In Vitro Bioactivity of Leaf Extract Fractions and Quercetin-3-O-Rhamnoside from Combretum elaeagnoides Against Staphylococcus Species Implicated in Causing Bovine Mastitis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study is interesting and could be relevant for veterinary practice, as it is well known that S. aureus easily developsantibiotic resistance and recurrent infection in cows are common. Therefore, there is an increased need for alternative solution. However, certain revisions are necessary for the manuscript to be acceptable.

The title of manuscript mentions Combretum elaeagnoides Klotzsch fraction, yet the name Klotzsch is not referenced elsewhere in the text. I would therefore request clarification of whether this denotes a specific strain of species or referes to something else.

The title also includes quarcetin-3-O-rhamnoside. For greater clarity, the authors should explicity state that quarcetin mentioned in the title is extracted from the leaves of the referenced plant species.

The title also refersto the use of fraction and extracts against mastitis-causing pathogens in cattle. However, the study evaluates inhibition only against staphylococci. Although staphylococci are among the primary etiological agents of mastitis, numerous other pathogens can also cause the disease. Therefore, it would be clearer if the title specificed that the activity was assessed against staphylococci as mastitis-causing bacteria.

Line 19: in vivo italic

Line 22: It woulf be helpful to clarify from which specific part of plant the component was isolated.

Line 22: The abbrevation S. aureus should be placed in parantheses, as this is the first time the species is mentioned.

Line 22-24: Throughout the manuscript, mastitis pathogens are mantioned, but experiments were performed solely with staphylococci. Clarifying this throughout the text would enhance accuracy and prevent potential misunderstanding. 

Line 29-30: In my opinion, this sentence is redundant in the abstract. It would be more appropriate to include it in the discussion section, with proper citation of the relevant authors.

Line 35: CeDCM and CeEtOAc - abbreviations should be used here, as they have already been introduced earlier.

Line 41-42: Abbreviations should also be used here, as they have already been introduced earlier.

Line 56: The abbreviation S. aureus should be placed in parentheses immediately after full name.

Line 62: It is unclear whether this paragraph refers to inflammationof the bovine udder. It would be helpful to describe the differences between subclinical and clinical mastitis in cows, especially since the experiment was conducted only with S. aureus isolates from milk of cows with clinical mastitis.

Line 76: Staphylococcus aureus possesses multiple mechanisms for developing antibiotic resistance. It would be valuable to also mention other mechanisms by which this bacterium protect itself, contributing to difficulty of treating bovine udder infections and the high incidence of recurrent infections.

Line 86: It would be interesting to highlight the traditional uses of this plant extract in African cultures, for example, for treating fever or diarrhea.

Line 93: Were these studies conducted using leaf extracts, and it would be interesting to specify which pathogens were tested.

Line 95: It should be clarified from which part of the plant guarcetin as the active compound was isolated.

Line 102: The abbreviations and use of parentheses are unclear.

Line 104: Is this deciduous tree

Line 105: Abbreviations should be used here, as they have already been introduced earlier in the manuscript.

Line 107: Please specify which microorganisms were tested and the number of strains used. Only Staphylococcus species were tested.

Table 1: The abbreviation MeOH should be explained, and its usage should be standardized throughout the manuscript, as it is inconsistently written in some places.

Line 139-142: It would be interesting to indicate in the text which strains were sensitive to which extracts, in order to more clearly observe whether certain strains are more susceptible to specific extracts.

Line 188: A space should be inserted between 28 and 29.

Line 190: Why was this measured only after 48 hours.

Line 193: It should be stated that gentamicin was used as the positive controle.

Line 212: It should be indicatedis that C. violaceum use as a model organism for anti-quorum experiments as they produce violacein pigment. Full name for C. violaceum.

Table 3. MeOH or MeoH, CEH or CEHx

Line 244: full name for LOX and NO

Section 2.4. It would be interesting to test these parameters on somatic cells from milk of cows with clinical mastitis, as was done with RAW macrophages.

Section 2.5. What is the rationale for using Vero cells in addition to bovine dermis cells? If this is only cited from a previous study, it might not need to be included as a separate subsection.

