Innovative Insights into In Vitro Activity of Colloidal Platinum Nanoparticles against ESBL-Producing Strains of Escherichia coli and Klebsiella pneumoniae

Round 1

Reviewer 1 Report

 

There is no doubt that authors are specialists in in-vitro work, this is good. However without the detailed characteristic of title (!!!) nanomaterial – sorry the work is worth nothing.

Authors could defend that this is commercial material, but, please believe me this is not an explanation.

Nanomaterials, especially metallic ones, tend to agglomerate/aggregate, this is natural.

To prevent this process some organic stabilizers are used. What about your material?

 

From my point of view, there is lack of two points, in the work. First is the mechanism explanation, "damage of bacterial cell wall" seems little not enough, and comparison with the Ag-nanoparticles.

It is important because a single path of cell death does not prevent multi-resistant

This is true that “platinum nanoparticles there are just a few ambiguous reports”, but the question is why? Maybe silver nanoparticles are much better?

 

 

Author Response

Response to reviewer (1) comments:

Ad1. There is no doubt that authors are specialists in in-vitro work, this is good. However without the detailed characteristic of title (!!!) nanomaterial – sorry the work is worth nothing.

Authors could defend that this is commercial material, but, please believe me this is not an explanation.

Response: We kindly thank you for your valuable comments. Regarding the concern on detailed characterization of nanomaterial used, we used available tools (mass spectrometry) and presented results in MS. Besides, we found several articles published and thus added additional explanation (i.e., characterization) of the nanoparticles in the abstract itself and appropriate part in the Methods section (lines 19-20 and 414-417 of the revised version of the manuscript).

We revised title according to reviewer suggestion.

 

Ad2. Nanomaterials, especially metallic ones, tend to agglomerate/aggregate, this is natural.

To prevent this process some organic stabilizers are used. What about your material?

Response: We kindly thank for valuable comment. In our study we did not use organic stabilizers because there is no established protocol or stabilizer recommended. Additionally, the stabilizers could affect the results (we elaborated this in the revised manuscript from line 372-375). Howver, we found that in the preparation process some researchers use PEG as stabilizing agent (ref Chlumsky et al, 2020 and 2022). With an idea of reducing the reproducibility bias, we decided to use commercially available nanoparticles especially since there are similar studies on nanoPt (Itohya et al 2019), or other commercially available metallic nanoparticles, without added stabilizers (Martinaga Pintarić et al 2020).

As aggregation of particles was one of our concerns we avoided the use of proteins in experiments (used media with no or low protein) as presence of proteins accelerate and enhance aggregation. Moreover, we pretreated suspension prior to use by ultrasonic bath.

Chlumsky, O., Purkrtova, S., Michova, H., Sykorova, H., Slepicka, P., Fajstavr, D., … Demnerova, K. (2021). Antimicrobial Properties of Palladium and Platinum Nanoparticles: A New Tool for Combating Food-Borne Pathogens. International Journal of Molecular Sciences, 22(15), 7892. doi:10.3390/ijms22157892

Chlumsky, O., Purkrtova, S., Michova (Turonova), H., Svarcova (Fuchsova), V., Slepicka, P., Fajstavr, D., … Demnerova, K. (2020). The effect of gold and silver nanoparticles, chitosan and their combinations on bacterial biofilms of food-borne pathogens. Biofouling, 36(2), 222–233. doi:10.1080/08927014.2020.1751132

Itohiya, H., Matsushima, Y., Shirakawa, S., Kajiyama, S., Yashima, A., Nagano, T., & Gomi, K. (2019). Organic resolution function and effects of platinum nanoparticles on bacteria and organic matter. PLOS ONE, 14(9), e0222634. doi:10.1371/journal.pone.0222634

Martinaga Pintarić, L.; Somogi Škoc, M.; Ljoljić Bilić, V.; Pokrovac, I.; Kosalec, I.; Rezić, I. Synthesis, Modification and Characterization of Antimicrobial Textile Surface Containing ZnO Nanoparticles. Polymers 2020, 12, 1210. https://doi.org/10.3390/polym12061210.

 

Ad3. From my point of view, there is lack of two points, in the work. First is the mechanism explanation, "damage of bacterial cell wall" seems little not enough, and comparison with the Ag-nanoparticles. It is important because a single path of cell death does not prevent multi-resistant.

Response: The aim of our study was to asses the possible effect of nanoplatinum on two differen bacteria and their resistant strains. This is also the part of a larger study in which we employ different nanoparticles in terms of incorporation in fabric and the possible use in different applications (medical and nonmedical).

