Fire Ant Venom Alkaloids Inhibit Biofilm Formation

Round 1

Reviewer 1 Report

The authors have taken the necessary steps to improve this manuscript overall. I only have minor comments:

Line 3: replace "of" with "for the"

Line 12: replace "depends" with "relies"

Lines 21 - 23: I would consider dividing into two complete sentences.

Line 59: replace "evidencing" with "providing evidence for"

Line 145: replace "looks" with "may be"

Author Response

The authors have taken the necessary steps to improve this manuscript overall. 

R: Thanks, all previous suggestions have helped strengthen your manuscript.

Line 3: replace "of" with "for the"

R: Done. 

Line 12: replace "depends" with "relies"

R: Done.

Lines 21 - 23: I would consider dividing into two complete sentences.

R: Thanks for the suggestion. We however opted to leave this phrase as it is. 

Line 59: replace "evidencing" with "providing evidence for"

R: Done.

Line 145: replace "looks" with "may be"

R: Done.

Additionally, we have proofread the final manuscript with the help of a friend. 

Thanks for your time dedicated to help our work!

Reviewer 2 Report

The chemical simplicity of solenopsins says it all about the potential of solenopsins as pharmaceuticals. A myriad of pharmacological properties of solenopsins have been reported and reviewed by Touchard et al. in Toxins journal (2016).  It was concluded, "Solenopsins, in general, are no exception with potent antimicrobial activity against fungi and gram-positive bacteria while solenopsin A was effective against gram-negative bacteria." What is reported in the current manuscript is not "novel" in any way. The authors cite a recent study of using solenopsins in dermatology. The study used solenopsins as ceramide mimics, which was not even relevant to the "harmful" aspects of solenopsins as toxins.

As shown in the video included in Fox's paper that was cited in the response to the reviewer's comments, the insecticidal activity of solenopsins is moderate at best, compared to many insecticidal spray formulations sold in garden stores. There are protein components in minute amounts in the fire ant venom that still need to be further studied. Some of those components may play more important roles in the severe toxicity of fire ant venom.

After taking into consideration the converted ug/disk concentrations for the disc-diffusion experiment, the solenopsins tested are weak antibiotics, comparing to the intermediate diameters of 10-15 mm observed at lower concentrations for most antibiotics. The observed antimicrobial properties appeared to be similar to the generic chemical properties of many small organic compounds. If the authors had studied the differential activities among the solenopsins and offered a structure-activity conclusion for the solenopsins, it would be more valuable for possible development of solenopsin-derived antibiotics.  

"Biofilm" in medicine and in microbiology has a specific definition, what the authors described can at best be "inhibition of bacterial growth on surfaces treated with solenopsins." The comparison should have been made with surfaces treated with triclosan or other disinfectants.

The authors should refer to the literature on biofilm formation, detection and evaluation for proper methodology in studying compounds that inhibit biofilm formation or promote biofilm dispersion and/or disruption. The authors stated "dipped in a nutrient broth containing 10⁹ CFU/mL P. fluorescens" without describing the length of time or conditions the test coupons were exposed to bacteria broth. It is obvious that prolonged exposure of the test coupon will result in lost of solenopsin to the broth.

Overall, the manuscript in its present form is not suitable for publication.

Author Response

The chemical simplicity of solenopsins says it all about the potential of solenopsins as pharmaceuticals. 

R: We do not understand the remark. Solenopsins are akin in 'chemical simplicity’ to top market drugs like acetylsalicylic acid and acetaminophen. Chemically related alkaloids include nicotine or ephedrine. Notwithstanding, the present manuscript is not about some pharmaceutical application.   

A myriad of pharmacological properties of solenopsins have been reported and reviewed by Touchard et al. in Toxins journal (2016).  It was concluded, "Solenopsins, in general, are no exception with potent antimicrobial activity against fungi and gram-positive bacteria while solenopsin A was effective against gram-negative bacteria." What is reported in the current manuscript is not "novel" in any way. 

R: Thanks for quoting a key conclusion from Touchard et al. (2016) — I have written it. Solenopsin A (= C11 in Table 1) remains to date the best-studied solenopsin. The venom of our focal species S. invicta has but trace amounts of solenopsin A (see in Table 1: 2.5%). Thus the potential of whole-venom preparations, and included solenopsins B-D remained largely unexplored. When writing about solenopsins in Touchard et al. (2016) I have not mentioned anti-biofilm activity precisely because the theme is novel: there are no published reports of surface-conditioning with solenopsins against biofilms.

The authors cite a recent study of using solenopsins in dermatology. The study used solenopsins as ceramide mimics, which was not even relevant to the "harmful" aspects of solenopsins as toxins.

R: There in fact exists a paper (Arbiser et al. 2017) about dermatological application of solenopsins, which is not included in the manuscript references. It is unrelated to the scope of present submission. Therefore we are not sure what was the meaning of this remark.

As shown in the video included in Fox's paper that was cited in the response to the reviewer's comments, the insecticidal activity of solenopsins is moderate at best, compared to many insecticidal spray formulations sold in garden stores.

R: We believe the reviewer here means Fox et al. (2019), which is also not within the scope of the present study and thus not included in the manuscript references. It was included in a previous response to a reviewer who seemed unfamiliar with literature about the bioactivities of solenopsins. Additionally, the paper Fox et al. (2019) deals with chemical ecology, which is also unrelated to commercial ‘insecticidal spray formulations’. Thus we are not sure of what was the point of the comment. 

There are protein components in minute amounts in the fire ant venom that still need to be further studied. Some of those components may play more important roles in the severe toxicity of fire ant venom.

R: Although we do agree with the specific remark — illustrated by our work into the subject in e.g. Zamith et al. (2018) —, we fail to grasp how it relates with the present submission. The tested solenopsins extract had no protein, and was not herein assayed for animal toxicity. 

