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Quantitative agar-invasion assay
Journal of Microbiological Methods 73 (2008) 100–104
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Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Quantitative agar-invasion assay Jure Zupan, Peter Raspor ⁎ Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
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Article history: Received 21 August 2007 Received in revised form 18 January 2008 Accepted 15 February 2008 Available online 10 March 2008 Keywords: Agar-invasion Clinical yeasts Invasiveness Quantitative assay Saccharomyces cerevisiae
a b s t r a c t A new method for quantification of yeast invasion into the agar medium was developed. Classical agarinvasion assays have been the methods of choice for determination of yeast invasion, but their main disadvantage is the lack of quantification. Our new Quantitative yeast agar-invasion test allows for quantitative measurements and enables sorting strains by their degree of invasiveness. The invasion abilities were measured for 10 clinical and non-clinical Saccharomyces cerevisiae strains and a strain of Candida albicans. Finally, the correlation between the degrees of strains invasiveness and their reported virulence was observed, proposing our assay as a method for quick determination of yeast virulence potential. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Saccharomyces cerevisiae can be found in food and feed and also as a saprophyte in the digestive, respiratory, and genitourinary tracts (Barnett et al., 2000; McCusker et al., 1994a,b). It is considered to be a relatively non-pathogenic yeast in healthy individuals (Aucott et al., 1990), even though it occasionally causes fungemia in immunocompetent adults and newborns (Casalone et al., 2005). However, with the progress in immunosuppressive therapies, there has been an increasing number of reports on systemic infection involving S. cerevisiae with an ill explored epidemiology (Malgoire et al., 2005). Symptoms are indistinguishable from those of invasive candidiasis (EnacheAngoulvant and Hennequin, 2005) and the fact that some strains can be resistant to fluconazole (Kontoyiannis and Rupp, 2000; Sobel et al., 1993) complicates treatment of invasive Saccharomyces infections. There are numerous reports of infections with S. cerevisiae that are seemingly related to treatment with probiotic S. cerevisiae (syn. Saccharomyces boulardii) (Enache-Angoulvant and Hennequin, 2005) necessitating the development of molecular markers for distinguishing S. cerevisiae strains (Malgoire et al., 2005; Riquelme et al., 2003). Some S. cerevisiae strains have the ability to invade agar upon nutrient deprivation, which could provide a selective advantage to invasive S. cerevisiae cells, as it would facilitate active foraging for scarce nutrients (Casalone et al., 2005). The mechanisms through which yeast cells sense these environmental cues and elicit invasive growth have been extensively reviewed by Gagiano et al. (2002). S. cerevisiae genome-wide expression profiling data indicate that invasive growth is not controlled solely by a dedicated invasion-
⁎ Corresponding author. Tel.: +386 1 423 11 61; fax: +386 1 257 40 92. E-mail address: [email protected] (P. Raspor). 0167-7012/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2008.02.009
specific transcriptional program and thus a variety of signals may contribute to initiate invasion (Breitkreutz et al., 2003). In this regard, quantitative invasion measurements in a broad range of different growth conditions can render important additional information, helping to clarify the exact role of relevant gene families. Classical plate-washing assay or agar-invasion assays are a wellknown methodology to study qualitative aspects of microbial invasive growth (Braus et al., 2003; Cullen and Sprague, 2000; Guo et al., 2000; Kontoyiannis et al., 2001; Lo and Dranginis, 1998; Roberts and Fink, 1994). Its major disadvantage is the apparent lack of quantification. It is necessary to develop a quantitative assay, which will give important phenotypic information required to link to gene expression studies of putative invasion genes in yeast and to differentiate invasive yeast strains as well as to determine the influence of various physiological conditions on their invasiveness. The idea to scan patches of invasion was first proposed in a study of genetic interaction networks (Drees et al., 2005), but the expression of invasiveness in exact units and adequate reproducibility of the assay would still be necessary. The aim of this study is to develop a simple, reproducible and rapid quantitative assay for determination of yeast invasion of solid growth media. 2. Materials and methods 2.1. Strains and cultivation S. cerevisiae strains were obtained from the Collection of Industrial Microorganisms (ZIM) at the Biotechnical Faculty, Slovenia and a set of 10 strains was established according to references on virulence, invasion and adhesion (Byron et al., 1995; Lo and Dranginis, 1998; McCusker et al., 1994a,b) and also according to preliminary experiments performed by qualitative plate-washing assay as described
