ABSTRACT
Candida albicans within the human host is well studied; however, identifying environmental reservoirs of pathogens is epidemiologically valuable for disease management. Oxygen-limited, carbohydrate-rich zones of wetlands, to which sewage-borne C. albicans is often exposed, are characteristically similar to the gastrointestinal reservoir. Consequently, using quantitative real-time PCR (qRT-PCR) and gas chromatography-mass spectrometry (GC-MS), we demonstrated that oxygen-limited zones in polluted wetlands may act as potential reservoirs of C. albicans.
TEXT
One-liter water samples originating from different zones at a sampling site (33°55′50″S, 18°51′10″E) in the sewage-polluted Plankenburg River (Western Cape, South Africa) were periodically analyzed for coliforms and yeasts by using classical and molecular microbiological methods. The three river zones (W [shallow], R [rock filtered], and P [plant filtered]) differed regarding depth (Fig. 1) and redox potential. The redox potential of the P zone, as determined using a Waterproof pH/ORP meter H1905 microcomputer (Hanna Instruments), measured between 10 and −80 mV. The redox potentials of the W and R zones were consistently ca. 80 to 100 mV higher than that of the P zone. The estimated volumetric flow rates of the respective zones, as determined by routing the current with a submerged pipe and measuring it with the bucket-and-stopwatch method (4), ranged from 0.2 liters/s to 0.4 liters/s for the W zone and from 0.0 liters/s to 0.1 liters/s for the R and P zones.
The different zones in the Plankenberg River that were studied included a shallow zone (W), 50 cm below the surface comprising clear, flowing water, and two deeper zones, situated 70 cm below the river surface. The latter included a rock-filtered zone (R) comprising slow-flowing water devoid of macrophytes situated on the rocky river bed and a plant-filtered zone (P) situated within slow-flowing water, between macrophytes and plant debris, on the river bed near the bank. Since the redox potentials of the W and R zones were notably higher than that of the P zone, the latter was referred to as the oxygen-limited, reducing zone and the W and R zones as the aerobic, oxidizing zones.
Coliform and total culturable yeast concentrations in the different zones were monitored on MacConkey agar (1) and Sabouraud glucose agar (SGA; 600 mg/liter chloramphenicol, pH 5.6, 30°C), respectively. The counts were all conducted in triplicate, and correlations (r) between coliform and total culturable yeast concentrations per zone (W, R, and P) were tested for significance (P) by using Statistica version 9.0 (Statsoft), also used in all subsequent statistical analyses. It was found that fecal pollution levels in the Plankenburg River were notably higher during the dry season than during the rainy season (Fig. 2). Also, there was a positive correlation between coliform and culturable yeast concentrations in the three zones that were studied. The r values obtained for the W, R, and P zones were 0.85 (P = 0.008), 0.92 (P = 0.0001), and 0.96 (P = 0.0009), respectively. These findings support the results of others who found a positive correlation between culturable yeast numbers, including those of C. albicans, and coliform counts in polluted waters (3).
Total coliform counts (A) and total yeast counts (B) in different zones of the Plankenburg River monitored over rainy and dry seasons. The rainy season is indicated; the rest of the year is predominantly dry. The dashed line represents clear, flowing water (W), the solid line represents rock-filtered zones (R), and the dotted line represents plant debris-filtered zones (P). All zones are ca. 50 cm below the water surface level. Counts were determined with three replicates per sample (n = 3). Total yeast and coliform concentrations in the respective zones were compared with a box-and-whisker plot showing means (♦, ●, or ○), standard deviations (□), and 1.96 × standard deviation (error bars).
On three occasions, quantitative real-time PCR (qRT-PCR) was used to enumerate C. albicans in the different zones (W, R, and P) of the Plankenburg River. These counts were all conducted in triplicate using fluorigenic 5′ nuclease (TaqMan) chemistry according to the method of Brinkman et al. (2). In each case, a 100-ml aliquot of the water sample was vortexed (Vortex Genie 2, set at 8) for 10 min to separate yeasts from particulate matter. The particulate matter in these subsamples was subsequently removed by passing each of them stepwise through a sterile sieve (0.1 mm), Whatman no. 1 filter paper (Schleicher and Schuell, Keene, NH), and a sterile 0.45-μm cellulose-nitrate filter disk (Sartorius Stedim Biolab Products, Aubagne, France). To remove the yeast cells from each filter disk, it was vortexed intermittently for 5 min (Vortex Genie 2, set at 8) with acid-washed glass beads (0.3 g; Sigma) in 1 ml physiological saline solution (PSS) (8.9 g/liter sodium chloride; Kimix, Cape Town, South Africa), contained in a 2-ml Eppendorf tube. After the filter disks were removed from the Eppendorf tubes, the cell suspension in each tube was centrifuged (5,000 × g, 10 min) and the pellet was resuspended in 100 μl PSS. The resulting cell suspension was examined microscopically to ensure that the yeast cells remained intact during the procedures described above. DNA was subsequently extracted from the cell suspension (Zymo fungal/bacterial DNA kit; Zymo Research, Irvine, CA). Subsequent PCRs were performed in 20-μl LightCycler capillaries (Roche Diagnostics, Basel, Switzerland) with the addition of 12.5 μl TaqMan universal master mix (Roche Diagnostics), 5 μl forward and reverse primers (5 μM each; Inqaba Biotechnical Industries, Pretoria, South Africa) with 400 nM TaqMan probe (3′ 6-carboxyfluorescein [FAM] labeled and 5′ 6-carboxytetra-methylrhodamine [TAMRA] quencher labeled; Inqaba Biotechnical Industries), 2.5 μl bovine serum albumin (2 mg/ml; Roche Diagnostics), and 5 μl DNA template. The forward (Calb F1) and reverse (Calb R1) primers used, as well as the probe (Calb P1), were designed by Brinkman et al. (2). Thermal cycling was initiated with 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C with a final cooling stage of 30 s at 40°C. Geotrichum candidum CBS 109.12 was used as reference strain and the ΔΔCT (comparative cycle threshold) method was used to determine the quantities of C. albicans cells (LightCycler software, version 4.05; Roche Diagnostics).
