**2. Microsatellite typing**

#### **2.1 Material and methods**

The 123 *C. albicans* (64 [52%] of them biofilm positive) strains for this study were collected during a study in biofilm forming abilities of yeast on indwelling devices (Paulitsch et al., 2009). The strains were stored at -70°C until examination.

Strains were subcultured on Sabouraud agar plates for 24 h at 35°C. For DNA extraction the PrepManTM Ultra Kit (Applied Biosystems [AB], Foster City, California) was used. For the microsatellite typing three different primer pairs were used (Botterel et al., 2001). The unmarked primers were HIS3R, CDC3R, and EF3 (Invitrogen, Lofer, Austria). The fluorescence labeling of the primers HIS3 (NEDTM, yellow), CDC3 (VICTM, green), and EF3R (6-FAMTM, blue) (all AB) was fitted to the DyeSet DS-33 (AB) which is recommended for 5-dye custom primer analyses. PCR was performed using the 96 well GeneAmp PCR System 9700 or the 96 well 2700 Thermal Cycler (both AB). PCR reactions were carried out as singleplex reactions for each primer pair. The samples were initially incubated for 2 minutes at 94°C to activate the Taq Polymerase (Eppendorf, Hamburg, Germany) and to denature the DNA. After thermal cycling (30 cycles; 94°C for 45 s, 48°C for 45 s, 68°C for 90 s) samples were kept at 68°C for another 5 minutes to complete partial polymerization.

Sample preparation for the injection in the 3100 Automatic Sequencer (AB) was done following the instructions. For analysis 1 µL of PCR product, 0.3 µL of size standard (GeneScanTM 500-LIZ®, AB) and 10 µL Hi-DiTM Formamide (AB) were mixed and transferred into a 96 well plate. The samples were denatured for 4 minutes at 94°C in a thermal cycler and immediately placed on ice. In every run three samples were used as internal control. The plate was transferred in the sequencer and processed using the Foundation Data Collection 3.0 software of the sequencer.

Data analysis was done with the GeneMapper® v3.7 software. Therefore it was necessary to set up the microsatellite analyses following the instructions of the manual (AB, 2005). The peaks were automatically detected (Auto Binning) with the created bin set, low quality data were checked manually and corrected. The results were exported in a Microsoft Excel sheet for documentation.

#### **2.2 Results**

4 Epidemiology Insights

strains was based on scanning electron microscopical findings (Paulitsch et al., 2009). Different primer pairs and also different combinations of primer pairs for the subtyping of *C. albicans* were reported elsewhere, see e.g. the works of Sampaio et al. (2005) or Fan

For each marker and for a given isolate one or two bands were observed, and each observed band was assigned to an allele. Because *C. albicans* is diploid each strain can be characterized

The discriminatory power (DP) is a numerical index to describe the probability that two unrelated samples of a test group are placed in two different typing groups. The DP of EF3 is 0.86, the DP of CDC3 is 0.77, and the DP of HIS3 is 0.91 (Botterel et al., 2001). The combined DP of all three markers was 0.97. In order to get reliable results, this index has to

The 123 *C. albicans* (64 [52%] of them biofilm positive) strains for this study were collected during a study in biofilm forming abilities of yeast on indwelling devices (Paulitsch et al.,

Strains were subcultured on Sabouraud agar plates for 24 h at 35°C. For DNA extraction the PrepManTM Ultra Kit (Applied Biosystems [AB], Foster City, California) was used. For the microsatellite typing three different primer pairs were used (Botterel et al., 2001). The unmarked primers were HIS3R, CDC3R, and EF3 (Invitrogen, Lofer, Austria). The fluorescence labeling of the primers HIS3 (NEDTM, yellow), CDC3 (VICTM, green), and EF3R (6-FAMTM, blue) (all AB) was fitted to the DyeSet DS-33 (AB) which is recommended for 5-dye custom primer analyses. PCR was performed using the 96 well GeneAmp PCR System 9700 or the 96 well 2700 Thermal Cycler (both AB). PCR reactions were carried out as singleplex reactions for each primer pair. The samples were initially incubated for 2 minutes at 94°C to activate the Taq Polymerase (Eppendorf, Hamburg, Germany) and to denature the DNA. After thermal cycling (30 cycles; 94°C for 45 s, 48°C for 45 s, 68°C for 90 s) samples were kept at 68°C for another 5 minutes to complete partial

