Antimicrobial Use and Resistance in Plant Agriculture: A One Health Perspective

Round 1

Reviewer 1 Report

Revised version should be acceptable for publication.

Author Response

We would like to take this opportunity to thank the reviewer for his work, and positive feedback. 

Reviewer 2 Report

The authors are commended for their significant improvements of the manuscript.  

Specific comments:

1) The sentences from line 279 to 282 need some minor edits for clarity.  

It is not clear what is meant by a 'pesticide that contains organisms with AMR determinants.'  Do you mean that pesticides (e.g. copper) may enrich environmental populations of microbes harboring AMR determinants, as suggested in the article by Heuer, or are you referring to low-quality fish- or animal-feed grade antibiotics contaminated with Streptomycetes DNA (e.g. Webb and Davies, 1993, Antimicrob. Agents Chemother. 37:2379–2384.)?  The inclusion of biocontrol organisms in this sentence (line 279) could be removed because that point is addressed later in Line 383 (reference 56). 

2) Line 302: Delete "an allergy to streptomycin" or replace with the text from citation #43 "known anaphylaxis to penicillin and IgE-mediated cow's milk allergy."  The authors speculated that she may have been sensitized by "consumption of residues in food products" such as meat and dairy products, but she didn't have an allergy to streptomycin. An accurate description is recommended because access to the article is blocked by a pay-wall. 

3) Line 824: Change to past tense, ...penicillin 'was' explored...   Actually penicillin was 'explored' for fire blight control in the 1950's, but these antibiotics haven't been approved for plant disease control. 

4)  Figure 3: The Y-axis is incomplete.  Could the figure be enlarged so that each of the bars are associated with a country?

5) The following are minor typographical errors: 

Line 166: typo: also

Lines 679-698.  Gentamicin misspelled.

The Literature cited section should be scanned for typos (Reference 37: typos ‘Effect’ and ‘different’) and formats (e.g. Reference 56: change authors to Kang, Y., et al…).

Author Response

Specific comments:

1) The sentences from line 279 to 282 need some minor edits for clarity. 

It is not clear what is meant by a 'pesticide that contains organisms with AMR determinants.'  Do you mean that pesticides (e.g. copper) may enrich environmental populations of microbes harboring AMR determinants, as suggested in the article by Heuer, or are you referring to low-quality fish- or animal-feed grade antibiotics contaminated with Streptomycetes DNA (e.g. Webb and Davies, 1993, Antimicrob. Agents Chemother. 37:2379–2384.)?  The inclusion of biocontrol organisms in this sentence (line 279) could be removed because that point is addressed later in Line 383 (reference 56).

As suggested, the inclusion of biocontrol organisms in the “first” (earlier) sentence was removed.

2) Line 302: Delete "an allergy to streptomycin" or replace with the text from citation #43 "known anaphylaxis to penicillin and IgE-mediated cow's milk allergy."  The authors speculated that she may have been sensitized by "consumption of residues in food products" such as meat and dairy products, but she didn't have an allergy to streptomycin. An accurate description is recommended because access to the article is blocked by a pay-wall.

As suggested, “with an allergy to…” was deleted.

3) Line 824: Change to past tense,...penicillin 'was' explored...   Actually penicillin was 'explored' for fire blight control in the 1950's, but these antibiotics haven't been approved for plant disease control.

Correct. We edited the sentence in line 512, mentioning that there is renewed interest in the use of penicillin for the treatment of plants.

4)  Figure 3: The Y-axis is incomplete.  Could the figure be enlarged so that each of the bars are associated with a country?

Done.

5) The following are minor typographical errors:

Line 166: typo: also

Fixed.

Lines 679-698.  Gentamicin misspelled.

Fixed.

The Literature cited section should be scanned for typos (Reference 37: typos ‘Effect’ and ‘different’) and formats (e.g. Reference 56: change authors to Kang, Y., et al…).

Done. Names of microorganisms were italicized and titles converted to sentence case.  

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

The authors present an excellent, comprehensive review on antimicrobial use and resistance in plant agriculture, including the use and impacts of antibiotics, heavy metals such as copper, and fungicides. Very well done.

