Metabolic perturbations and key pathways associated with the bacteriostatic activity of Clitoria ternatea flower anthocyanin fraction against Escherichia coli
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Clitoria ternatea flowers are rich in anthocyanins and possess various biological activities. Specifically, the antibacterial mechanism of action of C. ternatea anthocyanins remains unknown and was investigated in Escherichia coli. Time-kill assay was used to assess the antibacterial activity and the metabolic perturbations in E. coli were investigated utilising LC-MS-based metabolomics. Pathway analyses were carried out for metabolites showing ≥ 2-fold changes. The anthocyanin fraction remarkably reduced the growth of E. coli at 4 h by 95.8 and 99.9% at minimum inhibitory concentration (MIC) and 2 × MIC, respectively. The antibacterial activity of the anthocyanin fraction (MIC) was shown to have perturbed glycerophospholipids (1-Acyl-sn-glycero-3-phosphoethanolamine, phosphatidylglycerol, diacylglycerol and cardiolipin), amino acids (valine, tyrosine and isoleucine) and energy (ubiquinone and NAD) metabolites at 1 and 4 h. This study demonstrated significant metabolic perturbations of the glycerophospholipid, amino acid and energy metabolism being the key pathways involved in the antibacterial activity of anthocyanins from C. ternatea and has promising potential as antibacterial agents for E. coli-related infections.
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Thank you very much for your efforts in implementing all the suggestions and corrections to improve your manuscript. This work is now suitable for publication. Congratulations!
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I would like to thank the authors for their efforts in applying the comments and suggestions in their manuscript. However, there are still some minor points to amend. Please find them below: ·L393-394: “Amino acids in the form of enzymes” reads as if amino acids had catalytic activity themselves. Please rephrase to properly convey that enzymes are proteins, and thus are formed by amino acids ·L394-396: The glycolytic pathway, the TCA cycle and the pentose phosphate pathway are oxidative pathways to obtain energy, reduced coenzymes and metabolic precursors that are streamed to other metabolic branches, such as amino acid anabolic reactions (as illustrated in Figure 4A). However, this reads as if those oxidative pathways were used for making amino acids. Please rephrase this. ·L467: I would really like to commend the authors for availing …
I would like to thank the authors for their efforts in applying the comments and suggestions in their manuscript. However, there are still some minor points to amend. Please find them below: ·L393-394: “Amino acids in the form of enzymes” reads as if amino acids had catalytic activity themselves. Please rephrase to properly convey that enzymes are proteins, and thus are formed by amino acids ·L394-396: The glycolytic pathway, the TCA cycle and the pentose phosphate pathway are oxidative pathways to obtain energy, reduced coenzymes and metabolic precursors that are streamed to other metabolic branches, such as amino acid anabolic reactions (as illustrated in Figure 4A). However, this reads as if those oxidative pathways were used for making amino acids. Please rephrase this. ·L467: I would really like to commend the authors for availing their data to the public. As an Open Research Platform, we really value these efforts for transparency. However, some more explanation on the content that is shared would be needed in this “Data summary” section. Additionally, it would be ideal if the content that is shared had any sort of legend that allowed the reader to precisely know what they will find in the different spreadsheets. If this modification cannot be made directly on the shared document, please add the explanation related to the different data sheets in this section as well.