Line 283: in vivo - italic

Table 6: Explanations for the abbreviations are missing below the table.

Line 295: Which test was used to confirm the identification of S. aureus

Line 407: FOX - full name

Line 412: DSMO - full name

Line 514: In the conclusions, the authors should not be cited.

Are further studies planned on strains isolated from milk of cows with subclinical mastitis? Additionaly, it is unclear whether the tested strains originated from cows on the same farm or from different farms. 

It would be interesting to provide an explanation on how the extracts could be administered intramammarily to cows.

In my opinion, since the results and discussion are combined, the discussion should be expanded by comparing with the findings of other authors.

Additionally, the conclusion is too long and contains repetitions of the results. It should be shorter, clearer, and more concise.

 

 

 

 

Author Response

Comment 1: The title of manuscript mentions Combretum elaeagnoides Klotzsch fraction, yet the name Klotzsch is not referenced elsewhere in the text. I would therefore request clarification of whether this denotes a specific strain of species or referes to something else

Response 1: Thank you for this comment. In this context “Klotzsch” refers to the botanical authority for the plant species as recommended by the International Code of Nomenclature for plants, referencing the authority or person who described the plant name. We have included the authority (Klotzsch) in the abstract (first paragraph) and the introduction (third paragraph). We have deleted it from the title as it is not necessary to include it there as it may lead to confusion among readers unfamiliar with botanical nomenclature.

Comment 2: The title also includes quarcetin-3-O-rhamnoside. For greater clarity, the authors should explicity state that quarcetin mentioned in the title is extracted from the leaves of the referenced plant species.

