Galleria mellonella as a superficial model for Malassezia globosa and its treatment

  1. Maritza Torres
  2. Juliana Diaz-Ortiz
  3. Michael G. Davis
  4. James R. Schwartz
  5. Adriana Marcela Celis Ramírez

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Abstract

Introduction. Malassezia globosa is a yeast species that belongs to the mycobiota of humans and animals, associated with dermatological disorders, such as dandruff. This is a chronic scalp skin disorder characterized by flaking and itching. Treatments include commercial shampoo with different formulations that contain antifungal activities like zinc pyrithione (ZPT) or piroctone olamine (PO). The effectiveness of these formulations has been evaluated for decades for dandruff symptom relief of volunteers. To date, non-mammalian, in vivo methods exist to test formulations of these actives.

Aim. To evaluate in vivo in Galleria mellonella larva, two commercial antifungal shampoos (shampoo with 1 % ZPT and 1.6 % zinc Carbonate and shampoo with 0.5 % PO) against this species.

Methodology. G. mellonella larvae were inoculated with M. globosa on abraded cuticular surface. Then, integument cell viability, histological changes, and fungal burden were evaluated.

Results. Larvae inoculated with M. globosa showed higher lesion melanization and tissue damage. In addition, M. globosa population showed to increase over time. Concerning the shampoo’s effectiveness, both formulations significantly reduced M. globosa burden and tissue damage.

Conclusion. G. mellonella larvae were allowed to evaluate M. globosa superficial infection and antifungal effectiveness. Shampoos with ZPT and PO showed a positive effect on inoculated larvae.

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  1. Version published to 10.1099/acmi.0.000745.v3 on Access Microbiology
  3. Version published to 10.1099/acmi.0.000745.v2 on Access Microbiology
  5. Access Microbiology

    Comments to Author

    Comments to the Author This article is interesting and informative, can add useful information to the existing data on Malssezia infection and treatment. The following comments should be done: Line 18: Zinc pyrithione (ZPT) or Piroctone Olamine (PO) Lines 20&21&57&59 and through the manuscript "in vivo": should be italicized. Line 85 "M. globose" : should be italicized. Lines 94-95" Here it was demonstrated that G. mellonella could be a suitable model for assessing M. globosa infection establishment and the effectiveness of topic antifungal actives." Should be omitted from introduction. Line 99: please write the source of Malassezia globosa CBS 7986 strain Lines 124 & 127"dishes" should be replaced with "plates" Line 142 "no-inoculated" should be replaced with "non-inoculated" Line 146" PBS" please write in full at the first mention Line 151" (2mmdiameter) please taka a space Lines 172-173 "Abraded, inoculated, and treated with shampoo larvae and incubated for 24 hours, 48 hours, and 72 hours at 33C were taken to -20C for 5 minutes." Please rewrite this sentence Line 185 "' agar Dixon" should be replaced with "Dixon agar" Line 186" scored as one and cero" should be replaced with "scored as one and zero" Line 188: please revise the equation Line 201" under" should be "less than" Line 207" previously described [24] with modifications" should be omitted as it was previously mentioned in the methods section. Line 313-314 "for M. furfur y M. pachydermatis" please revise to be "for M. furfur and M. pachydermatis"

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  6. Access Microbiology

    Comments to Author

    This is a study evaluating the use of G. mellonella as a model to study M. globosa and evaluate topical treatments for this species and in general for this genus of yeasts. I have some concerns and disagreements with the conceptualisation of the project, hence I have to recognise that these points are maybe not possible to address by the authors but I'll share them in case they are useful: a. This is my major point: I disagree with the authors that G. mellonella is a good model to study Malassezia superficial infections. I'll give more details: Authors affirm in the document from page 2 line 56 to the discussion that "Due to the high prevalence of dandruff, poor knowledge of the host-pathogen interactions, and the need to evaluate commercial antifungal safety, it is necessary to implement in vivo models that allow for the determination of infection mechanisms, the virulence factors and the interaction of Malassezia with antifungals during a superficial infection" This statement is not accurate and don't think is a valid point to explore G. mellonella as a model to study superficial Malassezia infections. It is recognised worldwide that in order to study host-pathogen interactions of superficial infections and to evaluate superficial therapies (effectivity and safety mainly), you DONT need in vivo models. This is one of the foundations of 3R research. It is well recognised that this type of research questions have excellent models already, much closer to human contexts such as ex-vivo human skin or reconstructed 3D skin models, even the use of pig skin has been used as a good alternative model. None of this is acknowledged by the authors and it should as are the models that G.mellonella should be as good as. In addition, the use of mice in the study of Malassezia diseases is only accepted to study in deep detail immune responses that can't be evaluated with ex vivo or in vitro models like studying adaptive immunity. This is another misconception mentioned in the document, G. mellonella used due to its immune system, this is only partially true as it is only innate immunity what you could evaluate. Possibly this is the reason (plus evaluation of systemic tx) why the use of these larvae in systemic Malassezia infection makes more sense as you want to evaluate survival but definitively not much if you want to study superficial infections as there are definitively best models. I would also recommend moderation on the statement where authors affirm teguments in these larvae are a proxy for Human skin. Even Mice skin is not a proxy for Human skin as major biological differences exist. b. Methods and results - The inoculum used here is not specific, and I doubt is reproducible then making the evaluation of the shampoo tx difficult and potentially biased. I would recommend using more standardised methods where the inoculum can be objectively measured. - I disagree with the authors when they don't use CFUs as a method to evaluate the effect of the shampoo, it is true that quantifying and working with CFUs is challenging in Mallassezia but that is not a major impediment do it. Please see methods published by others, I would recommend Inari et al and LeibundGut-Landmann et al previous studies. - In their "Tegumental fungal burden" method, I doubt the use of the larvae would bring a benefit in evaluating the effect of shampoos compared to just using in vitro assays without the larvae. Authors don't seem to have done these comparison to provide evidence that having larvae as model to test this topical treatment is better than only testing the same tx in vitro. There are probably better models or similarly good where actual human or mice stratum corneum is used to carry out the same experiments and even local immune responses can be evaluated. Not sure if that yield also applies for G. mellonella. It is something that, to demonstrate this model is adding a value might be required. c. General observations 1. I would recommend the authors to check on several typos and English Grammar along the document. 2. Some of the species names are not in Italics

    Please rate the manuscript for methodological rigour

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  7. Version published to 10.1099/acmi.0.000745.v1 on Access Microbiology