Investigating Klebsiella pneumoniae biofilm preservation for scanning electron microscopy
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Klebsiella pneumoniae biofilm formation is associated with chronic and relapsing infections. Scanning electron microscopy (SEM) is a powerful tool for characterizing biofilm structure and studying their formation. Reliable visualization of biofilm structure requires careful sample preservation, otherwise there may be loss of non-covalent interactions that are susceptible to damage during the dehydration and washing preparation steps. However, no standard procedure has been adopted in the literature to fix K. pneumoniae biofilm for scanning electron microscopy studies. This lack of standardization makes it challenging to compare results between studies and determine the degree to which native structures have been preserved. To advance this critical area of study, we investigated different scanning electron microscopy fixation methods for K. pneumoniae biofilm preservation. Our study reveals the impact preparation steps can have on retaining in biofilm architecture observed using scanning electron microscopy. Using fixation methods developed through our studies, we show that although species that overproduce capsular extracellular polysaccharides produced more robust biofilms, K. pneumoniae can form a developed biofilm in the absence of capsular polysaccharides.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. All comments by the reviewers were satisfactorily addressed.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
This paper was a really interesting read. It is becoming increasingly obvious that the biofilm architecture is so important and even more so with the capsular and hypervirulent variants of Klebsiella. I am surprised to see how these small adjustments to the fixation protocol make such a difference, the optimised protocol presented here is clearly effective for maintaining the biofilm matrix. I would love to see this technique used with different isolates of Klebsiella, especially with new or alterative therapeutics. I think it would inform a lot of research and reveal secrets of the biofilm lifestyle! Very important for this manuscript to be shared. The paper was written in a bit of a different style from the traditional manuscript, but I enjoyed how detailed and reasoned the explanations were. Thought …
Comments to Author
This paper was a really interesting read. It is becoming increasingly obvious that the biofilm architecture is so important and even more so with the capsular and hypervirulent variants of Klebsiella. I am surprised to see how these small adjustments to the fixation protocol make such a difference, the optimised protocol presented here is clearly effective for maintaining the biofilm matrix. I would love to see this technique used with different isolates of Klebsiella, especially with new or alterative therapeutics. I think it would inform a lot of research and reveal secrets of the biofilm lifestyle! Very important for this manuscript to be shared. The paper was written in a bit of a different style from the traditional manuscript, but I enjoyed how detailed and reasoned the explanations were. Thought the way the chemistry information and previous microbiology/tissue sample microscopy was brought together really strengthens the manuscript. It is very well-evidenced and clearly explained. A couple of very minor comments where I would like a little more detail; Line 292 - was the polysaccharide precipitated onto the glass cover slip? I am unclear on what surface the isolated polysaccharides were imaged? Figure 4 - are images A and B at the same magnification? I don't know if I am just mistaking matrix for cells in image A - the exposed cells in image B seem to be bigger? Can you please confirm if these experiments were performed in biological replicates?
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very 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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Comments to Author
Reviewer summary: The Authors have presented a workflow towards a standardised method of fixation for observation of Klebsiella biofilms by electron microscopy (EM). The Authors note that there is no current standardised fixation method for biofilm observation using EM, and that their work addresses the knowledge gap and could go on to assist other researchers. The Authors use an iterative process of altering the composition of their fixative solution to preserve the exopolysaccharide matrix (EPS) which is commonly removed by harsh chemical fixatives. Ultimately, this leads to non-representative imaging or misreporting of biofilm morphology/composition in EM studies. The Authors' methodologies proceed logically, and each step is informed by the previous result. The manuscript is well written and …
Comments to Author
Reviewer summary: The Authors have presented a workflow towards a standardised method of fixation for observation of Klebsiella biofilms by electron microscopy (EM). The Authors note that there is no current standardised fixation method for biofilm observation using EM, and that their work addresses the knowledge gap and could go on to assist other researchers. The Authors use an iterative process of altering the composition of their fixative solution to preserve the exopolysaccharide matrix (EPS) which is commonly removed by harsh chemical fixatives. Ultimately, this leads to non-representative imaging or misreporting of biofilm morphology/composition in EM studies. The Authors' methodologies proceed logically, and each step is informed by the previous result. The manuscript is well written and clearly communicated. However, there is no indication as to whether the data they show is representative (i.e., no indication of replicates - biological and technical) - the Authors may consider adding these as supplementary data and including replicate numbers in the main text. Moreover, I believe that if the Authors mean to present this as a fully optimised method of fixation, then the manuscript requires additional experiments to support their claims: quantitative analyses of the EM images, titration experiments for the components of their fixative, and biochemical assays to quantify the preservation of EPS resulting from their fixation method. If not, it should caveated that further optimisation may be required according to factors such as pH and fixative concentration. Based on the above, I recommend this manuscript for Major Revisions prior to further review. I include some revision