J. Zupan, P. Raspor / Journal of Microbiological Methods 73 (2008) 100–104
before (Roberts and Fink, 1994). Consecutively, 3 groups of strains were introduced: highly virulent and/or invasive, moderately virulent and non-invasive and/or non-virulent. Additionally, Candida albicans ATCC 10261, was also assayed for comparison (Table 1). Strains were inoculated from frozen stocks on YPD plates (1% yeast extract (Biolife), 2% peptone (Oxoid), 2% glucose (Kemika), 2% agar (Biolife)) and incubated 2 days at 26 °C before performing the invasion assay. 2.2. Optimization of the Quantitative agar-invasion assay 2.2.1. Choosing the inoculation technique We performed and compared three inoculation techniques using YJM311: a) micromanipulator (1 cell per spot); b) pipetting small volumes (0.2 µl) of defined number of cells in suspension; and c) inoculation from plates with a loop. The appropriate concentrations of cell suspensions for the pipetting technique were made by counting the cells of overnight incubation (YPD broth, 30 °C) in a haemocytometer. To determine the influence of inoculating cell number on final colony size, 4 different concentrations were compared (100, 500, 1000 and 1500 cells per 0.2 µl). When inoculating with a loop, strains were transferred from pre-grown colonies and spotted on YPD plates, transferring approximately 100–3000 cells. This was achieved by forming a “needle shape” of the culture on the loop tip and precise inoculation. Amounts of inoculums spotted on the agar were checked with inverted microscope and the numbers of cells were estimated. 2.2.2. The geometry of the array We noticed that colonies close to each other reach smaller diameters than single colonies without neighbors. Therefore, the assay with distances between the center of adjacent colonies of 8, 10, 15, 20, 25 and 30 mm was performed using YJM311. Further, to determine the optimal geometry of colony spotting balancing high throughput and minimum negative influence of adjacent colonies on invasion and surface growth, spotting in 4 different forms was performed: in hexagonal form with a central point on round plates (7 colonies per plate), in octagonal form with central point on round (9 colonies per plate) and square plates (9 colonies per plate) and in 4 × 4 form on square plates (16 colonies per plate) with distances between the center of adjacent colonies (according to the geometry) 2.7, 2.3, 3.4 and 2.4 cm, respectively. Distances of colonies from the edge of the agar were kept at least at 0.5 cm. Each plate was inoculated with a loop with one strain in replicates. Table 1 Yeast strains used in the study and their reported virulence potential Species (strain) C. albicans (ATCC 10261) S. cerevisiae (56) S. cerevisiae (YJM311) S. cerevisiae (YJM128) S. cerevisiae (YJM309) S. cerevisiae (YJM273) S. cerevisiae (YHUM272) S. cerevisiae (YJM308) S. cerevisiae (YJM222) S. cerevisiae (ZIM2273) S. cerevisiae (S288c)
Origin
Reported virulence or Reference invasiveness Highly virulent
(Zink et al., 1996)
Danish blue veined cheese Man, bile tube
Highly invasive
Lung of man with AIDS Man, blood
Highly virulent
Man, peritoneal fluid Σ1278b derived
Moderately virulent
Based on our preliminary tests. (Byron et al., 1995; McCusker et al., 1994a,b) (Byron et al., 1995; McCusker et al., 1994a,b) (Byron et al., 1995; McCusker et al., 1994a,b) (Byron et al., 1995; McCusker et al., 1994a,b) (Lo and Dranginis, 1998)
Highly virulent
Highly virulent
Moderately virulent
Man, paracentesis Moderately virulent fluid Man Non-virulent/ non-invasive Σ1278b derived, Non-invasive flo11Δ::lacZ Laboratory strain Non-virulent/ non-invasive
(Byron et al., 1995; McCusker et al., 1994a,b) (McCusker et al., 1994a,b) (Lo and Dranginis, 1998) (Liu et al., 1996; McCusker et al., 1994a,b)
