Promising Photocytotoxicity of Water-Soluble Phtalocyanine against Planktonic and Biofilm Pseudomonas aeruginosa Isolates from Lower Respiratory Tract and Chronic Wounds

Round 1

Reviewer 1 Report

Dear Editor and Authors

I reviewed the paper entitled “Promising photocytotoxicity of water-soluble phtalocyanine against planktonic and biofilm Pseudomonas aeruginosa isolates from lower respiratory tract and chronic wounds” and submitted to Applied Sciences Journal.

The article is well written, it is logical and the English language fulfills the highest standards

However, there are some questions that need to be addressed before being accepted for publication.

 

General comments

In general, I would change the way that some of the data is introduced. As an example, Table 1 should include all the information of all the strains (sensibility to antibiotics and biofilm formation). I don’t know if this is introduced in the Supplementary Material (which was not available for me), but this is actually crucial information that needs to be available for the readers.

Furthermore, Table 1 first part can actually be better visualized with a Venn diagram, clearly stating the tendencies of the resistance patterns found.

The conclusions should be addressed both, in the effectiveness of the treatment and the strain variability effect. Do the authors observe a sustancial difference among the different strains tested? What is the correlation between “resistance” to the PACT effect and previously known antibiotic resistance patterns?

 

Particular comments

Section 2, page 3, line 94. I think the authors forgot to erase the instructions provided by the editors, as the phrase “The Materials and Methods should be described with sufficient details to allow others to replicate 2.1. Photosensitizer and light source” appears altogether

 

Section 2, page 3, line 101. I think it’s important to express the light dose (J/cm²) and the light irradiance (mW/cm²), allowing the readers to compare these results with other examples found in the literature.

 

Section 2, page 3, line 123. Itallics are missing for Klebsiella pneumoniae’s name

 

Section 3.1, page 4, line 184. I couldn’t find the Supplementary Information mentioned

 

Table 1. Maybe another form to represent at least the first part of Table 1 (Resistance patterns) would be with a Venn diagram. This would allow the reader to understand better the distribution of the resistance patterns. This is just a suggestion, as it’s not clear for me if, for example, a strain MEM resistant is taken in account in the IPM-MEM. If so, it must be stated clearly.

Also, did you find non-resistant strains at all? If not, I think it should be clearly stated in Table 1.

How many strains were isolated? The numbers of Table 1 don’t add up: Total of biofilm formation tested strains (29) =/= total of resistance patterns tested strains (20).

 

Figure 2. Number of tested strains = 29.

Did you test the PACT effect over control strains (PAO1, ATCC 27853, NCTC 6749)?

As the number of tested strains is not clearly stated elsewhere, it is confusing why the strain numbers IDs go up to 75.

Also, it would improve the visibility of your figure to increase the width of the bars and draw the x-axis with a thicker line.

 

Section 3.3, page 6, line 244. What concentration did you use? 1X10E-4 µM is 100 pM, which is 1,000,000 times lower than the concentration used to eradicate the planktonic cells (1x10E-4 M). Maybe it would help more to use µM or nM rather than the concentration and scientific notation. If this is accurate, do the authors have an explanation on why biofilm requires 6 orders of magnitude less photosensitizer than the planktonic cells to be eradicated?

 

Section 3.3. It has been previously described in the literature that P. aeruginosa prefers to form its bifilm rather in the walls than in the bottom of well (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/pdf/jove-47-2437.pdf). How does this simmetry could affect the irradiation effectiveness in the sample? What were your observation in this respect?

 

Figure 3. The use of a white gradient makes difficult for the reader to interpret where is the limit of the bar. I would advice the use of a solid background. Also, I suggest to use the same scale for the four graphs, in order to make a better comparison. Additionally, I suggest to include part of the information mentioned in the footnote in the figure, to facilitate the comparison between the different groups. Also, I think it would be interesting to include the resistance profiles of the tested strains, as this is not further mentioned in the figure. If D is irradiated zero times, why is this not before A, which was irradiated only one time? Wouldn’t it be better to express this as total light dose received? 0, 3.6, 7.2. 10.8 J/cm²?

 

Section 4, page 7, line 275. “Among the strains with strong biofilm biomass production there were both multi-drug resistant as well as antibiotic sensitive strains present”. Neither the readers not me can know or analyze this, as Table 1 does not describe the characteristics (antibiotic resistance and biofilm formation) of each strain individually.