points for the Authors' attention below. Major points: The findings referring to Figure 1 and Lines 113-121: The Authors state that 'Cells fixed with only glutaraldehyde had very little matrix material covering the cells and adhering to the background'. The cells in Figure 1A & B look objectively similar between the figure panels. The small clusters in Fig1A have approximately the same adhesive matrix levels as the larger microcolony shown in Fig1B per area. How can the Authors confirm that there is more or less EPS without any biochemical testing or image analysis? I see that Fig 1B has more cells present, but that does not equate necessarily to the retention of EPS by using PFA. I suggest that the Authors conduct a simple experiment to quantify the levels of EPS between the two groups presented in Fig1A and Fig1B. Following fixation, the biofilm should be stained using fluorescent EPS dyes (e.g., WGA-conjugates), and each sample imaged under the same acquisition parameters using a widefield or, preferably, a confocal microscope. Comparative fluorescence intensity analysis should determine if there is more/less EPS per area when fixed with GAH alone or GAH + PFA. The Authors should not claim higher/lower EPS levels based on images where this could be subjective without additional quantification. Moreover, relating to Figure 1, there are some cropping artefacts along the bottom and sides of the images. The Authors should resubmit this figure without the cropping artefacts... when zooming in on the high-res .EPS files, it appears to be because there is a diagonal rotation/crop on the images. Lines 193-212 referring to 'detailed analyses': The manuscript contains no direct quantitative analyses, but does present some high-quality electronmicrographs as figures. Line 200 & 211 convey that 'detailed analysis' has been conducted on the data to report these findings - but the findings are based on subjective observations. The Authors should either adopt quantitative image analysis to back up their claims or should completely redraft this section while refraining from the use of 'analysis(es)'. I do not disagree that, for example, Figure 6 shows marked changes in biofilm organisation under different fixation conditions. However, the observation is not quantitative or detailed. It is an objective interpretation of the image data, or a qualitative assessment. This is completely fine, but it should not be communicated as 'detailed analysis' unless the Authors modify the workflow to include quantitative computational or biochemical analysis of EPS levels. As above, statements such as in Line 204, '…considerably more biofilm matrix.' should be revoked unless measured. Ensuring this is truly the optimal method: Chemical fixation is also an imperative stage of some optical microscopy methods, particularly in SMLM imaging applications like dSTORM. Optical microscopists have developed many universal and sample-specific fixation protocols based on, as is here, aldehyde (with or without PFA) fixation or alcohol dehydration. One notable finding from these studies has been that the pH and concentration of fixative components (particularly PFA) can have a drastic effect on specimen integrity. The Authors have not presented any data from titration experiments that show the integrity of the biofilm following fixation using different concentrations of individual fixatives. Moreover, the Authors do not provide any indication as to why 2% PFA was selected, versus 1%, 3%, 4%, or even 0.5% which are all common for microbiological specimen preparation. How can the Authors be sure that this method is indeed optimal when they have not determined if there is a concentration-dependent relationship or dependency on the pH of the fixative? Minor points: Line 66 - 'Gram' should be capitalised in 'gram-positive'. Line 66-67 - The Authors suggest that references 6 & 18 only provide methods for Gram-positives, however, ref 18 also presents methods for Mannheimia haemolytica, a Gram-negative. Line 67/426 - A. Mukherjee is not listed as an author on this citation, please update the reference (https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0233973&type=printable). Line 80 - To avoid confusion, the Authors should consider changing the shorthand of 'S. wiggsiae and S. mutans' to 'Sc. wiggsiae and St. mutans' or 'Scar. wiggsiae and Strep. mutans'. Lines 126-129 - The authors provide a succinct summary of the structure of AB and RR. The inclusion of the chemical structures as a figure panel in Figure 2 may add non-specialist readers. Line 129 - please check tenses throughout. 'hypothesize' should be past tense here. Line 132 - A comma is required between 'that' and 'although' to complete the parenthesis. Line 138 - 'aclar' should be in all capitals with a '®' symbol afterwards. Line 155-159 - The previous figure had RR in panel A and AB in panel B. Could the Authors please change the order of all figure panels to be consistent with RR and AB, since that is the key change in many figures? If so, please update the relevant figure notations throughout the manuscript. Line 198 - Typo; the word 'formation' is missing from '…increased capsule <formation> may be an impediment…'. Line 235 - Have the Authors explored a 'best of both worlds' approach by testing a combined method with RR and AB? Methods Section - as this is a methods paper, I request that the Authors provide all reagent supplier details/CAT numbers to aid readers in replicating the method as closely as possible. Can the Authors please clarify that they are using EM-grade glutaraldehyde, as I understand this has a substantial effect specifically on fixation for EM-quality specimens. Line 292 - the Authors mentioned that they quantified the polysaccharide purified for their imaging experiments, would the Authors consider including this as Supplementary Info? Line 294 - Typo; 'aclar' should be in all capitals with a '®' symbol afterwards. Lines 299, 301, 302 - 'tetroxide' should be included after 'osmium'. Lines 303 & 306 - 'Osmium' should be lowercase, in keeping with the rest of the Methods section. Line 316 - Typo; 'Queens's' should be 'Queen's'. Line 317 - Typo; there were no hypervirulent strains used, I believe this should be 'hypermucovisous'. Table 1 - Can the Authors please provide the concentrations (%) for the graded alcohols and HMDS referenced in the table?
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Good
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?
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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