A total of 55 strains of C. albicans isolated from the blood samples of 55 patients with C. albicans septicemia who were admitted to the hospital from January 2017 to December 2018 were collected. The patients had not received antifungal therapy before the diagnosis of C. albicans septicemia and had not received catheter therapy. All C. albicans strains were sensitive to common antifungal agents.

A small number of clinically isolated C. albicans was selected via inoculation ring. C. albicans were inoculated on Sabouraud dextrose agar (SDA) using a three-zone scribing method and placed into a temperature box at 37°C overnight. The following day, one of the growing strains was transplanted into 5 mL of yeast peptone glucose (YPG) medium and cultured overnight in a shaking table at a speed of 200 rpm at 35°C. On the third day, the medium was removed from the shaker and centrifuged for 5 min at 3,000 rpm to collect the thalli. The obtained thalli were rinsed with saline solution three times and diluted with RPMI-1640 medium. C. albicans were then subcultured in yeast peptone and glucose medium at 150 rpm and 35°C in a shaking table overnight (Deng et al., 2016, 2017). The concentration of C. albicans solution was adjusted to 1 × 10⁷ cells/mL.

The susceptibilities of C. albicans strains to antifungal agents, including fluconazole (Diflucan, Pfizer Manufacturing Deut.), voriconazole (Vfend, Pfizer Manufacturing Deut.), itraconazole (Sporanox, Xian-Janssen Pharmaceutical Ltd.), caspofungin (Concidas, Merck & Co., Inc.), and micafungin (Mycamine, Astellas Pharma Tech Co., Ltd), were assayed in vitro using a microliquid-based dilution method M27-A3 [Clinical and Laboratory Standards Institute (CLSI), 2008]. The ATCC control used in this study was AT0CC10231.

To detect ALS3 expression levels, total RNA extracted from grinded fungi with TRIzol reagent (Invitrogen, Carlsbad, CA, United States) was used as the template for all reverse transcriptase reactions. The cDNA was synthesized with random priming using 10 μL of total RNA and RevertAid First Strand cDNA Synthesis Kit (Fermentas, CA, United States) following the manufacturer’s protocol. The upstream primer of ALS3 was 5′-CCGGTTTCATCTGAATCATTTAGTT-3′. The downstream primer of ALS3 was 5′-ACGACAAGGTGTACGAATTAACATCT-3′. The upstream primer of the internal gene (ACT1) was 5′-TGGGCCAAAAGGATTCTTATG-3′. The downstream primer of the internal gene was 5′-AGATCTTTTCCATATCATCCCAG-3′. The housekeeping gene fragments of C. albicans consisted of AAT1A, ACC1, ADP1, MPIB, SYA1, VPS13, and ZWF1B (Bougnoux et al., 2003). Quantitative expression of ALS3 levels was conducted by real-time RT-PCR with a LightCycler 96 system (Roche, Switzerland). The amplification consisted of denaturation at 95°C for 30 s (s), annealing at 95°C for 3 s, and extension at 60°C for 30 s (40 cycles). Each reaction was run in triplicate. ALS3 expression was normalized to ACT1. Expression levels of the regulatory gene and ALS3 were determined using the delta-delta Ct (2^–△△Ct) method. Ct indicates the average threshold period of the genes obtained in three independent experiments (Escribano et al., 2017). Data are presented as mRNA transcripts (arbitrary units) relative to ACT1 (Hosseini et al., 2019).

All 55 strains C. albicans were added to 12-well culture plates at 2 mL/well. A 10 mm sterile indwelling catheter was placed in each well. The culture plates were incubated in a 150 rpm shaking table at 35°C for 90 min (min). Then, culture plates and catheters were washed three times using aseptic saline and cultured in YPG medium for 72 h (h) at 37°C. Biofilm formation in vitro was completed in the 12-well culture plates (Deng et al., 2016, 2017).

