Clinical Candida isolates

Candida isolates recovered from clinical specimens were identified at the TASMC Clinical Microbiology Laboratory by growth characteristics on CHROMagar Candida (CHROMagar, Paris, France) and the Vitek 2 YST ID system (bioMérieux, Marcy-l’Étoile, France). The Vitek 2 database does not include C. auris, and this species is routinely misidentified as C. haemulonii (13). We therefore reviewed all isolates identified as C. haemulonii during January 2009–August 2015. Isolates recovered during May 2014–August 2015 were stored at −20°C and subjected to further analyses. We assessed thermotolerance by plating serial dilutions of yeast culture on Sabouraud dextrose agar (SDA) plates and assessing growth after 24 h incubation at 35°C–42°C.

Sequence-Based Species Identification

Candida isolates were streaked on SDA plates to ensure purity. We extracted DNA by using PrepMan Ultra solution (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions and amplified and sequenced the internal transcribed spacer (ITS) and D1/D2 large subunit (LSU) ribosomal DNA segments by using primer pairs ITS1/ITS4 and LSU1/LSU2 (14), respectively. PCR was performed in 0.2-mL tubes with 0.4 μmol/L or 0.2 μmol/L of each primer for ITS and LSU, respectively; 10 μL Larova Red Load Taq Master Mix (5×) (Larova, Jena, Germany); and ≈25 ng of template. PCR conditions were 95°C for 4.5 min (denaturation), 40 cycles of 95°C for 30 s (denaturation), 55°C (ITS) or 48°C (LSU) for 30 s (annealing), 72°C for 1 min (extension), and a final extension stage of 72°C for 7 min. PCR products were resolved on 0.7% agarose gel and stained with SERVA DNA stain clear G (Tamar, Mevaseret Zion, Israel). Products were cleaned with QIAquick PCR purification kit (QIAGEN, Hilden, Germany) and sequenced at Hy-Labs (Rehovot, Israel). We then aligned ITS and LSU sequences with the matching type strain sequences for CBS5149^T (C. haemulonii), CBS7798^T (C. duobushaemulonii), CNM-CL7239^T (C. haemulonii var. vulnera), CBS10099^T (C. pseudohaemulonii), and CBS10913^T (C. auris). A similarity score of >98% in both ITS and LSU sequences was required for species-level identification. All new sequences were deposited in GenBank (Table 1).

Phylogenetic Analyses

We aligned ITS and LSU sequences of C. haemulonii and C. auris isolates by using MUSCLE (15) and generated phylogenetic trees with the neighbor-joining method (16), using the Kimura 2-parameter method to compute evolutionary distances (17). We tested phylogeny with the bootstrap method (500 replicates) and used Schizosaccharomyces pombe strains ATCC 38366 and CBS 356 as outgroups. Evolutionary analyses were performed in MEGA7 (18).

Patient Characteristics

We retrospectively reviewed the medical records of patients from whom Vitek-identified C. haemulonii was recovered from any site and recorded patient demographics, hospital unit, co-morbidities, medications, and clinical characteristics by using a structured form. We found 40 patient-specific C. haemulonii isolates, 20 (50%) of which originated from patients receiving care at an outpatient peripheral vascular disease clinic (clinic A). We therefore conducted an investigation at clinic A, which included review of patient treatment protocols, observed patient care, and surveillance mycologic cultures from environmental surfaces, wound irrigation solutions, dressings, and the hands of medical staff. To define risk factors for C. haemulonii colonization, we conducted an unmatched case–control study using 40 noncolonized patients followed at clinic A as controls.

Antifungal Susceptibility Testing

We determined MICs of fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, anidulafungin, micafungin, caspofungin, and flucytosine by broth microdilution using Clinical and Laboratory Standards Institute methods (19). Results were read after 48 h for azoles, amphotericin B, and flucytosine and after 24 h for echinocandins.

Rhodamine 6G Efflux

To assess ABC-type drug transporter activity, we determined glucose-induced efflux of rhodamine 6G, as described previously (20,21). We grew Candida isolates to log-phase in liquid yeast extract glucose at 35°C. We then collected yeast cells by centrifugation, transferred 10⁹ cells to 20 mL fresh yeast extract glucose, and incubated them at 27°C for an additional 2 h. Next, we collected yeast cells by centrifugation, washed them twice in phosphate-buffered saline (PBS), and added 10 mL PBS containing 15 μM rhodamine 6G without glucose to the pellets. Suspensions were vortexed and incubated at 27°C for 90 min to enable rhodamine 6G uptake under carbon source–depleted conditions. We then collected cells by centrifugation, washed them twice in PBS, and suspended them in 750 μL PBS in microfuge tubes. To start rhodamine 6G efflux, we added 250 μL PBS with 8 mmol/L glucose. We prepared control tubes with glucose-free PBS, removed them after 5, 15, and 25 min of incubation at 35°C, and measured fluorescence in 200-μL aliquots of supernatant by using a spectrophotometer at excitation 527 nm and emission 555 nm (Synergy HT, BioTek, Winooski, VT, USA).

