The Enterococcus secretome inhibits the growth of Mycobacterium tuberculosis complex mycobacteria
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A corrigendum of this article has been published full details can be found at https://doi.org/10.1099/acmi.0.000843
Enterococcus mundtii , a commensal intestinal bacterium, was demonstrated to inhibit the growth of some Mycobacterium tuberculosis complex (MTC) species that cause tuberculosis in humans and mammals. To further explore this preliminary observation, we cross-investigated five E. mundtii strains and seven MTC strains representative of four MTC species using a standardized quantitative agar well diffusion assay. All five E. mundtii strains, calibrated at 10 MacFarland, inhibited the growth of all M. tuberculosis strains with various susceptibility profiles, but no inhibition was observed with lower inoculums. Further, eight E. mundtii freeze-dried cell-free culture supernatants (CFCS) inhibited the growth of M. tuberculosis , Mycobacterium africanum, Mycobacterium bovis and Mycobacterium canettii , the most susceptible MTC species (inhibition diameter 25±1 mm), proportionally to CFCS protein concentrations. The data reported here indicate that the E. mundtii secretome inhibited growth of all MTC species of medical interest, which broadens previously reported data. In the gut, the E. mundtii secretome may modulate the expression of tuberculosis, exhibiting an anti-tuberculosis effect, with some protective roles in human and animal health.
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The work presented is clear and the arguments well formed.
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The work presented is clear and the arguments well formed. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
The manuscript by Achache et al describes experiments which tested the secretome of Enterococci on Mycobacterium tuberculosis complex mycobacteria. The methodology involved culture supernatants in agar diffusion assays and measurement of the zones of clearance. I was excited to read this manuscript as the topic is interesting and the research questions timely and important. However, I encourage the authors to develop their manuscript for clarity and robustness of data. Major Concerns: It is not clear to me that the experiments reported have been replicated as would be the standard in biology and a requirement for good statistics. Have these experiments been performed multiple independent times? Could the authors provide details? If replicates have been performed, could the data for these replicates …
Comments to Author
The manuscript by Achache et al describes experiments which tested the secretome of Enterococci on Mycobacterium tuberculosis complex mycobacteria. The methodology involved culture supernatants in agar diffusion assays and measurement of the zones of clearance. I was excited to read this manuscript as the topic is interesting and the research questions timely and important. However, I encourage the authors to develop their manuscript for clarity and robustness of data. Major Concerns: It is not clear to me that the experiments reported have been replicated as would be the standard in biology and a requirement for good statistics. Have these experiments been performed multiple independent times? Could the authors provide details? If replicates have been performed, could the data for these replicates be included. Overall, I found the manuscript confusing to read. Perhaps treating the Enterococcus and Mycobacterium separately and giving them separate methods sections might help? Also see comments below on Figure one. Many of the diffusion assays presented show zones of clearance that breach the agar plate and I don't believe these can be used to measure inhibition zones reliably. These agar plate might inform the authors, but the formal analysis should only relate to complete zones of clearance. Statistics: Can the authors confirm the number of independent replicates? Can the authors discuss their statistical approach? It is not clear to me what value a one-way ANOVA brings to this story, without multiple comparisons within the ANOVA. Further comments: Line 87 all strains were identified … if they authors wish to include this statement, can they also include the data or reference if this has already been published. Suggestion: the extensive list of strains might be better placed in a table than text. This might aid readability. Line 126 the abbreviation DPBS is explained here but has already been cited at an earlier line without explanation. Please define abbreviation at the first use. Line 137 could the authors please clarify why cell free culture supernatant was pH adjusted? Line 141 could the authors please indicate why 1/5 dilution was chosen? I find the descriptions of CFU/ mL and/or MacFarland standars equivalants difficult to follow when their use is interchangeable in the text. Could the authors please use both or just CFU / mL alone. Line 173 - 177 I find this sentence confusing. The texts describes a similar inhibitory effect of CSURQ1712 and CSURP7988 but cites p values of 0.1 and 0.2. These p values do not indicate significant differences. Could the authors please correct this. I suspect there is either a typo here, or the p value doesn't relate to the inhibition zones shown. This section required clarification. Line 187 The protein values cited in the text should have standard deviations. Multiple mentions of standardisation of the well diffusion assay to the 1/5 dilution. The methods would benefit from detailing exactly what this standardisation is and how its performed. Line 218 "negativity of negative controls" Rephrasing this to "absence of zones of clearance around the negative controls" or similar scientific language is recommended. Line 219 The inhibitory effect was proportional to the inoculum… but this contradicts line X which states an effect was only observed at Y. I understood the statement in line X to indicate the effect was an on/off type effect. Please clarify these statements for accuracy. Line 231 "at large" suggestion to remove "at large" Line 257 is "validation of manuscript" correct here? It is surprising that the protein concentration differs so little between the 1/8 dilution and the 1/10 dilution, however the zone of clearance effect is completely lost. Could the authors speculate or discuss this in the discussion? Figure 1 I commend the authors for including a schematic figure describing their methodology and I would strongly encourage the authors to improve this figure. I have the following suggestions: 1. remove all shadows from boxes and pictures 2. revise colour scheme from a red/green to a colour-blind friendly pallet 3. slightly reduce the size of the agar plates at the bottom of the figure 4. use the exact same agar plate diagram and size for each plate - change the colour to indicate media and insert circles to indicate well. 5. Make all test tubes the same size and if you must have them at an angle, use the exact same angle for each test tube. 6. Make arrows uniform in size and colour 7. Make text uniform - don't present text in a coloured box or circle and remove coloured boxes. 