Six-week-old male C57 mice weighing 20.24 g ± 1.78 g (n = 50, Beijing Vitonlihua Experimental Animal Technology Co., Ltd., Beijing, China) were randomly divided into three groups. On the basis of ALS3 expression in C. albicans, mice were grouped into either the high ALS3 expression group (20 mice), low ALS3 expression group (20 mice), or blank control group (10 mice). A 20 mm long sterile indwelling catheter was placed into the abdominal cavity of each mouse. Mice in the two experimental groups received 1 × 10⁷ C. albicans with either high or low ALS3 expression by intraperitoneal injection 48 h later. Mice in the control group did not receive C. albicans injection. In all three groups, catheters were kept in the abdominal cavity.

After feeding under the same conditions for 2 weeks, all the three groups of mice were sacrificed by cervical dislocation. Catheters were taken out, washed three times using saline solution, then cultured in YPG medium for 48 h at 37°C. After catheters were cultured, the growing colonies of C. albicans were isolated. The randomly amplified polymorphic DNA (RAPD) method (Perrone et al., 2009) was used to determine whether C. albicans isolated in vitro were the same as those inoculated in abdominal cavities of the mice. Four arbitrary promoters (LEG2, CDL6s, Leptopatho, and CDL6as) were selected for the RAPD method (Vrioni and Matsiota-Bemard, 2001). Finally, ALS3 expression in C. albicans strains was detected, also we performed a drug sensitivity test.

Six-week-old male C57 mice weighing 21.02 ± 1.12 g (n = 40, Beijing Vitonlihua Experimental Animal Technology Co., Ltd, Beijing, China) were divided into four groups in the prevention of biofilm formation experiment. The mice were divided into the voriconazole group, caspofungin group, micafungin group, and control group, with 10 mice in each group. Ten C. albicans strains with high ALS3 expression were selected for the prevention experiment. A 20 mm long sterile indwelling catheter was placed into the abdominal cavity of each mouse. Ten mice in each group received a dose of 1 × 10⁷ C. albicans by intraperitoneal injection 48 h later. All mice in the experiment groups received preventive therapy consisting of different antifungal agents, which included voriconazole (10 mg/kg per day), caspofungin (1.5 mg/kg per day), and micafungin (1.5 mg/kg per day) in each group. After feeding under the same conditions for 2 weeks, C. albicans were extracted from the in vitro cultures.

SPSS 17.0 (SPSS, Inc., Chicago, IL, United States) statistical software was used for statistical analysis. Data were expressed as the mean ± standard error and analyzed by t-tests and F-tests. Susceptibility tests for antifungal agents were compared using non-parametric tests for independent samples. P < 0.05 was considered a statistically significant difference.

In the biofilm formation experiment, 29 C. albicans strains out of the 55 total strains formed biofilms in vitro. The C. albicans budded and began to form mycelium after 6 h of cell culture in vitro under observation with an inverted microscope. C. albicans colonies fused with each other and arranged into a network after 12 h of culture. C. albicans then clumped along the mycelium formation after 24 h of culture. Finally, the mycelium of C. albicans interlaced and formed a membrane network structure inside the entire catheter after 48 h of culture in vitro.

Cell walls of C. albicans were broken in the 26 strains that did not form biofilms. The thickness of most cell walls was about 110–220 nm. Electron density in the strains was higher in structures with spores that sprouted (Figure 1A). In the 29 strains that formed biofilms in vitro, cell walls were about 200–350 nm. Electron density in these strains and in structures with spores were the same as those that did not form biofilms. However, the strains that formed biofilms were rich in mitochondria (Figure 1B).

There were obvious differences among ALS3 expression in the 55 C. albicans strains after they were collected from patients. The ratio of ALS3 expression to the median of the 55 C. albicans strains is shown in Figure 2.

Objective stripes were presented at 100–200 bp after electrophoresis of gene amplification products with 2% sepharose gel. The average ALS3 expression was higher in the 29 C. albicans with biofilm formation than that in the 26 C. albicans without biofilm formation (P = 0.000). The average ALS3 expression declined after culture in vitro in the 29 strains with biofilm formation (P = 0.013). However, there were no differences in the 26 strains without biofilm formation before and after culturing (P = 0.167; Figure 3).