 

Section 4, page 8, line 287. “It is worth noting that for all tested strains the 3 log border for bactericidal activity for antibiotics was achieved” maybe this needs to be rephrased, as it can be understood as if the strains were sensitive to all the antibiotics, showing a 3-log reduction

 

Section 4, page 9, line 355. “The herein studied Pc+ at the concentration of 1.0×104 M” careful with the units. So this confirms that you used 100 µM rather than 100 pM, I guess.

 

Section 4, page 9, line 357. “The herein studied Pc+ at the concentration of 1.0×104 M after irradiation presented diverse activity against P. aeruginosa cells in biofilm and the reduction of bacterial cells showed strain-dependent variability” How do you sustentate the strain dependent variability? This was never discussed along the text.

 

References. There’s a double numeration in the references, and after reference 14 they’re different. Please verify this

 

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you for your valuable comments and suggestions. We appreciate your efforts in reviewing our paper and increasing its quality.

We have taken into consideration all suggestions

Best regards

Wojciech Szczolko

Author Response File: Author Response.pdf

Reviewer 2 Report

Authors utilized the previously reported phtalocyanine derivate in the literature, namely Pc+, as photosensitizer to assess its PDT sterization activity against multidrug-resistant P. aeruginosa isolates from lower respiratory tract and chronic wounds. This work seems to be of importance for possible clinical transition of Pc+ in PDT antibacterial therapy. Other comments were listed as follows:

1) Since the research results show that Pc+ can kill P. aeruginosa effectively at a low concentration of 1.0×10^-4 M, plate photographs of P. aeruginosa interacted with Pc+ in dark and under light, respectively should be added to verify these results visually.

2) SEM images of P. aeruginosa treated with Pc+ in dark and under light should be provided to demonstrate the damage of cell membranes.

Author Response

Dear Reviewer,

Thank you for your valuable comments and suggestions. We appreciate your efforts in reviewing our paper and increasing its quality.

We have taken into consideration all suggestions

Best regards

Wojciech Szczolko

Author Response File: Author Response.pdf

Reviewer 3 Report

Although currently the world is still struggling with the covid 19 pandemic, it is inevitable that we will face the problem of multidrug resistance infections in the near future. Thus, chronic wounds and lower respiratory tract infections are among one of the most serious health and social problems due to the problem of MDR and the presence of pathogens in biofilm form, which is up to 10 times more difficult to treat than planktonic forms.

The evaluated work is part of a trend of interdisciplinary topic such as the photodynamic disinfection/inactivation of microorganisms.

I believe that in this interesting paper the study is designed and executed appropriately and the manuscript is written correctly. The tested cationic photosesnsitizer after excitation with  light activation, showed a significant photodynamic efficacy leading up 6 logs reduction of P. aeruginosa (in planktonic form) and more than 4 logs in biofim. It is no doubt reasonable results, however, I suggest a few changes to improve the quality of the paper before accepting it.

1. I miss the discussion of the mechanisms of ROS generation. This is actually very important because ROS (as the authors themselves wrote in the introduction), are the phototoxic agents that lead to the destruction of microorganisms. Interestingly, while in photodynamic therapy of cancer mechanism I is gaining importance, in PDI, mechanism II seems to be the most desirable, due to the fact that microorganisms do not develop resistance and singlet oxygen.
2. It would be useful to add illustrations that would confirm the effectiveness of PDI (control groups and after treatment with Ps and world, respectively).

Author Response

Dear Reviewer,

Thank you for your valuable comments and suggestions. We appreciate your efforts in reviewing our paper and increasing its quality.

We have taken into consideration all suggestions

Best regards

Wojciech Szczolko

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear Authors and Editor

 

Again, thanks for letting me review this work.

After reading the response of the author, I’m still not satisfied, as I think this work can shine more. I think what lacks the most is a comparison of the efficiency of the treatment, based on the resistance profile of the isolated strains. This comparison would make of this a reference work for the future development of PACT.

 

General comments

In general, I would change the way that some of the data is introduced. As an example, Table

1 should include all the information of all the strains (sensibility to antibiotics and biofilm

formation). I don’t know if this is introduced in the Supplementary Material (which was not

available for me), but this is actually crucial information that needs to be available for the

readers.

Furthermore, Table 1 first part can actually be better visualized with a Venn diagram, clearly

stating the tendencies of the resistance patterns found.

We changed the Table in the way that it contains information about biofilm formation and

sensibility of all strains to antibiotics. The resistance profiles of all the tested strains are also

marked in Table 1.

Change addressed

 

The conclusions should be addressed both, in the effectiveness of the treatment and the strain

variability effect. Do the authors observe a sustancial difference among the different strains

tested? What is the correlation between “resistance” to the PACT effect and previously known

antibiotic resistance patterns?

