Phenotypic Switching and Filamentation in Candida haemulonii, an Emerging Opportunistic Pathogen of Humans

The capacity to switch between distinct cell types, known as phenotypic switching, is a common strategy adopted by Candida species to adapt to diverse environments. Despite considerable studies on phenotypic plasticity of various Candida species, Candida haemulonii is considered to be incapable of phenotypic switching or filamentous growth.

Candida duobushaemulonii: An Old But Unreported Pathogen

Candidiasis caused by species of the Candida haemulonii complex (Candida haemulonii and Candida duobushaemulonii) and closely related species, Candida auris and Candida pseudohaemulonii are increasing. These species often show reduced susceptibility to antifungal drugs, such as azoles and amphotericin B or, less frequently, echinocandins. However, conventional phenotypic identification methods are unable to accurately differentiate these species and, therefore, their prevalence may have been underestimated. In this study, 150 isolates that were probably misidentified were reanalyzed using two novel PCR approaches. We found that one isolate previously identified in 1996 as Candida intermedia was C. duobushaemulonii, being one of the oldest isolates of this species described to date. We also found that this isolate had reduced susceptibility to fluconazole, itraconazole, and amphotericin B.

Insights into the Multi-Azole Resistance Profile in Candida haemulonii Species Complex

The Candida haemulonii complex (C. duobushaemulonii, C. haemulonii, and C. haemulonii var. vulnera) is composed of emerging, opportunistic human fungal pathogens able to cause invasive infections with high rates of clinical treatment failure. This fungal complex typically demonstrates resistance to first-line antifungals, including fluconazole. In the present work, we have investigated the azole resistance mechanisms expressed in Brazilian clinical isolates forming the C. haemulonii complex. Initially, 12 isolates were subjected to an antifungal susceptibility test, and azole cross-resistance was detected in almost all isolates (91.7%). In order to understand the azole resistance mechanistic basis, the efflux pump activity was assessed by rhodamine-6G. The C. haemulonii complex exhibited a significantly higher rhodamine-6G efflux than the other non-albicans Candida species tested (C. tropicalis, C. krusei, and C. lusitaneae). Notably, the efflux pump inhibitors (Phe-Arg and FK506) reversed the fluconazole and voricolazole resistance phenotypes in the C. haemulonii species complex. Expression analysis indicated that the efflux pump (ChCDR1, ChCDR2, and ChMDR1) and ERG11 genes were not modulated by either fluconazole or voriconazole treatments. Further, ERG11 gene sequencing revealed several mutations, some of which culminated in amino acid polymorphisms, as previously reported in azole-resistant Candida spp. Collectively, these data point out the relevance of drug efflux pumps in mediating azole resistance in the C. haemulonii complex, and mutations in ERG11p may contribute to this resistance profile.

Susceptibility of the Candida haemulonii Complex to Echinocandins: Focus on Both Planktonic and Biofilm Life Styles and a Literature Review

Candida haemulonii complex (C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera) is well-known for its resistance profile to different available antifungal drugs. Although echinocandins are the most effective class of antifungal compounds against the C. haemulonii species complex, clinical isolates resistant to caspofungin, micafungin and anidulafungin have already been reported. In this work, we present a literature review regarding the effects of echinocandins on this emergent fungal complex. Published data has revealed that micafungin and anidulafungin were more effective than caspofungin against the species forming the C. haemulonii complex. Subsequently, we investigated the susceptibilities of both planktonic and biofilm forms of 12 Brazilian clinical isolates of the C. haemulonii complex towards caspofungin and micafungin (anidulafungin was unavailable). The planktonic cells of all the fungal isolates were susceptible to both of the test echinocandins. Interestingly, echinocandins caused a significant reduction in the biofilm metabolic activity (viability) of almost all fungal isolates (11/12, 91.7%). Generally, the biofilm biomasses were also affected (reduction range 20–60%) upon exposure to caspofungin and micafungin. This is the first report of the anti-biofilm action of echinocandins against the multidrug-resistant opportunistic pathogens comprising the C. haemulonii complex, and unveils the therapeutic potential of these compounds.

Increasing Prevalence of Multidrug-Resistant Candida haemulonii Species Complex among All Yeast Cultures Collected by a Reference Laboratory over the Past 11 Years

There is worldwide concern with the increasing rates of infections due to multiresistant Candida isolates reported in tertiary medical centers. We checked for historical trends in terms of prevalence rates and antifungal susceptibility of the Candida haemulonii species complex in our yeast stock culture collected during the last 11 years. The isolates were identified by sequencing the rDNA internal transcribed spacer (ITS) region, and antifungal susceptibility tests for amphotericin B, voriconazole, fluconazole, anidulafungin, and 5-fluorocytosine were performed by the Clinical and Laboratory Standards Institute (CLSI) microbroth method. A total of 49 isolates were identified as Candida haemulonii sensu stricto (n = 21), followed by C. haemulonii var. vulnera (n = 15) and C. duobushaemulonii (n = 13), including 38 isolates cultured from patients with deep-seated Candida infections. The prevalence of the C. haemulonii species complex increased from 0.9% (18 isolates among 1931) in the first period (December 2008 to June 2013) to 1.7% (31 isolates among 1868) in the second period (July 2014 to December 2019) of analysis (p = 0.047). All isolates tested exhibited high minimum inhibition concentrations for amphotericin B and fluconazole, but they remained susceptible to 5-fluorocytosine and anidulafungin. We were able to demonstrate the increased isolation of the multiresistant Candida haemulonii species complex in our culture collection, where most isolates were cultured from patients with deep-seated infections.

Pathogenicity Levels of Colombian Strains of Candida auris and Brazilian Strains of Candida haemulonii Species Complex in Both Murine and Galleria mellonella Experimental Models

Candida auris and Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) are phylogenetically related species that share some physiological features and habits. In the present study, we compared the virulence of these yeast species using two different experimental models: (i) Galleria mellonella larvae to evaluate the survival rate, fungal burden, histopathology and phagocytosis index and (ii) BALB/c mice to evaluate the survival. In addition, the fungal capacity to form biofilm over an inert surface was analyzed. Our results showed that in both experimental models, the animal survival rate was lower when infected with C. auris strains than the C. haemulonii species complex. The hemocytes of G. mellonella showed a significantly reduced ability to phagocytize the most virulent strains forming the C. haemulonii species complex. Interestingly, for C. auris, it was impossible to measure the phagocytosis index due to a general lysis of the hemocytes. Moreover, it was observed a greater capability of biofilm formation by C. auris compared to C. haemulonii species complex. In conclusion, we observed that C. auris and C. haemulonii complex have different levels of pathogenicity in the experimental models employed in the present study.