 

As expected in a manuscript with scope so varied and extensive, I found a few minor errors, typos etc which are detailed below. Otherwise, this is a needed current document of the resistance problem in plant agriculture.

 

Minor comments

 

Pg 1, ln 12 – suggest adding “Likewise,” before The recent rise …

 

Pg 1, lns 43-44 – suggest adding a reference here (Stuart Levy review possibly). Also, it is important to state that this one pesticide selecting for multiple resistance is observed in clinical medicine. I don’t know of any examples of this in plant agriculture situations. Thus, I believe that the authors should make this distinction since, as written, this could lead readers to believe that this selection process is ongoing in plant situations when there isn’t real evidence that it is.

 

Pg 2, lns 52-58 – same premise here, needs a citation and the statement that this is known to occur in clinical medicine situations and not in plant agriculture situations.

 

Pg 4, ln 166 – ref 16 is to a Burkholderia paper, I didn’t see a reference nearby on Aspergillus.

 

Pg 5, ln 203 – phyllosphere, typo

 

Pg 8, ln 385 – the strA-strB gene cassette is carried within the Tn3-family transposon Tn5393. There is no integron element located within this transposon; Tn5393 is a classic Tn3- type transposon with a tnpA (transposase), tnpR (resolvase), res site, and strA-strB located within inverted repeats.

            This sentence should be corrected and the next sentence on integrons should be deleted as that story is separate from the Tn5393 story.

 

            Streptomycin resistance associated with an integron has been characterized in Xanthomonas oryzae from China, but I believe that that is the only occurrence reported for plant pathogens. Almost everything else from plant pathogens is the association with strA-strB on Tn5393.

 

Pg 8, ln 387 – sulII, not sulI is the gene linked with strA-strB. A better reference for this is the review Sundin and Bender, 1996, Mol Ecol 5:133-143.

 

Table 3 – there are two references to Walsh’s work (ref 100) in this table, although the first entry says the first author is Tolba.

 

Pg 13, ln 568 – this sentence sounds like it is referring to Behlau’s work, but reference 123 is to an Aspergillus paper.

 

Pg 16, ln 702 – regarding fungicide resistance, the concern is selection of resistance in potential human pathogenic strains correct? Not the transfer of resistance, as I’m not sure that this has been demonstrated to occur in these fungi. Also, “development” should be changed to “evolution”.

 

Author information is incorrect for a couple of references:

 

62, should be Forster, H.

64, should be Xu, Y.

100, should be Walsh, F.

Author Response

The authors present an excellent, comprehensive review on antimicrobial use and resistance in plant agriculture, including the use and impacts of antibiotics, heavy metals such as copper, and fungicides. Very well done.

We are thankful for the positive feedback.

As expected in a manuscript with scope so varied and extensive, I found a few minor errors, typos etc which are detailed below. Otherwise, this is a needed current document of the resistance problem in plant agriculture.

 Responses bellow.

Minor comments

 

Pg 1, ln 12 – suggest adding “Likewise,” before The recent rise …

Edited as suggested.

Pg 1, lns 43-44 – suggest adding a reference here (Stuart Levy review possibly). Also, it is important to state that this one pesticide selecting for multiple resistance is observed in clinical medicine. I don’t know of any examples of this in plant agriculture situations. Thus, I believe that the authors should make this distinction since, as written, this could lead readers to believe that this selection process is ongoing in plant situations when there isn’t real evidence that it is.

As suggested, a reference has been added.

 Pg 2, lns 52-58 – same premise here, needs a citation and the statement that this is known to occur in clinical medicine situations and not in plant agriculture situations.

Examples are now provided of co and cross resistance in bacteria in plant production environment.

Pg 4, ln 166 – ref 16 is to a Burkholderia paper, I didn’t see a reference nearby on Aspergillus.

Reference fixed, Latge and Camilos.

Pg 5, ln 203 – phyllosphere, typo

Fixed.