After taking into consideration the converted ug/disk concentrations for the disc-diffusion experiment, the solenopsins tested are weak antibiotics, comparing to the intermediate diameters of 10-15 mm observed at lower concentrations for most antibiotics.

R: This is an interesting debatable point, which is unfortunately outside of the scope of the present manuscript. We have not specifically tested antibiotics in the present manuscript, as these are not recorded as industrial anti-biofilm chemicals. The disk-diffusion results we present merely illustrate the antibiotic activity of solenopsins against P. fluorescens as a main cause behind *anti-biofilm activity* — which is scope of this manuscript. 

The observed antimicrobial properties appeared to be similar to the generic chemical properties of many small organic compounds.

R: Please specify 'small organic compounds' and 'antimicrobial properties'. There are countless compounds which are used as antimicrobials, e.g. ethanol, formic acid, bleach. However we are not aware of reports of anti-biofilm surface-conditioning applications using any of these. We hereby present the first report of such application using animal-derived alkaloids.  

If the authors had studied the differential activities among the solenopsins and offered a structure-activity conclusion for the solenopsins, it would be more valuable for possible development of solenopsin-derived antibiotics.  

R: We agree with the idea, as screening the antimicrobial activity of synthetic & natural solenopsins is within our medium-term plans. However, this lies far outside the scope of the present manuscript, which deals with anti-biofilm effects from surface conditioning with alkaloids. We have added some lines to section 3.2 about this suggestion, also based on an insight by R#5.

"Biofilm" in medicine and in microbiology has a specific definition, what the authors described can at best be "inhibition of bacterial growth on surfaces treated with solenopsins.”

R: There is perhaps a misinterpretation: we have assayed against biofilm formation and growth, senso stricto, in three different experiments. Antimicrobial activity is illustrated as one of the factors behind the described anti-biofilm activity.  

 The comparison should have been made with surfaces treated with triclosan or other disinfectants.

R: Thanks for the suggestion. We ought to highlight that the specific background of biofilms in the present submission is in industrial and food facilities. We have now included a summary of the main preventive cleaning actions used in such facilities, as suggested by another reviewer. Triclosan isn’t mentioned. Triclosan is successfully employed against dental plaque and lung infections, which are other kinds of biofilms from a different nature. Antiseptic soaps (which often contain some triclosan) used in food facilities are reported as inefficient against biofilm-formation by Pseudomonas spp. Moreover, we are unaware of surface-conditioning reports using triclosan, which is ultimately the focus of the present investigation. 

The authors should refer to the literature on biofilm formation, detection and evaluation for proper methodology in studying compounds that inhibit biofilm formation or promote biofilm dispersion and/or disruption. 

R: Thanks for the suggestion. As also suggested by another reviewer, we have now expanded comments on the main methods currently employed in controlling biofilms in food facilities. The pertaining literature is thus referenced, thanks.

The authors stated "dipped in a nutrient broth containing 109 CFU/mL P. fluorescens" without describing the length of time or conditions the test coupons were exposed to bacteria broth. It is obvious that prolonged exposure of the test coupon will result in lost of solenopsin to the broth.

R: Thanks for drawing our attention to the methods section. Firstly, we ought to remark that a number of minor grammar adjustments were now applied to make some phrases clearer. Specifically concerning this remark, we should emphasise two important points: (i) Surface conditioning is a modification of surface chemistry based on molecular adsorption (Kanematsu & Barry 2015); (ii) solenopsins are not water-soluble. The principle anti-biofilm activity in the case of conditioned surfaces is that adsorbed molecules somehow prevent bacterial adhesion. Our results clearly illustrate that conditioning with solenopsins has an effect against biofilm formation, even after washing this surface. This demonstrates surface conditioning, where the chemical adsorption implies that molecules get physically bound to the surface in a way that they will not evaporate or wash away so easily. 

Overall, the manuscript in its present form is not suitable for publication.

R: We hope to have hereby clarified a number of key points which were perhaps misinterpreted: scope of the present submission, solenopsins chemistry, biofilms, surface conditioning. We hope the final version looks more acceptable, in view of this discussion and applied modifications. As we intend to publish the peer review alongside the manuscript, we feel this discussion may also prove of great use to forthcoming readers.

Thank you for your time and an insightful discussion!

Reviewer 3 Report

Thankyou for considering and addressing the points I raised in response to the first submission of this manuscript. I am satisfied they have all been addressed satisfactorily and recommend it is now suitable for publication.

Author Response

Thank you for considering and addressing the points I raised in response to the first submission of this manuscript. I am satisfied they have all been addressed satisfactorily and recommend it is now suitable for publication.

R: Thank you for the time spent improving on our work.

Reviewer 4 Report

Dear Authors,

Overall manuscript is fine. However, please explain how fire ant venom can be commercialized for use. Also, discuss some pros and cons of existing options and proposed solution for biofilm formation. 

Author Response

Overall manuscript is fine. 

R: Thanks !

However, please explain how fire ant venom can be commercialized for use. 

R: Given the current early state of research on solenopsins as natural products, we believe the last paragraph of section 3.1 is already elucidative enough regarding feasibility of commercial exploitation. Akin to the recent papers by Arbiser et al. (e.g. 2017) which sparked speculations & expectations over some impending “fire ant venom skin lotion” already years ago (e.g. see Marker 2017), we feel that a technical discussion into actual production and commercialisation lies beyond the reach of the present manuscript and could lead to premature expectations. Nonetheless, we emphasise mass-extraction and production techs for solenopsins are indeed needed for several applications, which is the topic of investigation of a few colleagues (see Fox et al. 2014) . 

Also, discuss some pros and cons of existing options and proposed solution for biofilm formation. 

