Antifungal susceptibility pattern and biofilm-related genes expression in planktonic and biofilm cells of Candida parapsilosis species complex

Document Type : Original Articles

Authors

1 Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

2 Department of Microbiology, Faculty of Science, Islamic Azad University, Varamin-Pishva, Iran

Abstract

Background and Purpose: Candida parapsilosis complex isolates are mainly responsible for nosocomial catheter-related infection in immunocompromised patients. Biofilm formation is regarded as one of the most pertinent key virulence factors in the development of these emerging infections. The present study aimed to compare in vitro antifungal susceptibility patterns and biofilm-related genes expression ratio in planktonic and biofilm’s cells of clinically C. parapsilosis complex isolates.
Materials and Methods: The current study was conducted on a number of 17 clinical C. parapsilosis complex (10 C. parapsilosis sensu stricto, 5 C. orthopsilosis, and 2 C. metapsilosis). The antifungal susceptibility patterns of amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, and caspofungin in planktonic and biofilm forms were closely examined using CLSI M27-A3 broth microdilution method. The expression levels of biofilm-related genes (BCR1, EFG1, and FKS1) were evaluated in planktonic and biofilm’s cells using Real-time polymerase chain reaction (PCR) technique.
Results: The obtained results indicated that all C. parapsilosis complex isolates were able to produce high and moderate amounts of biofilm forms. In addition, the sessile minimum inhibitory concentrations were reported to be high for fluconazole (≥ 64 μg/ml), itraconazole, voriconazole, and posaconazole (≥ 16 μg/ml), as compared to planktonic minimum inhibitory concentrations. Moreover, a significant difference was observed between antifungal susceptibility patterns for all azole antifungal agents (p <0.05). Furthermore, the BCR1 overexpression was considered significant in biofilms with regard to planktonic cells in C. parapsilosis species complex (P=0.002).
Conclusion: C. parapsilosis complex isolates were found susceptible to most of the tested antifungal drugs, while biofilms demonstrated a noticeable resistant to azoles. The marked discrepancy noted in antifungal susceptibility patterns among these species should be highlighted to achieve effective therapeutic treatment.