To the Conclusion section, the following sentence was added:

P. aeruginosa strains applied in this study exhibited various antibiotic resistance patterns and

varying degrees of biofilm formation. Nevertheless, no effects were observed related to the

biofilm formation ability on the sensitivity of P. aeruginosa towards antimicrobial

photodynamic therapy as well as related to the resistance to antibiotics.

Although a new comment was added to the conclusions, I think there’s still more to discuss. As an example, the authors don’t discuss, if any, the differences found between the different clinical isolates. Could PACT and this particular strategy be an alternative for the treatment of highly resistant clinical isolates? This is barely discussed in the conclusions and does not efficient and clearly compares the results presented in Figure 3, where at least all the strains were subject to a photodynamic treatment. Do all the strains are sensitive in the same magnitude? A statistical comparison was never mentioned along the whole manuscript and I think it’s missing. I’m sorry to emphasize in this, but you’ve made a monumental work and this comparison will make your work interesting for readers familiar with PACT, but also for readers unfamiliar with PACT and that, maybe, can be convinced of its effectiveness.

 

Particular comments

Section 2, page 3, line 94. I think the authors forgot to erase the instructions provided by the

editors, as the phrase “The Materials and Methods should be described with sufficient details

to allow others to replicate 2.1. Photosensitizer and light source” appears altogether

We deleted the phrase “The Materials and Methods should be described with sufficient details

to allow others to replicate”.

Change addressed

 

Section 2, page 3, line 101. I think it’s important to express the light dose (J/cm2) and the light

irradiance (mW/cm2), allowing the readers to compare these results with other examples

found in the literature.

We changed mW/cm2 to J/cm2.

Change addressed

 

Section 2, page 3, line 123. Itallics are missing for Klebsiella pneumoniae’s name

We corrected.

Change addressed

 

Section 3.1, page 4, line 184. I couldn’t find the Supplementary Information mentioned

We have added supplementary materials to the main text (Table 1).

Change addressed

 

Table 1. Maybe another form to represent at least the first part of Table 1 (Resistance

patterns) would be with a Venn diagram. This would allow the reader to understand better the

distribution of the resistance patterns. This is just a suggestion, as it’s not clear for me if, for

example, a strain MEM resistant is taken in account in the IPM-MEM. If so, it must be stated

clearly.

We changed the Table in the way that it contains information about biofilm formation and

sensibility of all strains to antibiotics. The resistance profiles of all the tested strains are also

marked in Table 1.

Change addressed

 

Figure 2. Besides the evident reduction on the number of colonies per plate, it appears that there’s a change in the colony morphology when the compound is added. Is this just my imagination or did you observed a change?

 

Also, did you find non-resistant strains at all? If not, I think it should be clearly stated in

Table 1.

How many strains were isolated? The numbers of Table 1 don’t add up: Total of biofilm

formation tested strains (29) =/= total of resistance patterns tested strains (20).

Figure 2. Number of tested strains = 29.

There were 29 tested strains. Nine strains were sensitive to the tested antibiotics.

Change addressed

 

Did you test the PACT effect over control strains (PAO1, ATCC 27853, NCTC 6749)?

As we observed the effects of preincubation and illumination time on the sensitivity of P.

aeruginosa standard strains on the PACT, these strains were used in preliminary studies to

determine the optimal exposure conditions. Thus, P. aeruginosa PAO1, P. aeruginosa ATCC

27853, P. aeruginosa NCTC 6749 were used to determine optimal conditions of the

photodynamic inactivation of biofilm. In addition, the PAO1 strain as well characterized for

biofilm formation and often used as a control strain in biofilm studies, was used to evaluate the

effect of PACT on P. aeruginosa biofilm.

Ok, thanks for the clarification

 

As the number of tested strains is not clearly stated elsewhere, it is confusing why the strain

numbers IDs go up to 75.

In section "2.2. Bacterial strains" the number of strains tested was completed – (n = 29). We

also added: “Strains were isolated from the lower respiratory tract (n = 13) and chronic

wounds (n = 16)”.

Changed addressed

 

Also, it would improve the visibility of your figure to increase the width of the bars and draw

the x-axis with a thicker line.

We changed the Figure 2 in new version of Figure 3.

In the legend of Figure 3, it says “Values od reduction…” instead of “Values of reduction…”.

 

Section 3.3, page 6, line 244. What concentration did you use? 1X10E-4 µM is 100 pM,

which is 1,000,000 times lower than the concentration used to eradicate the planktonic cells

(1x10E-4 M). Maybe it would help more to use µM or nM rather than the concentration and

scientific notation. If this is accurate, do the authors have an explanation on why biofilm

requires 6 orders of magnitude less photosensitizer than the planktonic cells to be eradicated?