Response 2: We agree with this comment and have rephrased the title to clarify this point. The new title reads “In vitro bioactivity of leaf extract fractions and quercetin-3-O-rhamnoside from Combretum elaeagnoides against Staphylococcus species implicated in causing bovine mastitis”. Comment 3: The title also refers to the use of fraction and extracts against mastitis-causing pathogens in cattle. However, the study evaluates inhibition only against staphylococci. Although staphylococci are among the primary etiological agents of mastitis, numerous other pathogens can also cause the disease. Therefore, it would be clearer if the title specificed that the activity was assessed against staphylococci as mastitis-causing bacteria. Response 3: The title has been adjusted to specify the organisms studied as per the above response. Comment 4: Line 19: in vivo italic Response 4: Changed as suggested. Comment 5: Line 22: It would be helpful to clarify from which specific part of plant the component was isolated. Response 5: The leaves have been mentioned as the plant part of interest. Comment 6: Line 22: The abbrevation S. aureus should be placed in parantheses, as this is the first time the species is mentioned. Response 6: This has been done as suggested. Comment 7: Line 22-24: Throughout the manuscript, mastitis pathogens are mantioned, but experiments were performed solely with staphylococci. Clarifying this throughout the text would enhance accuracy and prevent potential misunderstanding. Response 7: Thank you for pointing this out. The term “mastitis pathogens” has been replaced with “S. aureus”. Comment 8: Line 29-30: In my opinion, this sentence is redundant in the abstract. It would be more appropriate to include it in the discussion section, with proper citation of the relevant authors. Response 8: Thank you for this comment – we agree and have deleted it from the abstract as it is referenced in the Discussion section. Comment 9: Line 35: CeDCM and CeEtOAc - abbreviations should be used here, as they have already been introduced earlier. Response 9: The full names have been replaced as suggested where appropriate. Comment 10: Line 41-42: Abbreviations should also be used here, as they have already been introduced earlier. Response 10: Thank you, this has been changed. Comment 11: Line 56: The abbreviation S. aureus should be placed in parentheses immediately after full name. Response 11: The abbreviation has been incorporated as recommended (line 59). Comment 12: Line 62: It is unclear whether this paragraph refers to inflammation of the bovine udder. It would be helpful to describe the differences between subclinical and clinical mastitis in cows, especially since the experiment was conducted only with S. aureus isolates from milk of cows with clinical mastitis. Response 12: A general definition of inflammation has been described in this section, and the different forms of mastitis (clinical and sub-clinical) have been highlighted in the next paragraph of the manuscript. Comment 13: Line 76: Staphylococcus aureus possesses multiple mechanisms for developing antibiotic resistance. It would be valuable to also mention other mechanisms by which this bacterium protect itself, contributing to difficulty of treating bovine udder infections and the high incidence of recurrent infections. Response 13: The different mechanisms of resistance developed by S. aureus have been included in the Introduction. Comment 14: Line 86: It would be interesting to highlight the traditional uses of this plant extract in African cultures, for example, for treating fever or diarrhea. Response 14: Thank you for this comment. We have included a sentence on the traditional medicinal use of the plant to treat various infectious diseases in Africa (line 121 – 123). Comment 15: Line 93: Were these studies conducted using leaf extracts, and it would be interesting to specify which pathogens were tested. Response 15: Thank you – this omission has been addressed by including mention of the leaf extract and fractions, and the different strains of Staphylococcus aureus. Comment 16: Line 95: It should be clarified from which part of the plant guarcetin as the active compound was isolated. Response 16: It has been clarified in the Introduction and revised title that the compound was isolated from the leaves. Comment 17: Line 102: The abbreviations and use of parentheses are unclear. Response 17: This sentence has been rewritten to enhance the clarity (lines 132-136). Comment 18: Line 104: Is this deciduous tree Response 18: Yes, the tree is deciduous, and this has been included in the Introduction (line 121). Comment 19: Line 105: Abbreviations should be used here, as they have already been introduced earlier in the manuscript. Response 19: Thank you, this has been corrected as suggested. Comment 20: Line 107: Please specify which microorganisms were tested and the number of strains used. Only Staphylococcus species were tested. Response 20: This sentence has been revised for clarity. Comment 21: Table 1: The abbreviation MeOH should be explained, and its usage should be standardized throughout the manuscript, as it is inconsistently written in some places. Response 21: Thank you, this has been addressed. Comment 22: Line 139-142: It would be interesting to indicate in the text which strains were sensitive to which extracts, in order to more clearly observe whether certain strains are more susceptible to specific extracts. Response 22: Thank you for this comment – some information has been included as suggested (line 178-179). Comment 23: Line 188: A space should be inserted between 28 and 29. Response 23: For consistency, we have followed the journal instructions concerning referencing. Comment 24: Line 190: Why was this measured only after 48 hours. Response 24: Unfortunately, there was insufficient quantity of the isolated compound to test at other time periods and this has been mentioned in the text. Comment 25: Line 193: It should be stated that gentamicin was used as the positive control. Response 25: This has been included as suggested. Comment 26: Line 212: It should be indicated that C. violaceum use as a model organism for anti-quorum experiments as they produce violacein pigment. Full name for C. violaceum. Response 26: Thank you for this suggestion which has been incorporated in the text (Lines 262-264, and the full name of Chomobacterium violaceum has been included. Comment 27: Table 3. MeOH or MeoH, CEH or CEHx Response 27: Thank you for pointing this out – the error have been corrected for uniformity. Comment 28: Line 244: full name for LOX and NO Response 28: The full names have been included in this paragraph. Comment 29: Section 2.4. It would be interesting to test these parameters on somatic cells from milk of cows with clinical mastitis, as was done with RAW macrophages. Response 29: This is a novel point and the sentence “It would be interesting to evaluate activity of the plant samples against somatic cells found in milk of cows affected with bovine mastitis.” has been included in this section. Comment 30: Section 2.5. What is the rationale for using Vero cells in addition to bovine dermis cells? If this is only cited from a previous study, it might not need to be included as a separate subsection. Response 30: Thank you, this is a valid point and we have revised this section to remove the emphasis on Vero cells. It is more relevant to assess potential toxicity to bovine dermis cells as this is the intended site of application. The lack of toxicity to Vero cells serves merely to support the low cytotoxicity of the extract and fractions. Comment 31: Line 283: in vivo - italic Response 31: This has been corrected. Comment 32: Table 6: Explanations for the abbreviations are missing below the table. Response 32: Thank you, these have been added. Comment 33: Line 295: Which test was used to confirm the identification of S. aureus Response 33: The test used to confirm identification of the strains at the Milk Laboratory has been referenced in Line 362-363 as these were the same strains used in the current study. The method is as follows: “Using a sterile 10 µL plastic loop (Quality Biological, USA), milk samples were plated on blood tryptose agar and incubated at 37°C for 24 h. Presumptive Staphylococcus spp. colonies were initially identified based on phenotypic morphology, and biochemical tests as described by Quinn et al. (1994) and the presence of coagulase using the slide agglutination test kit (Staphaurex® kit, Oxoid, Thermo Fisher Scientific, USA). Coagulase-positive isolates were streaked onto a mannitol salt agar (Oxoid, Thermo Fisher Scientific, USA) and incubated for 24 h at 37°C [16]. Mannitol positive isolates were confirmed to be S. aureus using the analytical profile index (API)® Staph™ kit (API Staph test kit, Biomerieux, South Africa).” (Mphahlele et al., 2020). Comment 34: Line 407: FOX - full name Response 34: The full name of the FOX reagent (ferrous oxidation-xylenol orange) has been included (Line 470). Comment 35: Line 412: DSMO - full name Response 35: The full name of DMSO is given in Line 455 (section 3.4.2). Comment 36: Line 514: In the conclusions, the authors should not be cited. Response 36: Thank you, this has been corrected. Comment 37: Are further studies planned on strains isolated from milk of cows with subclinical mastitis? Additionally, it is unclear whether the tested strains originated from cows on the same farm or from different farms.  Response 37: Thank you, this is a useful suggestion to test the samples against subclinical mastitis isolates and we are currently incorporating this into future plans. The strains originated from different farms and this has been mentioned in the Methods section (Lines 358-359) Comment 38: It would be interesting to provide an explanation on how the extracts could be administered intramammarily to cows. Response 38: At this stage, it is only intended to use the extracts as a teat dip to reduce the population of infectious mastitis-causing bacteria on the surface of the skin and in the teat canal if possible. Comment 39: In my opinion, since the results and discussion are combined, the discussion should be expanded by comparing with the findings of other authors. Response 39: Combretum elaeagnoides has not been widely studied; however, the discussion was enriched by comparing the available data with other relevant and recent studies (e.g., Masuku et al., 2025; Orlandi et al., 2024). Comment 40: Additionally, the conclusion is too long and contains repetitions of the results. It should be shorter, clearer, and more concise. Response 40: The conclusion has been revised to be more concise and focused.

