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Article
Peer-Review Record

Synthetic Human Lactoferrin Peptide hLF(1-11) Shows Antifungal Activity and Synergism with Fluconazole and Anidulafungin Towards Candida albicans and Various Non-Albicans Candida Species, Including Candidozyma auris

Antibiotics 2025, 14(7), 671; https://doi.org/10.3390/antibiotics14070671
by Carlo Brouwer 1,2, Youp van der Linden 3, Maria Rios Carrasco 3, Saleh Alwasel 2, Tarad Abalkhail 2, Fatimah O. Al-Otibi 2, Teun Boekhout 2,3 and Mick M. Welling 4,*
Reviewer 1: Anonymous
Reviewer 2:
Reviewer 3: Anonymous
Antibiotics 2025, 14(7), 671; https://doi.org/10.3390/antibiotics14070671
Submission received: 21 May 2025 / Revised: 23 June 2025 / Accepted: 28 June 2025 / Published: 2 July 2025
(This article belongs to the Special Issue Bioactive Peptides and Their Antibiotic Activity)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present their results on the susceptibility testing and potential added value of AMP (e.g. lactoferrin) to treat Candida infections, specifically antifungal resistance Candida infections. 

That lactoferrin has antifungal activities is not new, but to assess if it could be of value in the combination with currently used antifungals to improve the treatment of drug-resistant Candida infections such as Cz. auris, is novel. 

The manuscript suffers from not being focussed, only descriptive data, results are not well organized, and numerous erros and incorrect statements throughout the manuscript. Therefore it is difficult to distil the main message from this work. 

Specific comments: 

The title is incorrect, synergisms was not tested by using amphotericin B.

First sentence abstract, not the Cz. auris as such, but diseases caused by it are associated with mortality

Decimals should be with written with a ‘full stop’ and not a ‘comma’

‘intervention group’ to indicate the Cz. auris strains reads a bit odd

Be consequent to use italic font for names of fungi

Add flucytosine to line 53-54

In the introduction (line 58-72) the authors should refer to the increase in invasive infections observed globally with a huge burden in Africa and India.

Line 75, Cz. auris infections are not being treated with azole, rephrase.

Resistance to Cz. auris is not due to poor antifungal stewardship (line 77), needs to be corrected.

Cz. auris is intrinsically resistant, so line 79-81 need to be rephrased.

Please detail in the introduction the mechanism of action of lactoferrin (line 90-92).

It is a bit surprising why hLF was not tested in the combination with an echinocandin, can the authors provide the argumentation for that and add this to the last sentences of the introduction.

Table 1, I only count 16 species and not 18?

Tables, provide the units/entities for the MIC results, and no more than 2 decimals are recommended.

Why are references added to the tables? The origin is already provided of the isolates used.

Table 2 is redundant and can be removed.

Table 3 is showing strains from 5 countries, not 6, and why didn’t the authors provide information on which clade these Cz. auris belong to?

Table 3, you either present mean and st.dev. OR median and range; in this table presenting the range doesn’t make much sense for the individual isolates.

Line 123-128 need to be rephrased, sentences are not running well and it is unclear what is ‘AND’

In the introduction there was no mentioning of anidulafungin being tested.

Table 4, the top row is not readable.

Line 139, 17 strains of 16 species were tested, not 18 strains of 17 species.

Methods:

I don’t understand the sentence stated for subheading 4.1

Not sure why line 263-275 are in italic font?

Material & methods not formatted well.

4.5: the authors can not refer to previous performed susceptibility testing for the single agents, and now comparing their synergy data including those agents to that reported by others.

Line 315-323 is a repeat of Line 282-292

How was determined which concentration to use for the hLF?

Line 335-337, can the authors refer to a paper in which this method is published and validated? I am not familiar with this methodology.

The discussion is not focussed and should be more concise and in line with the research done. To add a conclusion after the methods section seems a bit odd. 

Comments on the Quality of English Language

can be improved

Author Response

To the Editor:

Reply: We thank the editor for the opportunity to revise our manuscript and resubmit it for review in Antibiotics. The valuable comments and suggestions we received from the reviewers are much appreciated and have significantly improved the revised version of the manuscript. We have fully considered all points and have changed the manuscript accordingly. Below, you will find our point-by-point responses to the questions and remarks of all reviewers.

