Candida haemulonii species complex: emerging fungal pathogens of the Metschnikowiaceae clade

Abstract

Candida species represent the most common fungal pathogens of humans, causing not only superficial infections but also life-threatening invasive infections, especially in immunocompromised individuals. While Candida albicans remains the most frequent cause of candidiasis, infections caused by non-albicans Candida species have been increasingly reported in clinical settings over the past two decades. Recently, species of the Metschnikowiaceae clade including the “superbug” Candida auris and other members of the Candida haemulonii species complex have attracted significant attention due to their multidrug resistance and high rates of transmission in clinical settings. In this review, we summarize the epidemiology, biology, virulence, and drug resistance of the C. haemulonii species complex and discuss potential reasons for the recent increase in prevalence of infections caused by non-albicans species in clinical settings.

Introduction

Pathogenic fungi cause not only superficial infections but also life-threatening invasive infections, especially in immunocompromised individuals¹. It is estimated that 1.7 billion people suffer from superficial fungal infections and 2.5 million people suffer from serious invasive fungal infections annually worldwide^1,2. Of the latter group, approximately 1.5 million people die from these invasive fungal infections every year^1,3. Fungal infections have been gaining worldwide attention recently as notable spikes in the incidence rates of these infections have significantly increased over the past several years likely due to surges in the use of clinical invasive procedures, such as central venous catheters and shunts, the overuse of broad-spectrum antibiotics, the increase in prolonged hospital and intensive care unit (ICU) stays, the prevalence of human immunodeficiency virus (HIV) infections and other immunocompromised conditions, and a shift towards more aging populations^3–5. In addition, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has further exacerbated the situation⁶. Aspergillus, Mucorales and Candida species, including Candida auris, for example, have all been reported to cause coinfections with SARS-Cov-2, resulting in higher morbidity and mortality rates compared to single infections caused by of any of these pathogens alone⁶.

Candida species are the most common fungal pathogens that cause mucosal candidiasis, deep organ and bloodstream infections⁷. Despite receiving treatment with antifungal drugs, over 40% of patients with invasive Candida infections die^1,8,9. Candida albicans is the most frequently isolated Candida species in clinical settings, and as such has drawn significant research attention^3,10. Over the past two decades, however, epidemiological surveillance has indicated a shift towards the isolation of non-albicans Candida species, likely due to an increase in the use of antifungal drugs in clinical practices^9,11. Specially, non-albicans Candida species of the Candida haemulonii complex and the closely related species C. auris, which some studies have classified into the C. haemulonii complex^12,13, have garnered significant attention from both clinical and basic research communities. Multidrug resistance is a notably common characteristic of the C. haemulonii species complex.

Most pathogenic Candida species (except for Candida glabrata) phylogenetically belong to the CTG clade, where species translate the CTG codon to serine rather than the canonical leucine¹⁴. The CTG clade is composed of the Metschnikowiaceae and Debaryomycetaceae clades¹⁵. C. albicans, Candida tropicalis, and Candida parapsilosis belong to the Debaryomycetaceae clade, while C. auris and the C. haemulonii species complex are classified into the Metschnikowiaceae clade. Species in the C. haemulonii complex are particularly concerning in hospital settings due to their rapid and increased emergence worldwide and intrinsic resistance to existing antifungal drugs^16,17. Here, we review the research progress made over the past two decades on the epidemiology, biology, virulence, and drug resistance of the C. haemulonii species complex.

Epidemiology

C. haemulonii was first isolated from the seawater the Atlantic Ocean and the intestines of the Haemulon sciurus fish in 1962¹⁸. The first clinical isolate of C. haemulonii was recovered from the blood of a patient with renal failure in 1984¹⁹. In 2007, the first outbreak of C. haemulonii fungemia was reported in a neonatal intensive care unit (NICU) in Kuwait, where seven isolates were identified from the blood of four neonates²⁰. Fungemia caused by C. haemulonii was later reported in hospitalized patients in China and Korea in 2009^21–23. Other species of the C. haemulonii complex including the Candida haemulonii sensu stricto (ss) species, Candida duobushaemulonii, Candida pseudohaemulonii, Candida haemulonii var. vulnera, and Candida vulturna have been sporadically identified from patients in hospital settings over the years (Figure 1)^16,24–27.

Figure 1.

Representative species of the Candida CTG Clade.

A. Dates of the first isolate report of species in the Candida haemulonii complex. B. Maximum-likelihood phylogenetic tree of C. haemulonii species complex and closely related species was constructed based on ITS sequences and 1,000 bootstrap replicates. All sequences were acquired from the NCBI GenBank database. General time reversible (GTR) and gamma distribution with invariant sites (G+I) models were used.