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2.2.3. The incubation and measurements Plates were loosely wrapped in a plastic bag to minimize desiccation but still allowing for efficient aeration. The plates were incubated 4 days at 30 °C when optimizing the method and at 37 °C in the final assay where the invasiveness between strains was compared and correlated with virulence. After incubation, grayscale images of plates were generated with Gel Doc 2000 gel documentation system (Bio-Rad, Italy). Colonies above the agar surface were then washed off with a gentle stream of deionized water, as described before (Roberts and Fink, 1994) and the plates were recorded again. To exclude influences of different brightness (aperture) and focal lengths used during image recording, a square of white paper (1 cm2) was used as a standard for data normalization at every photographed plate. 2.2.4. Calibration The system was first calibrated by milk (3.5% fat) pippetted on parafilm. A scale of volumes from 0.6–30 µl was measured in eight replicates and a calibration curve was made. 2.2.5. Processing of data and quantification A volume tool of Quantity One 4.0.3 (Bio-Rad, Italy) software, originally designed for the analysis of DNA amounts in gels, was used for measuring pre-wash or total (T), post-wash or invasive (I) colony volumes (VT and VI, respectively) and milk drop volumes. Measurements of colony volumes and subtraction of local background intensity were performed according to software instructions, which define the volume as “sum of the intensities of the pixels within the volume boundary × pixel area”. A data output of the software yields adjusted volumes (X) with volume units as “intensity units × mm2”. Normalization was done by division of adjusted volumes of spots (XT,I) with an adjusted volume of standardized areas (XS), which yielded normalized values NT,I = XT,I/XS. Using the equations obtained from the calibration curve, pre-wash (VT) and invasive (VI) colony volumes were calculated. 2.2.6. Invasion and relative invasion Based on different colony sizes due to different relative growth rates, the measurements of absolute invasion (VI) specific for a strain were performed. However, with the intention to compare the invasion ability excluding the parameter of relative growth rate, or in other words, to determine the strain specific tendency to invade rather than grow on the surface, relative invasion was introduced as RI = VI × V−T 1 × 100%. 2.2.7. Validation of the method and validation parameters The measurements were validated indirectly by counting methods. For this purpose, YJM311 was assayed as described, except that the surface colony and the invasive colony were separately suspended in appropriate volume of physiological salt solution and the number of the cells was determined using CFU and haemocytometer (Bürker– Türk) counting methods. Validation parameters describing the assay were also defined. They included: the limit of detection (LOD) and the limit of quantification (LOQ) of the assay, determined statistically and empirically; the assay precision (reproducibility) by calculating relative standard deviations (coefficient of variations, CV) for the 10 S. cerevisiae strains in 7 replicates; and the accuracy/recovery by comparing the measured and the pipetted volumes of milk. 3. Results and discussion When comparing the inoculation techniques, in all three cases we obtained almost the same reproducibility (coefficients of variations, CV): around 10% for VT, and around 20% for VI. However, for various reasons only the inoculation with a loop was satisfactory. Firstly, the inoculation with a micromanipulator was found very time consuming,
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as inoculation of one plate with seven replications lasted 30 min. Secondly, the inoculation from a suspension of cells was also found inappropriate due to numerous disadvantages. A pipetted droplet of a cell suspension characteristically spreads on the surface of agar and forms a much larger spot than in the case of inoculation from colonies with a loop. This is important because our results indicate that an inoculation area has a more significant influence on the final colony volume than an inoculating cell number. Additionally, we noticed that the size and shape of drops are greatly influenced by the agar surface characteristics, which differs between different agar media. As expected, these variations significantly influenced the final colony size. Also, due to the preparation of inocula, the technique was found to be time consuming. By a careful inoculation of strains with a loop, relatively equal colonies were obtained in replicates (Fig. 1A). No significant correlation between the inoculum cell number (100–3000 cells) and the final colony size was observed in practice (data not shown). Rather than a number of cells, a diameter of the inoculum spotted on the agar is important. However, low CV of colony volumes confirm that the precisely defined amounts of inoculums are not needed. Additionally, this inoculating technique was found rapid (one plate with 7 spots per minute) and simple.
Fig. 2. Calibration of the assay. A comparison of yeast colonies (YJM273) incubated on YPD 2, 3 and 4 days at 30 °C (upper row) and corresponding volumes of pipetted milk (lower row). Note that milk volume corresponds to whole colony (surface and invasive part).
