Protective Effect of Probiotics against Pseudomonas aeruginosa Infection of Human Corneal Epithelial Cells

Round 1

Reviewer 1 Report

The manuscript provides interesting findings concerning the role of probiotics against bacteria infecting corneal tissues. The authors achieved well the results which clearly demonstrated that probiotic films aided prevent and treat eye infections. Therefore, I recommend the publication of this manuscript after a minor revision would be done. Comments are presented below:

-The abstract lacks numerical data from results. I suggest to improve this section with numerical values which demonstrate interesting findings. As an example, the percentages of HCE viability can be included in this section.

-All figures are not legible. Please increase the font size.

-Equations must be numbered and presented in the correct form. Line 459: please present the formula in the correct form.

-Please add a Conclusions section.

Author Response

Reviewer 1

The manuscript provides interesting findings concerning the role of probiotics against bacteria infecting corneal tissues. The authors achieved well the results which clearly demonstrated that probiotic films aided prevent and treat eye infections. Therefore, I recommend the publication of this manuscript after a minor revision would be done. Comments are presented below:

-The abstract lacks numerical data from results. I suggest to improve this section with numerical values which demonstrate interesting findings. As an example, the percentages of HCE viability can be included in this section.

The authors thank the reviewer for the comments.

As suggested by the reviewer, the authors improved the abstract section by adding the percentages of HCE cell viability, mean, SD and p values.

-All figures are not legible. Please increase the font size.

As suggested by the reviewer, the authors increased the font size of all figures.

-Equations must be numbered and presented in the correct form. Line 459: please present the formula in the correct form.

As suggested by the reviewer, the authors corrected the equation (Line 469).

-Please add a Conclusions section.

As suggested by the reviewer, the authors added the conclusion section (New paragraph 5).

Reviewer 2 Report

Reviewer Comments (ijms-2827963-peer-review)

This manuscript addresses a timely topic and makes a relevant contribution to the field. In its current form, this paper cannot be considered for publication. However, some major revisions are needed before it can be published.

1.      In the results sections 2.1.1. and 2.1.2, lines 105-109 and 122-130 should be moved to the introduction part, since these sentences provide general information.

2.      In section 2.1.3, figure 3(A-C). Probiotic treatment with HCE requires different concentrations probiotics (low to high CFU), and its growth curves (MTT, LDH) must be shown in line graphs in a concentration-dependent manner. Similarly, figure 4 needs to use line graph with growth curves.

3.      Section 2.1.5. In figures 5 and 6, immunocytochemistry staining of mucin and occludin is required to confirm Western blotting and PCR quantification of gene and protein expression. It may increase the strengthens to this manuscript.

4.      Figures 7 and 8 in section 2.2, both results need to be confirmed with cell morphological images from the Live/Dead staining assay.

5.      The caption of Table 1 states: "Results are expressed as Log10 + Standard Deviation (SD)". They should move their result part and check what "UFC" means. Likewise, p.a. + (L. r.) etc... should be represented in the table captions in their full form.

6.      Figure 9A&B groups 3 and 4 of section 2.2.3 should either be revised or removed from the results, since the short incubated medium supernatant cannot elicit significant levels of growth factors. Similarly, figure 10A in 1h graph need to be revise.

7.      The discussion line-313 “Several experimental and clinical results showed the benefits of probiotics in the prevention of P. aeruginosa infection [37]” missing the some more references.

8.      HCE cell viability needs to be combined with 4.6 and 4.7 methodology and shortened. Additionally, 4x10⁵ cells/well in 96-well plates is not an acceptable concentration for cell viability and cytotoxicity studies. 1-5x10³ to 10⁴ is the maximum concentration used in MTT assays for cell viability and cytotoxicity studies.

Author Response

Reviewer 2

This manuscript addresses a timely topic and makes a relevant contribution to the field. In its current form, this paper cannot be considered for publication. However, some major revisions are needed before it can be published.

1. In the results sections 2.1.1. and 2.1.2, lines 105-109 and 122-130 should be moved to the introduction part since these sentences provide general information.

The authors thank the reviewer for the comments.

As suggested by the reviewer, the authors moved these sentences to introduction section (Lines 58-64; 74-79).