The concentration of Pc used in our studies is 1x10-4 M. Usage of µM was an editorial mistake.

Thanks for the clarification

 

Section 3.3. It has been previously described in the literature that P. aeruginosa prefers to

form its bifilm rather in the walls than in the bottom of well

(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/pdf/jove-47-2437.pdf). How does

this simmetry could affect the irradiation effectiveness in the sample? What were your

observation in this respect?

In our research, we also observed biofilm formation on the walls rather than on the bottom of

the wells. Therefore, we used 200 µl of broth for the biofilm cultivation, and the same volume

of Pc was used to assess the PACT effect on P. aeruginosa biofilm. The procedure allowed the

contact of the entire biofilm with the studied Pc.

This comment does not address my question. As you’re irradiating from above, how can you make sure that all the cells are being irradiated in the same manner? It’s even possible that the results are better, but due to the irradiation geommetry this is not seen.

 

Figure 3. The use of a white gradient makes difficult for the reader to interpret where is the

limit of the bar. I would advice the use of a solid background. Also, I suggest to use the same

scale for the four graphs, in order to make a better comparison. Additionally, I suggest to

include part of the information mentioned in the footnote in the figure, to facilitate the

comparison between the different groups. Also, I think it would be interesting to include the

resistance profiles of the tested strains, as this is not further mentioned in the figure. If D is

irradiated zero times, why is this not before A, which was irradiated only one time? Wouldn’t

it be better to express this as total light dose received? 0, 3.6, 7.2. 10.8 J/cm2?

Figure 3 was corrected and in a new version of manuscript the numbering was changed to 4.

The new version of the Figure has been improved, but there are still some small details about it. In Fig. 4C the strain number has a dark line crossing it. In the legend of Figure 4, it says “Values od reduction…” instead of “Values of reduction…”

 

Section 4, page 7, line 275. “Among the strains with strong biofilm biomass production there

were both multi-drug resistant as well as antibiotic sensitive strains present”. Neither the

readers not me can know or analyze this, as Table 1 does not describe the characteristics

(antibiotic resistance and biofilm formation) of each strain individually.

These information was completed in Table 1.

The information was completed and a new question has arised. How the authors choose which strains to use? As an example, why didn’t they choose to analyze the efficiency of biofilm destruction of strains 33 and 38? These seems to be interesting strains, which are likely to be difficult to eradicate with any traditional antibiotic treatment. It’s not evident why did the authors choose one or another strain.

 

Section 4, page 8, line 287. “It is worth noting that for all tested strains the 3 log border for

bactericidal activity for antibiotics was achieved” maybe this needs to be rephrased, as it can

be understood as if the strains were sensitive to all the antibiotics, showing a 3-log reduction

The sentence was modified. “Bactericidal activity of antibacterial agent in vitro is usually

defined as a reduction by 3-log of the number of viable cells. It is worth noting that for all tested

strains, the parameter was exceeded.”

Change addressed

 

Section 4, page 9, line 355. “The herein studied Pc+ at the concentration of 1.0×104 M”

careful with the units. So this confirms that you used 100 µM rather than 100 pM, I guess.

The concentration of Pc used in our studies is 1x10-4 M. Usage of µM was an editorial mistake.

Change addressed

 

Section 4, page 9, line 357. “The herein studied Pc+ at the concentration of 1.0×104 M after

irradiation presented diverse activity against P. aeruginosa cells in biofilm and the reduction

of bacterial cells showed strain-dependent variability” How do you sustentate the strain

dependent variability? This was never discussed along the text.

The sentence in the section 3.3. was modified. “As revealed in Fig 4, after single irradiation a

decrease of the number of bacteria in biofilm varied between tested strains in the range of 1.3

- 4.2 log”.

 

Figure 5. In the legend is missing the concentration and light dose applied on the biofilm. Furthermore, the legend of the scale bar of the images is not clear enough.

 

References. There’s a double numeration in the references, and after reference 14 they’re

different. Please verify this.

It was corrected

Change addressed

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you for your valuable comments and suggestions. We appreciate your efforts in reviewing our paper and increasing its quality.

We have taken into consideration all suggestions

Best regards

Wojciech Szczolko

Author Response File: Author Response.docx

Reviewer 2 Report

The authors have responded to all my concerns and completely revised the manuscript. So I suggest that it can be accepted for publication in its current form. 

Author Response

Dear Reviewer,

Thank you for accepting the changes.

Best regards

Wojciech Szczolko