During the biofilm formation experiment in mice, two mice in the high ALS3 expression group died on the third and fourth days after the sterile indwelling catheter was placed into the abdominal cavity. No mice died in the low ALS3 expression group or control group. After 2 weeks, the catheters were removed. A total of 14 C. albicans strains (14/18, 77.8%) in the high ALS3 expression group formed biofilms with membrane structures attached to the catheters walls when observed under a microscope.

Three C. albicans strains (3/20, 15%) in the low ALS3 expression group formed biofilms. The rate of biofilm formation in the high ALS3 expression group was higher than that in the low ALS3 expression group (P = 0.000; Figure 4A). After the catheters were cultured, growing colonies of C. albicans were extracted. There were 12 C. albicans strains from catheters of the high ALS3 expression group and eight C. albicans strains from catheters of the low ALS3 expression group that were extracted from culture in vitro (Figure 4B). All C. albicans strains that were isolated from culture in vitro were identified by RAPD as having the same origins as C. albicans inoculated in the abdominal cavities of the mice.

In the prevention of biofilm formation experiment, eight C. albicans strains (8/10, 80%) in the voriconazole group, two C. albicans strains (2/10, 20%) in the caspofungin group, and three C. albicans strains (3/10, 30%) in the micafungin group formed biofilms with membrane structures attached to the catheter walls when observed under a microscope (Figure 5). The rates of biofilm formation in the caspofungin and micafungin groups were lower than that in voriconazole group (P_caspofungin = 0.007 and P_micafungin = 0.025). However, there were no differences in the biofilm formation rate between the caspofungin and micafungin groups (P = 0.606).

After the catheters removed from the mice were cultured in vitro, growing colonies of C. albicans were isolated. A total of 12 C. albicans strains in the high ALS3 expression group were isolated from the culture system, while eight C. albicans strains in the low ALS3 expression group were isolated. All 12 C. albicans strains in the high ALS3 expression group formed biofilms, while only one C. albicans strain formed biofilm in the low ALS3 expression group. The average ALS3 expression of 12 C. albicans strains in the high ALS3 gene expression group declined from 13,710 ± 1,839 to 8,828 ± 1,680. The average ALS3 expression of eight C. albicans in the low ALS3 expression group was 1,860 ± 706, while it was 1,607 ± 324 in the same group before the biofilm formation experiment. There were no differences in average ALS3 expression before and after the biofilm formation experiment in the low ALS3 expression group (Figures 4C,D).

The susceptibility of C. albicans to antifungal agents (e.g., fluconazole, voriconazole, itraconazole, caspofungin, and micafungin) in vitro was assayed. The activities of antifungal agents against the planktonic form of 55 C. albicans strains isolated from blood samples of patients with candidaemia were examined. All antifungal agents showed high antifungal activity against the 55 C. albicans strains (Table 1). After the biofilm formation experiment in vitro, activities of antifungal agents against the 26 C. albicans strains without biofilm formation and 29 C. albicans strains with biofilm formation were compared. The C. albicans strains with biofilm formation were more resistant to fluconazole, voriconazole, and itraconazole. However, these strains remained sensitive to caspofungin and micafungin. The C. albicans strains without biofilm formation were sensitive to all antifungal agents (Table 2).

After the biofilm formation experiment in mice, activities of antifungal agents against the 12 C. albicans strains from the catheters in high ALS3 expression group and the eight C. albicans strains from the catheters in low ALS3 expression group were compared. The C. albicans from the catheters in the low ALS3 expression group were sensitive to all antifungal agents. The C. albicans strains from the catheters in high ALS3 expression group were more resistant to fluconazole, voriconazole, and itraconazole. These strains remained sensitive to caspofungin and micafungin (Table 3).