Candida species distribution, genotyping and virulence factors of Candida albicans isolated from the oral cavity of kidney transplant recipients of two geographic regions of Brazil

During a 3 months-period, 154 patients from two different geographic regions of Brazil (111 from Natal, Rio Grande do Norte and 43 from Maringa, Parana) were subjected to clinical examination of the oral cavity for candidiasis diagnosis, according to the criteria established for oral lesions in HIV patients recommended by the EC-Clearinghouse and World Health Organization classifications [14]. Only patients who agreed to take part on a surveillance confidential study, in accordance to the Local Research Ethics committee from The Onofre Lopes University Hospital, approved under the number 152/07, were enrolled in this study. Strains isolates from oral colonization (without any symptoms) or during episodes of oral candidiasis were evaluated in this study. For genotyping and virulence studies, we randomly selected 48 clinical strains of C. albicans (38 from Natal, Rio Grande do Norte and 10 from Maringa, Parana). All the isolates are described in Table 1. The samples were stocked on YPD glycerol (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose and 3% glycerol) at -80°C at the Laboratório de Micologia Médica e Molecular, Universidade Federal do Rio Grande do Norte, Brazil. C. albicans ATCC90028 and SC5314 were included in this study as reference strains.

Samples containing 2 mL of saliva were collected from all patients, by previous stimulation with chewing gums. When lesions were present, a sterile swab was rubbed on the mucosal surface of the buccal cavity. Subsequently, 100 μL of cells suspensions were inoculated on the surface of Sabouraud Dextrose Agar (SDA; Oxoid, UK) added 300 μg/mL of cloramphenicol (Park-Davis), by using a Drigalsky loop. The plates were incubated at 37°C for 48 h. Yeast colonies were plated on CHROMagar Candida® (CHROMagar Microbiology, Paris, France) to check for purity and screening for different color colonies. Species identification was based on the characteristics of the cells observed microscopically after cultivation on cornmeal agar added Tween 80, as well as assimilation and fermentation testing and ID32C System (bioMérieux Marcy l’Etoile, France), whenever it was necessary [15]. Strains belonging to the Candida parapsilosis species complex were identified previously with molecular methods [13].

Microsatellite typing was performed using the primer M13 (5’ GAGGGTGGCGGTTCT--3’) (IDT) as previously described [16]. ABC genotyping was performed with the following primers: CAINT- L (5′-ATAAGGGAAGTCGGCAAAATAGATCCGTAA-3′) and CA-INT-R (5′-CCTTGGCTGTGGTTTCGCTAGATAGTAGAT-3′) [17]. Briefly, 1.0 μL of DNA 40 ng/μL was added to 29 PCR Master Mix (Promega) to a final volume of 25 μL. The samples were amplified in a Thermocycler (Amplitherm TX96, USA) using the following cycling parameters: one initial cycle of 94°C for 3 min followed by 30 cycles of 1 min at 94°C, 1 min at 57°C, 1 min at 72°C and a final cycle of 5 min at 72°C. For Microsatellite typing, 1.0 μL of DNA 40 ng/μL, 2.5 μL of 10 × PCR buffer (100 mM Tris–HCl, pH 8.3, 500 mM KCl, 3.5 mM MgCl₂), 5 μL of dNTPmix (100 mM each dNTP), 1.0 μL of each primer (50 pmol/μL), 0.13 μL of tween 20 and 1.0 unit of Taq DNA polymerase were added to a final volume of 25 μL. Forty-five cycles of amplification were performed using the same cycling parameters described previously, except that the annealing temperature was 36°C for the Microsatellite typing. PCR products were size-separated by agarose gel electrophoresis, and the gel was stained in a 0.5 μg/mL ethidium bromide buffer solution (TAE).

Gel images were analyzed with the GelCompar II software, version 4.5 and BioNumerics (Applied Maths, Kortrijk, Belgium). The similarities between the profiles were calculated using the Dice coefficient to generate the matrixes of similarity coefficients to dendrogram constructions. For profile clustering, the unweighted pair-group method with arithmetic averages was used with a tolerance of 2%.

An initial amount of 10⁶ cells/mL in YPD + 20% FBS (Fetal Bovine Serum, Sigma-Aldrich®, Brazil) was incubated at 37°C, with gyratory shaking at 200 rpm. After 3 h incubation, samples of the cultures were mixed with an equal volume of 10% formaldehyde to arrest further development and the mean morphology index (MI) was determined [18]. Values close to 1 indicate a population of spheroidal yeast cells and values close to 4 indicate a population of true hyphal cells, with values between 1 and 4 indicating mixed or pseudohyphal morphologies.

Phospholipase activity was estimated by the egg yolk agar method [19], with inocula prepared from overnight NGY (Neopeptone (Difco, Detroit, MI) 1 g/L, glucose 4 g/L and yeast extract 1 g/L) cultures standardized to 2 × 10⁵ cells/mL.

PMN freshly isolated from blood samples of a single healthy volunteer on the day of the experiment [20] were suspended in Eagle’s minimal essential medium (Gibco) + 20 mM HEPES, pH 7.2, and standardized to 8 × 10⁵ PMN/mL. C. albicans cells grown overnight in NGY washed and resuspended at 5 × 10⁶ yeasts/mL in HEPES-buffered Eagle’s minimal essential medium containing one-tenth volume of fresh human plasma. Equal volumes of PMN and yeast suspensions were mixed and incubated at 37°C for 3 h with rotation at 50 rpm. The phagocytosis of C. albicans were established by counting in triplicate the number of yeasts inside or attached in 100 PMNs [21].

Data were analyzed using the statistical software “GraphPad”, version 3.0. Results were presented as mean ± standard deviation, and differences were analyzed by the Mann–Whitney test. For all the analyses, P was considered a default value of 0.05 and the confidence interval of 95%.