C. duobushaemulonii was first isolated from a patient with a foot ulcer in 1990²⁸. A retrospective study reported that a C. duobushaemulonii strain from the toenail of a patient in Spain was misidentified as Candida intermedia in 1996²⁹. Several invasive C. duobushaemulonii infections were reported in China by the China Hospital Invasive Fungal Surveillance Net (CHIF-NET) between 2009 and 2017^26,27. Overall, although known cases of C. duobushaemulonii infection are rare to date, occasional hospital outbreaks have been reported²⁸.

C. pseudohaemulonii was first isolated in 2006 from the blood of a patient in Thailand²⁵. Between 2004 and 2006, seven isolates of C. pseudohaemulonii were recovered from the blood of seven Korean patients²². Interestingly, however, hospital outbreaks caused by C. pseudohaemulonii have not yet been reported. C. haemulonii var. vulnera, which was first identified in 2012, is a rare variant of C. haemulonii. Infections caused by C. haemulonii var. vulnera have been reported in Brazil, India, Argentina, Peru, and China^30–34. The first strain of C. vulturna was isolated from flowers in Cagayan de Oro, Philippines in 2016, and as such, C. vulturna was thought to be associated with plants and the environment²⁴. That same year, a C. vulturna strain was then isolated from the blood of a patient who died of pneumonia in Malaysia²⁴. In 2022, a case of catheter-related C. vulturna fungemia was identified from a septic patient with an infected intractable retroperitoneal cyst in Malaysia³⁵. Candida khanbhai is the newest member of the C. haemulonii complex, and was isolated from clinical samples obtained from patients in Kuwait and Malaysia in a study published this year¹³. Overall infections caused by the C. haemulonii species complex have been increasing in prevalence recently with new reports of cases in China and Brazil, and have garnered considerable attention due to the multidrug-resistant properties of the isolates involved^36,37. Both clinical and basic research communities are urged to keep a close watch on these globally emerging fungal pathogens.

Biology

Similar to the major human fungal pathogen C. albicans, species of the C. haemulonii complex can undergo morphological transitions and form biofilms, which are important processes contributing to fungal pathogenesis^38–41. Clinical isolates of C. haemulonii produce smooth and round colonies under standard laboratory culture conditions, and can form colonies with light to dark violet coloration CHROMagar, a particular chromogenic medium used to isolate and differentiate certain clinically relevant Candida species⁴². C. haemulonii phenotypic transitions have been recently characterized⁴⁰. C. haemulonii displays different phenotypes (white, pink, or filamentous) in response to specific growth conditions. The transition between the white and pink phenotypes appears to be the primary switching system in C. haemulonii. Clinical isolates of C. haemulonii often form both white and pink colonies at 25°C on yeast peptone dextrose (YPD) agar plates containing the red dye phloxine B. Cells from pink colonies are larger than those from white colonies. Similar to the white-opaque switch of C. albicans, the white-pink switch of C. haemulonii appears to be heritable and reversible. Moreover, C. haemulonii pink cells can form wrinkled colonies containing elongated filaments at 25°C on yeast peptone glycerol (YPG) agar plates. This C. haemulonii filamentous phenotype is relatively stable, suggesting that it is regulated through genetic or epigenetic mechanisms. These distinct C. haemulonii phenotypes also differ in gene expression profiles, metabolic profiles, the production of secreted aspartyl proteases (Saps), and virulence⁴⁰.

In addition to morphological transitions, the ability to form biofilms is another important virulence factor for pathogenic Candida species^8,41, A fungal biofilm is a coordinated and functional community of fungal cells that are encased in an extracellular matrix. Fungal biofilms have increased drug resistance properties compared to free-floating fungal cells. Like C. albicans, species of the C. haemulonii complex can form biofilms on indwelling medical devices⁸, which significantly increases the morbidity and mortality of hospitalized patients^43,44. For example, C. haemulonii has been shown to cause serious bloodstream infections that originate from biofilms formed on the surface of indwelling intravascular catheters⁴¹. It has been reported that proteins and carbohydrates are the main components of the extracellular matrix of biofilms formed by clinical isolates of the C. haemulonii species complex⁴¹. Interestingly, species of the C. haemulonii complex can form biofilms of different biomasses on several types of catheter surfaces, including vascular (polystyrene), urinary (siliconized latex), nasoenteric (polyurethane), and nasogastric (polyvinyl chloride)⁴¹. While the structure and function of biofilms formed by the C. haemulonii species complex have been described, the underlying mechanisms of biofilm formation in these species remain unclear^23,41,45,46. In general, the abilities to undergo phenotypic transitions and to form biofilms are important in the environmental adaptation and virulence of the C. haemulonii species complex. Future exploration into the regulatory mechanisms involved in these processes will provide new insights into the development of novel strategies for the prevention and treatment of fungal infections.