Sample preparation for the injection in the 3100 Automatic Sequencer (AB) was done following the instructions. For analysis 1 µL of PCR product, 0.3 µL of size standard (GeneScanTM 500-LIZ®, AB) and 10 µL Hi-DiTM Formamide (AB) were mixed and transferred into a 96 well plate. The samples were denatured for 4 minutes at 94°C in a thermal cycler and immediately placed on ice. In every run three samples were used as internal control. The plate was transferred in the sequencer and processed using the

Data analysis was done with the GeneMapper® v3.7 software. Therefore it was necessary to set up the microsatellite analyses following the instructions of the manual (AB, 2005). The peaks were automatically detected (Auto Binning) with the created bin set, low quality data were checked manually and corrected. The results were exported in a Microsoft Excel sheet

et al. (2007).

by six alleles with the method used.

be greater than 0.90 (Botterel et al., 2001).

2009). The strains were stored at -70°C until examination.

Foundation Data Collection 3.0 software of the sequencer.

**2. Microsatellite typing 2.1 Material and methods** 

polymerization.

for documentation.

Typing of 123 *C. albicans* strains was done with the above mentioned three primer pairs. Only from strain number 85 (sample W60) no data from the EF3 locus was producible. Although the DNA was isolated a second time and several PCR reactions were done for this locus, no peaks could be generated. In table 1 detailed information of all three loci for each strain is listed.


Microsatellite Typing of Catheter-Associated *Candida albicans* Strains 7

**CDC3/CDC3R EF3/EF3R HIS3/HIS3R** 

**sample allele 1 allele 2 allele 1 allele 2 allele 1 allele 2**


**CDC3/CDC3R EF3/EF3R HIS3/HIS3R** 

**sample allele 1 allele 2 allele 1 allele 2 allele 1 allele 2**


Microsatellite Typing of Catheter-Associated *Candida albicans* Strains 9

a

b

Fig. 1. (a) Biofilm of *C. albicans* (W65) in catheter lumen. (b) Biofilm detail of *C. albicans* (W91). : yeast cells; +: bud scars; m: matrix material; arrow: hyphae. SEM micrographs were taken with a Philips XL30 ESEM scanning electron microscope using the high vacuum mode (emission electrons detection, acceleration voltage 20 kV, operating distance 10 mm).


(samples in italic letters: biofilm positive)

Table 1. Microsatellite data for 123 *C. albicans* strains.

A comparison of the results did not reveal information of typical microsatellite models for *C. albicans* strains which produced biofilms in this study. Only 41 of the investigated strains showed a similarity with one or up to six other strains (Table 2).


(+: biofilm positive; -: biofilm negative)

Table 2. Microsatellite models.

The most convergent data were generated with the CDC3 primer pair, only 12 different allele pairs were; found, with the EF3 primer pair 25 different pairs were located, and HIS3 primers provided 50 different pairs of alleles.

From six patients two strains were available, each of them originated from different samples and showed *C. albicans* infections in routine diagnostics. Both samples from one patient were biofilm positive, from another patient both samples were negative. The microsatellite data of these catheters are listed in table 3. When only HIS3 and CDC3 alleles were compared, five out of the six patients showed the same strain two times, when they were also compared with EF3 primer alleles, only one patient had the same strain two times.

The comparison of the genotyping of biofilm forming *C. albicans* strains (e.g. see figure 1) with non-biofilm forming *C. albicans* species shows also a consistent distribution of genotypes.

**CDC3/CDC3R EF3/EF3R HIS3/HIS3R** 

120 W107 117 129 129 139 154 158 121 W108 117 129 123 133 162 202 122 *W110* 121 125 123 137 154 166 123 *W113* 117 125 129 133 178 178

A comparison of the results did not reveal information of typical microsatellite models for *C. albicans* strains which produced biofilms in this study. Only 41 of the investigated strains

**CDC3/CDC3R EF3/EF3R HIS3/HIS3R n allele 1 allele 2 allele 1 allele 2 allele 1 allele 2 + - total**  113 117 123 129 150 162 2 2 117 129 129 139 154 154 1 4 5 117 129 130 139 154 154 7 4 11 121 129 130 139 154 154 2 2 121 125 123 137 154 166 1 3 4 117 125 120 129 162 162 2 2 117 125 126 126 162 186 1 1 2 117 125 120 120 162 198 2 2 117 125 120 129 162 198 2 2 4 117 125 120 129 162 214 2 2 125 125 123 133 166 182 3 3 125 125 126 133 166 182 1 1 2 23 18 41