Patient’s clinical and demographic data are summarized in Table 1. For both regions, the majority of the patients had arterial hypertension as the most frequent underlying disease. In addition, the most frequent age class was 35 to 64 years old (Table 1).

From the strains obtained from Natal, RN (Northeast), it was possible to isolate yeasts from 70 out of 111 patients (63.1%), while from Maringa, 12 out of 43 (27.5%) Candida spp positive cultures were obtained. Species distribution for both regions is described in Table 2. C. albicans was the most predominant species found in both cities, followed by C. tropicalis. In only a single patient from Maringa it was possible to isolate three strains belonging to different Candida species (C. albicans, C. glabrata, C. tropicalis).

DNA from all the 76 strains of C. albicans and the reference strains ATCC90028 and SC5314 was submitted to ABC typing. Genotype A was the most prevalent with 58 isolates (76.4%) followed by Genotype C, with 15 strains (19.27%) and Genotype B, with 3 isolates (3.9%; Table 3). The same trend was observed when the strains obtained from each region were analyzed separately: In the Northeast of Brazil, C. albicans genotype A was the most prevalent, with 42 isolates (71.2%), followed by Genotype C (n = 14 isolates; 23.7%) and Genotype B, with 3 strains (5.1%). The higher incidence of genotype A was also found in the South of the country with 16 isolates (94.1%) followed by genotype C (n = 1 isolate; 5.9%). C. albicans genotype B was not observed for the isolates obtained from this region (Table 3).

We randomly selected 48 isolates of C. albicans including some strains belonging to genotype A (21 from Natal and 10 from Maringa) an all the other strains belonging to genotypes B and C from both regions and performed Microsatellite genotyping. The genomic DNA of all of the 48 isolates was successfully amplified with the M13 primer, which targets genome repetitive sequences. The DNA amplification generated well-defined band patterns, ranging from 100 pb to 2 kb. The microsatellite technique showed sufficient discriminatory power for recognizing intraspecific variation (Figure 1). As expected, the external control strains of C. albicans, SC5314 and ATCC90028, were placed in completely different clusters by the dendrogram analysis performed using GelCompar II, proving the efficiency of the technique in separating strains isolated from different geographic areas. These control strains showed 75% similarity and were placed together within a different cluster of all the other clinical isolates (Figure 1).

We could not detect any particular cluster of either colonizing or infecting strains. In addition, in both cases, strains with 100% similarity obtained from different patients were found. Regarding to the comparisons of genetic relatedness within the two different geographic regions of Brazil, we could observe an enriched cluster for Maringa (South), where 80% of the strains were placed together. In addition, they ranged from 80% up to 93% similarity among them.

Other interesting finding is associated with some degree of correlation observed between the ABC type of C. albicans and the high percentage of similarity among these isolates when Microsatellite primer M13 was used. In most of the cases, strains with identical percentage of similarity determined with primer M13, also had the same A or C genotype (except for strains 02 and 85 from Natal as well as 15 T and 18A from Maringa; A and C genotypes in both cases). In addition, C. albicans genotype C strains from Natal were placed within two well-defined separated clusters with proven high relatedness (more than 90% similarity within the same cluster). In relation to genotype B, two out of three isolates showed a similarity of 94%, being placed in the same cluster (Figure 1).

Furthermore, when different strains were obtained from different samples of the same patient, they all had the same ABC genotype (genotype A). Nevertheless, Microsatellite using primer M13 showed higher discriminatory power, because these strains were not considered identical with the latest technique, showing some degree of genetic variability, when this method was used.

For all the virulence factors tested, we only performed statistical analysis for comparisons among strains belonging to genotypes A and C, because of the low number of isolates belonging to genotype B found. In relation to phospholipase production, all the strains were able to produce the enzyme, regardless if they were obtained from episodes of colonization or infection. For genotype A, a Mean Pz of 0.51 ± 0.04 was obtained, while for genotype C, a Mean of 0.55 ± 0.05, while for the genotype B, the Mean Pz was 0.57 ± 0.07. No statistical differences were found when strains belonging to genotypes A and C were compared (Table 3).

When the morphology index (MI) was used to score cells morphology, no clear differences could be observed for the three different A, B or C genotypes. The ability of the strains to change the morphology from bud cells to hyphae when grown in the presence of YPD + 20% FBS was verified for strains belonging to the three referred genotypes. Cells were predominantly found as pseudohyphae, with a mean MI ranging from 2.66 ± 0.61 (genotype C) to 2.77 ± 0.69 (genotype A). The mean MI isolates of genotype B was 2.67 ± 0.71. No statistical differences were observed among the isolates (Table 3).

We also investigated the ability of the different strains to resist to phagocytosis by PMNs. Therefore, the number of C. albicans cells phagocytosed by 100 PMNs was determined after three hours of co-incubation of both cells. C. albicans genotype C was more resistant to PMNs attack. Strains belonging to genotype B had an average of 141.33 ± 14.27 cells phagocytosed by 100 PMNs. Strains belonging to genotype C were significantly more resistant to the attack of phagocytic cells than the isolates belonging to genotype A (Mean of 109.93 ± 12.29 versus 121.67 ± 16.06, respectively; Table 3).

When the pathogenicity factors were compared among all the strains obtained from the two geographic regions, the strains obtained from Maringa (South) expressed more efficiently all the virulence factors evaluated in vitro. This difference was considered statistically significant for resistance to phagocytosis by PMN (Table 4). Interestingly, 40% of the isolates obtained from Maringa were obtained from lesions, whereas only 18.4% of the isolates from Natal were infecting (Tables 3 and 4).