 

Reviewer 1

Comments and Suggestions for Authors

The authors present their results on the susceptibility testing and potential added value of AMP (e.g. lactoferrin) to treat Candida infections, specifically antifungal resistance Candida infections. That lactoferrin has antifungal activities is not new, but to assess if it could be of value in the combination with currently used antifungals to improve the treatment of drug-resistant Candida infections such as Cz. auris, is novel.

The manuscript suffers from not being focussed, only descriptive data, results are not well organized, and numerous erros and incorrect statements throughout the manuscript. Therefore it is difficult to distil the main message from this work.

Reply: We thank the reviewer for acknowledging our work. Concerning the comments on our manuscript, we changed them according to the suggestions made by the reviewer. We apologize for all our inconsistencies throughout the manuscript.

Specific comments:

The title is incorrect, synergisms was not tested by using amphotericin B.

Reply: we corrected the title of the document. Synergism was tested against anidulafungin and not amphotericin B. We apologize for the inconvenience.

First sentence abstract, not the Cz. auris as such, but diseases caused by it are associated with mortality

Reply: We thank the reviewer for this suggestion, we corrected this sentence.

Decimals should be with written with a 'full stop' and not a 'comma'

Reply: We corrected this issue throughout the entire manuscript. We apologize for the inconvenience.

'intervention group' to indicate the Cz. auris strains reads a bit odd

Reply: We corrected this into the treatment cohort. We apologize for the inconvenience.

Be consequent to use italic font for names of fungi

Reply: We corrected this throughout the entire manuscript. We apologize for the inconsistency.

 

 

Add flucytosine to line 53-54

Reply: We thank the reviewer for this suggestion and have corrected this sentence accordingly. There are five classes of antifungal agents: triazoles, echinocandins, polyenes, flucytosine, and allylamine. Flucytosine (5-fluorocytosine or 5-FC) is a systemic antifungal antimetabolite agent belonging to a class of its own.

In the introduction (line 58-72) the authors should refer to the increase in invasive infections observed globally with a huge burden in Africa and India.

Reply:  We thank the reviewer for this suggestion. We corrected this sentence.

Cz. auris has been detected in 61 nations spanning six continents. Reports indicate its presence in all subregions of Africa, with over 2500 cases. Significant outbreaks have been noted in specific healthcare environments in South Africa and India, Cz. auris has been linked to as much as 25% and 40% of candidemia occurrences.

Bhargava, A.; Klamer, K.; Sharma, M.; Ortiz, D.; Saravolatz, L. Candida auris: A Continuing Threat. Microorganisms 2025, 13, 652. https://doi.org/10.3390/microorganisms13030652.

Line 75, Cz. auris infections are not being treated with azole, rephrase.

Reply: We corrected this sentence. We apologize for the inconvenience. Treatment of infections with Cz. auris is challenging. Symptomatic infections are treated preferably with echinocandins, such as caspofungin, because of the relatively mild side effects.

Resistance to Cz. auris is not due to poor antifungal stewardship (line 77), needs to be corrected.

Reply: We corrected this sentence. We apologize for the inconvenience.

Resistance of Cz. auris to commonly used antifungal drugs is caused by a variety of factors, such as mutations in the ERG11 gene ( increased copy number of the ERG11 gene), overexpression of efflux pumps such as CDR1 and MDR1 and excessive and prophylactic use of antifungal agents.

Cz. auris is intrinsically resistant, so line 79-81 need to be rephrased.

Reply: We corrected this sentence. We apologize for the inconvenience.

An increasing problem is the development of drug resistance, Cz. auris is recognized for its intrinsic resistance to a broad spectrum of antifungal agents, encompassing azole, echinocandin, and polyene categories. This resistance renders it a challenging pathogen to manage against fluconazole in combination with echinocandins and/or the more toxic polyene amphotericin B.

Please detail in the introduction the mechanism of action of lactoferrin (line 90-92).

Reply: We corrected this sentence. We apologize for the inconvenience.

The hLF1-11 peptide's antifungal mechanism involves disrupting fungal cell membranes and targeting mitochondrial function, leading to cell death. It interacts with and enhances immune cell function, fostering a more vigorous antifungal response.