Keywords


1. Weems JJ Jr. Candida parapsilosis: epidemiology, pathogenicity, clinical manifestations, and antimicrobial susceptibility. Clin Infect Dis. 1992; 14(3):756-66.
2. Spiliopoulou A, Dimitriou G, Jelastopulu E, Giannakopoulos I, Anastassiou E, Christofidou M. Neonatal intensive care unit candidemia: epidemiology, risk factors, outcome, and critical review of published case series. Mycopathologia. 2012; 173(4):219-28.
3. Tavanti A, Davidson AD, Gow NA, Maiden MC, Odds FC. Candida orthopsilosis and Candida metapsilosis spp. nov. to
replace Candida parapsilosis groups II and III. J Clin Microbiol. 2005; 43(1):284-92.
4. Arastehfar A, Daneshnia F, Kord M, Roudbary M, Zarrinfar H, Fang W, et al. Comparison of 21-Plex PCR and API 20C AUX, MALDI-TOF MS, and rDNA sequencing for a wide range of clinically isolated yeast species: Improved identification by combining 21-Plex PCR and API 20C AUX as an alternative strategy for developing countries. Front Cell Infect Microbiol. 2019; 9:176.
5. Gácser A, Schäfer W, Nosanchuk JS, Salomon S, Nosanchuk JD. Virulence of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis in reconstituted human tissue models. Fungal Genet Biol. 2007; 44(12):1336-41.
6. Ramage G, Martínez JP, López-Ribot JL. Candida biofilms on implanted biomaterials: a clinically significant problem. FEMS Yeast Res. 2006; 6(7):979-86.
7. Modiri M, Khodavaisy S, Barac A, Dana MA, Nazemi L, Aala F, et al. Comparison of biofilm-producing ability of clinical isolates of Candida parapsilosis species complex. J Mycol Med. 2019; 29(2):140-6.
8. Coenye T, Nelis HJ. In vitro and in vivo model systems to study microbial biofilm formation. J Microbiol Methods. 2010; 83(2):89-105.
9. Araújo D, Henriques M, Silva S. Portrait of Candida species biofilm regulatory network genes. Trends Microbiol. 2017; 25(1):62-75.
10. Holland LM, Schröder MS, Turner SA, Taff H, Andes D, Grózer Z, et al. Comparative phenotypic analysis of the major fungal pathogens Candida parapsilosis and Candida albicans. PLoS Pathog. 2014; 10(9):e1004365.
11. Ding C, Vidanes GM, Maguire SL, Guida A, Synnott JM, Andes DR, et al. Conserved and divergent roles of Bcr1 and CFEM proteins in Candida parapsilosis and Candida albicans. PloS One. 2011; 6(12):e28151.
12. Connolly LA, Riccombeni A, Grózer Z, Holland LM, Lynch DB, Andes DR, et al. The APSES transcription factor Efg 1 is a global regulator that controls morphogenesis and biofilm formation in Candida parapsilosis. Mol Microbiol. 2013; 90(1):36-53.
13. Aslani N, Janbabaei G, Abastabar M, Meis JF, Babaeian M, Khodavaisy S, et al. Identification of uncommon oral yeasts from cancer patients by MALDI-TOF mass spectrometry. BMC Infect Dis. 2018; 18(1):24.
14. Arastehfar A, Khodavaisy S, Daneshnia F, Najafzadeh MJ, Mahmoudi S, Charsizadeh A, et al. Molecular identification, genotypic diversity, antifungal susceptibility, and clinical outcomes of infections caused by clinically underrated yeasts, candida orthopsilosis, and candida metapsilosis: an Iranian multicenter study (2014-2019). Front Cell Infect Microbiol. 2019; 9:264.
15. Pierce CG, Uppuluri P, Tristan AR, Wormley FL Jr, Mowat E, Ramage G, et al. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc. 2008; 3(9): 1494-500.
16. Silva S, Henriques M, Martins A, Oliveira R, Williams D, Azeredo J. Biofilms of non-Candida albicans Candida species: quantification, structure and matrix composition. Med Mycol. 2009; 47(7):681-9.
17. Stepanović S, Vuković D, Hola V, Bonaventura GD, Djukić S, Ćirković I, et al. Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS. 2007; 115(8):891-9.
18. Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts: third edition (M27-A3). Wayne, PA: Clinical and Laboratory Standards Institute; 2008.
19. Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts: fourth informational supplement (M27S4). Wayne, PA: Clinical and Laboratory Standards Institute; 2012.
20. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antifungal susceptibility testing of yeasts. CLSI supplement M60. Wayne, PA: Clinical and Laboratory Standards Institute; 2017.
21. Pfaller M, Diekema D. Progress in antifungal susceptibility testing of Candida spp. by use of Clinical and Laboratory Standards Institute broth microdilution methods, 2010 to 2012. J Clin Microbiol. 2012; 50(9):2846-56.
22. Ramage G, Vande Walle K, Wickes BL, López-Ribot JL. Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother. 2001; 45(9):2475-9.
23. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65(1-2):55-63.
24. Rodrigues CF, Gonçalves B, Rodrigues ME, Silva S, Azeredo J, Henriques M. The effectiveness of voriconazole in therapy of Candida glabrata’s biofilms oral infections and its influence on the matrix composition and gene expression. Mycopathologia. 2017; 182(7-8):653-64.
25. Chomzynski P, Mackey K. Modification of the TRIZOL reagent procedure for isolation of RNA from polysaccharide-and proteoglycan-rich sources. Biotechniques. 1995; 19(6):942-5.
26. Khodavaisy S, Badali H, Meis J, Rezaie S, Hagen F, Afshari S, et al. Microsatellite genotyping of Candida parapsilosis from Iranian clinical isolates. Curr Med Mycol. 2017; 3(4):15-20.