Reviewer 2 Report

Comments and Suggestions for Authors

General Assessment

The manuscript entitled “In vitro bioactivity of Combretum elaeagnoides Klotzsch fractions and quercetin-3-O-rhamnoside against bovine mastitis-causing pathogens” addresses a highly relevant topic, namely the search for alternative antimicrobial and anti-inflammatory agents for the management of bovine mastitis in the context of increasing antibiotic resistance. The study is comprehensive, combining antibacterial, antibiofilm, anti-quorum-sensing, anti-inflammatory, and cytotoxicity assays, and focuses on clinically relevant Staphylococcus aureus isolates.

Overall, the manuscript presents a substantial amount of experimental data and demonstrates promising biological activities, particularly for the ethyl acetate fraction and quercetin-3-O-rhamnoside. However, several major issues related to experimental design clarity, data interpretation, statistical treatment, and manuscript organization must be addressed before the work can be considered for publication.

 

Major Revisions

-The description of concentrations used across different assays (MIC, antibiofilm, metabolic activity, QS inhibition, anti-inflammatory assays) lacks consistency and clarity. For example, antibiofilm assays are reported at both 1 mg/mL and 2 mg/mL in different sections and tables. The authors should clearly justify the choice of concentrations for each assay and ensure consistency between the Methods section, Tables, and Results narrative.

-Although mean values and standard deviations are reported, the manuscript does not adequately describe the statistical analyses used to compare treatments.

The authors should specify:

• The number of biological and technical replicates.
• The statistical tests applied (e.g., ANOVA, post hoc tests).
• The criteria for significance (p-values).

Where appropriate, statistical comparisons between extracts/fractions and positive controls should be included to strengthen the conclusions.