It is a bit surprising why hLF was not tested in the combination with an echinocandin, can the authors provide the argumentation for that and add this to the last sentences of the introduction.

Reply: We appreciate the reviewer's suggestion. However, anidulafungin is generally considered a first-line treatment in our hospital for Cz. auris infections, and its effectiveness can be enhanced when combined with other antifungals like fluconazole.

Table 1, I only count 16 species and not 18?

Reply: The reviewer is correct. We corrected this throughout the entire manuscript. We apologize for the inconvenience.

Tables, provide the units/entities for the MIC results, and no more than 2 decimals are recommended.

Reply: We thank the reviewer for this suggestion We corrected this in all tables. We apologize for the inconvenience.

Why are references added to the tables? The origin is already provided of the isolates used.

Reply: Many isolates' taxonomy (nomenclature, classification) has changed or been adapted in recent years. Not everyone is aware of these new names. It is, therefore, a helpful reminder for novice readers to mention the references.

Table 2 is redundant and can be removed.

Reply: We disagree with the reviewer's remark. We indicate that this control peptide, where substitution of 4 amino acids (alanine replacements) in the strains used in this manuscript, does not give signs of growth inhibition. Structure relationship studies have shown that strains can show different activity against substitutions.

Table 3 is showing strains from 5 countries, not 6, and why didn't the authors provide information on which clade these Cz. auris belong to?

Reply: The reviewer is correct. In the first version, six countries were represented. Because the exact origin of a strain could not be retrieved, it was removed from the table. We apologize for the inconvenience.

Table 3, you either present mean and st.dev. OR median and range; in this table presenting the range doesn't make much sense for the individual isolates.

Reply: We thank the reviewer for this suggestion We corrected this in the  table. We apologize for the inconvenience.

Line 123-128 need to be rephrased, sentences are not running well and it is unclear what is 'AND'

Reply: We changed the abbreviation of anidulafungin from AND to ANI throughout the entire manuscript. Furthermore, six strains of Cz. auris (MIC values between 12.5-25 μg/mL) and three strains of C. parapsilosis (MIC values between 25-50 μg/mL). This piece of text has been removed as it does not contribute to the explanation. We apologize for the inconvenience.

In the introduction there was no mentioning of anidulafungin being tested.

Reply: We corrected this sentence. We apologize for the inconvenience.

Table 4, the top row is not readable.

Reply: We corrected the table. We apologize for the inconvenience.

Line 139, 17 strains of 16 species were tested, not 18 strains of 17 species.

Reply: The reviewer is correct. We corrected this. We apologize for the inconvenience.

Methods:

I don't understand the sentence stated for subheading 4.1

Reply:  We corrected this sentence. All chemicals used for the experiments described in this manuscript were purchased from commercial sources and were used without further purification. We apologize for the inconvenience.

Not sure why line 263-275 are in italic font?

Reply: The reviewer is correct. We corrected this. We apologize for the inconvenience.

Material & methods not formatted well.

Reply: The reviewer is correct. We corrected this issue. We apologize for the inconvenience.

4.5: the authors can not refer to previous performed susceptibility testing for the single agents, and now comparing their synergy data including those agents to that reported by others.

Reply: We corrected this. The strains shown in Table 4 were tested for susceptibility to fluconazole (FLU) and anidulafungin (ANI) using a modified EUCAST broth protocol described in the Discussion section. We apologize for the inconvenience.

Line 315-323 is a repeat of Line 282-292

Reply: The reviewer is correct. We corrected this and removed repeated lines. We apologize for the inconvenience

How was determined which concentration to use for the hLF?

Reply: Many papers have already extensively determined the concentration range of the hLF1-11 peptide and tested it against many different strains. This is also referred to in the revised manuscript. See references by Brouwer et al., Lupetti et al., Nibbering et al., (References 25, 26, 33, 35, 36, and 37).

Line 335-337, can the authors refer to a paper in which this method is published and validated? I am not familiar with this methodology.

Reply:  This protocol for drug combination studies is described in the following paper: Bellio P, Fagnani L, Nazzicone L, Celenza G. New and simplified method for drug combination studies by checkerboard assay. MethodsX. 2021 Oct 11;8:101543. doi: 10.1016/j.mex.2021.101543. PMID: 34754811; PMCID: PMC8563647.