8. The figure required further explanation in the legend - what are 1, 2 and 3 in the schematic referring to? Figure 2 1. Perhaps the deviation bars or data points could be slightly different colours or thicknesses to aid visualisation? Currently, it is difficult to see individual data points that sit on the error bars. Perhaps grey error bars would help? 2. Remove the shadow from behind the agar plate picture. Also, the blue box outline is unnecessary and could be removed. 3. Have the ANOVA tests been performed to test significance of individual strain combinations? Could the authors explain the logic of performing an ANOVA on this group of strains (S10, S14, S15, S16) without making individual comparisons. This can easily be done in Graphpad and would add value to these data. For example, I suspect S15 treated individually might be significantly different from S14 (if only slightly and probably not meaningfully) in the CSURQ1712 graph. 4. Each graph should be it's own panel and therefore have an identifying letter. E.g. Figure 2 B-F. 5. Line 426 the first description of this strain being obtained from human stool should not be in the figure legend. Please remove this detail from the legend and document this in the methods. Figure 3 1. It is not advisable to have figure 3, panel a sub-panel a,… please relabel these sub panels (i, ii… etc) or make each plate it's own panel. 2. Panel A (a) here an asymmetric diffusion pattern is shown. Ideally, this assay should be performed with either an agar plate large enough or a well small enough to allow a symmetric and entire diffusion pattern occur. Could the authors please explain why this hasn't been possible? I believe the 1/8 dilution shown in (c) is sufficient to measure the zone of clearance, but the authors have indicated they used the 1/5 which also doesn't show a full diffusion pattern. 3. B I believe this graph would benefit from larger data points and perhaps a grey error bar. 4. I have concerns about how the data for dilution 1/2 and 1/5 were recorded in light of the images presented in this figure. It appears only the 1/5 dilution has error bars, but I would expect error in these types of data. Please clarify. 5. C The legend indicates a 1/10 dilution that is not present on the graph. It is difficult to see which bars correspond to the patterns shown in the legend. Perhaps increasing the bar size would help. Are there again no errors bars from these data? If not, please explain. 6. Please remove the blue boxes surrounding the figure. Figure 4 Remove all shadows from boxes The panel labels (A, B…) are not aligned and make the figure difficult to interpret. These panel labels could also benefit from being bigger. I would again strongly advise the red/green colour scheme and encourage a colour-blind friendly scheme. The legend for the red and green colours is poorly placed and closer to the strain names than the colour label. Figure 5 This figure is missing panel labels, while the wells seem to be labelled C and D. I have concerns about how this diffusion was measured given that the agar plate is too small to measure the full zone of clearance. Table 1 Each cell showing a measurement in the table should be the same size and appear uniformly in this table.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
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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Comments to Author
This short paper describes inhibition of growth of Mycobacteria with cell free supernate (CFS)of Enterococcus species. The methodology to demonstrate inhibition of growth of Mycobacterial strains is simple but effective. Growth of Mycobacteria is inhibited in the presence of CFS but not when sterile culture broth i is used. The authors suggest that the Enterococcus strains are producing a bacteriocin which is inhibiting growth of Mycobacteria. However the factor that is responsible for the inhibition is not characterised at all. Some simple experiments such as testing the effect of heat treatment of the CFS and/or protease treatment of the CFS would go a long way to strengthening this manuscript. Some further controls could be also be included. Are these effects specific for Mycobacteria or do you …
Comments to Author
This short paper describes inhibition of growth of Mycobacteria with cell free supernate (CFS)of Enterococcus species. The methodology to demonstrate inhibition of growth of Mycobacterial strains is simple but effective. Growth of Mycobacteria is inhibited in the presence of CFS but not when sterile culture broth i is used. The authors suggest that the Enterococcus strains are producing a bacteriocin which is inhibiting growth of Mycobacteria. However the factor that is responsible for the inhibition is not characterised at all. Some simple experiments such as testing the effect of heat treatment of the CFS and/or protease treatment of the CFS would go a long way to strengthening this manuscript. Some further controls could be also be included. Are these effects specific for Mycobacteria or do you see similar effects with other bacteria? Presentation of the results could be improved in places. Labelling of some of the figures needs attention. Figure 2 - it is confusing to have individual figure parts labelled A and B and then parts of the figure A labelled A and B. Please use different letters here. Figure 3 M and S are explained in the figure legend but no M and S is on parts a, b or c of part A Figure 5 No A and B labels on the figure but A and B referred to in the legend. For the diffusion assay label wells M and S for consistency. Line 183 and throughout - replace the word fractions with dilutions The conclusion statement needs to be modified. Rather than the work showing that Enterococci are likely to be key players in the control of Mycobacteria in the gut a more realistic conclusion might be that the results of this study suggest that further investigations are warranted to see if enterococci play any role in vivo in the control of Mycobacteria in the gut.
Please rate the manuscript for methodological rigour
Very 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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