-The antibiofilm and metabolic activity results are extensive but sometimes descriptive rather than mechanistic. The authors should better distinguish between:

• Inhibition of initial adhesion (T0),
• Disruption of immature biofilms (T24),
• Effects on mature biofilms (T48).

A more critical discussion is needed to explain why certain fractions lose activity at T48 and how this relates to biofilm maturation and compound stability.

-While violacein inhibition is clearly demonstrated, the biological relevance of Chromobacterium violaceum QS inhibition to bovine mastitis pathogens should be discussed more explicitly. The authors should clarify whether QS inhibition occurred independently of growth inhibition for each fraction and discuss the implications for anti-virulence strategies.

-The manuscript frequently attributes activity to quercetin-3-O-rhamnoside, particularly in the ethyl acetate fraction, but no quantitative phytochemical analysis (e.g., relative abundance) is provided.

The authors should either:

• Provide quantitative data supporting this claim, or
• Clearly state that the attribution is speculative and based on previous isolation studies.

-Statements suggesting direct application of C. elaeagnoides extracts as “herbal treatments” for mastitis are premature. The conclusions should be moderated to emphasize that the results are in vitro and that in vivo efficacy, safety, formulation, and pharmacokinetics remain to be established.

 

Minor Revisions

-The Results and Discussion sections are combined, but some paragraphs are purely descriptive and would be clearer if separated or more critically discussed. Repetitive statements comparing results to previous studies by the same authors should be reduced.

-Tables are dense and difficult to read. Consider simplifying or splitting large tables (e.g., antibiofilm and metabolic activity data).

-Units should be consistently reported in table headers.

-Figure captions should be more descriptive and self-explanatory.

-The rationale for choosing gentamicin, ciprofloxacin, amphotericin B, and quercetin as controls should be briefly justified in the Methods or Discussion. The performance of positive controls should be more explicitly discussed relative to plant extracts.

-The selectivity index (SI) is an important strength of the study but is under-discussed. The authors should more clearly highlight which fractions present the most favorable balance between antimicrobial activity and cytotoxicity.

-Some references are outdated or inconsistently formatted. Ensure uniform citation style and verify that all references cited in the text appear in the reference list.

Author Response

Comment 1: The description of concentrations used across different assays (MIC, antibiofilm, metabolic activity, QS inhibition, anti-inflammatory assays) lacks consistency and clarity. For example, antibiofilm assays are reported at both 1 mg/mL and 2 mg/mL in different sections and tables. The authors should clearly justify the choice of concentrations for each assay and ensure consistency between the Methods section, Tables, and Results narrative.

Response 1: The concentrations used in this study are those established in our laboratory for the different assays, following published methods to allow for comparison of results. The compound for the antibiofilm assay was tested at the same concentration as the positive controls (1 mg/mL), while the 2 mg/mL was a stock concentration prepared for the antibiofilm assay.

Comment 2: Although mean values and standard deviations are reported, the manuscript does not adequately describe the statistical analyses used to compare treatments.

The authors should specify:

• The number of biological and technical replicates.
• The statistical tests applied (e.g., ANOVA, post hoc tests).
• The criteria for significance (p-values).

Response 2: The statistical tools used and number of experimental replicates has been clarified

Comment 3: Where appropriate, statistical comparisons between extracts/fractions and positive controls should be included to strengthen the conclusions.

Response 3: There was no statistical significance between the samples in the anti-inflammatory assays; furthermore, the crude MeOH extract had no statistical significance against the different fractions and the compound in terms of both NO and Lox-15 activity. This was added to the conclusion section,

Comment 4: The antibiofilm and metabolic activity results are extensive but sometimes descriptive rather than mechanistic. The authors should better distinguish between:

• Inhibition of initial adhesion (T0),
• Disruption of immature biofilms (T24),
• Effects on mature biofilms (T48).

Response 4: The different mechanistic antibiofilm results have been clarified in the Results to aid proper understanding of the results in Table 2. The results of the different stages (T0, T24, T48) are clarified in the Results section (Lines 169-170).

Comment 5: A more critical discussion is needed to explain why certain fractions lose activity at T48 and how this relates to biofilm maturation and compound stability.