Pg 8, ln 385 – the strA-strB gene cassette is carried within the Tn3-family transposon Tn5393. There is no integron element located within this transposon; Tn5393 is a classic Tn3- type transposon with a tnpA (transposase), tnpR (resolvase), res site, and strA-strB located within inverted repeats.

            This sentence should be corrected and the next sentence on integrons should be deleted as that story is separate from the Tn5393 story.

 

            Streptomycin resistance associated with an integron has been characterized in Xanthomonas oryzae from China, but I believe that that is the only occurrence reported for plant pathogens. Almost everything else from plant pathogens is the association with strA-strB on Tn5393.

 Edited as suggested by the reviewer.

Pg 8, ln 387 – sulII, not sulI is the gene linked with strA-strB. A better reference for this is the review Sundin and Bender, 1996, Mol Ecol 5:133-143.

Reference added:

Sundin, G. and C. Bender, Dissemination of the strA‐strB streptomycin‐resistance genes among commensal and pathogenic bacteria from humans, animals, and plants. Molecular ecology, 1996. 5(1): p. 133-143.

Table 3 – there are two references to Walsh’s work (ref 100) in this table, although the first entry says the first author is Tolba.

Fixed.

Pg 13, ln 568 – this sentence sounds like it is referring to Behlau’s work, but reference 123 is to an Aspergillus paper.

Reference has been replaced.

Pg 16, ln 702 – regarding fungicide resistance, the concern is selection of resistance in potential human pathogenic strains correct? Not the transfer of resistance, as I’m not sure that this has been demonstrated to occur in these fungi. Also, “development” should be changed to “evolution”.

 Edited as suggested.

Author information is incorrect for a couple of references:

 

62, should be Forster, H.

Fixed.

64, should be Xu, Y.

Fixed.

100, should be Walsh, F.

Fixed.

Reviewer 2 Report

The review manuscript called ‘Antimicrobial use and resistance in plant agriculture: A One Health perspective’ by Miller, Ferreira, and Le Jeune is a summary of antimicrobial and select antifungal compounds that are used in plant production and possible linkages between animal, plant, and environmental sources of AMR.  These subjects have been covered in several other review articles.  The strength of this manuscript are sections 5.1 and 5.2 which is a clear call for worldwide data on pesticide use and best practices.

Comments:

