Quantification of Pseudomonas aeruginosa biofilms using electrochemical methods
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Currently 2.29% of deaths worldwide are caused by antimicrobial resistance (AMR), compared to 1.16% from malaria, and 1.55% from human immunodeficiency virus and acquired immunodeficiency syndrome (HIV/AIDs). Furthermore, deaths resulting from AMR are projected to increase to more than 10 million per annum by 2050. Biofilms are common in hospital settings, such as medical implants and pose a particular problem as they have shown resistance to antibiotics up to 1000-fold higher than planktonic cells because of dormant states and reduced growth rates. This is compounded by the fact that many antibiotics target mechanisms of active metabolism and are therefore less effective. The work presented here aimed to develop a method for biofilm quantification which could be translated into the clinical setting, as well as used in the screening of antibiofilm agents. This was carried out alongside crystal violet staining, as a published point of reference. Using electrochemical impedance spectroscopy and square wave voltammetry, P. aeruginosa biofilm formation was detected within an hour after seeding P. aeruginosa on the sensor. A 40% decrease in impedance modulus was shown when P. aeruginosa biofilm had formed, compared to the media only control. As such, this work offers a starting point for the development of real-time biofilm sensing technologies, which can be translated into implantable materials.
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During the peer review process, confusion has arisen about the similarity between the results presented in this manuscript and that of some previously published work (Dunphy et al. Combining hyperspectral imaging and electrochemical sensing for detection of Pseudomonas aeruginosa through pyocyanin production. Sensors & Diagnostics. 2022;1(4):841–50.). After carefully checking the methods in the two manuscripts, I believe them to be slightly different (HIS vs EIS), but the lack of clarity in this submission on how this method builds upon the previously published method- why is it different? Why is it needed in addition to the other method? etc gives rise to this confusion. Additionally, Figure 4 presents an SWV standard curve for pyocyanin as part of method development for using this pigment as a measure of biofilm formation- this method …
During the peer review process, confusion has arisen about the similarity between the results presented in this manuscript and that of some previously published work (Dunphy et al. Combining hyperspectral imaging and electrochemical sensing for detection of Pseudomonas aeruginosa through pyocyanin production. Sensors & Diagnostics. 2022;1(4):841–50.). After carefully checking the methods in the two manuscripts, I believe them to be slightly different (HIS vs EIS), but the lack of clarity in this submission on how this method builds upon the previously published method- why is it different? Why is it needed in addition to the other method? etc gives rise to this confusion. Additionally, Figure 4 presents an SWV standard curve for pyocyanin as part of method development for using this pigment as a measure of biofilm formation- this method development is already published as part of the Dunphy et al paper. The conclusions in this submission state that you have developed a standardised method for biofilm quantification using CV staining (line 434-5)- I don’t see this in this manuscript. Did the CV staining method require standardisation? Is it not already a well developed and widely used technique with wash steps already included? The stated benefit to this new method is to show that biofilm can be measured in real time vs endpoint only readings via the CV staining method, but a coherent comparison has not been presented to show the limitation of the CV method for measuring 4-hour biofilm vs this new method. The presentation of the data in figures 2-6 is confusing- why set the OD600 = 1 seeding density point as 100% and normalise the other data points to it, rather than present the raw absorbance/current readings for each seeding density? That way the ability of the two methods to detect cells at a lower density would be comparable. Please clarify if the data are from 3 biological or technical replicates. There are many issues with the presentation of the results in this submission. However, I hope that these can be revised to result in a manuscript that contains a useful comparison between this methodology and the standard CV 96-well assay. If you wish to proceed with revising this submission, please respond to the points above along with all peer review comments and include a tracked changes version with the revisions clearly outlined. In line with Access Microbiology’s Open Data policy, please deposit the raw data underlying the work in the Society’s data repository Figshare account here: https://microbiology.figshare.com/submit. Please also cite this data in the Data Summary of the main manuscript and list it as a unique reference in the References section. When you resubmit your article, the Editorial staff will post this data publicly on Figshare and add the DOI to the Data Summary section where you have cited it. This data will be viewable on the Figshare website with a link to the preprint and vice versa, allowing for greater discovery of your work, and the unique DOI of the data means it can be cited independently. Details on our Open Data policy can be found here- https://www.microbiologyresearch.org/open-data
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Comments to Author
