ANOVA, but you have not compared them one by one, as mentioned above, a Tukey HSD test would work well here.

R: Done

7. Line 339: " Furthermore, each exo-polymer in combination with CS moderately inhibited the biofilm formation of S. aures (< 40%), being BPS B3-15 the most active (39%)." - There seems to be no difference between EPS B3-15, BS B3-15 and BPS B3-15, as shown in the graph, but how is that more active?

R: the sentence has been modified

 

Round 2

Reviewer 1 Report

The questions have been answered and the manuscript is fine.