Mouse Model of Disseminated Candidiasis

To assess the relative virulence of C. haemulonii and C. auris, we determined the lethality and tissue fungal loads of representative strains in a mouse model of hematogenous disseminated candidiasis. Experiments were approved by the TASMC Institutional Animal Care and Use Ethics Committee. We used cyclophosphamide (150 mg/kg intraperitoneally) to immunosuppress 6-week-old female BALB/c mice weighing 16–20 g (Harlan, Rehovot, Israel) 3 days before and on the day of infection. Candida cells were collected from log-phase culture on the day of infection and washed twice in sterile PBS. Mice were infected in groups of 10 with C. haemulonii strain TA001-14, C. auris strain TA005-14, and C. albicans strain CBS 8837. We injected 100 μL of PBS containing 7 × 10⁷ yeast cells intravenously into the lateral tail vein of each animal. A control group received intravenous injection of cell-free PBS. Death was assessed over 12 days. Kidney tissue fungal loads were determined in separate experiments where mice were similarly immunosuppressed and infected intravenously with 4 × 10⁷ yeast cells/100 μL PBS. Seven days after infection, mice were killed by CO₂ inhalation, and kidneys were excised aseptically, weighed, and homogenized in a TissueLyser (QIAGEN). Homogenates were serially diluted 10- to 1,000-fold in sterile saline and plated on SDA. We calculated fungal loads (CFU per gram of tissue) from colony counts after 48 h incubation at 35°C.

Statistical Analyses

We compared continuous variables between case and control patients using the Student t test for normally distributed variables and the Wilcoxon rank-sum test for non–normally distributed variables. We compared dichotomous variables using Fisher’s exact test. Rhodamine 6G efflux, expressed as relative fluorescence units, was computed for each Candida strain and compared by using 1-way analysis of variance. We used Dunnett’s multiple comparisons test to compare relative fluorescence unit values of specific C. haemulonii and C. auris strains with averaged control values of 5 C. glabrata strains. Survival curves of mice infected with different Candida strains were plotted by using the Kaplan-Meier method and compared with the log-rank test. We considered 2-tailed p values <0.05 statistically significant.

Sequence-Based Identification

We identified 40 patient-specific Candida strains as C. haemulonii by the Vitek-2 YST ID system during January 2009–July 2015. Isolates were recovered from wounds (n = 24), urine (n = 9), blood (n = 5), and central venous catheter tips (n = 2). Of these, 9 isolates recovered during May 2014–May 2015 were available for analysis; we identified 6 (including the 5 blood isolates) as C. auris and 3 as C. haemulonii by ITS and LSU sequencing (Table 1). Sequences were 100% identical among strains of each species. C. auris strains from Israel shared 98.6% and 98.3% similarity of ITS and LSU sequences, respectively, with the C. auris type strain CBS10913^T. C. haemulonii strains were 100% identical to C. haemulonii CBS5149^T on the basis of ITS and LSU sequences.

Figure 1

Figure 1. Phylogenetic relationships of Candida auris and C. haemulonii strains isolated in Tel Aviv, Israel, compared with reference strains. Phylogenetic trees were generated from internal transcribed spacer (A) and D1/D2 domain of...

Phylogenetic trees based on ITS and LSU sequences showed that the C. auris isolates from Tel Aviv are distinct from other isolates from East Asia, Africa, and the Middle East. Specifically, isolates from Israel showed 98.6% similarity of ITS and LSU sequences with the India clone, represented by CBS12768, 96.2% similarity with the South Korea clone, and 96.7% similarity with strain CH1 from Kuwait. In contrast, ITS and LSU sequences from Israel C. haemulonii strains were 100% homologous with C. haemulonii from South Korea, Brazil, and Kuwait, suggesting worldwide predominance of a single C. haemulonii clone (Figure 1).

Clinical Features

Eight of 9 patients with sequence-validated isolates were hospitalized at TASMC (Table 1). An additional patient with C. auris infection was hospitalized at the Wolfson Medical Center, but was receiving regular care for HIV infection at TASMC. All 3 C. haemulonii isolates were recovered from chronic leg ulcers of patients with peripheral vascular disease, 2 of whom were treated at vascular outpatient clinic A. Five of 6 C. auris isolates represented BSI: 3 patients had vascular catheter–related candidemia, and 2 had primary nosocomial candidemia of unclear origin. Two of 5 patients with C. auris BSI died during hospitalization.