In our experiments (YJM311, YPD agar, 30 °C), a significant decrease of the pre-wash colony volumes at colony distances below 25 mm were observed after 4 days and therefore four different geometries were tested. The pre-wash colony volumes on round plates with 9 and on square plates with 16 colonies (the distances between adjacent colonies were 23 and 24 mm, respectively), were considerably smaller (30%) than on round plates with 7 and on square plates with 9 colonies (the distances between adjacent colonies were 27 and 34 mm, respectively). Considering these results, we propose an inoculation of strains in the hexagonal form with a central point using round plates (7 colonies per plate). In the proposed geometry, the distances between all adjacent colonies are equivalent and therefore the error between replications is minimized (Fig. 1A). In order to confirm that post-wash recordings really represent invasive colonies, blocks of such colonies in agar were sliced and photographed laterally (Fig. 1B). When the invasive part of the colonies was checked microscopically, elongated cells and pseudohyphal growth were observed for C. albicans, 56 and YJM311. Other strains showed a pseudohyphal growth to a lesser extent or not at all. The macro-morphology of invasive colonies was strain specific and ranged from a homogenous invasion to the formations of peg-like structures (Scherz et al., 2001). These variations in the colony shape actually do not influence the accuracy of measurement, since the depth of invasion is reflected in pixel intensities — the deeper the invasion, the higher is the intensity (whiteness) of the pixels. Deep invasions result in higher values of signals (area × pixel intensities) in colony area and therefore the calculated volume of invasion is proportionately increased. Milk was found appropriate as a calibrating substance, because it allows accurate pipetting of small volumes and it forms bright spherical drops on parafilm, which well resemble the yeast colony shape (Fig. 2). The average CV of the measured milk drops' volumes ranging from 0.6–30 µl was 5% (data not given). Using “the least squares fit through points”, two calibration curves, one for volumes below 10 µl (Eq. (1)) and the other for volumes above this value (Eq. (2)), sufficiently fit the data, with high R-squared value 0.9999 and 0.9967, respectively. Two calibration curves were used because in this way, more accurate results were obtained. 2 VT;I ðbelow 10 μ1Þ ¼ a NT;I þ b NT;I þ cðc was set to 0Þ
ð1Þ
and VT;I ðabove 10 μlÞ ¼ p NT;I q:
Fig. 1. The Quantitative agar-invasion assay of Saccharomyces cerevisiae YJM273. A: The array of the assay. Strain was assayed on YPD agar plate in 7 replicates and incubated 4 days at 30 °C. A standardized area (1 cm2) of white printing paper was used beside every photographed plate for normalization of processed data. B: The side view of the invasive colony growing into agar.
ð2Þ
Haemocytometer and CFU methods were used to determine the cell numbers in the pre-wash and post-wash colonies. To enable a comparison with the Quantitative agar-invasion assay, we transformed the cell number values obtained by the counting methods to the colony volumes — this was done by multiplying the pre-wash and post-wash cell numbers obtained with the counting methods by the average volume of the pre-wash and post-wash cells, respectively, determined with a microscope. The results clearly show that the
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methods are comparable when colony volumes are considered and especially when the relative invasion is compared (Fig. 3). The standard deviations of both counting methods are besides their characteristic error, additionally increased on account of the errors assigned to the cell volume measurements with a microscope. It has to be mentioned that invasion cannot be measured with the counting techniques unless cell volumes are measured for each strain at each assay — this would be inevitable because the volume of colonization and invasion is dependent on the invasive morphology. Limits of detection and limits of quantification were determined statistically and empirically. When the post-wash plate of the S288c was used as a negative control, LOD and LOQ were calculated 0.01 and 0.04 mm3, respectively. When LOD and LOQ were determined empirically, higher but more realistic values were obtained. Empirical LOQ was determined as the minor invasive colony volume detected in 7 studied replications that contemplated CV acceptable (below 30%). Among the strains assayed at 30 °C, YHUM272 showed the lowest detectable invasion volume with acceptable CV. All 7 measurements of the replicates were above zero and the average VI was 0.19 ± 0.05 mm3 with CV = 25%. Biological LOD and empirical LOQ were then calculated 0.1 mm3 and 0.2 mm3, respectively. To determine the precision of the method, 10 S. cerevisiae strains and C. albicans ATCC 10261 were assayed at 37 °C in 7 replicates per plate and then the standard deviation was calculated (Fig. 4). The results show that the method is highly reproducible considering this generally variable biological system. The average coefficient of variation for VT values of the replicas was 8%, with the maximum and minimum value 16% (ZIM2273) and 3% (YJM273) respectively. The average CV for VI above LOQ (N0.2 mm3) was 15%, maximum 23% (YJM308) and minimum 7% (YJM311). For the relative invasion, CV values were expectably decreased; for the strains, where VI was above LOQ, the average CV for RI was 14%, with the maximum 24% (YJM308) and minimum 7% (strain 56). Strongly invasive strains turn out to be highly reproducible (CV for VI in three independent repeats with YJM311 at 30 °C was 8%). Recovery was calculated as a measured volume divided by the pipetted volume of milk drops. In a separate experiment, the volumes of milk 0.40, 0.50, 0.75, 1, 2, 4, 6, 10, 15, 20, 25 and 40 µl were measured in 6 replicates. The minimum recovery was 84% (at 0.75 μl), maximum 123% (at 0.5 µl) and the average recovery was 104%. To estimate the optimal incubation time, 9 assays with the incubation times from 1 to 9 days were performed with YJM311 grown in 7 replicates on YPD agar plates at 30 °C. It was observed that CV for VI was decreasing with incubation time and growth of colonies.