The discussion is not focussed and should be more concise and in line with the research done. To add a conclusion after the methods section seems a bit odd.

Reply: We thank the reviewer for this suggestion. We corrected this accordingly in the revised manuscript. We apologize for the inconvenience.

Comments on the Quality of English Language can be improved

Reply: The manuscript has been extensively checked for English grammar.

Reviewer 2 Report

Comments and Suggestions for Authors

Excellent paper. Exciting to have possibility of new antifungals from the AMP region of hLF. 

  1. The one major clarification needed is that in the title and throughout the manuscript the authors mention that amphotericin B is being tested for synergy with hLF1-11 (title, lines 18,25, 256, and 385) but in Figure 4 it appears that anidulafungin (and fluconaozle) were evaluated for syngerism. It appears anidulafungin should replace amphotericin B in the title, lines 18,25, 256, and 385. Please clarify. 
  2. Recommend to modify in the abstract the sentence starting at line 14, that hLF1-11 is  potential new therapy against all yeast:
    "A synthetic peptide, consisting of 11 amino acids of human lactoferrin (hLF1-11) offers a new therapy that is active against Candida albicans, non-albicans Candida yeasts, as well as Cz. auris." 
  3. "AND" is a confusing abbreviation to use for anidulafungin. Is there another one?
  4. Table 4. Horizontal top row confusing and not lining up. Would recommend having text vertically instead of horizontal.  Also this particularly confusing with the abbreviation of "AND" as it makes one think it is one agent and another agent vs meaning anidulafungin if that makes sense. 
  5. In discussion might be of interest to have one sentence if it is a future possibility to use this AMP region as a potential therapy for specific bacterial pathogens.  
  6. In discussion might be of interest to have one sentence if it is a future possibility to use the other AMP region as a potential therapy.  

Author Response

Reviewer 2

Comments and Suggestions for Authors

Excellent paper. Exciting to have possibility of new antifungals from the AMP region of hLF.

Reply: We thank the reviewer for acknowledging our work. We changed the revised manuscript according to the suggestions.

The one major clarification needed is that in the title and throughout the manuscript, the authors mention that amphotericin B is being tested for synergy with hLF1-11 (title, lines 18,25, 256, and 385), but in Figure 4, it appears that anidulafungin (and fluconazole) were evaluated for synergism. It appears anidulafungin should replace amphotericin B in the title, lines 18,25, 256, and 385. Please clarify.

Reply: The reviewer is correct. We corrected this throughout the entire manuscript. The confusion arose because we were working on two different manuscripts simultaneously. We apologize for the inconvenience.

Recommend to modify in the abstract the sentence starting at line 14, that hLF1-11 is  potential new therapy against all yeast:

"A synthetic peptide, consisting of 11 amino acids of human lactoferrin (hLF1-11) offers a new therapy that is active against Candida albicans, non-albicans Candida yeasts, as well as Cz. auris."

Reply: We thank the reviewerf or this suggestion. We changed this in the manuscript.

"AND" is a confusing abbreviation to use for anidulafungin. Is there another one?

Reply: We changed the abbreviation of anidulafungin from AND to ANI throughout the entire manuscript

Table 4. Horizontal top row confusing and not lining up. Would recommend having text vertically instead of horizontal. Also this particularly confusing with the abbreviation of "AND" as it makes one think it is one agent and another agent vs meaning anidulafungin if that makes sense.

Reply: We thank the reviewer for this suggestion. We changed the table. This was already mentioned by other reviewer as well.

In discussion might be of interest to have one sentence if it is a future possibility to use this AMP region as a potential therapy for specific bacterial pathogens. 

Reply: We thank the reviewer for this suggestion. We added extra sentences related to this particular subject. See below in the last reply for our answer.

In discussion might be of interest to have one sentence if it is a future possibility to use the other AMP region as a potential therapy. 

Reply: We thank the reviewer for this suggestion. We added some sentences.