R: Thanks for a sound suggestion. We have incorporated some lines discussing the most commonly-employed food industry measures against biofilm formation into the Discussion, adding to the rationale that the incorporation of less aggressive surface-adhering chemicals are expected contribute to the solutions. 

Thank you for the constructive comments!

References: 

Arbiser et al. Scientific Reports, 2017; 7 (1) DOI: 10.1038/s41598-017-10580-y

Chen, L. & Fadamiro, H.Y. (2009) Toxicon, 53, 469–478.

Fox 2014 DOI 10.1007/978-94-007-6649-5_38-1

Fox et al. Toxicon. 2019 Feb;158:77-83. doi: 10.1016/j.toxicon.2018.11.428 

Kanematsu, H., & Barry, D. M. (2015). Conditioning films. In Biofilm and Materials Science (pp. 9-15). Springer, Cham.

Liu et al. 2017. Florida Entomologist, 100(3):522-527 (2017). https://doi.org/10.1653/024.100.0305

Marker, 2017. https://www.labroots.com/trending/immunology/6870/fire-ant-venom-ingredient-skin-care-products

Park, J.; Kaufmann, G.F.; Bowen, J.P.; Arbiser, J.L.; Janda, K.D. (2008) Solenopsin A, a venom alkaloid from the fire ant Solenopsis invicta, inhibits quorum sensing signaling in Pseudomonas aeruginosa. Journal of Infectious Diseases, v. 198, n. 8, p.1198–1201.

Shi et al. 2015 Front. Ecol. Evol. 3:76. doi: 10.3389/fevo.2015.00076 

Touchard et al. 2016 Toxins 8 (1), 30 https://doi.org/10.3390/toxins8010030

Reviewer 5 Report

Fire Ant Venom Alkaloids Inhibit Biofilm Formation.

The authors extracted the alkaloids from venom of fire ants and evaluated its potency for inhibiting the biofilm formation of Pseudomonas fluorescens on polystyrene and stainless surface. The article demonstrated a new application of alkaloids to these material, but I think the significance of this study appears very small. Besides, the article was not well written and it must be professionally proofread as it contains many grammatical mistakes and sentences with unclear structure. In my opinion, the paper would be acceptable for publication if the authors present more scientific information of this material convincingly. Therefore, I would recommend major revision for this manuscript.

1.      Experimental section 5.1: Identification of venom composition is critical in the study, but these analytical conditions are missed in the article. In the section, the condition for GCMS analysis is not complete that is difficult to follow or repeat by the audience. For example, how much volume of biphasic liquid was used for extraction? how much amount of sample was injected for GCMS analysis? What kind of GC column was used for sample analysis? What is the database used for compound identification? the extraction method is important to the bioactivity evaluation. the authors can demonstrate the consistency of method used in the article. please provide the consistency of the extraction method.

2.      Line 58-59: “The obtained inhibition haloes were largest for the highest tested concentrations of solenopsins, evidencing antimicrobial activity against P. fluorescens. Do the authors try to indicate the dose dependent effect? What is the MIC based on the antimicrobial results? Please clarify.

3.      Figure 1: the index and unit of y-axis is missed.

4.      Figure 2 and 3, the meaning of abbreviation is not found in the figure legend, like CTL, abc and poly. Please provide.

5.      Line 80-82, solenopsins had some capacity of reducing a mature biofilm (i.e. preformed biofilm). Please clearly descript the meaning of mature biofilm/preformed biofilm. What is the difference between it with the biofilm shown in Figure 2? What is it correlation with the antiadhesive effect? Please clarify.

6.      In Figure 3, the data showed that extracted solenopsins cannot inhibit the formation of “mature film” on the steel surface with solenopsins conditioning (blue dot). However, authors also indicated that no viable cells were recovered from the conditioning stainless steel (Line 99-100, and Figure 5D). it seems these two data cannot match well. Please clarify.

7.      Line 110, the rhamnolipids was shown in the result but there is no description about this compound in the introduction section. Why the rhamnolipids is included in the comparative test? The author can have more discussion about it in the article.  

8.      Line 120, where is the appendix and reference in the article?

9.      Line 131: The term of aka’C13’ and aka’C15’ in this sentence was not able to be found in the Table 1. Please make indication and match the description in the aticle.

10.  Section 3.2. the authors compared the antimicrobial effect of synthetic and extracted solenopsins and indicate a better efficacy raised by the extracted one. Basically, the comparison of inhibition effect should base on the same condition, for example, using the same bacterium as testing targets. The authors could repeat the inhibition assay using the same bacterium for comparing the effect.

11.  Line 163-164. The author discussed the inhibition of sole opsin against the gram-positive bacterial of C. michiganensis, but such data is not included in the article. please add.

12.  Line 181, “See negative results reported in Supplementary Materials”, where is the “supplemental material” in the article? please indicate data in the sentence.

13.  Line 210-211, “the solenopsins extract did not display any effect on water surface tension”. The hydrophobicity of solenopsins on coating surface is very critical for its inhibition effect? Do authors analysis the contact angle on the polystyrene and stainless surface, and compare it with water molecule? Please clarify.

Author Response

The authors extracted the alkaloids from venom of fire ants and evaluated its potency for inhibiting the biofilm formation of Pseudomonas fluorescens on polystyrene and stainless surface. The article demonstrated a new application of alkaloids to these material, but I think the significance of this study appears very small. Besides, the article was not well written and it must be professionally proofread as it contains many grammatical mistakes and sentences with unclear structure. In my opinion, the paper would be acceptable for publication if the authors present more scientific information of this material convincingly. Therefore, I would recommend major revision for this manuscript.

R: Thanks for your insights. We have incorporated suggestions by other reviewers and proofread the manuscript while adjusted several passages. During this process we have emphasised on the findings of this study. Hopefully now the corrected version is clearer and acceptable. 