Fig. 3. Validation of the Quantitative agar-invasion assay. Colony volumes of strain YJM311 were indirectly determined by CFU method (a) and Haemocytometer method (b) in 2 replicates and compared to colony volumes determined by the Quantitative agar-invasion assay (c) in 7 replicates. The error bars indicate standard deviation values. □ pre-wash (total) colony volume; ■ post-wash (invasive) colony volume; ● Relative invasion.
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Fig. 4. Invasion profile of C. albicans and 10 S. cerevisiae strains incubated on YPD agar plates 4 days at 37 °C. Strains are sorted by invasion volumes (VI) from most invasive to non-invasive. The results are the average of 7 replicates. The error bars indicate standard deviation values. □ pre-wash (total) colony volume; ■ post-wash (invasive) colony volume; ● Relative invasion.
After 4 days of incubation time, CV for VT and VI reached reasonably low value of 12 and 14%, respectively. The lowest variations were observed after 7 days of incubation (CV for VT and VI were 6 and 12%, respectively). However, in order to shorten the assay time and to minimize time-dependent changes in invasiveness due to changes in structure and composition of growth medium (Scherz et al., 2001), the optimal incubation time chosen was 4 days. The experiments were done in addition with fresh (1 day) and old (21 days) colonies of YJM311 to check for the possible influence of the age of pre-grown colonies on the assay. The average VI of 7 replicates for the fresh and the old colonies was 2.6 ± 0.3 mm3 and 2.5 ± 0.4 mm3, respectively, suggesting no significant influence of pre-grown colony age on invasion. However, a significant decrease in invasion volumes and changes in macro-morphology were observed when YJM311 was frequently re-inoculated. The possible influence of media thickness on the assay was considered and tested as well in this work. It was observed that more media poured in the plates resulted generally in increased yeast growth and larger colonies (high VT values). On the contrary, measured invasion (VI) in this regard was found to be non-affected (data not shown). However, the differences of VT and VI values on plates with 30 and 35 ml of media were found statistically insignificant, and therefore approximately constant volume of media of around 30 ml was set as default in our work. The most invasive yeast in the performed experiments was C. albicans ATCC 10261, which was expected because of its known virulence and powerful penetration abilities (Zink et al., 1996). Among S. cerevisiae strains, 56, was determined as the most invasive (VI = 6.9 ± 1.1 mm3). Moreover, it showed the highest relative invasion on the
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YPD agar at 30 °C (RI = 31% ± 2%) among all strains used. Interestingly, the strain was originally isolated from the Danish blue veined cheese. Among clinical strains used in this study, YJM311 was found to be the most invasive at 37 °C, resulting in VI = 6.2 ± 0.5 mm3 (Fig. 4). If the results are compared with the references on the virulence of assayed strains (Byron et al., 1995; McCusker et al., 1994a,b), a high correlation can be observed (Table 1, Fig. 4). All strains used in our study that were previously reported as avirulent or non-invasive, were reliably determined as non-invasive by our assay and vice versa; The group of the moderately virulent strains (YJM308, YHUM272 (Σ1278b derived), YJM273) was in accordance with their moderately expressed invasion. Three strains out of four highly virulent/invasive S. cerevisiae strains showed the strongest invasion. The most virulent, S. cerevisiae strain (YJM311) reported previously, was also determined as the most invasive clinical S. cerevisiae strain used in our study. Moreover, at 42 °C, it showed also the highest invasion among all clinical and non-clinical S. cerevisiae strains (data not shown). Beside virulence, also a high correlation between the invasiveness on YPD at 37 °C and flocculation (Byron et al., 1995) can be observed. These data suggest that the Quantitative agar-invasion assay can be used as a method for quick determination of the virulence/flocculation potential. Low coefficients of variations presented in our assays and a wide variety between strains used are indicating that VT,I and even better RI are good phenotypic markers for the strain-specific invasiveness. To our knowledge, the newly-presented assay format for the first time allows determination of invasiveness quantitatively in exact units. The Quantitative agar-invasion assay represents an alternative approach to determine the virulence potential of yeast strains as well as to determine the effects of environmental/external factors. Due to simplicity and short analytical times of the method, it can be successfully used for screening of clinically isolated and mutant strains of S. cerevisiae, other yeasts and yeast-like fungi. Acknowledgements We thank Lene Jespersen from KVL University, Copenhagen, Denmark and Collection of Industrial Microorganisms (ZIM) at the Biotechnical Faculty, Slovenia for providing us the S. cerevisiae strains. This work was financially supported by the Slovenian Ministry of higher education, science and technology under grant No. 1000-05-310064 and EC financial contribution — EU 6-FP-IP PATHOGENCOMBAT FOOD-CT2005-007081. J.Z. also thanks Jan Mavri and Urška Lešnik for their assistance with the review. References Aucott, J.N., Fayen, J., Grossnicklas, H., Morrissey, A., Lederman, M.M., Salata, R.A., 1990. Invasive infection with Saccharomyces cerevisiae: report of three cases and review. Rev. Infect. Dis. 12, 406–411. Barnett, J.A., Payne, R.W., Yarrow, D., 2000. Yeasts: Characteristics and Identification. Cambridge University Press, Cambridge. Braus, G.H., Grundmann, O., Bruckner, S., Mosch, H.U., 2003. Amino acid starvation and Gcn4p regulate adhesive growth and FLO11 gene expression in Saccharomyces cerevisiae. Mol. Biol. Cell 14, 4272–4284.