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Dear Authors, Thank you very much for your efforts in implementing the reviewers’ suggestions and comments in your revised manuscript. It has undoubtedly gained in quality and methodological soundness. However, there are still some points that need amendment. Please address the following: • Although it was suggested by Reviewer 1 and fixed to some extent, a clean-cut definition of the difference between bactericidal and bacteriostatic activity, which happens to be important for the interpretation of the results, is not entirely achieved. Please provide it supported with the appropriate references. • In line with the previous comment, as the conclusion is that the flower fraction of study is specifically bactericidal, I strongly recommend to consider a change of the title including this, rather than the generic “antibacterial …
Dear Authors, Thank you very much for your efforts in implementing the reviewers’ suggestions and comments in your revised manuscript. It has undoubtedly gained in quality and methodological soundness. However, there are still some points that need amendment. Please address the following: • Although it was suggested by Reviewer 1 and fixed to some extent, a clean-cut definition of the difference between bactericidal and bacteriostatic activity, which happens to be important for the interpretation of the results, is not entirely achieved. Please provide it supported with the appropriate references. • In line with the previous comment, as the conclusion is that the flower fraction of study is specifically bactericidal, I strongly recommend to consider a change of the title including this, rather than the generic “antibacterial activity” • In comment 19 from Reviewer 1, there is a discussion on the toxicity of the anthocyanin fraction on eukaryotic cells and rats. However, this is not included in the manuscript. I strongly suggest introducing this in the manuscript where appropriate, as it will contribute to enrich the discussion. • Remove brackets from E. coli strain name (e.g. L161, L220, L279) • L230: use “respectively” to match each CFU count to each MIC-based concentration • L207, L208, L215: include citations for the different databases • Statistical analysis (L154-158): state which statistical test is applied to which dataset, as well as the significance threshold (p-value), for example in the respective figure legends • L381: “Amino acids being the basic building blocks of proteins, are also biomolecules that are essential in the biological functions of microorganisms…” Please correct the grammar in this sentence • In the interest of transparency and reproducibility, consider making the whole metabolomics dataset publicly available, either as supplementary material or uploading to the appropriate database and citing it in the manuscript. Please refer to the Microbiology Society Open Data Policy for more information (https://www.microbiologyresearch.org/open-data) • An exhaustive revision of the references is needed, especially with respect to appropriateness and format. Examples of this are: o L309-314: reference 25 is used for explaining the differences between OM and IM. However, this is explained in the introduction of reference 25, it is not a result of that work. Please use the original refences when possible and appropriate throughout the manuscript. o L314-317: reference 26 has to do with the trafficking of phospholipids and CL in mitochondria, not in bacteria. Please refer to a more content-appropriate article to support this information. o L390-394: The TCA cycle is not directly involved in the oxidation of lipids, carbohydrates and amino acids. It is involved in the oxidations of different metabolic intermediates that derive from the catabolism of those via multiple branches of the metabolism (e.g. pyruvate, alpha-ketoglutarate). Please correct and be as precise as possible in general statements throughout the manuscript. Furthermore, reference 34, used for supporting this, refers to the TCA cycle in skeletal muscle, whilst there is plenty of information directly related to the TCA cycle in bacteria. o L394-395: reference 35 is unrelated to the TCA and/or bacterial respiration, and thus to this statement o L414: reference “Sun”
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In this manuscript, the authors describe the consequences at the metabolic level of a flowers extract on E. coli, discussion a possible mechanism of action for its antimicrobial activity. In the current global context, the description of possible new antimicrobials represents a meaningful contribution to multiple fields. The manuscript has been reviewed by two experts in the field and their reviews are enclosed. Both reviewers have a number of major issues that need to be addressed. Most importantly, for the sake of clarity, the methodology section needs substantial improvement. Furthermore, the consistency with previous literature, the coherence between different section of the manuscript and the clarity of a number of concepts used in this work need improvement. Please consider the reviewers’ comments thoroughly and address their …
In this manuscript, the authors describe the consequences at the metabolic level of a flowers extract on E. coli, discussion a possible mechanism of action for its antimicrobial activity. In the current global context, the description of possible new antimicrobials represents a meaningful contribution to multiple fields. The manuscript has been reviewed by two experts in the field and their reviews are enclosed. Both reviewers have a number of major issues that need to be addressed. Most importantly, for the sake of clarity, the methodology section needs substantial improvement. Furthermore, the consistency with previous literature, the coherence between different section of the manuscript and the clarity of a number of concepts used in this work need improvement. Please consider the reviewers’ comments thoroughly and address their concerns point by point in a separate document. A revised manuscript should include appropriate revisions.