Response 5: The comment above has also been clarified (lines 191- 193).

Comment 6: While violacein inhibition is clearly demonstrated, the biological relevance of Chromobacterium violaceum QS inhibition to bovine mastitis pathogens should be discussed more explicitly. The authors should clarify whether QS inhibition occurred independently of growth inhibition for each fraction and discuss the implications for anti-virulence strategies.

Response 6: Although S. aureus produces auto-inducing peptides which encourage virulency and biofilm formation, this was not targeted in this study. Chromobacterium violaceum is a useful model for cell-to-cell communication between bacteria, providing a visible indication in terms of the purple pigment produced. The mechanism of quorum sensing was explained in the manuscript.

This statement ‘The authors should clarify whether QS inhibition occurred independently of growth inhibition for each fraction and discuss the implications for anti-virulence strategies’ was explained in the methodology section “MICs were determined as the lowest concentration at which the samples inhibited growth and prevented purple pigmentation. The minimum quorum-sensing inhibitory concentration (MQSIC) was determined by the presence of growth (turbidity) with no visible purple pigmentation.” (Lines 470 – 472.)

Comment 7: The manuscript frequently attributes activity to quercetin-3-O-rhamnoside, particularly in the ethyl acetate fraction, but no quantitative phytochemical analysis (e.g., relative abundance) is provided.

The authors should either:

• Provide quantitative data supporting this claim, or
• Clearly state that the attribution is speculative and based on previous isolation studies.

Response 7: Thank you for highlighting this omission. We have included a statement that this attribution is based on previous isolation studies and that “Further research is needed to quantify the amount of the compound in the extract, and also to isolate further potentially active compounds.” (Lines 147-149).

Comment 8: Statements suggesting direct application of C. elaeagnoides extracts as “herbal treatments” for mastitis are premature. The conclusions should be moderated to emphasize that the results are in vitro and that in vivo efficacy, safety, formulation, and pharmacokinetics remain to be established.

Response 8: This was clarified in the Conclusion section with the addition of the sentence “However, it is essential that further safety and pharmacokinetic experiments, as well as appropriate formulation and in vivo studies, are conducted.” (Lines 611-613.)

 

Minor revisions

Comment 9: The Results and Discussion sections are combined, but some paragraphs are purely descriptive and would be clearer if separated or more critically discussed. Repetitive statements comparing results to previous studies by the same authors should be reduced.

Response 9: There are limited studies on C. elaeagnoides, which is why comparison of the results was done only with available data/information. However, we have also improved and enriched the discussion referring to similar studies on other Combretum species (e.g., Masuku et al., 2025; Orlandi et al., 2024) (Lines-).

Comment 10: Tables are dense and difficult to read. Consider simplifying or splitting large tables (e.g., antibiofilm and metabolic activity data).

Response 10: Thank you for your feedback. We have separated the tables and presented them as suggested by the reviewer.

Comment 11: Units should be consistently reported in table headers.

Response 11: Thank you for pointing this out – it has been rectified.

Comment 12: Figure captions should be more descriptive and self-explanatory.

Response 12: The captions have been carefully revised to improve them.

Comment 13: The rationale for choosing gentamicin, ciprofloxacin, amphotericin B, and quercetin as controls should be briefly justified in the Methods or Discussion. The performance of positive controls should be more explicitly discussed relative to plant extracts.

Response 13: This has been justified in the Results and Conclusion sections (Line 579).

Comment 14: The selectivity index (SI) is an important strength of the study but is under-discussed. The authors should more clearly highlight which fractions present the most favorable balance between antimicrobial activity and cytotoxicity.

Response 14: The fractions that present the most favorable balance between antimicrobial activity and cytotoxicity have been highlighted in the Results and Discussion (line 359-60).

Comment 15: Some references are outdated or inconsistently formatted. Ensure uniform citation style and verify that all references cited in the text appear in the reference list.

Response 15: References were formatted according to the journal's guidelines, and all were checked in both the text and the reference list.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I would like to thank the authors for addresing all the suggested comments in order to improve the quality of the manuscript. I recommend that the paper be accepted for publication in the journal.