Breitkreutz, A., Boucher, L., Breitkreutz, B.J., Sultan, M., Jurisica, I., Tyers, M., 2003. Phenotypic and transcriptional plasticity directed by a yeast mitogen-activated protein kinase network. Genetics 165, 997–1015. Byron, J.K., Clemons, K.V., McCusker, J.H., Davis, R.W., Stevens, D.A., 1995. Pathogenicity of Saccharomyces cerevisiae in complement factor five-deficient mice. Infect. Immun. 63, 478–485. Casalone, E., Barberio, C., Cappellini, L., Polsinelli, M., 2005. Characterization of Saccharomyces cerevisiae natural populations for pseudohyphal growth and colony morphology. Res. Microbiol. 156, 191–200. Cullen, P.J., Sprague Jr., G.F., 2000. Glucose depletion causes haploid invasive growth in yeast. Proc. Natl. Acad. Sci. U. S. A. 97, 13619–13624. Drees, B.L., Thorsson, V., Carter, G.W., Rives, A.W., Raymond, M.Z., Avila-Campillo, I., Shannon, P., Galitski, T., 2005. Derivation of genetic interaction networks from quantitative phenotype data. Genome Biol. 6, R38. Enache-Angoulvant, A., Hennequin, C., 2005. Invasive Saccharomyces infection: a comprehensive review. Clin. Infect. Dis. 41, 1559–1568. Gagiano, M., Bauer, F.F., Pretorius, I.S., 2002. The sensing of nutritional status and the relationship to filamentous growth in Saccharomyces cerevisiae. FEMS Yeast Res. 2, 433–470. Guo, B., Styles, C.A., Feng, Q., Fink, G.R., 2000. A Saccharomyces gene family involved in invasive growth, cell–cell adhesion, and mating. Proc. Natl. Acad. Sci. U. S. A. 97, 12158–12163. Kontoyiannis, D.P., Rupp, S., 2000. Cyclic AMP and fluconazole resistance in Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 44, 1743–1744. Kontoyiannis, D.P., Tarrand, J., Prince, R., Samonis, G., Rolston, K.V., 2001. Effect of fluconazole on agar invasion by Candida albicans. J. Med. Microbiol. 50, 78–82. Liu, H., Styles, C.A., Fink, G.R., 1996. Saccharomyces cerevisiae S288C has a mutation in FLO8, a gene required for filamentous growth. Genetics 144, 967–978. Lo, W.S., Dranginis, A.M., 1998. The cell surface flocculin Flo11 is required for pseudohyphae formation and invasion by Saccharomyces cerevisiae. Mol. Biol. Cell 9, 161–171. Malgoire, J.Y., Bertout, S., Renaud, F., Bastide, J.M., Mallie, M., 2005. Typing of Saccharomyces cerevisiae clinical strains by using microsatellite sequence polymorphism. J. Clin. Microbiol. 43, 1133–1137. McCusker, J.H., Clemons, K.V., Stevens, D.A., Davis, R.W., 1994a. Genetic characterization of pathogenic Saccharomyces cerevisiae isolates. Genetics 136, 1261–1269. McCusker, J.H., Clemons, K.V., Stevens, D.A., Davis, R.W., 1994b. Saccharomyces cerevisiae virulence phenotype as determined with CD-1 mice is associated with the ability to grow at 42 degrees C and form pseudohyphae. Infect. Immun. 62, 5447–5455. Riquelme, A.J., Calvo, M.A., Guzman, A.M., Depix, M.S., Garcia, P., Perez, C., Arrese, M., Labarca, J.A., 2003. Saccharomyces cerevisiae fungemia after Saccharomyces boulardii treatment in immunocompromised patients. J. Clin. Gastroenterol. 36, 41–43. Roberts, R.L., Fink, G.R., 1994. Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. Genes Dev. 8, 2974–2985. Scherz, R., Shinder, V., Engelberg, D., 2001. Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization. J. Bacteriol. 183, 5402–5413. Sobel, J.D., Vazquez, J., Lynch, M., Meriwether, C., Zervos, M.J., 1993. Vaginitis due to Saccharomyces cerevisiae: epidemiology, clinical aspects, and therapy. Clin. Infect. Dis. 16, 93–99. Zink, S., Nass, T., Rosen, P., Ernst, J.F., 1996. Migration of the fungal pathogen Candida albicans across endothelial monolayers. Infect. Immun. 64, 5085–5091.