However, we tried to decipher, using available methods, the possible mechanisms which underly the observed effect.

Since Ag nanoparticles are also noble nanoparticles and are among the most extensively and detailly researched in terms of their antimicrobial effects and mechanisms of antimicrobial action, we strongly believe it is reasonable to draw comparisons with them. We also made as many comparisons of our results with the results of studies which also used platinum or other noble metal nanoparticles (including Ag) in the context of examining their antibacterial effects.

Ad4. This is true that “platinum nanoparticles there are just a few ambiguous reports”, but the question is why? Maybe silver nanoparticles are much better?

Response: Thank you for the detailed insights and comments on this, and as reviewer thoughts are similar to ours, the question raised during the experimental process was similar. Perhaps silver nanoparticles are better and this is still a matter of debate. However, one needs to have in mind that first reports on the antimicrobial activity of platinum nanoparticles date only a decade ago while Ag nanoparticles are exploited for more decades. One of the reasons is its traditional and historical use what is not the case for platinum.

 

 

Reviewer 2 Report

This manuscript mainly reports the antibacterial activity of platinum nanoparticles, which makes it a new method to against ESBL-Producing strains of Escherichia coli and Klebsiella pneumoniae. The whole manuscript is very detailed and well organized. However, some defects should be corrected before acceptance.

1.    In order to make the chart look less cluttered, tables 2 and 3 can be centered and more concise.

2.    For lines 109 and 132, please note µg/ml and µg ml^-1.

3.    In part 2.3, there seems to be no good argument that the medium affects the biofilm.

4.    Please make the labels in figures 2 and 3 clearer.

5.    Part 2.5, the statistics part can be explained separately.

6.    In the third part, the results of biofilm experiment and cell permeation experiment of Klebsiella pneumoniae are not well explained in detail.

7.    The format of the fourth part of the hierarchical title should be consistent with the previous part.

Author Response

This manuscript mainly reports the antibacterial activity of platinum nanoparticles, which makes it a new method to against ESBL-Producing strains of Escherichia coli and Klebsiella pneumoniae. The whole manuscript is very detailed and well organized. However, some defects should be corrected before acceptance.

Dear reviewer,

we would like to thank you for your valuable comments and the critical review of our manuscript. We adressed all question raised and hope that MS will meet criteria for acceptance.

Ad1. In order to make the chart look less cluttered, tables 2 and 3 can be centered and more concise.

 

Response: We designed the tables in as requested from Pharmaceutics journal. However, we considered your suggestion and made a small corrections to the tables (visible in the revised version of manuscript). 

 

Ad2. For lines 109 and 132, please note µg/ml and µg ml^-1.

Response: We corrected these as suggested.

 

Ad3. In part 2.3, there seems to be no good argument that the medium affects the biofilm.

Response: We made some corrections to the text to make it informative. Also, we cited studies discussing the exact issue (Adamus-Białek W, Kubiak A, Czerwonka G. Analysis of uropathogenic Escherichia coli biofilm formation under different growth conditions. Acta Biochim Pol. 2015;62(4):765-71. doi: 10.18388/abp.2015_1127.; Lin Z, Long M, Liu W, Liu T, Li F, Wu Y. Distinct biofilm formation regulated by different culture media: Implications to electricity generation. Bioelectrochemistry. 2021 Aug;140:107826. doi: 10.1016/j.bioelechem.2021.107826.).

 

Ad4. Please make the labels in figures 2 and 3 clearer.

Response: Labels are corrected as suggested.

 

Ad5. Part 2.5, the statistics part can be explained separately.

Response: We accepted the suggestion and made corrections in the revised version of the manuscript.

 

Ad6. In the third part, the results of biofilm experiment and cell permeation experiment of Klebsiella pneumoniae are not well explained in detail.

Response: We made some corrections according to suggestions and hope that it is sound and clear now.

 

Ad7. The format of the fourth part of the hierarchical title should be consistent with the previous part.

Response: Format of the manuscript has been corrected.  

 

 

Reviewer 3 Report

As an alternative to antibiotics, nanoparticles are increasingly used to target different multi-drug resistant pathogens. In the presented manuscript, Vukoja and colleagues demonstrated that colloidal platinum nanoparticles exert bactericidal or bacteriostatic effects on selected E. coli and K. pneumoniae strains. The antibacterial mechanism may include the induction of reactive oxygen species (ROS) and the destabilization of the cell wall. My main concern is the method used to detect ROS - bacteria were incubated with DiBAC4(3) (see lines 480-482). This probe is used to monitor changes in membrane potential and exhibits enhanced fluorescence (excitation 490/emission 516 nm) upon binding to intracellular proteins or membranes. The authors wrote that fluorescence intensity was measured at 485 nm excitation and 535 nm emission. These wavelengths indicate that probably another probe, DCFDA, was (or should be) used. DCFDA is oxidized in the presence of ROS.