• Suggest spelling out PPP in the document.  It is defined on page 3 of the manuscript and is not used again until page 17.  Because it is only used 6 times in the document, I recommend writing ‘plant protection products’ or something similar instead of the abbreviation.
• The Literature Cited list ended at reference #133.  Consequently, no comments could be made about the supporting papers cited after page 15 (line 634) of the manuscript.
• The definition of acquired resistance “acquired resistance develops as a result of genetic changes or exchanges following is random mutation and horizontal gene transfer, respectively“ is very broad and uncommon (line 102).  To my understanding, acquired resistance does not include resistance due to spontaneous mutation in a chromosomal gene, such as the common single nucleotide polymorphism in rpsL which confers streptomycin resistance and is not considered transferrable. 
• What is the purpose of the two sentences in beginning at line 214? To quote:  ‘Moreover, some researchers have explored the use of antimicrobial washes and packaging, including the use of copper-containing products, penicillins, tetracyclines and ciprofloxacin, as methods to control post-harvest losses in vegetables [30]. Although these studies were experimental, it is possible that some processors are already including these approaches to control losses in the absence of cold chain control.’    Do the authors have evidence that these materials that were examined in 2012 are being used for packaging in India?  If so, which materials?  Why wasn’t mercuric chloride included in the list?  Is the message that we should be leery of packaged produce from LMIC countries?  Please delete or clarify the point that you are trying to make with these sentences.
• Recommend removing Lines 228 to 231 that refer to an article (reference 32 and a related article) about a child in Canada that experienced anaphylactic shock within several hours of consuming a slice of blueberry tart. “For example, in 1999, a young child with an allergy to streptomycin was rushed to the emergency room in anaphylactic shock [32]. The source of the allergen was eventually attributed to blueberries used in a pie that was consumed.”  The article states that they were unable to detect streptomycin in the pie materials.  They found that the pie filling inhibited bacterial growth in a plate assay.  They assumed that the antibiosis was due to streptomycin because streptomycin is used on fruits. Because blueberries are small fruits, they must have been treated with the antibiotic. But they were apparently unaware that streptomycin is not labeled for blueberries in Canada.  Additionally, streptomycin is ineffective as an oral antibiotic but is administered intramuscularly. Nonetheless, they did report that after injecting the child with increasing doses of streptomycin, she eventually experienced anaphylactic shock. It sounded torturous. Overall, the article does not provide a clear case of agricultural use (or possible misuse) of an antibiotic was responsible for the medical emergency experienced by the child.
• Recommend expanding Line 231 to 232 “Several species of plants can take up and concentrate antimicrobials, further exacerbating the problem [33-36].”  Are the plants of concern mainly leafy greens and root crops?  Or do these findings also apply to perennial crops and grains?
• In line 277, the authors state “Biocontrol agents intentionally added to crops to control diseases may harbor antimicrobial resistance genes and serve as a reservoir for transmission (49).”  The reference by Losasso et al did not examine biocontrol agents as reservoirs for AMR transmission to humans. They examined samples from omnivores, vegans, and vegetarians, diets and found that “tetA was frequently isolated among the tested samples without any relationship with the diet” and that there is an “involvement of animal-derived food in promoting the spread and stabilization of ARGs within the microbial community of the human gut.”
• For readability, consider combining the two paragraphs from lines 279 to 296. 
• Line 398: typo ‘aminoglycocides’
• Aren’t nystatin and pimaricin considered GRAS compounds? (line 604). They are commonly added as food preservatives for processed dairy and meat.
• Reference for instability of polyene macrolide resistance? (line 617).
• Line 641, consider changing ‘agents’ to ‘chemicals’
• Reference for line 656 about geraniol inhibition of efflux pumps?
• Line 665, Typo—add italics for Aspergillus nidulans.
• Line 824, suggest changing ‘preventing’ to ‘mitigating.’

Author Response

The review manuscript called ‘Antimicrobial use and resistance in plant agriculture: A One Health perspective’ by Miller, Ferreira, and Le Jeune is a summary of antimicrobial and select antifungal compounds that are used in plant production and possible linkages between animal, plant, and environmental sources of AMR.  These subjects have been covered in several other review articles.  The strength of this manuscript are sections 5.1 and 5.2 which is a clear call for worldwide data on pesticide use and best practices.

Comments:

• Suggest spelling out PPP in the document.  It is defined on page 3 of the manuscript and is not used again until page 17.  Because it is only used 6 times in the document, I recommend writing ‘plant protection products’ or something similar instead of the abbreviation.

Edited as suggested.

• The Literature Cited list ended at reference #133.  Consequently, no comments could be made about the supporting papers cited after page 15 (line 634) of the manuscript.

All references have been updated.

• The definition of acquired resistance “acquired resistance develops as a result of genetic changes or exchanges following is random mutation and horizontal gene transfer, respectively“ is very broad and uncommon (line 102).  To my understanding, acquired resistance does not include resistance due to spontaneous mutation in a chromosomal gene, such as the common single nucleotide polymorphism in rpsL which confers streptomycin resistance and is not considered transferrable. 

It is our understanding that “acquired resistance” includes that develop de novo as a result of mutation.  The reviewer is correct that this type of resistance is not horizontally transmissible. It may be transferable vertically.  No change has been made to this sentence, but we have added two references to support the satement. 

MacLean, R. C. and A. S. Millan (2019). "The evolution of antibiotic resistance." Science 365(6458): 1082-1083. 