The emergence of multidrug-resistant microbial pathogens has created an pressing want for brand new healing options, with LF and its derived peptides, lactoferricins (LFcins), rising as promising applicants because of their multifaceted roles in innate immunity and microbial pathogenesis. LF reveals bacteriostatic and bactericidal properties, promotes immune responses, and has been proven to inhibit viral access and counter microbial mechanisms of infection, making it a precious adjuvant in fighting antibiotic-resistant microorganisms and - fungi.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript describes the bactericidal activity of a N-terminal peptide of human lactoferrin against a whole range of pathogens. This peptide has previously shown a broad antimicrobial spectrum, but this study contains new information and a ample review of known data. The study is under the possibility of using antimicrobial peptides as alternatives to the commonly used antifungals for treating infections caused by Cz. Auris.

The manuscript is interesting, and it could be acceptable after addressing the following items:

  • Abstract: Provided thT MIC is defined at lines 19-20, FIC (line 26) should also be defined.
  • Heading of Table 1. The table is very useful and illustrative. I just suggest to (1) include the units of MIC values. (2) improve the format (i.e decreasing the letter size to avoid words of number truncations; (3) indicate the meaning of the strain: and abbreviations and the bold first line, (4) The last line has no reference. Complete.
  • Table 2, heading: Ala substituitions. Repair that word
  • Table 4: Same improvements that Table 1 about improving the format
  • Line 122 and so on, fluconazole and anidulafungin are abbreviated by FLU and AND. Abbreviations are defined at materials and methods, but this is too late, as material and methods are the last section. For instance, the sequence of the undecapeptide is found later (line 295), but Ala substitutions are discussed much earlier. I suggest the definition of any abbreviation the first time at it appears to facilitate comprehensive reading.
  • The letter type at Materials and Methods would be uniformed.
  • Section 5. Conclusion would be concise and brief. Avoid introductory sentences at that section.

Author Response

Reviewer 3

Synergism was tested against anidulafungin and not amphotericin B.

Comments and Suggestions for Authors

This manuscript describes the bactericidal activity of a N-terminal peptide of human lactoferrin against a whole range of pathogens. This peptide has previously shown a broad antimicrobial spectrum, but this study contains new information and a ample review of known data. The study is under the possibility of using antimicrobial peptides as alternatives to the commonly used antifungals for treating infections caused by Cz. Auris.

Reply: We thank the reviewer for acknowledging our work. We changed the revised manuscript according to the suggestions.

The manuscript is interesting, and it could be acceptable after addressing the following items:

Abstract: Provided thT MIC is defined at lines 19-20, FIC (line 26) should also be defined.

Reply: We thank the reviewer for this suggestion. We added some words to explain. In the combinatory tests, drugs acting together, the fractional inhibitory concentration indexes [FIC]< 1.0, showing a synergistic or additive effect on the efficacy of FLU and ANI when used in combination with hLF(1-11). We apologize for the inconvenience.

Heading of Table 1. The table is very useful and illustrative. I just suggest to (1) include the units of MIC values. (2) improve the format (i.e decreasing the letter size to avoid words of number truncations; (3) indicate the meaning of the strain and abbreviations and the bold first line, (4) The last line has no reference. Complete.

Reply: The reviewer is correct. We corrected this. We apologize for the inconvenience.

Table 2, heading: Ala substituitions. Repair that word

Reply: The reviewer is correct. We corrected this. The other reviewers also mentioned this issue. We apologize for the inconvenience.

Table 4: Same improvements that Table 1 about improving the format

Reply: We thank the reviewer for this suggestion. We changed the table. The other reviewer also mentioned this issue.

Line 122 and so on, fluconazole and anidulafungin are abbreviated by FLU and AND. Abbreviations are defined at materials and methods, but this is too late, as material and methods are the last section. For instance, the sequence of the undecapeptide is found later (line 295), but Ala substitutions are discussed much earlier. I suggest the definition of any abbreviation the first time at it appears to facilitate comprehensive reading.

Reply: The reviewer is correct. We corrected this throughout the entire manuscript. We apologize for the inconvenience.

The letter type at Materials and Methods would be uniformed.

Reply: The reviewer is correct. We corrected this. We apologize for the inconvenience.

Section 5. Conclusion would be concise and brief. Avoid introductory sentences at that section.

Reply: We thank the reviewer for this suggestion. We have corrected this. The manuscript has been extensively checked for English grammar. We apologize for the inconvenience.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have responded in a satisfactory manner to the reviewer's comments. 

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