2. In section 2.1.3, figure 3(A-C). Probiotic treatment with HCE requires different concentrations probiotics (low to high CFU), and its growth curves (MTT, LDH) must be shown in line graphs in a concentration-dependent manner. Similarly, figure 4 needs to use line graph with growth curves.

As suggested by the reviewer, the authors added the growth curves of probiotics incubated with HCE cells in line graphs in the figures 3 and 4.

3. Section 2.1.5. In figures 5 and 6, immunocytochemistry staining of mucin and occludin is required to confirm Western blotting and PCR quantification of gene and protein expression. It may increase the strengthens to this manuscript.

As suggested by the reviewer, the authors performed western blot analysis to confirm immunofluorescence data of occludin and mucin (Figures 5 and 6, panels D).

4. Figures 7 and 8 in section 2.2, both results need to be confirmed with cell morphological images from the Live/Dead staining assay.

The authors thank the reviewer for the suggestion.

Human corneal epithelial (HCE) cells are primary cells that require time (10-12 days) to be cultured after the cryopreservation. Due to limited time available and reagents lacking, the authors were unable to carry out the Live/Dead staining assay for each probiotic strain. However, considering the interesting data of this study, the authors consider performing this staining in the future for another manuscript that could represent the continuation of the present paper also using an in vivo model.

5. The caption of Table 1 states: "Results are expressed as Log10 + Standard Deviation (SD)". They should move their result part and check what "UFC" means. Likewise, p.a. + (L. r.) etc... should be represented in the table captions in their full form.

As suggested by the reviewer, the authors moved this sentence to the table caption and presented the name of probiotics in full form. “UFC” means “CFU”; the authors apologize for the typing error.

6. Figure 9A&B groups 3 and 4 of section 2.2.3 should either be revised or removed from the results, since the short incubated medium supernatant cannot elicit significant levels of growth factors. Similarly, figure 10A in 1h graph need to be revise.

The authors decided to remove the non-significant results obtained after 1h of co-incubation of P. aeruginosa with probiotic strains, concerning cytokines release and NO_x levels, from the figures 9 and 10 as suggested by the reviewer.

7. The discussion line-313 “Several experimental and clinical results showed the benefits of probiotics in the prevention of P. aeruginosa infection [37]” missing the some more references.

As suggested by the reviewer, the authors added more references (Ref: 38, 39, 40).

8. HCE cell viability needs to be combined with 4.6 and 4.7 methodology and shortened. Additionally, 4x10⁵ cells/well in 96-well plates is not an acceptable concentration for cell viability and cytotoxicity studies. 1-5x10³ to 10⁴ is the maximum concentration used in MTT assays for cell viability and cytotoxicity studies.

As suggested by the reviewer, the authors combined the paragraph 4.5 with the paragraphs 4.6 and 4.7 and shortened them.

The number of the cells seeded in 96-well plates for cell viability is 4x10⁴ cells/well. The authors apologize for the typing error.

Reviewer 3 Report

Good approach. Please consider a few points.

Minor points:

1. Abstract, include actual data with means, SEM, and p values of the most important results, and add a conclusion sentence.

2.  Introduction there are 2 paras with single sentence, provide a larger para.

3. Figure 1B, include means and SEM

4. From what humans/patients were HCE provided, clarify in the text.

Fair

Author Response

Reviewer 3

Good approach. Please consider a few points.

Minor points:

1. Abstract, include actual data with means, SEM, and p values of the most important results, and add a conclusion sentence.

The authors thank the reviewer for the comments.

As suggested by the reviewer, the authors added the means, SD and p values of the most important results and a conclusion sentence in the abstract section.

2. Introduction there are 2 paras with single sentence, provide a larger para.

As suggested by the reviewer, the authors revised these paragraphs (Lines 36-38, 55-64, 74-81, 86-89).

3. Figure 1B, include means and SEM.

As suggested by the reviewer, the authors revised the figure 1B.

4. From what humans/patients were HCE provided, clarify in the text.

Human corneal epithelial cells (HCE) were kindly provided from Sooft Research Center, Italy, SpA. HCE cells were isolated from the human cornea of a donor patient as described by Cristaldi and colleagues (Cristaldi et al., 2020).

As suggested by the reviewer, the authors clarified it in the Materials and methods section.

