Biofilm Inhibition, Antibacterial and Antiadhesive Properties of a Novel Biosurfactant from Lactobacillus paracasei N2 against Multi-Antibiotics-Resistant Pathogens Isolated from Braised Fish

Round 1

Reviewer 1 Report

The authors conducted an assessment of the antibiotic susceptibility and biofilm-forming capability of pathogens obtained from braised fish. Additionally, they characterized and evaluated the antibacterial, anti-adhesion, and anti-biofilm activities of a biosurfactant derived from Lactobacillus paracasei subsp. tolerans N2. These investigations were carried out effectively, and the manner of explanation and presentation of data was satisfactory.

Biochemical and elemental analyses, FTIR spectroscopy, GC-MS analysis, 1H NMR spectroscopy, and high-resolution mass spectrometry were employed by the authors to identify the biosurfactant. The biosurfactant was identified as a novel isomer comprising two isomers with masses of 482.28 and 507.27 m/z. Its characterization revealed it to be glycolipotentan.

The results section provides a comprehensive depiction of the FTIR spectra. Typically, when referring to an FTIR spectrum, it is accompanied by a database, enabling the search for similar compounds. However, in this instance, compounds exhibiting highly similar spectra were not mentioned. It would be beneficial to include a description regarding this aspect.

Regrettably, Table 5 appears to be missing. Therefore, it is requested to include the missing table in the document.

Author Response

Thank you for the comments to improve the quality of this paper. They were incorporated and changes made in the manuscript were highlighted in red. Below is a point-by-point response to your comments.

 

1. The results section provides a comprehensive depiction of the FTIR spectra. Typically, when referring to an FTIR spectrum, it is accompanied by a database, enabling the search for similar compounds. However, in this instance, compounds exhibiting highly similar spectra were not mentioned. It would be beneficial to include a description regarding this aspect.

As suggested by the reviewer, glycolipoprotein biosurfactants exhibiting similar spectra were added in the revised manuscript.

“Similar spectra for glycolipoprotein biosurfactants were reported in the literature by Mouafo et al. [14] with the biosurfactant from L. paracasei subsp. tolerans N2 using sugar cane molasses as substrate and Ferreira et al. [36] with the biosurfactant from L. paracasei using corn steep liquor as substrate.”

However, to our knowledge, FTIR spectra databases for biosurfactants are not yet available. That is the reason why it was not mentioned.

 

2. Regrettably, Table 5 appears to be missing. Therefore, it is requested to include the missing table in the document.

Corrected. Thank you for that remark. We are sorry and apologize for that mistake. Table 5 was added.

Reviewer 2 Report

The manuscript entitled “Biofilm inhibition, antibacterial and antiadhesive properties of a novel biosurfactant from Lactobacillus paracasei N2 against multi-antibiotics resistant pathogens isolated from braised fish” is interesting for the scientific community. My comments to the authors are listed below.

Keywords: Please give only the relevant keywords for the present work. 

Materials and methods: Please write in italics the name of the genus and species for the pathogenic strains. The Materials and methods should be presented in a more synthetic way. Please do not describe three times how you determine the biofilm formation (i.e., crystal violet method).

Is the 24-hour incubation of pathogenic strains sufficient to determine their ability to form biofilms?

Results and Discussion: Did the authors check if the tested pathogenic strains harbor any mutation (e.g.,  AmpC,  gyrA, gyrB, etc.)? In Figure 5, the authors present only two images of E. coli EM2 cells treated or not with biosurfactant. Please add also SEM images for the other pathogenic strains studied. Please correct this statement “This result was different to those reported in the literature as Gram positive bacteria due to their membrane constitution are generally more sensitive to antimicrobials compared to Gram positive bacteria [49,50].”

Please correct this statement “….. S. aureus CMCC26003 as well as genes dltB and cidA involved in the adhesion and release of eDNA, respectively [60].”  

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Author Response

The authors thank the reviewer for the quality of the comments for the improvement of the paper. The comments were incorporated and changes made in the manuscript were highlighted in red. Below is a point-by-point response to your comments.

 

1. Keywords: Please give only the relevant keywords for the present work.

Corrected as suggested by the reviewer.

2. Materials and methods: Please write in italics the name of the genus and species for the pathogenic strains.

Corrected. It was forgotten while formatting the paper according to fermentation template.

“Pathogenic strains of Escherichia coli EM2, Staphylococcus aureus SA1, Salmonella enteritidis PE1, Pseudomonas aeruginosa CT3, Yersinia enterolitica MH5, Proteus mirabilis MR2 and Klebsiella pneumoniae AG5 were isolated and identified from braised fish sold in the city of Yaoundé [5].”

3. The Materials and methods should be presented in a more synthetic way. Please do not describe three times how you determine the biofilm formation (i.e., crystal violet method).

As suggested by the reviewer, it was corrected in the revised version of the manuscript.

4. Is the 24-hour incubation of pathogenic strains sufficient to determine their ability to form biofilms?

Thank you for that remark. We have performed preliminary tests on biofilms formation by the pathogenic strains at different incubation times. We found that, all the pathogens were positive to biofilm after 24 h. That is the reason why 24 h was selected for this study.

5. Results and Discussion: Did the authors check if the tested pathogenic strains harbor any mutation (e.g.,  AmpC,  gyrA, gyrB, etc.)?

Thank you for that deep remark. In this study, the pathogenic strains were not screened for the presence of mutation genes. That is the reason why during the interpretation of results, the resistance due to these genes was suggested as a possible resistance mechanism based on data available in the literature. This interesting idea of the reviewer will be considered in our future studies.

6. In Figure 5, the authors present only two images of E. coli EM2 cells treated or not with biosurfactant. Please add also SEM images for the other pathogenic strains studied.

Thank you for that remark. SEM analysis was performed only on E. coli EM2 cells because the strain showed the highest multiple resistance to antibiotics. Modifications were added in the revised version of the paper.

7. Please correct this statement “This result was different to those reported in the literature as Gram positive bacteria due to their membrane constitution are generally more sensitive to antimicrobials compared to Gram positive bacteria [49,50].”

Corrected.

“This result differs from those generally reported in the literature. Indeed, Gram negative bacteria are known to be more resistant to antimicrobials because of the lipopolysaccharide layer present on their outer surface membrane, which acts as an effective barrier [49,50]. However, in this study, they were more sensitive to biosurfactant than the Gram positive bacteria.”

8. Please correct this statement “….. S. aureus CMCC26003 as well as genes dltB and cidA involved in the adhesion and release of eDNA, respectively [60].” 

Corrected as suggested by the reviewer.

“Indeed, biosurfactants from lactic acid bacteria such as L. plantarum and P. acidilactici were reported to be effective at 12.5 mg/mL in reducing of the expression of agrA and icaA genes involved in biofilm formation by S. aureus CMCC26003 [60]. Inhibition of the expression genes dltB and cidA involved in the adhesion and release of eDNA, which are involved in the biofilm formation by S. aureus CMCC26003 has also been observed [60].”

 

Round 2

Reviewer 1 Report

The authors changed the previous manuscript and improved considerably the manuscript. I recommend this revised version.

 

Author Response

Thank you for your valuable comments that have significantly improved the quality of the manuscript.