Other comments:

Tables 2 and 3 could be combined  to demonstrate better the influence of media on the MIC values.

Lines 407-409, 446-447, maybe it is worth including this important information in the "Results" section, in paragraph 2.1?

If the same cultures were described in paragraphs 2.3 (biofilms) and 2.4 (planktonic cells), it would be better to present these results in one section/paragraph.

Lines 328-329 and 521-522. The K. pneumoniae  strains practically did not form a biofilm, therefore these conclusions should be limited to E.coli.

Author Response

Dear reviewer,

we would like to thank you for your valuable comments and overall affirmative review in the first place, and hope that, following our responses and revision, manuscript will be suitable for publication.

Sincerely

Authors

 

Reviewer

As an alternative to antibiotics, nanoparticles are increasingly used to target different multi-drug resistant pathogens. In the presented manuscript, Vukoja and colleagues demonstrated that colloidal platinum nanoparticles exert bactericidal or bacteriostatic effects on selected E. coli and K. pneumoniae strains. The antibacterial mechanism may include the induction of reactive oxygen species (ROS) and the destabilization of the cell wall. My main concern is the method used to detect ROS - bacteria were incubated with DiBAC4(3) (see lines 480-482). This probe is used to monitor changes in membrane potential and exhibits enhanced fluorescence (excitation 490/emission 516 nm) upon binding to intracellular proteins or membranes. The authors wrote that fluorescence intensity was measured at 485 nm excitation and 535 nm emission. These wavelengths indicate that probably another probe, DCFDA, was (or should be) used. DCFDA is oxidized in the presence of ROS.

Response: Due to mistake we mixed two experiments and presented it as one. We conducted both experiments, using DiBAC4(3) to asses membrane potential changes as well as DCFDA assay to adress possible involvement of ROS following nanoparticle treatment. Both experiments are now described and the results incorporated in manuscript.

 

Other comments:

Tables 2 and 3 could be combined to demonstrate better the influence of media on the MIC values.

Response: We considered this suggestion and tried to make a combination of both tables but the clarity of the created table was not satisfactory. 

 

Lines 407-409, 446-447, maybe it is worth including this important information in the "Results" section, in paragraph 2.1?

Response: We accept the suggestion and the correction is incorporated in the revised version of the manuscript.

 

 

If the same cultures were described in paragraphs 2.3 (biofilms) and 2.4 (planktonic cells), it would be better to present these results in one section/paragraph.

Response: We considered the suggestion, but due to clarity of the present concept, we leave these paragraphs separated.

 

Lines 328-329 and 521-522. The K. pneumoniae  strains practically did not form a biofilm, therefore these conclusions should be limited to E.coli.

Response: We agree with the reviewers opinion, so we made corrections in the revised version of manuscript.

 

Reviewer 4 Report

Dear Authors:

 

In my viewpoint the manuscript pharmaceutics-1824297 titled "A Comprehensive Insight Into In Vitro Activity of Colloidal Platinum Nanoparticles Against ESBL-Producing Strains of Escherichia coli and Klebsiella pneumonia" can be accepted to publication after minor revision.

Suggestions to the minor revision are listed at each major topic of manuscript.

Title:

I suggest:

Innovative Insigths from in vitro Activity of Colloidal Platinum Nanoparticles Against ESBL-Producing Strains of Escherichia coli and Klebsiella pneumonia.

 

Abstract:

Delete: Further investigation of platinum nanoparticles' antibacterial properties and their potential biomedical application is needed.

Re-write:

"The key mechanism of their antibacterial effect could be the damage of bacterial cell wall, but ROS induction may also have an important role."

 

I suggest:

Both, The key mechanism of their antibacterial effect  and ROS induction are discussed.

In the last sub-paragraph seems that indicate something that does not belong to the manuscript, hen should be deletade, "Further investigation of platinum nanoparticles' antibacterial properties  and their potential biomedical application is needed."

In the item Introduction:

 

The last paragraph would be re-write.

Ex. In this study, from authors viewpoint, the investigating antibacterial
properties of colloidal platinum nanoparticles towards both clinically multidrug-resistant E. coli and K. pneumoniae is carriedby the first time. The antibacterial properties and action mechanism of nPt towards tom mentioned bacterial species is discussed.