1.      Experimental section 5.1: Identification of venom composition is critical in the study, but these analytical conditions are missed in the article. In the section, the condition for GCMS analysis is not complete that is difficult to follow or repeat by the audience. For example, how much volume of biphasic liquid was used for extraction? how much amount of sample was injected for GCMS analysis? What kind of GC column was used for sample analysis? What is the database used for compound identification? the extraction method is important to the bioactivity evaluation. the authors can demonstrate the consistency of method used in the article. please provide the consistency of the extraction method.

R: As mentioned in the methods, we have effected venom extraction following procedures detailed in Fox et al. (2017), wherein all equipment settings and steps concerning chemical species identifications can be found. We opted not to repeat all procedures for the sake of brevity, as procedures have been published and discussed in details in the cited source. Regarding the consistency of the extraction method, there are already a number of papers reporting on solvent alkaloids extraction from fire ants, most notably Chen & Fadamiro (2009), Fox et al. (2013), Shi et al. (2015), and Liu et al. (2017). In fact, the paper by Liu et al. (2017) has already specifically dealt with reproducibility and consistency of alkaloidal extraction through hexane-soaking of fire ants. Therefore, we believe re-describing these matters would elongate the present submission unnecessarily, as it is built on previously established protocols. 

2.      Line 58-59: “The obtained inhibition haloes were largest for the highest tested concentrations of solenopsins, evidencing antimicrobial activity against P. fluorescens. Do the authors try to indicate the dose dependent effect? What is the MIC based on the antimicrobial results? Please clarify.

R: Thanks for your precise suggestions. As illustrated by Figure 1, there is a significantly stronger inhibitory effect achieved by the highest concentrations of solenopsins, which is suggestive of a dose-dependent activity. We have now included an estimation of MIC by linear regression using the results of mean inhibition haloes. This was an excellent addition. 

3.      Figure 1: the index and unit of y-axis is missed.

R: Thanks for pointing out. A legend for y-axis is now added.

4.      Figure 2 and 3, the meaning of abbreviation is not found in the figure legend, like CTL, abc and poly. Please provide.

R: Well spotted, thanks! We have corrected these flaws.

5.      Line 80-82, solenopsins had some capacity of reducing a mature biofilm (i.e. preformed biofilm). Please clearly descript the meaning of mature biofilm/preformed biofilm. What is the difference between it with the biofilm shown in Figure 2? What is it correlation with the antiadhesive effect? Please clarify.

R: The secretion of a microbial biofilm follows surface adhesion by the microbes. The biofilm matrix is made of factors secreted by adhered bacteria. The principle of surface-conditioning involves a focal molecule that get adsorbed to the exposed surface, which will somehow later affect microbial adhesion. A preformed biofilm, as described in our methods, is the biofilm allowed to grow freely on a non-conditioned surface. This is therefore fundamentally different from attempting to grow a biofilm on a conditioned surface. We made tests to verify whether solenopsins could affect preformed biofilms, and only obtained recorded effects on polystyrene. In principle there’s no relationship between surface conditioning and our tests with mature biofilms. However exposure to solenopsins may affect bacterial adhesion to already-existent, preformed biofilm. We hope this explanation clarifies some doubts. Nonetheless we have included a short introductory paragraph to the relevant discussion section as to refresh readers on biofilm formation and the employed terms, and emphasised on the terminology difference wherever fitting in the narrative.

6.      In Figure 3, the data showed that extracted solenopsins cannot inhibit the formation of “mature film” on the steel surface with solenopsins conditioning (blue dot). However, authors also indicated that no viable cells were recovered from the conditioning stainless steel (Line 99-100, and Figure 5D). it seems these two data cannot match well. Please clarify.

R: We believe the impression of results mismatching derives of the confusion concerning surface conditioning vs. preformed biofilm on an untreated surface. Our results demonstrate surface conditioning with solenopsins will delay biofilm formation, whilst exposure to solenopsins doesn’t seem to affect a biofilm already grown on steel. The two situations are fundamentally different to what regards previous surface conditioning with solenopsins. We believe the addition of a note clarifying the difference between a mature biofilm vs. a biofilm growing on a conditioned surface as done in the discussion above was sufficient to clarify this issue. 

7.      Line 110, the rhamnolipids was shown in the result but there is no description about this compound in the introduction section. Why the rhamnolipids is included in the comparative test? The author can have more discussion about it in the article.  

R: Originally, the physico-chemical tests employing the rhamnolipids as positive controls were presented in an appendix to the main manuscript, as negative results. Two previous reviewers suggested we incorporated the appendix analysis into the main manuscript, thus significant structural changes took place. The reviewer is right about the necessity of some mention to rhamnolipids being made in the intro. We have now incorporated some lines from the previous versions of the manuscript to the introduction and methods as to make this point clearer. Thanks for the suggestion. 

8.      Line 120, where is the appendix and reference in the article?

R: Sorry, but these mentions have remained unchecked from a previous version of this manuscript before to a first round of peer review, as mentioned above. The instances were now corrected. Thanks for pointing out.

9.      Line 131: The term of aka’C13’ and aka’C15’ in this sentence was not able to be found in the Table 1. Please make indication and match the description in the aticle.

R: Thanks for pointing this out. We have added a column with trivial names to Table 1.

10.  Section 3.2. the authors compared the antimicrobial effect of synthetic and extracted solenopsins and indicate a better efficacy raised by the extracted one. Basically, the comparison of inhibition effect should base on the same condition, for example, using the same bacterium as testing targets. The authors could repeat the inhibition assay using the same bacterium for comparing the effect.