Virulence

Secreted aspartyl proteases (Saps), phospholipases, and esterases are important virulence factors that are known to facilitate the colonization and survival of pathogenic Candida species in their hosts⁴⁷. The genomes of the C. haemulonii species complex genomes contain multiple genes encoding Sap-like conserved domains, and the production of Saps by both clinical and environmental C. haemulonii species complex isolates has been reported^33,48–51. In fact, polyclonal antibodies specific to C. albicans Sap1, Sap2, and Sap3 are able to recognize C. haemulonii Sap-like proteins⁴⁹. Other hydrolytic enzymes, including serine proteases, phospholipases and esterases, have also been found in species of the C. haemulonii complex^{33,48,50,52,53}.

Compared to other pathogenic Candida species, the dose-dependent virulence of species of the C. haemulonii complex has been reported to be relatively low in an immunocompetent murine model of disseminated infection⁵⁴. In this model, all mice inoculated with C. haemulonii survived; however, half of the mice died when the inoculum of C. haemulonii cells was increased by 1 to 2-logs. Yeast cells of C. haemulonii were recovered from different organs of infected mice on days 5 and 10 post-infection regardless of inoculum size⁵⁴. Another study demonstrated that C. haemulonii was completely nonvirulent in an immunosuppressed mouse model of disseminated infection and no viable yeast cells were recovered from the kidneys of infected mice 12 days post-infection⁵⁵. In a Galleria mellonella infection model, reduced fungal burdens and prolonged host survival was observed for species of the C. haemulonii complex compared to C. auris⁵⁶. Indeed, in both the mouse and G. mellonella infection models, host survival rates when infected with species of the C. haemulonii complex were higher compared to when the host was infected with C. auris or C. albicans⁵⁶. Future virulence studies will be important to shed new light on the mechanisms of pathogenesis of the C. haemulonii species complex compared to other Candida species.

Drug resistance

Species of the C. haemulonii complex are often resistant to multiple antifungal drugs, which is a common reason for treatment failure¹⁷. A majority of clinical isolates of the C. haemulonii complex exhibit limited susceptibility to the triazoles and to amphotericin B with elevated minimum inhibitory concentrations (MICs)^{20,22,26,30,42,57,58}. Several clinical isolates of the C. haemulonii complex also exhibit resistance to the echinocandins and to flucytosine^{16,26,27,31,33,34,59,60}.

It has been reported that mutations in the lanosterol 14α-demethylase-encoding gene ERG11 and upregulation of the efflux pump-encoding genes, such as CDR1, are associated with azole resistance in the C. haemulonii species complex^28,61,62. Interestingly, efflux pump inhibitors have been shown to reverse the observed azole resistance caused by these mutations⁶². The mechanisms of echinocandins and amphotericin B resistance in the C. haemulonii species complex remain to be investigated. Given the common multidrug resistant characteristics of the C. haemulonii complex species, the associated mechanisms are likely to be complex and significant research effort should be focused on this area in the future.

Perspectives

The prevalence of historically “rare” fungal pathogens, such as C. auris and the C. haemulonii species complex, has been increasingly observed in clinical settings over the past two decades. Given the difficulties in accurately identifying these species using conventional phenotypic or standard biochemical methods, infections caused by these species are likely underestimated. The more widespread use of molecular identification techniques, such as metagenomic next-generation sequencing (mNGS) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) in clinical settings will greatly improve the accuracy of identification and the characterization of these fungal species.

The emergence of new fungal pathogens and the increase in reports of multidrug resistant C. auris and species of the C. haemulonii complex from different countries can be potentially explained by several reasons. First, the widespread use of antifungal drugs in clinical settings and of fungicides in the environment (for example, for agricultural use and for wood preservation) may promote the evolution and emergence of drug resistant fungal species. Second, the increase of immunocompromised and more elderly populations, combined with clinical antifungal drug treatment regimens, provides an additional avenue for the evolution of antifungal drug resistance. Third, ecological factors, including climate change, combined with exposure to fungicides in the environment, provide yet another avenue for new emerging multidrug resistant fungal pathogens to infect the human host.

Although the global prevalence of the C. haemulonii species complex remains relatively low compared to other fungal pathogens, nosocomial outbreaks have been reported with increasing frequency in many countries. To limit infections caused by the C. haemulonii species complex, future studies should focus on understanding the epidemiology, pathogenesis, and drug resistance of these important emerging fungal pathogens. In particular, the transmission of the C. haemulonii species complex is a notable area for future research, as how transmission to the host occurs is not clearly understood. Given the increase in reported cases and the multidrug resistance of the associated isolates, the C. haemulonii species complex should be considered to be a serious public health threat worldwide.

Biography

Chengjun Cao, Professor

Chengjun Cao graduated from Zhengzhou University with Bachelor of Science in 2011. She received her doctoral degree in microbiology from Institute of Microbiology, Chinese Academy of Sciences in 2017. She got post-doctoral training at Rutgers University during 2017 to 2022. Dr. Cao currently works at Southwest University (Chongqing, China). Her research interest is the regulatory mechanism of pathogenicity and antifungal drug resistance of human fungal pathogens, including Cryptococcus and Candida species.