Our results obtained with qRT-PCR indicated that the numbers of C. albicans within the Plankenburg River, enumerated with a standard error of 0.0196 and an amplification efficiency of 1.879, differed in the W, R, and P zones in the river (Table 1). Despite the presence of culturable yeasts and coliforms in all three zones (Fig. 2), C. albicans was not detected with qRT-PCR in the shallow W zone. It could be found only in the two deeper zones; the R zone demonstrated approximately 0 cells/100 ml during the rainy periods and 102 cells/100 ml during the drier period (Table 1). Importantly, C. albicans levels remained at 102 to 103 cells/100 ml in the oxygen-limited, reducing P zone, irrespective of fluctuating coliform and culturable yeast concentrations (Fig. 2). These findings, together with the apparent absence of C. albicans in the shallow W zone, indicated that the yeast is capable of sustainable growth in the oxygen-limited, reducing P zone.
C. albicans concentrations in triplicate samples taken each month from the Plankenburg River zones and analyzed by qRT-PCR
To further investigate the ability of C. albicans to grow in the P zone, in vitro studies were conducted to test C. albicans for anaerobic utilization of nutrients, particularly simple saccharides, in plant debris typically found on river banks. Thus, 60 g dead plant debris (Scirpus and Typha leaves, stems, and roots) was first cut into 2-mm-by-2-mm fragments, suspended in 600 ml distilled water, and autoclaved (121°C, 2 kg/cm2, 15 min). Subsequently, the particulate matter was filtered (Whatman no. 2 filter paper) from the resulting extract, and the filtrate was sterilized via further filtration (cellulose acetate, 0.22 μm; Sartorius Stedim Biolab Products). Aliquots (9.9 ml each) of the sterile extract were aseptically dispensed into test tubes and inoculated with 0.1 ml PSS containing 1 × 104 washed stationary phase cells of one of four different C. albicans strains (CAB 628-1, CAB 628-2, TH 8908, or TH 8912). The resulting cultures were anaerobically incubated at 30°C in an upright position within anaerobic jars containing Anaerocult A sets (Merck, Darmstadt, Germany) and strapped onto a rotary shaker (150 rpm, 25-mm throw). Preliminary growth curves under these conditions (hourly readings at an optical density of 600 nm [OD600]) demonstrated log phase at 6 to 8 h. It was therefore decided to harvest the culture fluids of the above-mentioned cultures after 7 h via sterile filtration (0.22-μm syringe filters; GVS Filter Technologies, Bologna, Italy). All cultures were prepared in triplicate, and negative controls, consisting of 9.9 ml extract that received 0.1 ml PSS without yeast cells, were also included in the experimentation. The concentrations of mono- and disaccharides remaining in the culture filtrates were determined with gas chromatography-mass spectrometry (GC-MS) (6). It was found that, under anaerobic conditions, all the C. albicans strains were able to assimilate glucose and fructose, present in the wetland plant debris extract (Fig. 3). Although these experiments were not conducted under the same culture conditions, our results are in accordance with the findings of others recorded in literature, which state that C. albicans is capable of aerobically assimilating many mono- and disaccharides, as well as being able to ferment glucose, galactose, and sucrose (5).
Carbohydrate concentrations, determined using GC-MS, remaining in a wetland plant debris extract after anaerobic culturing with C. albicans strains (CAB 628-1, CAB 628-2, TH 8908, and TH 8912), in comparison with uninoculated controls. The yeast was cultured in the plant extract for ca. 7 h at 30°C. The sugars assessed for assimilation by C. albicans include glucose (A), fructose (B), and galactose (C). The results are the means (■) of three repetitions. Standard deviations (□) and (1.96 × standard deviation) (error bars) are depicted.
Using qRT-PCR, we found that C. albicans is able to persist within the deeper oxygen-limited, reducing zones of wetlands, despite its apparent absence in shallow aerobic, oxidizing zones. In addition, we demonstrated that carbon sources present in wetland plant debris may act as nutrient sources for the yeast under anaerobic conditions. These findings suggest oxygen-limited, reducing zones of wetlands as a niche for C. albicans outside its human host and a potential external reservoir for this human commensal.
ACKNOWLEDGMENTS
We acknowledge the assistance of Annatjie Hugo and Fletcher Hilten.
We acknowledge the Water Research Commission (WRC) and the National Research Foundation (NRF) of South Africa for financial support.
FOOTNOTES
- Received 29 July 2011.
- Accepted 9 January 2012.
- Accepted manuscript posted online 13 January 2012.
- Copyright © 2012, American Society for Microbiology. All Rights Reserved.