Cloeckaert A, Zygmunt MS, Doublet B. Editorial: Genetics of Acquired Antimicrobial Resistance in Animal and Zoonotic Pathogens. Front Microbiol. 2017;8:2428. Published 2017 Dec 5. doi:10.3389/fmicb.2017.02428 

• What is the purpose of the two sentences in beginning at line 214? To quote:  ‘Moreover, some researchers have explored the use of antimicrobial washes and packaging, including the use of copper-containing products, penicillins, tetracyclines and ciprofloxacin, as methods to control post-harvest losses in vegetables [30]. Although these studies were experimental, it is possible that some processors are already including these approaches to control losses in the absence of cold chain control.’    Do the authors have evidence that these materials that were examined in 2012 are being used for packaging in India?  If so, which materials?  Why wasn’t mercuric chloride included in the list?  Is the message that we should be leery of packaged produce from LMIC countries?  Please delete or clarify the point that you are trying to make with these sentences.

Fungicides are widely applied post-harvest to fruits and vegetables to extend shelf life and reduce post-harvest loss. Post-harvest application of antimicrobials.

• Recommend removing Lines 228 to 231 that refer to an article (reference 32 and a related article) about a child in Canada that experienced anaphylactic shock within several hours of consuming a slice of blueberry tart. “For example, in 1999, a young child with an allergy to streptomycin was rushed to the emergency room in anaphylactic shock [32]. The source of the allergen was eventually attributed to blueberries used in a pie that was consumed.”  The article states that they were unable to detect streptomycin in the pie materials.  They found that the pie filling inhibited bacterial growth in a plate assay.  They assumed that the antibiosis was due to streptomycin because streptomycin is used on fruits. Because blueberries are small fruits, they must have been treated with the antibiotic. But they were apparently unaware that streptomycin is not labeled for blueberries in Canada.  Additionally, streptomycin is ineffective as an oral antibiotic but is administered intramuscularly. Nonetheless, they did report that after injecting the child with increasing doses of streptomycin, she eventually experienced anaphylactic shock. It sounded torturous. Overall, the article does not provide a clear case of agricultural use (or possible misuse) of an antibiotic was responsible for the medical emergency experienced by the child.

At the reviewer’s suggestion, the sentence has be reworded to decrease the perceived certainty of the strength of association between the consumption of the berries and the disease outcome.

• Recommend expanding Line 231 to 232 “Several species of plants can take up and concentrate antimicrobials, further exacerbating the problem [33-36].”  Are the plants of concern mainly leafy greens and root crops?  Or do these findings also apply to perennial crops and grains?

Edited as: “If used improperly, antimicrobial residues may remain on fruits at the time of consumption at levels that poses a threat to human health.   Although rare, antimicrobial residues in food can also cause life-threating allergic reactions among susceptible individuals [40,41]”.

• In line 277, the authors state “Biocontrol agents intentionally added to crops to control diseases may harbor antimicrobial resistance genes and serve as a reservoir for transmission (49).”  The reference by Losasso et al did not examine biocontrol agents as reservoirs for AMR transmission to humans. They examined samples from omnivores, vegans, and vegetarians, diets and found that “tetA was frequently isolated among the tested samples without any relationship with the diet” and that there is an “involvement of animal-derived food in promoting the spread and stabilization of ARGs within the microbial community of the human gut.”

Replaced with appropriate reference.

• For readability, consider combining the two paragraphs from lines 279 to 296. 

Edited as suggested.

• Line 398: typo ‘aminoglycocides’

Fixed.

• Aren’t nystatin and pimaricin considered GRAS compounds? (line 604). They are commonly added as food preservatives for processed dairy and meat.

Related reference has been updated.

• Reference for instability of polyene macrolide resistance? (line 617).

Added : Ahmed M.Z. et al. (2021) Antifungal Drugs: Mechanism of Action and Resistance. In: Ahmed S., Chandra Ojha S., Najam-ul-Haq M., Younus M., Hashmi M.Z. (eds) Biochemistry of Drug Resistance. Springer, Cham. https://doi.org/10.1007/978-3-030-76320-6_5

• Line 641, consider changing ‘agents’ to ‘chemicals’

Edited as suggested.

• Reference for line 656 about geraniol inhibition of efflux pumps?

Reference [149] has been added.

• Line 665, Typo—add italics for Aspergillus nidulans.

Fixed.

• Line 824, suggest changing ‘preventing’ to ‘mitigating.’

Edited as suggested.