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Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Quantitative agar-invasion assay Jure Zupan, Peter Raspor ⁎ Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
a r t i c l e
i n f o
Article history: Received 21 August 2007 Received in revised form 18 January 2008 Accepted 15 February 2008 Available online 10 March 2008 Keywords: Agar-invasion Clinical yeasts Invasiveness Quantitative assay Saccharomyces cerevisiae
a b s t r a c t A new method for quantification of yeast invasion into the agar medium was developed. Classical agarinvasion assays have been the methods of choice for determination of yeast invasion, but their main disadvantage is the lack of quantification. Our new Quantitative yeast agar-invasion test allows for quantitative measurements and enables sorting strains by their degree of invasiveness. The invasion abilities were measured for 10 clinical and non-clinical Saccharomyces cerevisiae strains and a strain of Candida albicans. Finally, the correlation between the degrees of strains invasiveness and their reported virulence was observed, proposing our assay as a method for quick determination of yeast virulence potential. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Saccharomyces cerevisiae can be found in food and feed and also as a saprophyte in the digestive, respiratory, and genitourinary tracts (Barnett et al., 2000; McCusker et al., 1994a,b). It is considered to be a relatively non-pathogenic yeast in healthy individuals (Aucott et al., 1990), even though it occasionally causes fungemia in immunocompetent adults and newborns (Casalone et al., 2005). However, with the progress in immunosuppressive therapies, there has been an increasing number of reports on systemic infection involving S. cerevisiae with an ill explored epidemiology (Malgoire et al., 2005). Symptoms are indistinguishable from those of invasive candidiasis (EnacheAngoulvant and Hennequin, 2005) and the fact that some strains can be resistant to fluconazole (Kontoyiannis and Rupp, 2000; Sobel et al., 1993) complicates treatment of invasive Saccharomyces infections. There are numerous reports of infections with S. cerevisiae that are seemingly related to treatment with probiotic S. cerevisiae (syn. Saccharomyces boulardii) (Enache-Angoulvant and Hennequin, 2005) necessitating the development of molecular markers for distinguishing S. cerevisiae strains (Malgoire et al., 2005; Riquelme et al., 2003). Some S. cerevisiae strains have the ability to invade agar upon nutrient deprivation, which could provide a selective advantage to invasive S. cerevisiae cells, as it would facilitate active foraging for scarce nutrients (Casalone et al., 2005). The mechanisms through which yeast cells sense these environmental cues and elicit invasive growth have been extensively reviewed by Gagiano et al. (2002). S. cerevisiae genome-wide expression profiling data indicate that invasive growth is not controlled solely by a dedicated invasion-
⁎ Corresponding author. Tel.: +386 1 423 11 61; fax: +386 1 257 40 92. E-mail address: [email protected] (P. Raspor). 0167-7012/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2008.02.009
specific transcriptional program and thus a variety of signals may contribute to initiate invasion (Breitkreutz et al., 2003). In this regard, quantitative invasion measurements in a broad range of different growth conditions can render important additional information, helping to clarify the exact role of relevant gene families. Classical plate-washing assay or agar-invasion assays are a wellknown methodology to study qualitative aspects of microbial invasive growth (Braus et al., 2003; Cullen and Sprague, 2000; Guo et al., 2000; Kontoyiannis et al., 2001; Lo and Dranginis, 1998; Roberts and Fink, 1994). Its major disadvantage is the apparent lack of quantification. It is necessary to develop a quantitative assay, which will give important phenotypic information required to link to gene expression studies of putative invasion genes in yeast and to differentiate invasive yeast strains as well as to determine the influence of various physiological conditions on their invasiveness. The idea to scan patches of invasion was first proposed in a study of genetic interaction networks (Drees et al., 2005), but the expression of invasiveness in exact units and adequate reproducibility of the assay would still be necessary. The aim of this study is to develop a simple, reproducible and rapid quantitative assay for determination of yeast invasion of solid growth media. 2. Materials and methods 2.1. Strains and cultivation S. cerevisiae strains were obtained from the Collection of Industrial Microorganisms (ZIM) at the Biotechnical Faculty, Slovenia and a set of 10 strains was established according to references on virulence, invasion and adhesion (Byron et al., 1995; Lo and Dranginis, 1998; McCusker et al., 1994a,b) and also according to preliminary experiments performed by qualitative plate-washing assay as described