In the item Conclusion:

 

Delete the phrase " In order to find their biomedical applications, there is a need for additional research and not just under in vitro conditions but in vivo settings as well Translational studies should be a step further in the research of this scientific field, since without them many of these important findings will not find their way to be applied in practice." In this phrase, nothing is added, yet seems some kind of fail corrections.

 

 

 

Author Response

Dear reviewer,

we would like to thank you for your valuable comments and affirmative review; we corrected manuscript according to suggestions and hope that it will be accepted for publication in Pharmaceutics.

Dear Authors:

 In my viewpoint the manuscript pharmaceutics-1824297 titled "A Comprehensive Insight Into In Vitro Activity of Colloidal Platinum Nanoparticles Against ESBL-Producing Strains of Escherichia coli and Klebsiella pneumonia" can be accepted to publication after minor revision.

Suggestions to the minor revision are listed at each major topic of manuscript.

 

Title:

I suggest:

Innovative Insigths from in vitro Activity of Colloidal Platinum Nanoparticles Against ESBL-Producing Strains of Escherichia coli and Klebsiella pneumonia.

Response: We considered this suggestion along with other reviewers comments and decided to accept it.

 

Abstract:

Delete: Further investigation of platinum nanoparticles' antibacterial properties and their potential biomedical application is needed.

Response: We also accepted the suggestion and made correction visible in the revised version of the manuscript.

Re-write:

"The key mechanism of their antibacterial effect could be the damage of bacterial cell wall, but ROS induction may also have an important role."

I suggest:

Both, The key mechanism of their antibacterial effect  and ROS induction are discussed.

Response: We considered this suggestion as well and made changes visible in the revised version of the manuscript.

In the last sub-paragraph seems that indicate something that does not belong to the manuscript, hen should be deletade, "Further investigation of platinum nanoparticles' antibacterial properties  and their potential biomedical application is needed."

Response: Suggestion accepted.

 

In the item Introduction:

The last paragraph would be re-write.

Ex. In this study, from authors viewpoint, the investigating antibacterial
properties of colloidal platinum nanoparticles towards both clinically multidrug-resistant E. coli and K. pneumoniae is carriedby the first time. The antibacterial properties and action mechanism of nPt towards tom mentioned bacterial species is discussed.

Response: We considered the suggestion and made corrections accordingly.

 

In the item Conclusion: 

Delete the phrase " In order to find their biomedical applications, there is a need for additional research and not just under in vitro conditions but in vivo settings as well Translational studies should be a step further in the research of this scientific field, since without them many of these important findings will not find their way to be applied in practice." In this phrase, nothing is added, yet seems some kind of fail corrections.

Response: We made corrections to the revised version of manuscript as suggested.

Reviewer 5 Report

• The quality of English writing is good, but some grammatical and structure errors should be revised.
• Abbreviations should be written fully at first appearance in text only then abbreviate

·       Abbreviate platinum nanoparticles all over the manuscript as you mentioned the full name at the beginning of the manuscript and abbreviation in between brackets

·       Replace Enterobacteriaceae by Enterobacterales along the manuscript

• Supplement the results in your abstract with numerical values for accuracy.

·       Capitalize the keywords

·        Line 419: MBC was determined as the lowest concentration at which there was no visible bacterial growth or even kill 99.9% of bacteria on solid media, please correct

·        Lines 449-460: Inhibition of biofilm formation assay: is a method for biofilm formation not its inhibition, please correct

·        Did the authors test only 4 bacterial strains? At least 3 strains of each bacteria should be tested

• Please add the following relevant references to your manuscript:

Line 44: https://doi.org/10.1016/j.cimid.2018.09.013

                  https://doi.org/10.3390/biology10050373

Line 333: https://doi.org/10.1186/s12917-021-02842-9

 

Line 303: https://doi.org/10.3390/antibiotics11040440

 

Author Response

Dear reviewer,

we would like to thank you for your valuable comments. We considered suggestions and made corrections accordingly, and sincerely hope that the revised version of the manuscript will meet the criteria to be published in Pharmaceutics.

Sincerely

Authors.

 

 

Ad1. The quality of English writing is good, but some grammatical and structure errors should be revised.

Response: We made grammatical checkout by native English speaker.

 

Ad2. Abbreviations should be written fully at first appearance in text only then abbreviate. Abbreviate platinum nanoparticles all over the manuscript as you mentioned the full name at the beginning of the manuscript and abbreviation in between brackets.