The work presented in the manuscript refers to a quantification method of P. aeruginosa biofilms with electrochemical methods. While this paper is presented in another angle, it is very similar to an already published paper (reference 38). This other paper, in which all the authors of the current manuscript are co-authors, presents much more detail to the methods - which I though was somewhat lacking in the current manuscript. Overall, I think this paper lacks adequate depth, and its structure could be improved. For instance, the introduction lines 108-118 have some concepts repeated. Also, the authors explore pyocyanin as a proxy for biofilm formation, and electrochemical monitoring without citing other papers with relevant information (for instance https://pubmed.ncbi.nlm.nih.gov/22123963/ and …
Comments to Author
The work presented in the manuscript refers to a quantification method of P. aeruginosa biofilms with electrochemical methods. While this paper is presented in another angle, it is very similar to an already published paper (reference 38). This other paper, in which all the authors of the current manuscript are co-authors, presents much more detail to the methods - which I though was somewhat lacking in the current manuscript. Overall, I think this paper lacks adequate depth, and its structure could be improved. For instance, the introduction lines 108-118 have some concepts repeated. Also, the authors explore pyocyanin as a proxy for biofilm formation, and electrochemical monitoring without citing other papers with relevant information (for instance https://pubmed.ncbi.nlm.nih.gov/22123963/ and https://pubmed.ncbi.nlm.nih.gov/36509018/). Authors also did not use the most common P. aeruginosa laboratory strain (PAO1), not clearly stating the importance of the two strains selected. The methods are described in a very direct way, however sometimes they are a bit confusing, and it's hard to understand why authors decided to do normalisations when the raw values would work well. A clear example of this is Figures 5 and 6 in which points were normalised but raw data (Figure S1) allows easier comparison. Data in Table 1 was over analysed - particularly the "10-fold reduction" mentioned in lines 239-241. As an example, note that if the treatment changed (PBS wash vs no wash) you can't say that replicates are comparable, and as such they should not be numbered 1-6. There were some oddities in the data as well, like cells seeded at OD600=0.2 then decreasing after 4h incubation (line 236). Currently, best practice in plotting data, particularly when no biological replicates are present, just the minimum 3 technical replicates, is that in the plots authors should display all the points to allow better interpretation. Note that increasing technical replicates and including biological replicates could help with what the authors mention in line 294 (challenges with accurate and repeatable measurements). Furthermore, I was somewhat disappointed that after authors presented a good calibration curve for pyocyanin (r2 = 99.9%), they then did not extrapolate an equation to quantify the concentration of pyocyanin from the current measurements, which they say correlates with P. aeruginosa density (line 325). In another part of the discussion, authors note that LESB58 has been shown by other studies to be a superior biofilm former (without references) due to lack of motility. This does not match with the raw data displayed in Figure S1 - one possible explanation is that the experiment was done in a static environment so absence of motility might not have been as relevant, but the authors did not mention this, instead normalising the values and saying they were not different.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
Yes: Possible duplication of published results
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Riordan et al. present a useful method for P. aeruginosa biofilm quantification, which addresses a current research challenge and will be beneficial for a large number of researchers. Major comments: * A wider range of P. aeruginosa biofilm contexts added to the introduction would strengthen this section and better emphasise the importance of new methods for biofilm quantification (e.g. chronic lung infection, chronic wounds). Similarly, addition of more infection contexts outside of medical devices and implants in the discussion would strengthen the manuscript. Researchers focused on these P. aeruginosa biofilm infections are likely to be interested in the method. * A diagram providing an overview of the electrochemical methods described in the introduction would improve the clarity, particularly …
Comments to Author
Riordan et al. present a useful method for P. aeruginosa biofilm quantification, which addresses a current research challenge and will be beneficial for a large number of researchers. Major comments: * A wider range of P. aeruginosa biofilm contexts added to the introduction would strengthen this section and better emphasise the importance of new methods for biofilm quantification (e.g. chronic lung infection, chronic wounds). Similarly, addition of more infection contexts outside of medical devices and implants in the discussion would strengthen the manuscript. Researchers focused on these P. aeruginosa biofilm infections are likely to be interested in the method. * A diagram providing an overview of the electrochemical methods described in the introduction would improve the clarity, particularly for those who may be less familiar with these technologies. * Further discussion of how this method could be used for more clinically relevant P. aeruginosa biofilm models would improve the discussion section, as well as the potential limitations (e.g. would components of artificial sputum media or chronic wound mimicking media be likely to interfere with measurements or is this method a viable option?) Minor comments: * L229-252. The purpose of this section is slightly unclear as just OD is not a typical method to measure biofilm growth. This section would be improved with a clearer and more concise explanation. * Figures 2,3,5 & 6 - improve x axis labels to make it clear this is the seeding optical density.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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