R: We thoroughly agree in that including a number of synthetic solenopsins would generate further, interesting insights into the phenomenon. However we currently do not have synthetic solenopsins available. There are very few companies which will synthesise solenopsins, and for a steep price. The choice of a P. fluorescens as focal species for the present paper was based on the fact that it is a major species involved in biofilm formation in industrial and food facilities. Testing other bacteria exploited by other authors is surely a theme of our interest, however outside the scope of the present report. Thanks for the nice suggestions! 

11.  Line 163-164. The author discussed the inhibition of sole opsin against the gram-positive bacterial of C. michiganensis, but such data is not included in the article. please add.

R: Thanks for pointing out this issue. Unclear phrase construction conveyed an incorrect message on this point: we have not tested C. michiganensis but only P. fluorescens. In the appointed paragraph, we discuss the previous finds by other authors, mainly regarding tested solenopsins concentrations and observed inhibition halo diameters by disk-diffusion tests. On this specific phrase we highlighted a possible correlation of bacterial wall structure (as gram-negative or gram-positive) and their resistance to solenopsins exposure. It was thus unclear, and now we have re-rewritten it as to make our meaning more evident. 

12.  Line 181, “See negative results reported in Supplementary Materials”, where is the “supplemental material” in the article? please indicate data in the sentence.

R: As explained above, this mention has inadvertently remained from a previous version of this manuscript. Sorry. We have replaced the outdated mention with the relevant section reference.

13.  Line 210-211, “the solenopsins extract did not display any effect on water surface tension”. The hydrophobicity of solenopsins on coating surface is very critical for its inhibition effect? Do authors analysis the contact angle on the polystyrene and stainless surface, and compare it with water molecule? Please clarify.

R: Previous authors have believed that solenopsins might be amphipathic molecules, meaning they’d be surface-active compounds, as a possible main mechanism for their antimicrobial and anti-biofilm activities. We have decided to directly test their surface activity, and a hydrophobicity test is a standard procedure in such case. This is explained in discussion of section 3.5. Perhaps the reviewer lost track of our rationale because of the issues pointed out in questions 12, 8, 7, 6, which we have now revisited and corrected. “The hydrophobicity of solenopsins on coating surface is very critical for its inhibition effect?” This is exactly the question raised by this set of experiments concerning surface-activity, and the answer is no. Other mechanisms of biofilm inhibition from surface conditioning with solenopsins remain to be tested for P. fluorescens, possibly related to a quorum-sensing disruption mechanism demonstrated by Park et al. (2008) for P. aeruginosa.

Thank you very much for the insightful comments which have improved our paper!

References: 

Arbiser et al. Scientific Reports, 2017; 7 (1) DOI: 10.1038/s41598-017-10580-y

Chen, L. & Fadamiro, H.Y. (2009) Toxicon, 53, 469–478.

Fox et al. Toxicon. 2019 Feb;158:77-83. doi: 10.1016/j.toxicon.2018.11.428 

Kanematsu, H., & Barry, D. M. (2015). Conditioning films. In Biofilm and Materials Science (pp. 9-15). Springer, Cham.

Liu et al. 2017. Florida Entomologist, 100(3):522-527 (2017). https://doi.org/10.1653/024.100.0305

Marker, 2017. https://www.labroots.com/trending/immunology/6870/fire-ant-venom-ingredient-skin-care-products

Park, J.; Kaufmann, G.F.; Bowen, J.P.; Arbiser, J.L.; Janda, K.D. (2008) Solenopsin A, a venom alkaloid from the fire ant Solenopsis invicta, inhibits quorum sensing signaling in Pseudomonas aeruginosa. Journal of Infectious Diseases, v. 198, n. 8, p.1198–1201.

Shi et al. 2015 Front. Ecol. Evol. 3:76. doi: 10.3389/fevo.2015.00076 

Round 2

Reviewer 2 Report

Solenopsins have many interesting properties in bio-warfare employed by fire-ants. If the authors want to claim that the two surfaces were permanently modified by the 2,4-dialkyl piperidine compounds, they need to provide evidence for that claim.   

As indicated by the author there was no amphipathic surface property, how can they state that the compound is not water soluble. 

Most importantly, the authors did not provide convincing evidence supporting inhibition of biofilm as defined by researchers in the field of Microbiology and Medicine. 

Author Response

Rev#2

- Solenopsins have many interesting properties in bio-warfare employed by fire-ants. 

R: Agreed.

- If the authors want to claim that the two surfaces were permanently modified by the 2,4-dialkyl piperidine compounds, they need to provide evidence for that claim. 

R: We never made these claims in the manuscript. Please mind what the key points below: 

A. Firstly, “we are sorry as apparently the reviewer may have missed the context: the alkaloids prevalent in our samples (known as ‘solenopsins’) actually present the chemical structure 2,6-dialkyl-piperidines.” [Phrase recycled from a previous response to the reviewer]

B. Surface conditioning is not about permanent modifications, nor did we test the surfaces for indeterminate conditioning effects. Surface conditioning relies on adsorbed molecules causing an effect many hours after the initial exposure to and washing away of active compounds. Antimicrobial and anti-biofilm effects from conditioning surfaces with solenopsins are directly demonstrated by the experiments described in Methods 5.2.3., 5.2.4., 5.2.5. which generated the results shown in Figures 2, 3, 4, and 5. 

C. We have tested for possible modifications of conditioned surfaces physicochemical properties, and clearly state we have *not* detected physical modifications in the surfaces. 

D. A main point of our manuscript is to report the antibiofilm effects from conditioning industrial surfaces with solenopsis. The contents of this manuscript describe evidence for this effect, as perfectly acknowledged by the other reviewers.

- As indicated by the author there was no amphipathic surface property, how can they state that the compound is not water soluble. 