The most convergent data were generated with the CDC3 primer pair, only 12 different allele pairs were; found, with the EF3 primer pair 25 different pairs were located, and HIS3

From six patients two strains were available, each of them originated from different samples and showed *C. albicans* infections in routine diagnostics. Both samples from one patient were biofilm positive, from another patient both samples were negative. The microsatellite data of these catheters are listed in table 3. When only HIS3 and CDC3 alleles were compared, five out of the six patients showed the same strain two times, when they were also compared with EF3 primer alleles, only one patient had the same strain two

The comparison of the genotyping of biofilm forming *C. albicans* strains (e.g. see figure 1) with non-biofilm forming *C. albicans* species shows also a consistent distribution of genotypes.

(samples in italic letters: biofilm positive)

(+: biofilm positive; -: biofilm negative) Table 2. Microsatellite models.

times.

primers provided 50 different pairs of alleles.

Table 1. Microsatellite data for 123 *C. albicans* strains.

showed a similarity with one or up to six other strains (Table 2).

**sample allele 1 allele 2 allele 1 allele 2 allele 1 allele 2**

Fig. 1. (a) Biofilm of *C. albicans* (W65) in catheter lumen. (b) Biofilm detail of *C. albicans* (W91). : yeast cells; +: bud scars; m: matrix material; arrow: hyphae. SEM micrographs were taken with a Philips XL30 ESEM scanning electron microscope using the high vacuum mode (emission electrons detection, acceleration voltage 20 kV, operating distance 10 mm).

Microsatellite Typing of Catheter-Associated *Candida albicans* Strains 11

Totally different data were provided from Shi et al. (2007) who collected isolates by female and male patients with genital infection, rectal and oral samples. The authors reported 54.9% of the strains investigated to show the same multilocus genotype, these results were

The CDC3 locus showed 12 different allele pairs, the EF3 locus 25 allele pairs, and the HIS3 locus 50 allele pairs. This is convergent with the data within the three loci and leads to 94 multilocus genotypes. When compared with the results of Botterel et al. (2001) who reported 65 different multilocus genotypes with different allele associations of 10 for CDC3, 22 for EF3, and 25 for HIS3, it is obvious that the HIS3 locus was clearly more divergent within the current study. However, it remains unclear whether this variation is typical for *C. albicans* strains collected from BSI, or if the discriminatory power (DP) of the HIS3 locus (0.91) is not strong enough. The calculated overall DP for the CDC3, EF3, and HIS3 multilocus genotyping was 0.97. It is worth noting that the DP of HIS3 alone was the highest of the three loci (CDC3: 0.77, EF3: 0.86) (Botterel et al., 2001). Nevertheless, a comparison of the typing information without the HIS3 locus showed that the groups of strains sharing the same genotype do not increase significantly (data

The comparison of the genotyping of biofilm forming *C. albicans* strains with non-biofilm forming *C. albicans* species shows also a consistent distribution of genotypes. There is no literature to compare these specific results with, but as aforementioned, a consistent contribution of genotype data collected with the CDC3, EF3, and HIS3 multilocus

The collected information about strains from the same patients are worth a closer look: Only one patient out of six showed 2 strains sharing the multilocus genotype. Using the same typing system, Beretta et al. (2006) investigated 14 isolates of eight patients and reported 4 strains with the same genotype for one patient out of three. Another patient had 2 of 3 strains sharing the genotypes (Beretta et al., 2006). When only HIS3 and CDC3 alleles were compared, five out of the six patients in the current study show the same strain twice. Because of these findings, the typing was done without EF3 locus information, and as it is mentioned above for the typing without HIS3 allele information, no significant increase in the numbers of strains sharing the same multilocus genotype could be seen (data not

Recapitulating the multilocus genotyping with the CDC3, EF3, and HIS3 system during this study, the data presented here is in good agreement with the authors mentioned

The multilocus genotyping with the CDC3, EF3, and HIS3 system during this study did work well and provided data comparable to former studies. Therefore it is strongly indicated that the genotyping of *C. albicans* strains should be continued in future studies. Aditionally the results give possible evidence that genotypes do not matter in the connection to biofilm forming abilities, so that potentially all *C. albicans* strains are able to

genotyping system seems to be normal for *C. albicans* strains.

clearly different from all other studies.

not shown).

shown).

above.

**4. Conclusion** 


(+: biofilm positive; -: biofilm negative)

Table 3. Microsatellite models of 12 strains from six patients (two strains each).
