Demonstration of Mutation Development and Virulence Change in Reference Candida Strains Exposed to Caspofungin

Abstract

Purpose: Echinocandin resistance is causing problems in the treatment. FKS gene mutations were detected on the genomes of the resistant Candida strains exposed to echinocandins. The aim of this study was to compare the resistance of reference Candida strains exposed to caspofungin and to investigate whether there was a difference in virulence factors between exposed and non-exposed with caspofungin. Methods: Caspofungin susceptible reference strains, exposed to caspofungin during their incubation in caspofungin containing agar plates at concentrations of 16-0.03 µg/ml. MIC values of caspofungin were determined all strains. FKS gene regions of the caspofungin exposed strains were sequenced in order to reveal the difference between the reference strains. Relationship between caspofungin exposure and virulence properties were investigated in vivo and in vitro methodologies. The in vivo virulence study on the mini-experimental animal model Galleria mellonella (wax moth) was performed. Results: MIC values of the caspofungin exposed strains were found to be increased as one/two fold dilution when compared to the sensitive reference strains. The result of FKS gene sequence no mutation is detected in the reference strains, while a point mutation in caspofungin exposed C. glabrata strains were detected. No significant difference was found in virulence properties of reference strains and caspofungin exposed strains. Conclusions: In conclusion, it was understood that the fact that sensitive strains were exposed to caspofungin even at low concentrations caused an increase in MIC, and that virulence and drug resistance should be evaluated independently.

Keywords

• Caspofungin
• FKS mutation
• Galleria mellonella
• Candida
• Virulence

References

1. 1. Touil HF., Boucherit K, Boucherit-Otmani Z et al. Optimum Inhibition of Amphotericin-B-Resistant Candida albicans Strain in Single-and Mixed-Species Biofilms by Candida and Non-Candida Terpenoids. Biomolecules 2020; 10: 342.
2. 2. Perlin DS. Mechanisms of echinocandin antifungal drug resistance. Ann New York Acad Sci 2015; 1354: 1-11.
3. 3. Beyer R, Spettel K, Zeller I et al. Antifungal susceptibility of yeast bloodstream isolates collected during a 10‐year period in Austria. Mycoses 2019; 62: 357-367.
4. 4. Pfaller MA, Diekema DJ, Turnidge JD et al. Twenty Years of the SENTRY Antifungal Surveillance Program: Results for Candida Species From 1997–2016. Open Forum Infect Dis 2019;6 Suppl 1: 79-94.
5. 5. Toutounji M, Tokajian S, Khalaf RA. Genotypic and phenotypic characterization of Candida albicans Lebanese hospital isolates resistant and sensitive to caspofungin. Fungal Genet Biol 2019; 127: 12-22.
6. 6. Bordallo-Cardona MÁ, Escribano P, de la Pedrosa EGG et al. In vitro exposure to increasing micafungin concentrations easily promotes echinocandin resistance in Candida glabrata isolates. Antimicrob Agents Ch 2017; 61: e01542-16.
7. 7. Chassot F, Venturini TP, Piasentin FB et al. Exploring the in vitro resistance of Candida parapsilosis to echinocandins. Mycopathologia 2016; 181: 663-70.
8. 8. Shields RK, Kline EG, Healey KR, et al. Spontaneous mutational frequency and FKS mutation rates vary by echinocandin agent against Candida glabrata. Antimicrob Agents Ch 2019; 63: e01692-18.

9. 9. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts- Third Edition: M27–A3. NCCLS, Wayne, PA.2008.
10. 10. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts, Third Edition informational supplement M27–S3. NCCLS, Wayne, PA. 2008.
11. 11. Lackner M, Tscherner M, Schaller M et al. Positions and numbers of FKS mutations in Candida albicans selectively influence in vitro and in vivo susceptibilities to echinocandin treatment. Antimicrob Agents Ch 2014; 58: 3626-35.
12. 12. Castanheira M, Woosley LN, Diekema DJ et al. Low prevalence of fks1 hot spot 1 mutations in a worldwide collection of Candida strains. Antimicrob Agents Ch. 2010; 54: 2655-9.
13. 13. Cheung Y-Y, Hui M. Effects of Echinocandins in combination with Nikkomycin Z against invasive Candida albicans bloodstream isolates and the fks mutants. Antimicrob Agents Ch 2017; 61: e00619-17.
14. 14. Dudiuk C, Gamarra S, Leonardeli F et al. Set of classical PCRs for detection of mutations in Candida glabrata FKS genes linked with echinocandin resistance. J Clin Microbiol 2014; 52: 2609-14.
15. 15. de Melo AV, Zuza-Alves D, da Silva-Rocha W et al. Virulence factors of Candida spp. obtained from blood cultures of patients with candidemia attended at tertiary hospitals in Northeast Brazil. J Mycol Med 2019; 292: 132-139.
16. 16. Kalkancı A, Fouad A, Erdoğan M et al. Bazı bakteri ve mantarların virülansının araştırılmasında Galleria mellonella’nın in vivo model olarak kullanılması. Mikrobiyol Bul 2015; 49: 366-76.
17. 17. Pappas PG, Kauffman CA, Andes DR et al. Clinical practice guideline for the management of candidiasis: 2016 update by the Infectious Diseases Society of America. Clin Infect Dis 2015; 62: e1-50.
18. 18. Grosset M, Desnos-Ollivier M, Godet C et al. Recurrent episodes of Candidemia due to Candida glabrata, Candida tropicalis and Candida albicans with acquired echinocandin resistance. Med Mycol Case Rep 2016; 14: 20-3.
19. 19. Khan Z, Ahmad S, Mokaddas E et al. Development of echinocandin resistance in Candida tropicalis following short-term exposure to caspofungin for empiric therapy. Antimicrob Agents Ch 2018; 62: AAC. 01926-17.
20. 20. Chassot F, Venturini TP, Piasentin FB et al. Activity of antifungal agents alone and in combination against echinocandin-susceptible and-resistant Candida parapsilosis strains. Rev Iberoam Micol 2019; 36: 44-7.
21. 21. Pham CD, Iqbal N, Bolden CB et al. The role of FKS mutations in C. glabrata: MIC values, echinocandin resistance and multidrug resistance. Antimicrob Agents Ch 2014; 58: 4690-6.
22. 22. Ellepola AN, Khajah R, Jayatilake S et al. Impact of brief exposure to antifungal agents on the post-antifungal effect and hemolysin activity of oral Candida albicans. J Appl Oral Sci 2015; 23: 412-8.
23. 23. Ripeau J-S, Aumont F, Belhumeur P et al. Effect of the echinocandin caspofungin on expression of Candida albicans secretory aspartyl proteinases and phospholipase in vitro. Antimicrob Agents Ch 2002; 46: 3096-100. 24. Ellepola ANB, Chandy R, Khan ZU et al. Caspofungin‐induced in‐vitro post‐antifungal effect and its impact on adhesion related traits of oral Candida dubliniensis and Candida albicans isolates. J Microbiol Immunol 2016; 60: 160-7.
24. 25. Papp C, Kocsis K, Tóth R et al. Echinocandin-Induced Microevolution of Candida parapsilosis Influences Virulence and Abiotic Stress Tolerance. mSphere. 2018; 3: e00547-18.
25. 26. Rodrigues CF, Rodrigues ME, Henriques M. Susceptibility of Candida glabrata biofilms to echinocandins: Alterations in the matrix composition. Biofouling. 2018; 34: 569-7