R: We are sorry as we cannot grasp the logic behind this statement. Maybe the reviewer is not aware of what is an amphipathic compound (e.g. the relationship with water solubility), and unfamiliar with solenopsins literature. We clarify these points:

A. We did not attempt to establish whether solenopsins are water insoluble, as this is already known as fact for decades (see [1] [2] [3], echoed by key literature reviews [4] [5] [6] [7]);

B. We have tested solenopsins (i.e. not “surfaces”) for amphipathic properties and detected none, compared to positive controls rhamnolipids, which are amphipathic natural compounds.

C. Non-amphipathic compounds are not expected to be water soluble (nor vice-versa) as illustrated by a number of well-known pharmacological compounds ([8] [9]) and trivial chemicals (e.g. water-insoluble & nonamphipathic benzene). Table S2 may further clarify this point.

Thus, given that fact A retrieved from the literature does not conflict with facts B and C, we cannot understand what was the basis for the reviewer's statement.   

[1] Blum, M.S.; Robert Walker, J.; Callahan, P.S.; Novak, A.F. Chemical, insecticidal, and antibiotic properties of fire ant venom. Science. 1958, 128, 306–307.

[2] MacConnell, J.G.; Blum, M.S.; Fales, H.M. The chemistry of fire ant venom. Tetrahedron 1971, 27, 1129–1139.

[3] Jouvenaz, D.P.; Blum, M.S.; MacConnell, J.G. Antibacterial activity of venom alkaloids from the imported fire ant, Solenopsis invicta Buren. Antimicrob. Agents Chemother. 1972, 2, 291–293.

[4] Blum, M.S. Poisonous ants and their venoms. Insect poisons, allergens, other Invertebrate venoms; 1984, 2, 225–242.

[5] Chen, J.; Shang, H. Advances in research on the venom chemistry of imported fire ants. In Recent advances in entomological research: From molecular biology to pest management; 2012; pp. 417–433.

[6] Fox, E.G.P. Venom Toxins of Fire ants. In Venom genomics and proteomics; 2014; pp. 1–16 ISBN 978-94-007-6649-5.

[7] Touchard, A.; Aili, S.R.; Fox, E.G.P.; Escoubas, P.; Orivel, J.; Nicholson, G.M.; Dejean, A. The biochemical toxin arsenal from ant venoms. Toxins (Basel). 2016, 8, 1–28.

[8] Seeman, P.; Seeman The membrane actions of anesthetics and tranquilizers. Pharmacol. Rev. 1972, 24, 583–655.

[9] Conrad, M.J.; Singer, S.J. Solubility of amphipathic molecules in biological membranes and lipid bilayers and its implications for membrane structure. Biochemistry 1981, 20, 808–818.

- Most importantly, the authors did not provide convincing evidence supporting inhibition of biofilm as defined by researchers in the field of Microbiology and Medicine.

R: We are not sure what is the logic behind this claim, given the present paper reports on straightforward evidence for biofilm inhibition. Mind the points below:

A. Inhibition of biofilm formation is shown by Figure 2, and Figure 5 illustrates strong inhibition of bacterial adhesion, which is an essential step for biofilm formation; 

B. The model bacterium tested in our manuscript is Pseudomonas fluorescens, which is a biofilm-secreting microbe that is well known from “the field of Microbiology and Medicine”, particularly in industrial food facilities as discussed at length in the manuscript.

Thus contrasting A and B to a lack of technical arguments for why (i) wouldn't results from Figures 2 and 5 be considered convincing, or (ii) whether the biofilm secreted by P. fluorescens wouldn't be a true biofilm, it is hard to understand what was the basis for the remark.  

Nevertheless, we thank you for your time and the opportunity to further explain the technical background of our manuscript and research.  

Reviewer 4 Report

Authors resolved remaining queries from me.

Author Response

Rev#4

Authors resolved remaining queries from me.

R: Thank your for your suggestions that improved our manuscript, and for the time invested!

Reviewer 5 Report

The authors have provided the description to my comments and improved the quality of figures in the  article . I do not have any further comments on the manuscript. 

Author Response

Rev#5

The authors have provided the description to my comments and improved the quality of figures in the  article. I do not have any further comments on the manuscript. 

R: Thank you for your insightful comments, and constructive suggestions!

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

This manuscript reports the use of solenopsins as biofilm inhibitors. The chemical structures of the four “alkaloids” are known to be 2-alkyl-5-methyl-piperidines. There isn’t anything new to be discovered for these 4 simple dialkyl-piperidine compounds. The authors showed inhibition of P. fluorecens growth in paper disk assay on LB agar. The dose ranges for these chemicals are similar to what would be expected for most simple organic amines or piperidines. The concentrations required to see growth inhibition for P. fluorescens was much higher than that of most antibiotics. The authors also indicated these chemical extracts were not able to block bacterial growth completely on the surface of two types of materials. The authors also showed these compounds are not amphipathic as would be expected for most surfactants.

The manuscript also has major weaknesses in missing proper positive controls. There are labels unexplained in all the graphs. All the methods for generating the graphs were not clearly described.The supplemental material has little relevance to the manuscript.

Author Response

“This manuscript reports the use of solenopsins as biofilm inhibitors. The chemical structures of the four “alkaloids” are known to be 2-alkyl-5-methyl-piperidines. There isn’t anything new to be discovered for these 4 simple dialkyl-piperidine compounds.”

R: We are sorry as Reviewer #1 seems to have missed a fundamental points: the chemical structure of the alkaloids prevalent in our samples (aka ‘solenopsins’) is 2,6-dialkyl-piperidines. In reality, there is a solidly expanding body of original research about the solenopsins, as summarised in Fox (2014), and recently exampled by Arbiser et al. (2017, 2018), Fox et al. (2019). These are reports on novel activity and facets of the solenopsins from spheres as distant as ecology and biotechnology.

