All reviews of published articles are made public. This includes manuscript files, peer review comments, author rebuttals and revised materials. Note: This was optional for articles submitted before 13 February 2023.
Peer reviewers are encouraged (but not required) to provide their names to the authors when submitting their peer review. If they agree to provide their name, then their personal profile page will reflect a public acknowledgment that they performed a review (even if the article is rejected). If the article is accepted, then reviewers who provided their name will be associated with the article itself.
Well done on an improved manuscript which impressed the reviewers.
[# PeerJ Staff Note - this decision was reviewed and approved by Valeria Souza, a PeerJ Section Editor covering this Section #]
I feel that the overall quality of the manuscript is much improved. The manuscript feels much more focused, is less confusing and the limitations of the study are clearly described.
Methods now contain all relevant details for reproducibility. The authors have also clearly justified and explained their selection of using leukocytes as an overall measure for the degradation of blood over time.
Figures are improved and easier to interpret. Limitations of findings have been discussed.The work provides an important contribution for those wishing to develop ex vivo blood models for gene analysis and highlights some of the important experimental factors which may impact the variability of such studies.
I commend the authors on a much improved manuscript
The three reviewers have, in places, each commented on quite differing aspects of the manuscript, and so this will require major revision.
A good background is provided in this manuscript.
Hypotheses are clear and well explained. Results address the hypotheses and the article structure is well presented.
Literature references are comprehensive. One exception is on Line 138, where Turkey’s comparisons requires referencing.
The text needs some minor modifications particularly regarding English use:
Abstract
Consider replacing: ‘set-up conditions’ with ‘variables’.
In the second sentence limit the use of ‘however’ to once. (Lines 16-17)
Line 21: Plural of inoculum is inocula. Inoculums (used throughout text) is rarely used in English. Personally, I would consider replacing this with, ‘Higher bacterial concentrations produced a more significant loss of antimicrobial activity.’
Introduction
Line 32: Consider replacing originate with cause.
Line 33: Consider replacing urgent with important.
Line 34: Consider replacing endure with survive.
Line 37: Replace, ‘This model have’ with ‘This model has’
Line 40: Replace ‘faecallis’ with ‘faecalis’
Line 44-45: Consider replacing: ‘However, often, the implementation of these models lacks optimization’ with ‘However, the implementation of these models often lack optimization’
Line 52: Consider replacing: ‘Besides that’ with ‘Additionally’
Materials and Methods
Line 76-77: Consider replacing, ‘Negative control was accomplished’ with ‘Negative controls were achieved’.
Line 78-79: This sentence does not describe determination of bacterial concentration. Were the aliquots inoculated onto solid agar plates? Was this performed using replicates?
Line 90: Separate overtime to over time.
Line 94: Consider replacing ‘during 10 minutes’ with ‘for 10 minutes’.
Line 103: Replace ‘mixed, on’ with ‘mixed in’.
Line 107: Exclude comma after rpm
Line 111: Consider replacing ‘experimental set-ups’ with ‘experimental variables’.
Line 134: Replace ‘Reactions efficiency’ with ‘reaction efficiencies’.
Line 138: ?two-away ANOVA or two way ANOVA
Line 153: Consider replacing, ‘Some bacterial death’ with a more concise description.
Line 160: Consider replacing ‘rest of’ with ‘remaining’.
Line 171: Consider replacing ‘inoculums’ with ‘concentrations’.
Line 174: Consider replacing ‘the is inherent to particular traits’ to ‘the inherent traits’.
Line 173-174: The author explains that the source of donor blood (gender and age) may contribute to variability of experimental results. Genders of the donors are revealed in Raw data for Figure 1 but not the age. I noted marked variation in total leukocyte counts in donor A compared to the donors J and N at T0 and 8 hours. Perhaps adding age range could add to the date set since age was initially presented as a variable by the author.
Line 184: Exclude comma after survival.
Line 186: Exclude ‘it’s’ at the end of the line.
Line 189-190: Consider replacing ’this percentage was slightly’ with ‘cell death rate was slightly’.
Line 197-198: Consider replacing ‘’this is an extensive period of incubation could yield’ with ‘ 12 to 24 hours is an extensive period of incubation which may yield’.
Line 202: Consider replacing ‘An easy…………the blood under gentle agitation’ with ‘simple way to ……the blood using gentle agitation’.
Line 205: Replace ‘leukocytes’ with ‘leukocyte’
Line 214: Consider rewording ‘very long periods’.
Line 215: Consider replacing ‘4h of interval’ with ‘an interval of 4h’.
Conclusion
Line 239: Replace ‘evasion from’ to ‘evasion of’.
Line 240-241: Please consider replacing ‘help to plan a better and more reliable model’ with ‘contribute to standardization of experimental conditions and produce a more reliable model’.
Line 241-242: Consider replacing ‘These findings are’ with ‘The findings from the study’
Figures 1, 2 and 3: Turkey’s multiple comparison test – Turkey has been spelled ‘Turkey’ and ‘Tukey’.