Response: We understand your point but since the term of platinum nanoparticles repeats frequently throughout the manuscript, we believe that it would get to repetitive and harder to understand and follow the text if we use just the abbreviations all over the manuscript. As it is visible from other articles published in Pharmaceutics [1-3], authors here and then use full words for already abbreviated terms if there is a need to use them frequently throughout the text.

1. Eaton, D.M.; Martin, T.G.; Kasa, M.; Djalinac, N.; Ljubojevic-Holzer, S.; Von Lewinski, D.; et al. HDAC Inhibition Regulates Cardiac Function by Increasing Myofilament Calcium Sensitivity and Decreasing Diastolic Tension. Pharmaceutics 2022, 14, 1509. https://doi.org/10.3390/pharmaceutics14071509
2. Akhmadeev, B.S.; Nizameev, I.R.; Kholin, K.V.; Voloshina, A.D.; Gerasimova, T.P.; Gubaidullin, A.T.; et al. Molecular and Nano-Structural Optimization of Nanoparticulate Mn2+-Hexarhenium Cluster Complexes for Optimal Balance of High T1- and T2-Weighted Contrast Ability with Low Hemoagglutination and Cytotoxicity. Pharmaceutics 2022, 14, 1508. https://doi.org/10.3390/pharmaceutics14071508
3. Aquila, G.; Regin, Y.; Murgia, X.; Salomone, F.; Casiraghi, C.; Catozzi, C.; et al. Daily Intraperitoneal Administration of Rosiglitazone Does Not Improve Lung Function or Alveolarization in Preterm Rabbits Exposed to Hyperoxia. Pharmaceutics 2022, 14, 1507. https://doi.org/10.3390/pharmaceutics14071507.

 

Ad3. Replace Enterobacteriaceae by Enterobacterales along the manuscript

Response: We are aware of the novel nomenclature for the order of Enterobacterales from the work of Adelou and colleagues [4], but we cited the articles which were mentioning E. coli and K. pneumoniae as part of family of Enterobacteriaceae, and we as well wanted to mention them as part of this family rather than order because it is more precise classification. Therefore, we believe that we used proper nomenclature in this case and would suggest to leave it as it is if you agree.

4. Adeolu, Mobolaji, et al. "Genome-based phylogeny and taxonomy of the ‘Enterobacteriales’: proposal for Enterobacterales ord. nov. divided into the families Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov." International journal of systematic and evolutionary microbiology 66.12 (2016): 5575-5599.

 

 

 

Ad4. Supplement the results in your abstract with numerical values for accuracy.

Response: Although we would like as well to include the numerical values in the abstract, due to word count limitation for the abstract set by journal, we are limited in presenting the numerical data in the abstract. Also, since there are many numerical results worth mentioning we believe that the abstract would get overcrowded and would exceed significantly the upper word limit count set for the abstract by journal. We kindly thank you for understanding.

 

Ad5. Capitalize the keywords

Response: According to journal’s “instructions for authors”, in the manuscript template before submission the keywords are written in with small first letter.

https://www.mdpi.com/journal/pharmaceutics/instructions 

•  

Ad6. Line 419: MBC was determined as the lowest concentration at which there was no visible bacterial growth or even kill 99.9% of bacteria on solid media, please correct

Response: The suggestion is accepted and the correction is made in the revised version of manuscript.

•  

Ad7. Lines 449-460: Inhibition of biofilm formation assay: is a method for biofilm formation not its inhibition, please correct

Response: We made a correction in the revised version of manuscript as suggested.

 

Ad8. Did the authors test only 4 bacterial strains? At least 3 strains of each bacteria should be tested

Response: As this study intended to explore antibacterial efficacy of nanoplatinum on strains that have resistant correspondent and being relevant for human health we chosen KP and EC with their resistant (ESBL) strain. We do agree that in studies where overall antimicrobial activity is tested one must test more strains but in this particular experimental setup it was limited due to mentioned reasons.

 

Ad9. Please add the following relevant references to your manuscript:

Line 44: https://doi.org/10.1016/j.cimid.2018.09.013

https://doi.org/10.3390/biology10050373

Response: We appreciate the suggestion and read all references mentioned, but due to limitations set by journal we cited second reference since it is more recent.

 

Line 333: https://doi.org/10.1186/s12917-021-02842-9

Response: This article does not mention platinum nanoparticles but rather Ag nanoparticles. Since one of the referees elaborated against comparison with Ag nanoparticles read the paper and find it interesting and nice as future directive for our work.

 

Line 303: https://doi.org/10.3390/antibiotics11040440

Response: We read the paper and as it is in line with discussion we accept this suggestion and it is now incorporated in the revised version of manuscript.

 

Round 2

Reviewer 1 Report

thank you