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Comments to Author
The work has some value and significance, although the advantages and disadvantages should be considered: Advantages: the work investigates mechanism of action of anthocyanins fraction against E. coli - it is new Disadvantages: The phytochemical analysis of the obtained fraction was not presented, hence indeed we don't know what were the active compounds used in the experiment. The methodology lacks many details, and in general is not precise, strongly needs clarification, also in the section related to bacterial metabolites identification. In the discussion: it is suggested to rethink the presented observations in regard to the study "Cell Metab. 2019 Aug 6; 30(2): 251-259. doi: 10.1016/j.cmet.2019.06.009: 10.1016/j.cmet.2019.06.009". The authors of the manuscript under evaluation suggest that …
Comments to Author
The work has some value and significance, although the advantages and disadvantages should be considered: Advantages: the work investigates mechanism of action of anthocyanins fraction against E. coli - it is new Disadvantages: The phytochemical analysis of the obtained fraction was not presented, hence indeed we don't know what were the active compounds used in the experiment. The methodology lacks many details, and in general is not precise, strongly needs clarification, also in the section related to bacterial metabolites identification. In the discussion: it is suggested to rethink the presented observations in regard to the study "Cell Metab. 2019 Aug 6; 30(2): 251-259. doi: 10.1016/j.cmet.2019.06.009: 10.1016/j.cmet.2019.06.009". The authors of the manuscript under evaluation suggest that decreased energy production results in bacterial cell death, however in the earlier paragraphs they stated that observed effect of anthocynanins was bacteriostatic (lines 225-226) . It is contradictory. Also, if you analyse the suggested work, you will find the information that decreased metabolic activity in bacteria is strictly related to bacteriostatic effect of antibiotics. Hence it is mandatory to re-evaluate observations to make correct conclusions. Other remarks: Please make it clear: was the extraction yield 50%? The 10 g of plant material was used to prepare extract , than 5g of the extract was used for semi-purification. Was it possible to obtain 5g of the extract from 10g of plant material? Line 107: what was used for pH adjustment? Line 112: what was the size (weight) of the amberlite resin? Line 115: was water used to dissolve sample? Line 117: how the flow rate was controlled? What was the final yield of the purified anthocyanins fraction? Line 128: What was the volume of cultures treated with anthocyanins? It is not sated anywhere what solvent was used to dissolve the frozen anthocyanins for the antimicrobial experiments. Could you please clarify the volume of the experimental culture: line 159 states 50 ml, while line 167 states 20ml. Line 170: what was used to normalize cultures? Line 179: was the residue after centrifugation removed? Lines 181-185: the parameters of ion source and detector should be given Line 193: authors reported that retention time was used for identification, so were authentic standards used?, because in databases retention time is rather not reported. It also would be good to specify the allowed mass difference which was applied during identification (delta ppm). Can you please specify how the infernal standard was used in the calculations? Line 219: it is hard to believe that you can dissolve 100mg of plant extract in 1 ml! Rather suspension can be obtained. Line 220-223: Can you support this statement with reference? The other authors report also ternatin A1, A2, A3, B1, B2, B3, B4, preternatin A3 (https://www3.e-kenkyu.com/bpb-reports-online-journal/uploads/manuscript/file/137/4_136.pdf). Lines 241-251: it is not clear why the discussion about the structure of compounds present in used fraction is placed in the section "Metabolic profile of E. coli (ATCC 25922) treated with C. ternatea anthocyanin fraction" Lines: 394-395: this statement about glycoside residues is not supported by any proof in the evaluated work, firstly, the composition of the used fraction was not determined, secondly, the glucose moiety can be substituted by p-coumaroyl or malonyl residues.
Please rate the manuscript for methodological rigour
Poor
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?
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
1. Methodological rigour, reproducibility and availability of underlying data Good but the MIC section and the effect on growth need to explored in greater detail. 2. Presentation of results Good, particularly for the metabolomics although the data does not appear to have been uploaded to a metabolomics database which would support transparency. 3. How the style and organization of the paper communicates and represents key findings Good 4. Literature analysis or discussion Quite a few sections need to be more appropriately referenced as highlighted above. 5. Any other relevant comments In this manuscript, the authors present findings of the anthocyanin fractions from Clitoria ternatea flowers as having a bacteriostatic effect on E. coli. Through metabolome analysis they show the involvement of …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data Good but the MIC section and the effect on growth need to explored in greater detail. 2. Presentation of results Good, particularly for the metabolomics although the data does not appear to have been uploaded to a metabolomics database which would support transparency. 3. How the style and organization of the paper communicates and represents key findings Good 4. Literature analysis or discussion Quite a few sections need to be more appropriately referenced as highlighted above. 