We reviewed the medical records of 40 patients with Vitek-identified C. haemulonii cultures. Thirty-three (83%) were male. Median age was 74 years (range 37–91 years). Nineteen (48%) had peripheral vascular disease, 20 (50%) had diabetes mellitus, 22 (55%) had ischemic heart disease, and 11 (28%) had end-stage renal disease. Twenty patients (50%) were receiving regular care at clinic A, representing 8% (20/261) of all clinic patients. In all 20 patients, C. haemulonii had been recovered from chronic leg ulcers, and none had documented wound infection at the time of culture. Cultures of environmental surfaces, medical devices, dressings, irrigation solutions, and hands of medical staff were negative for yeast. Compared with 40 control patients who were not carriers of C. haemulonii, carriers were older, had a lower glomerular filtration rate, and were more likely to be male and to have ischemic heart disease (Table 2). Observations revealed a practice among medical staff of routinely applying topical miconazole cream to chronic ulcers without evidence of infection. Periodic wound cultures were obtained regularly, irrespective of signs of ulcer inflammation or purulence. Multiple social interactions were noted among patients in a single room where wound care was performed.

Antifungal Susceptibility

All C. haemulonii and C. auris isolates had fluconazole MICs >8 mg/L (range 16–64 mg/L; MIC₅₀ 32 mg/L). MICs of other azoles were also elevated: itraconazole, 0.25 to >16 mg/L (MIC₅₀ 0.5 mg/L); voriconazole, 0.25–1 mg/L (MIC₅₀ 0.5 mg/L); and posaconazole, 0.06 to >8 mg/L (MIC₅₀ 0.25 mg/L). Amphotericin B MIC ranged from 1 to 2 mg/L for C. auris isolates and from 2 to 8 mg/L for C. haemulonii isolates. All isolates appeared susceptible to anidulafungin (MIC 0.03 mg/L) and all isolates except 1 C. haemulonii were susceptible to micafungin (MIC 0.12–0.5 mg/L; MIC₅₀ 0.12 mg/L). Caspofungin MIC was 0.5 mg/L for all isolates. All isolates except 1 C. auris were susceptible to flucytosine (Table 3).

Rhodamine 6G Efflux

Figure 2

Figure 2. Comparison of rhodamine 6G efflux over time among Candida isolates from Tel Aviv, Israel. Rhodamine 6G efflux is expressed as relative fluorescence units measured in culture supernatants after the addition of...

Rhodamine 6G is a substrate of ATP binding cassette (ABC) type efflux pumps responsible for multiazole resistance in C. glabrata. C. haemulonii and C. auris strains exhibited robust rhodamine 6G efflux activity when glucose (8 mM) was present in the medium, consistent with ABC-type transport. Rhodamine 6G efflux of C. auris strains was significantly greater than that of C. glabrata strains (14.4-, 10-, and 6.7-fold higher at 5, 15, and 25 min, respectively; p<0.0001) and C. haemulonii (3.8-, 3.8-, and 3.6-fold higher at 5, 15, and 25 min, respectively; p<0.0001). C. haemulonii showed greater rhodamine 6G efflux than C. glabrata (3.8-, 2.7-, and 1.9-fold higher at 5, 15, and 25 min, respectively (p<0.0001) (Figure 2).

Thermotolerance

Figure 3

Figure 3. Differing thermotolerance of Candida auris and C. haemulonii. A) Sabouraud dextrose agar plates showing growth of representative Candida strains after 24 h incubation at 35°C–42°C; B) Thermal growth range of Candida...

Survival and growth at physiologic temperature are prerequisites for microbial invasion and pathogenicity. C. haemulonii isolates grew well at 35°C, but growth at 37°C was poor or absent, and no growth occurred at 40°C and 42°C. In contrast, growth of C. auris isolates at 37°C and 40°C was similar to that of C. albicans, and 4 of 6 isolates grew at 42°C (Figure 3).

Virulence in a Mouse Model of Disseminated Candidiasis

Figure 4

Figure 4. Differing virulence of Candida auris and C. haemulonii assessed in a mouse model of hematogenous disseminated candidiasis. Virulence was assessed in immunosuppressed BALB/c mice after intravenous injection of yeast cell suspension....

We compared the virulence of C. auris and C. haemulonii isolates in a mouse model of hematogenous disseminated candidiasis. C. haemulonii was completely nonvirulent in this model; 100% of mice survived 12 days after inoculation with no visible signs of illness. In contrast, inoculation with C. auris resulted in rapid death and only 20% survival 5 days after infection (p = 0.0002, log-rank test). Death of mice infected with C. auris was significantly less rapid than that of mice infected with C. albicans (median survival 4 d and 1 d, respectively; p = 0.01; Figure 4, panel A). Kidney tissue fungal load correlated with survival rates. Specifically, we recovered no viable yeast cells from kidneys of mice inoculated with C. haemulonii, whereas infection with C. auris and C. albicans yielded median tissue loads of 5.9 × 10⁴ CFU/g and 7.1 × 10⁵ CFU/g, respectively (p<0.0001; Figure 4, panel B). Histopathologic analysis showed yeast cell aggregates in kidneys of C. auris–inoculated mice, distinct from tissue invasive hyphae observed in C. albicans–infected kidneys (Figure 4, panel C).