No comment
No comment
I commend the authors for their experimental work and manuscript.
Most suggestions are related to use of the English language and will ensure that an international audience can clearly understand the text.
I look forward to seeing the publication of this article.
The authors clearly state the primary aim of the study, which is to identify the key parameters which could affect experimental outcomes using an ex vivo blood model for investigating s. epidermidis transcriptional responses. The authors rightly state that variation in experimental design between laboratories can result in varied results and understanding the parameters which have the greatest effect on experimental outcome could help guide future experiments and lead to standardised methods.
Although the manuscript is mostly well written, there are a number of grammatical errors and it would benefit from proof reading by a native speaker.
I feel the term “blood viability” needs to be clearly defined in the introduction as this term is ambiguous. Does it relate to the number of viable red blood cells, white blood cells, all cells, complement activity?
I also feel some of the claims made in abstract are too ambitious. For example “the volume of human blood could be reduced to as little as 0.18 ml without affecting the stability of gene transcription”. This sounds as if all genes within the genome were analysed. As only a small set of genes (three in total) were analysed, perhaps this could be rephrased to “the volume of human blood could be reduced to as little as 0.18 ml without affecting the stability of transcription of genes under investigation in the current study” or similar.
Similarly, the statement “Our data demonstrated that a loss of 2% of blood viability after 4h of collection resulted in a 5-fold loss of antimicrobial activity against S. epidermidis” It has not been confirmed that this loss of antimicrobial activity is directly related to the small reduction in leukocyte viability seen as the levels of other antimicrobial components of the blood were not measured during this time period. For example, a loss in complement activity may actually be responsible for, or contribute to, the reduction of antimicrobial activity. Therefore, this should perhaps be rephrased as “Our data demonstrated that a loss of 2% of blood viability after 4h of collection correlates with a 5-fold loss of antimicrobial activity against S. epidermidis”
Please include the number of biological and technical replicates performed in the section relating to the influence of anticoagulants on bacterial growth.
Regarding the section on human blood collection, was the blood used immediately following collection for further experiments? If so, please state this. If not, please state how it was stored.
Experimental details are lacking in places. For example:
Lines 61-62, “The suspension was then washed once and resuspended in 0.9% NaCl in order to obtain an inoculum of 1 × 109 CFU/mL” how was this achieved? Was it adjusted to a specific optical density which correlated to a known cfu/ml? As this is a method development paper and a major parameter affecting experimental outcome was inoculum size, this step should be described in detail.
Lines 72-74, “TSB (Liofilchem Teramo, Italy) was added to the different blood collection tubes and were shaken for the same duration as the tubes containing the human blood”. How long was the duration? Please be specific.
Lines 78-79, “bacterial growth was determined by CFU quantification”. Which method was used? Spread plating, Miles and Misra?
Lines 103-104, “Whole blood was collected and an aliquot (0.9 mL) was immediately taken and mixed, on a 2 mL tube, with 105 CFU/mL of bacteria”. Do the authors mean that the final concentration of bacteria was 105 CFU/mL when mixed with the blood or was a volume of bacterial suspension already at 105 CFU/mL added to the blood? If the latter, how much was added?
The authors aim to determine factors which influence experimental outcomes in an ex vivo blood model. One such parameter identified was the inoculum concentration. The authors hypothesise that at higher bacterial concentrations, bacterial survival and subsequent growth, is due to a corresponding loss of leukocyte viability, which presumably is not happening at lower bacterial concentrations. To prove this, the authors should show a reduction in leukocyte viability which directly correlates to bacterial inoculum size. The authors do show a reduction in leukocyte viability which correlates to time from blood collection, but this is different.
Furthermore, it is not made clear why the authors chose to measure leukocyte viability over other potentially important antimicrobial components of blood which are also known to degrade with time. Although leukocytes are an important antimicrobial component of the innate immune response, I feel the discussion does not acknowledge other relevant antimicrobial components of blood, such as complement. As there will be a limited amount of complement available in the blood samples for binding to bacteria, its antimicrobial capacity will be directly influenced by the concentration of bacteria present. In my opinion, this will much more likely have a greater effect on the variability of experimental outcomes with regards to transcription than leukocyte viability, which is unlikely to be altered by the presence of differing concentrations of S. epidermidis.
The authors aim to determine if the total volume of blood used in the model can alter gene transcription levels and therefore introduce variability within results. The authors conclude that “no significant differences were found in the expression of the selected genes using, in any of the different volumes of human blood tested. Noteworthy, a volume of blood as low as 0.18 ml per reaction can be used without impacting the transcription of the selected genes”. This is an important preliminary result and is useful to the field, but I feel it should be stressed that this is a small set of genes and that this may not hold true for all genes within the genome.
Lines 209-212, “As can be seen in Figure 2, the results showed that a higher antimicrobial activity was detected when blood was used immediately after collection, resulting in 97% of bacterial cell death. In contrast, when blood was used after 4h of its collection, only 84% of bacterial death was detected”. I’m assuming that “immediately after collection” relates to 0 hours on figure 2? However, there is no bar for bacterial death at 0 hours – Is the figure correct?
