Identification of Virulence Factors in Isolates of Candida haemulonii, Candida albicans and Clavispora lusitaniae with Low Susceptibility and Resistance to Fluconazole and Amphotericin B

Abstract

Emerging life-threatening multidrug-resistant (MDR) species such as the C. haemulonii species complex, Clavispora lusitaniae (sin. C. lusitaniae), and other Candida species are considered as an increasing risk for human health in the near future. (1) Background: Many studies have emphasized that the increase in drug resistance can be associated with several virulence factors in Candida and its knowledge is also essential in developing new antifungal strategies. (2) Methods: Hydrophobicity, adherence, biofilm formation, lipase activity, resistance to osmotic stress, and virulence 'in vivo' on G. mellonella larvae were studied in isolates of C. haemulonii, C. albicans, and C. lusitaniae with low susceptibility and resistance to fluconazole and amphotericin B. (3) Results: Intra- and interspecies variability were observed. C. haemulonii showed high hydrophobicity and the ability to adhere to and form biofilm. C. lusitaniae was less hydrophobic, was biofilm-formation-strain-dependent, and did not show lipase activity. Larvae inoculated with C. albicans isolates displayed significantly higher mortality rates than those infected with C. haemulonii and C. lusitaniae. (4) Conclusions: The ability to adhere to and form biofilms associated with their hydrophobic capacity, to adapt to stress, and to infect within an in vivo model, observed in these non-wild-type Candida and Clavispora isolates, shows their marked virulence features. Since factors that define virulence are related to the development of the resistance of these fungi to the few antifungals available for clinical use, differences in the physiology of these cells must be considered to develop new antifungal therapies.

Keywords: C. albicans; C. haemulonii; C. lusitaniae; Galleria mellonella; adherence; biofilm; hydrophobicity; resistance; stress; virulence factors.

Figure 1

Histograms of hydrophobicity and adherence on plastic surface of C. albicans, C. lusitanieae, and C. haemulonii isolates. (A) Percentage of hydrophobicity. (B) Percentage of adherence on plastic surface.

Figure 2

Histograms of biofilm formation after 24 and 48 h of C. albicans, C. lusitaniae, and C. haemulonii. * p-value < 0.0392, ** p-values < 0.004.

Figure 3

G. mellonella survival assays after infection with C. albicans (IMR-M-L 1462, 1463, 1464, ATCC 24433), C. haemulonii (IMR-M-L 785, 1293, 1375), and C. lusitaniae (IMR-M-L 301, 522, 1112, 1384). Results are expressed as % survival in comparison to uninfected (PBS-treated) larvae. Median values obtained per group (10 larvae) are presented. Larvae infected with C. albicans significantly decreased survival, as assessed using the Mantel–Cox log-rank test (** p < 0.0042).