“The authors showed inhibition of P. fluorecens [sic] growth in paper disk assay on LB agar. The concentrations required to see growth inhibition for P. fluorescens was [sic] much higher than that of most antibiotics.”

R: This is an intriguing point, thanks for bringing it up. In the absence of a reference to the claim about how much of another antibiotic would be necessary to create an inhibition halo around 6mm paper disks, we present our own, as follows. As mentioned per methods section, the paper disks were dipped in concentrations of solenopsins ranging 500–5,000 μg/mL. Based on lab estimations, saturating a paper disk takes up ca. 20 μl of solution, i.e. 5–100 μg/disk of solenopsins extract. Regarding antibiotics we took published references for compound amounts used in similar tests: “ampicillin (10 μg/disk), chloramphenicol (30 μg/disk), and streptomycin (10 μg/disk)” (Mith et al. 2014). The tested active doses are thus well within the same range of action of standard antibiotics used in similar assays. And that for a crude extract.

Therefore we disagree: the tested amounts are comparable in amounts employed for antibiotics in similar tests, reinforcing the potential of solenopsins as antibiotics originally demonstrated by Jouvenaz et al. (1972).

“The authors also indicated these chemical extracts were not able to block bacterial growth completely on the surface of two types of materials.”

R: Yes, we did not achieve complete inhibition. We do not think, however, that this demonstrates a lack of potential for the compounds: solenopsins are evidently active against biofilm formation, and within workable concentrations, as demonstrated above.

“The authors also showed these compounds are not amphipathic as would be expected for most surfactants.”

R: We are confused by this statement, as in our understanding, biosurfactants are actually typically amphipathic in organic chemistry. The term surfactant literally means “surface-active” which was exactly the chemical property for which solenopsins tested negative in this study. All amphipathic compounds are surface-active compounds, hence surfactants.

Another term for “amphipathic” would be “amphiphilic”, which perhaps would prove less confusing? We have thus decided to employ the word amphiphilic and amphophile in lieu of amphipathic in our manuscript — thank you for a suggestion.

“The manuscript also has major weaknesses in missing proper positive controls.”

R: The methods and assay designs relied on their context and published classical references, e.g. the paper-disk tests were designed based on Jouvenaz et al. (1972). Surface conditioning tests were made mimicking real applications. Unfortunately we had no compatible positive controls for surface conditioning with alkaloids, as this is a novel technology application. However, mind there are, actually, explicit positive controls for the surface-tension tests.

“There are labels unexplained in all the graphs.” — Another reviewer has identified one unexplained feature on one graph, which has now been corrected (see Reviewer#3). In case of any other pending issues, please indicate with specific details.

“All the methods for generating the graphs were not clearly described.The supplemental material has little relevance to the manuscript.”

R: I am sorry if perhaps the point was not made clear enough in the methods section or perhaps the file format not friendly. But the supplementary material consists precisely of the R code of data input and script behind all graphs. We have slightly increased emphasis on this disclosure to avoid a confusion among readers.

Arbiser et al. Scientific Reports, 2017; 7 (1) DOI: 10.1038/s41598-017-10580-y

Fox et al. Toxicon. 2019 Feb;158:77-83. doi: 10.1016/j.toxicon.2018.11.428

Jouvenaz et al. Antimicrob. Agents Chemother. 1972, 2(4):291. DOI: 10.1128/AAC.2.4.291

Karlsson et al. Vasc Cell. 2015 May 8;7:5. doi: 10.1186/s13221-015-0030-2

Mith et al. Food Sci Nutr. 2014 Jul;2(4):403-16. doi: 10.1002/fsn3.116

Reviewer 2 Report

The authors evaluate the use of venom alkaloids to inhibit the growth/accumulation of biofilms on polystyrene and stainless steel surfaces. Overall this study was straightforward and seems like it would be of wide interest to readers of Toxins and the medical/industrial fields. I particularly appreciated the inclusion of negative results.

Minor Concerns:

Is there a specific type of polystyrene tested? I'm not sure if there are variable types?

The authors should introduce Pseudomonas fluorescens at some point in the introduction, the reasons behind using it in this study is not made clear until the methods.

The authors should include some component of viability of the use of these for industrial or medical applications. How difficult is it to synthesize these? Would harvesting of fire ants be necessary (and potential biological control?). 

There are numerous grammar comments included in the attached pdf.

Comments for author File: Comments.pdf

Author Response

The authors evaluate the use of venom alkaloids to inhibit the growth/accumulation of biofilms on polystyrene and stainless steel surfaces. Overall this study was straightforward and seems like it would be of wide interest to readers of Toxins and the medical/industrial fields. I particularly appreciated the inclusion of negative results.

R: Thanks to Reviewer #2 for the kind comments.

Minor Concerns:

Is there a specific type of polystyrene tested? I'm not sure if there are variable types?

R: We have obtained the material purely defined as ‘polystyrene’ as described in the methods. To that matter we have added more specific details. We have further contacted the company and obtained no specific info on type of polysterene, excepting that it is the hard type.

The authors should introduce Pseudomonas fluorescens at some point in the introduction, the reasons behind using it in this study is not made clear until the methods.

R: Good point. We have added a paragraph on this point was applied as given in the appended revised version.

The authors should include some component of viability of the use of these for industrial or medical applications. How difficult is it to synthesize these? Would harvesting of fire ants be necessary (and potential biological control?).

R: There are in fact a number of synthesis methods published for solenopsins, however few companies currently sell these compounds. Collecting and killing fire ant nests where they are rated as invasive pests is feasible for obtaining the compounds in gram amounts (Fox et al. 2013) however it depends on special training and equipment. We have added a paragraph commenting in this direction as suggested.

Finally, we have perused the appended file with annotated comments and applied each and every modification as indicated. Thank you for all care taken with our manuscript.