27. Nakamura T, Takahashi H. Epidemiological study of Candida infections in blood: susceptibilities of Candida spp. to antifungal agents, and clinical features associated with the candidemia. J Infect Chemother. 2006; 12(3):132-8.
28. Lockhart SR, Messer SA, Pfaller MA, Diekema DJ. Geographic distribution and antifungal susceptibility of the newly described species Candida orthopsilosis and Candida metapsilosis in comparison to the closely related species Candida parapsilosis. J Clin Microbiol. 2008; 46(8):2659-64.
29. Cantón E, Pemán J, Quindós G, Eraso E, Miranda-Zapico I, Álvarez M, et al. Prospective multicenter study of the epidemiology, molecular identification, and antifungal susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis isolated from patients with candidemia. Antimicrob Agents Chemother. 2011; 55(12):5590-6.
30. Dagi HT, Findik D, Senkeles C, Arslan U. Identification and antifungal susceptibility of Candida species isolated from bloodstream infections in Konya, Turkey. Ann Clin Microbiol Antimicrob. 2016; 15(1):36.
31. Gonçalves S, Amorim C, Nucci M, Padovan ACB, Briones MR, Melo AS, et al. Prevalence rates and antifungal susceptibility profiles of the Candida parapsilosis species complex: results from a nationwide surveillance of candidaemia in Brazil. Clin Microbiol Infect. 2010; 16(7):885-7.
32. Lovero G, Borghi E, Balbino S, Cirasola D, De Giglio O, Perdoni F, et al. Molecular identification and echinocandin susceptibility of Candida parapsilosis complex bloodstream isolates in Italy, 2007-2014. PloS One. 2016; 11(2):e0150218.
33. Tay ST, Na SL, Chong J. Molecular differentiation and antifungal susceptibilities of Candida parapsilosis isolated from patients with bloodstream infections. J Med Microbiol. 2009; 58(Pt 2):185-91.
34. Neji S, Trabelsi H, Hadrich I, Cheikhrouhou F, Sellami H, Makni F, et al. Molecular study of the Candida parapsilosis complex in Sfax, Tunisia. Med Mycol. 2017; 55(2):137-44.
35. Feng X, Ling B, Yang G, Yu X, Ren D, Yao Z. Prevalence and distribution profiles of Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis responsible for
superficial candidiasis in a Chinese university hospital. Mycopathologia. 2012; 173(4):229-34.
36. Tosun I, Akyuz Z, Guler NC, Gulmez D, Bayramoglu G, Kaklikkaya N, et al. Distribution, virulence attributes and antifungal susceptibility patterns of Candida parapsilosis complex strains isolated from clinical samples. Med Mycol. 2013; 51(5):483-92.
37. Maria S, Barnwal G, Kumar A, Mohan K, Vinod V, Varghese A, et al. Species distribution and antifungal susceptibility among clinical isolates of Candida parapsilosis complex from India. Rev Iberoam Micol. 2018; 35(3):147-50.
38. Trabasso P, Matsuzawa T, Fagnani R, Muraosa Y, Tominaga K, Resende MR, et al. Isolation and drug susceptibility of Candida parapsilosis sensu lato and other species of C. parapsilosis complex from patients with blood stream infections and proposal of a novel LAMP identification method for the species. Mycopathologia. 2015; 179(1-2):53-62.
39. Chen YC, Lin YH, Chen KW, Lii J, Teng HJ, Li SY. Molecular epidemiology and antifungal susceptibility of Candida parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis in Taiwan. Diagn Microbiol Infect Dis. 2010; 68(3):284-92.
40. Gomez-Lopez A, Alastruey-Izquierdo A, Rodriguez D, Almirante B, Pahissa A, Rodriguez-Tudela J, et al. Prevalence and susceptibility profile of Candida metapsilosis and Candida orthopsilosis: results from population-based surveillance of candidemia in Spain. Antimicrob Agents Chemother. 2008; 52(4):1506-9.
41. Rizzato C, Poma N, Zoppo M, Posteraro B, Mello E, Bottai D, et al. CoERG11 A395T mutation confers azole resistance in Candida orthopsilosis clinical isolates. J Antimicrob Chemother. 2018; 73(7):1815-22.
42. Saracli MA, Gumral R, Gul HC, Gonlum A, Yildiran ST. Species distribution and in vitro susceptibility of Candida bloodstream isolates to six new and current antifungal agents in a Turkish tertiary care military hospital, recovered through 2001 and 2006. Mil Med. 2009; 174(8):860-5.
43. Ruiz LS, Khouri S, Hahn RC, da Silva EG, de Oliveira VK, Gandra RF, et al. Candidemia by species of the Candida parapsilosis complex in children’s hospital: prevalence, biofilm production and antifungal susceptibility. Mycopathologia. 2013; 175(3-4):231-9.
44. Katragkou Α, Chatzimoschou A, Simitsopoulou M, Dalakiouridou M, Diza-Mataftsi E, Tsantali C, et al. Differential activities of newer antifungal agents against Candida albicans and Candida parapsilosis biofilms. Antimicrob Agents Chemother. 2008; 52(1):357-60.
45. Brilhante RS, Sales JA, da Silva MLQ, de Oliveira JS, de Alencar Pereira L, Pereira-Neto WA, et al. Antifungal susceptibility and virulence of Candida parapsilosis species complex: an overview of their pathogenic potential. J Med Microbiol. 2018; 67(7):903-14.
46. Pannanusorn S, Ramírez-Zavala B, Lünsdorf H, Agerberth B, Morschhäuser J, Römling U. Characterization of biofilm formation and the role of BCR1 in clinical isolates of Candida parapsilosis. Eukaryotic Cell. 2014; 13(4):438-51.
47. Nikoomanesh F, Roudbarmohammadi S, Roudbary M, Bayat M, Heidari G. Investigation of bcr1 gene expression in Candida albicans isolates by RT-PCR technique and its impact on biofilm formation. Infect Epidemiol Med. 2016; 2(1):22-4.