Reviewer 4 Report

This interesting study concerns the possibility of protection of human corneal epithelial cells against P. aeruginosa infection by probiotics, Lactobacillus reuteri and Bifidobacterium longum subsp. infantis, demonstrating anti-inflammatory and antinitrosative activities, reducing TNF-α  level, NOx amount and re-establishing IL-10 level by the probiotic treatment.

Questions and Remarks:

Did the authors exclude the possibility of bacterial contribution to MTT reduction in the viability assay?

Was there no effect of the buffer with Triton X-100 on the viability of bacteria?

In the Material and methods the Authors use the term “aggregation coefficient” while when presenting results they write “aggregation %”. It would be desirable to use the same term throughout the text.

How can you reconcile 2% cell death with the 20% LDH release?

Line 95, 97, 99: do the authors mean “cysteine”?

Line 319: “without induce”, perhaps ”without induction” 

Line 410: “Ax and Ay are the individual aggregation properties”, “aggregation properties” is a general term, a measurable quantity should be listed 

Line 465: Please indicate the concentration of Trypan Blue

Fig. 9: The term “concentration” would be more appropriate than “quantity” since the dimension is pg/mL 

English generally OK, few spelling/grammar errors detected, so a final check would be desirable,

Author Response

Reviewer 4

This interesting study concerns the possibility of protection of human corneal epithelial cells against P. aeruginosa infection by probiotics, Lactobacillus reuteri and Bifidobacterium longum subsp. infantis, demonstrating anti-inflammatory and antinitrosative activities, reducing TNF-α level, NOx amount and re-establishing IL-10 level by the probiotic treatment.

Questions and Remarks:

Did the authors exclude the possibility of bacterial contribution to MTT reduction in the viability assay?

Scientific evidence has proven that cytotoxicity assay, such as MTT and XTT assay, are a useful diagnostic method for evaluating the cytotoxicity of bacterial cultures thanks their rapidity and sensitivity (Twaruzek et al., 2018; Elham et al., 2022). However, considering the possibility of a bacterial contribution to MTT reduction, the authors performed also LDH assay and Trypan blue staining to confirm the previous MTT data as shown in the figures 3, 7 and 8.

Was there no effect of the buffer with Triton X-100 on the viability of bacteria?

Several studies demonstrated that Triton X-100 at low concentrations (e.g. 0.1%, 0.25%, 0.5%) doesn’t affect the viability of bacteria (Zi et al., 2018; Miki et al., 2013). In our study, we used Triton X-100 at the concentration of 0.1%; this concentration didn’t exert any effect on the viability of bacteria.

In the Material and methods, the Authors use the term “aggregation coefficient” while when presenting results, they write “aggregation %”. It would be desirable to use the same term throughout the text.

As suggested by the reviewer, the authors revised it in the text (paragraph 4.2).

How can you reconcile 2% cell death with the 20% LDH release?

The authors reconcile the 20% of LDH release probably to sensitivity of HCE cells as demonstrated also in a previous study by Gao et al., 2016 where LDH release in immortalized HCE cells (control group) was almost 15%.

However, as described in the Materials and methods section, we calculated LDH release including the OD values of positive control, according to the formula:

% LDH release =100x Experimental LDH release (OD₄₉₀)

                                      (Maximum LDH release (OD₄₉₀)

Despite the low OD values of our experimental groups, LDH release reached 20% when we included the values of positive control.

Line 95, 97, 99: do the authors mean “cysteine”?

It means cysteine. The authors apologize for the typing error and revised it.

Line 319: “without induce”, perhaps” without induction”

As suggested by the reviewer, the authors revised it.

Line 410: “Ax and Ay are the individual aggregation properties”, “aggregation properties” is a general term, a measurable quantity should be listed.

As suggested by the reviewer, the authors clarified it in the text (paragraph 4.2).

Line 465: Please indicate the concentration of Trypan Blue.

As suggested by the reviewer, the authors indicated the concentration of Trypan blue in the Materials and methods section (Line 473).

Fig. 9: The term “concentration” would be more appropriate than “quantity” since the dimension is pg/mL.

As suggested by the reviewer, the authors revised it in the figure 9.

Round 2

Reviewer 2 Report

I accept the present form for publication.