5. Any other relevant comments In this manuscript, the authors present findings of the anthocyanin fractions from Clitoria ternatea flowers as having a bacteriostatic effect on E. coli. Through metabolome analysis they show the involvement of disrupted membrane integrity, perturbed glycerophospholipid and energy metabolism as the key pathways involved in bacteriostatic effects of the plant. The manuscript is concise and well written, and congratulations to the authors for these exciting and interesting findings. While it is clear that exposure to the anthocyanin fractions has an impact of E. coli, the precise nature of this impact needs some additional clarity and the focus of the findings of the metabolomic data needs to be sharpened. Important Revisions - The terms bacteriostatic and antibacterial are both used to describe the effects of C. ternatea throughout the paper. The authors should clarify the distinction between the two terms and select. - It needs to be made clear in the main text to the reader if the strain of E. coli is being tested is of clinical origin or is multidrug resistant. If the strain is not of clinical origin or MDR, do the authors think the extract will be effective against these more recalcitrant strains. - The authors need to explore the true nature of the effect of this fraction in more detail. The MIC was previously determined as 10mg/ml, however from Figure 1 it is quite clear that at 24 hours, the E. coli have overcome the inhibitory effect of the fraction and have recovered to the same CFU as the negative control. The other issue with this figure is that at 2xMIC there is a clear killing effect as stated in the text the CFU drops from 7.4 x 10^5 to 2 x10^2. This suggests that the fraction may be bactericidal. The other issue with this figure is that in all conditions it appears that there is an increase in bacterial growth from 0-1 hour, how can this be explained given the inhibitory nature of the fraction, you would expect the effect to be immediate. Given this is a fraction however, rather than a pure compound there could be synergistic and antagonistic interactions taking place at once. This section would benefit from a growth curve analysis with readings every 15 minutes. - The authors base a significant component on the MIC that was previously determined in Jeyaraj et al.,2022. However this MIC was determined in an agar dilution method, and as much of this study is based in broth cultures, it would be very beneficial to conduct an MIC assay in liquid broth. The benefit of this also is that there may be regional or temporal impacts on anthocyanin concentration that would lead to differences in the MIC from batch to batch. Findings should also be put in context with Uma et al., 2009 where the crude flower extract had an MIC of between .25-10 mg/mL. - For the time kill assays - what was the anthocyanin fraction resuspended in? o Is a vehicle control needed for the assay? - The authors need to state their definition of MIC within the text. - Is it known that the concentrations of anthocyanins are the same in each flower? (Dependant on the size/ stage of the flower) - Does the geographical location of flower impact the concentration of anthocyanins present? - The authors should provide a brief description of the agar dilution method used in this paper, as well as citing their previous paper, to make it easier for the reader. - Were the treated samples and untreated controls used for metabolomic analysis subjected to the same processes/vehicle as the treatment? - The text states (line 162) that the MIC is used for metabolome analysis - if the bacteria does not grow (bacteriostatic) at this concentration then is there likely to be pleiotropic effects of the bacteria being in different growth phases, particularly at the 4 hour timepoint. ? - Line 300 the authors state that there is a greater perturbation in glycerophospholipid metabolites at 1 hour compared to 4 hours. However, based on figure1 the bacteria has continued to grow at the 1 hour time point (although it is not clear if this increase in growth is statistically significant), how does this fit with the glycerophospholipid hypothesis? - The study would be greatly strengthened by some validation of the impact on membrane integrity using perhaps live cell imaging or a kit such as LIVE/DEAD BacLight Bacterial viability kit. - Line 354 how does this higher reduction at 4 hours as compared to 1 hour compared with the previous section on the impact on glycerophospholipids? - Authors should show the ubiquinol-6 and -8 at 1 hr to give a full picture of the results. - Greater weight should be added to the effect on the TCA cycle as this is strengthened by the previous work which the authors have cited by Sun et al., 2018. - If standards of Ternatins are available to purchase, authors should consider this as a control for their assays. - For the bacterial culture preparation for metabolome analysis - why was a different agar (nutrient agar) used to grow the colonies than in the time kill assay? - The authors state in line 402-404 that the anthocyanin fraction could be used to reduce E. coli related infections however this does not align with previous work published by the authors which states that the anthrocyanin rich extract displays toxicity at doses above 156.3 µg/mL against HEK-293 cells (Jeyaraj et al.,2022 , Figure 3), which is much lower the MIC described in this work. Greater context needs to added here. - Minor points - Refences needed for the following: o Line 63, second sentence o Line 71, second sentence o Line 246, second sentence o Line 248, second sentence o Line 308 o Line 251, reference does not support the statement. Needs to be rewritten to fit the reference or a new reference is needed. Reference paper states ''It was demonstrated that the sugars do not contribute to the binding affinity or the DNA cleavage selectivity, although the presence of the sugar enhances the DNA cleavage efficiency by 2-5 times'' - Lines 223-225, Findings from Gram positive bacteria are compared to Gram negative are not representative and thus cannot support the findings and perhaps more relevant examples could be used. - Figure 1, Line 237 - Are the replicates technical or biological?
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?
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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