Fox et al. Toxicon. 2013 Apr;65:5-8. doi: 10.1016/j.toxicon.2012.12.009

Reviewer 3 Report

Review: Fire Ant Venom Alkaloids Inhibit Biofilm Formation

Disclaimer: I am a venom researcher but not an expert in either alkaloids or biofilm formation.

General comments:

This manuscript describes antimicrobial and anti-biofilm activity of fire ant venom solenopsins. Strikingly, strong anti-biofilm activity is induced not only by adding solenopsins to living cells directly, but by conditioning surfaces with solenopsins that are then washed away with water. The authors note that this treatment did not alter the surface to make it amphipathic, and present empirical evidence in the Supplementary materials to back this statement up. Presumably, this is the way for the authors to argue that the molecular mechanism by which solenopsins act is not simply membrane disruption, but through some more specific pathway such as the activation of cell surface receptors or infiltration and signalling in the intracellular compartment. However, this is not clearly. Overall the manuscript contains important data and has been carefully prepared. My main comment is that the tests showing solenopsin treatment does not make the surface amphipathic are clearly important to the interpretation of the results and should appear in the main manuscript, in the Results section. They should not be put in the Supplementary Materials and only referred to for the first time in the Discussion. Also, if the authors are proposing that solenopsins act by another pathway other than disrupting membranes, they should clearly state that, e.g. “Since we found evidence that solenopsin treatment potently protected surfaces against biofilm formation, but not by inducing an amphipathic surface, we propose that solenopsin anti-biofilm activity is not mediated simply by amphipathic disruption of lipid membranes.” After revisions to address these comments and the minor comments below, I suggest the manuscript is suitable for publication.

Specific comments:

·      Line 76: I don’t think this is paradoxical

·      Figure 3 x-axis label: I don’t think you need the square brackets, or if you use them they should enclose ‘Solenopsins extract’

·      Line 93: Please italicize binomial name

·      Line 129: Steochiometry?

·      Line 136: Which is less than the effect reported in our study?

·      Line 149: This is a result and I think it should be reported in the Results section. For the Discussion, maybe discuss what this infers in terms of the molecular mechanism of antimicrobial action?

·      Line 160: According with should be According to

·      Line 160: Again, do not refer to this result for the first time in the Discussion. Also, if it is important to the way the main results of the paper are interpreted, why is it only Supplementary Material and not appear in the main document?

·      Table S1: What do a and b refer to in middle column?

Author Response

Disclaimer: I am a venom researcher but not an expert in either alkaloids or biofilm formation.

General comments:

This manuscript describes antimicrobial and anti-biofilm activity of fire ant venom solenopsins. Strikingly, strong anti-biofilm activity is induced not only by adding solenopsins to living cells directly, but by conditioning surfaces with solenopsins that are then washed away with water. The authors note that this treatment did not alter the surface to make it amphipathic, and present empirical evidence in the Supplementary materials to back this statement up. Presumably, this is the way for the authors to argue that the molecular mechanism by which solenopsins act is not simply membrane disruption, but through some more specific pathway such as the activation of cell surface receptors or infiltration and signalling in the intracellular compartment. However, this is not clearly.

R: Thanks for a description of the manuscript. The interpretation is overall right.

Overall the manuscript contains important data and has been carefully prepared. My main comment is that the tests showing solenopsin treatment does not make the surface amphipathic are clearly important to the interpretation of the results and should appear in the main manuscript, in the Results section. They should not be put in the Supplementary Materials and only referred to for the first time in the Discussion. Also, if the authors are proposing that solenopsins act by another pathway other than disrupting membranes, they should clearly state that, e.g. “Since we found evidence that solenopsin treatment potently protected surfaces against biofilm formation, but not by inducing an amphipathic surface, we propose that solenopsin anti-biofilm activity is not mediated simply by amphipathic disruption of lipid membranes.”

R: Thanks for sound comments. We agreed in incorporating the Appendix into the main manuscript file and amending the conclusions as suggested. We had originally kept it separately as the test results proved clearly negative.

After revisions to address these comments and the minor comments below, I suggest the manuscript is suitable for publication.

Specific comments:

• Line 76: I don’t think this is paradoxical

R: Thanks, we removed the comment.

• Figure 3 x-axis label: I don’t think you need the square brackets, or if you use them they should enclose ‘Solenopsins extract’

R: Agreed, we have removed the feature.

• Line 93: Please italicize binomial name

R: Thanks, done.

• Line 129: Steochiometry?

R: We originally meant “stoichiometry” but now decided this was not the best term. Readjusted as indicated.

• Line 136: Which is less than the effect reported in our study?

R: Sorry if not clear. We have limited our comments to Jouvenaz et al. tests using gram-negative bacteria, which are more resistant. Out of >10 species tested, they observed limited inhibition haloes with 4 species. We have added further details on their species and tested concentration relative to our results, directly in the narrative in this passage. Thanks for pointing out.

• Line 149: This is a result and I think it should be reported in the Results section. For the Discussion, maybe discuss what this infers in terms of the molecular mechanism of antimicrobial action?

R: Yes, we have agreed to include it in the main manuscript. However, as it is a direct negative result of a specific molecular property, it provides no other clue for the mechanism of action excepting that it is not related to some surface-active physical interaction as suggested by some authors.

• Line 160: According with should be According to

R: Fixed there and elsewhere, thanks!

• Line 160: Again, do not refer to this result for the first time in the Discussion. Also, if it is important to the way the main results of the paper are interpreted, why is it only Supplementary Material and not appear in the main document?

R: Yes, we have agreed to include it in the main manuscript, as discussed above. Thanks!

• Table S1: What do a and b refer to in middle column?

R: Thanks for pointing out; an explanation was added directly to the figure legend.