Line 231, “AMPS produced by the host”. Could you please define the acronym – I’m assuming antimicrobial peptides?
Figure S1 (A) which reports bacterial growth in the presence of different anticoagulants has significance values reported above specific time points but there is no figure legend to explain what these represent or what samples have been compared. Is this versus the TSB control?
Although I feel the manuscript does contain findings which may be useful to the field, especially those wishing to undertake transcriptional analyses in blood, I feel the manuscript is not acceptable in its current form. Too many statements are overly ambitious without discussing possible alternative reasons for the results achieved and experimental limitations are not fully acknowledged.
The authors investigate variations in the parameters of ex vivo models for Staphylococcus epidermidis (SE) blood infection, and report that different collection tubes, SE concentration, and blood processing time all have an impact. I commend the authors for highlighting the importance of optimisation, and agree that these factors are often not reported in the literature. I do however, have some major concerns regarding the manuscript, which are outlined below.
There are some grammatical errors throughout the manuscript, which should be corrected to improve clarity. For example, on lines 16/17 the word “however” is used twice, only the second is needed. Line 24, the phrase “ex vivo gene expression” is confusing. Line 37 should read “This model has”. Line 103 should read “in a 2 mL”. Line 173-174 “due to the is inherent” is confusing. Removing some of the commas in lines 103/104 would make the text flow better. Line 166 “lower stimuli” is confusing.
In addition, the text is ambiguous at times. For example, on line 43 it isn’t clear if “the transcriptional response” refers to the transcriptional response of human blood, or that of SE. Again, line 156 “regarding gene expression” should instead specifically state “expression of SE genes” to make it explicitly clear what is being measured. Similarly, line 167 “analysis of the transcriptional response” is ambiguous.
In Figure 1, there are 4 lines in the top right corner of the graph with stars indicating significance, but it isn’t clear what these comparisons are. This needs addressing.
In Figure 2, it isn’t clear from the legend which results relate to blood alone or blood plus SE. Further, it isn’t clear in the legend that results from two separate viability assays are presented; flow cytometry for blood, and CFU enumeration for bacteria.
In general, all figure legends should contain all necessary information to interpret the Figure. In Figure 3, the legend doesn’t provide sufficient detail; CFU? Length of incubation?
There are significant details missing in terms of the methodology. Specifically, more detail is required in how CFU was determined. The methods describe “CFU counting” but was this done by spotting serial dilutions of bacterial preparations onto plates followed by overnight incubation? Or counting under a microscope? Since much of the results and interpretation is based on assessment of bacterial viability, this needs to be described in sufficient detail.
In addition, the authors state that they used blood from five donors, but no demographic information is presented for these individuals. At a bare minimum, age and gender should be reported.
Regarding the flow cytometry analyses of leukocyte viability, some representative flow plots should be shown alongside a gating strategy including the exclusion of doublet cells, debris etc.
Regarding the primers used for detection of SE genes, were the PCR products sequenced to confirm that the correct product was being amplified by the designed primers?
The rationale for the results presented in Figure 2 isn’t clear from the text. Initially the authors state that this experiment was performed in light of the results in Figure 1; lines 184/185 “it was hypothesized the increase of bacteria survival, in the higher inoculum experiment, was related to the loss of viability of blood cells”. After reading this, I expected to see results relating the highest inoculum to blood cell viability. Instead, the authors performed this experiment using only 105 CFU/mL. Figure 1 clearly shows that at an inoculum of 105 CFU/mL, blood could control infection; only ~25% of bacteria were alive at 4 hours, and even less were alive by 8 hours. Essentially, the authors hypothesis was not tested by the experiment presented in Figure 2. The authors later describe the importance of time between blood collection and utilisation and in that regard, the experiment for which results are presented in Figure 2 are justified.
In addition, there seems to be a discrepancy in the results between Figure 1 and 2. In Figure 1, a 4-hour incubation with 105 CFU/mL resulted in ~25% bacterial survival, which is equivalent to ~75% death. However, in Figure 2, these same culture conditions (4-hour, 105 CFU/mL) resulted in close to 100% bacterial cell death across three independent experiments. Can the authors clarify this?
Finally, can the authors justify their choice in using the highest inoculum (108 CFU/mL) for the experiment presented in Figure 3? Since the data in Figure 1 shows that at this concentration, SE can happily survive in blood culture up to 8 hours, I would expect gene expression to be stable at this concentration, regardless of blood volume.
I would suggest changing the title slightly to “Optimizing a reliable ex vivo human blood model to analyse expression of Staphylococcus epidermidis genes”.
The authors conclude that blood has lost its antimicrobial properties following a period between collection and utilization. Can the authors validate this claim by measuring total protein or specific antimicrobial peptides in the blood serum following this period of time?
All text and materials provided via this peer-review history page are made available under a Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.