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before (Roberts and Fink, 1994). Consecutively, 3 groups of strains were introduced: highly virulent and/or invasive, moderately virulent and non-invasive and/or non-virulent. Additionally, Candida albicans ATCC 10261, was also assayed for comparison (Table 1). Strains were inoculated from frozen stocks on YPD plates (1% yeast extract (Biolife), 2% peptone (Oxoid), 2% glucose (Kemika), 2% agar (Biolife)) and incubated 2 days at 26 °C before performing the invasion assay. 2.2. Optimization of the Quantitative agar-invasion assay 2.2.1. Choosing the inoculation technique We performed and compared three inoculation techniques using YJM311: a) micromanipulator (1 cell per spot); b) pipetting small volumes (0.2 µl) of defined number of cells in suspension; and c) inoculation from plates with a loop. The appropriate concentrations of cell suspensions for the pipetting technique were made by counting the cells of overnight incubation (YPD broth, 30 °C) in a haemocytometer. To determine the influence of inoculating cell number on final colony size, 4 different concentrations were compared (100, 500, 1000 and 1500 cells per 0.2 µl). When inoculating with a loop, strains were transferred from pre-grown colonies and spotted on YPD plates, transferring approximately 100–3000 cells. This was achieved by forming a “needle shape” of the culture on the loop tip and precise inoculation. Amounts of inoculums spotted on the agar were checked with inverted microscope and the numbers of cells were estimated. 2.2.2. The geometry of the array We noticed that colonies close to each other reach smaller diameters than single colonies without neighbors. Therefore, the assay with distances between the center of adjacent colonies of 8, 10, 15, 20, 25 and 30 mm was performed using YJM311. Further, to determine the optimal geometry of colony spotting balancing high throughput and minimum negative influence of adjacent colonies on invasion and surface growth, spotting in 4 different forms was performed: in hexagonal form with a central point on round plates (7 colonies per plate), in octagonal form with central point on round (9 colonies per plate) and square plates (9 colonies per plate) and in 4 × 4 form on square plates (16 colonies per plate) with distances between the center of adjacent colonies (according to the geometry) 2.7, 2.3, 3.4 and 2.4 cm, respectively. Distances of colonies from the edge of the agar were kept at least at 0.5 cm. Each plate was inoculated with a loop with one strain in replicates. Table 1 Yeast strains used in the study and their reported virulence potential Species (strain) C. albicans (ATCC 10261) S. cerevisiae (56) S. cerevisiae (YJM311) S. cerevisiae (YJM128) S. cerevisiae (YJM309) S. cerevisiae (YJM273) S. cerevisiae (YHUM272) S. cerevisiae (YJM308) S. cerevisiae (YJM222) S. cerevisiae (ZIM2273) S. cerevisiae (S288c)
Origin
Reported virulence or Reference invasiveness Highly virulent
(Zink et al., 1996)
Danish blue veined cheese Man, bile tube
Highly invasive
Lung of man with AIDS Man, blood
Highly virulent
Man, peritoneal fluid Σ1278b derived
Moderately virulent
Based on our preliminary tests. (Byron et al., 1995; McCusker et al., 1994a,b) (Byron et al., 1995; McCusker et al., 1994a,b) (Byron et al., 1995; McCusker et al., 1994a,b) (Byron et al., 1995; McCusker et al., 1994a,b) (Lo and Dranginis, 1998)
Highly virulent
Highly virulent
Moderately virulent
Man, paracentesis Moderately virulent fluid Man Non-virulent/ non-invasive Σ1278b derived, Non-invasive flo11Δ::lacZ Laboratory strain Non-virulent/ non-invasive
(Byron et al., 1995; McCusker et al., 1994a,b) (McCusker et al., 1994a,b) (Lo and Dranginis, 1998) (Liu et al., 1996; McCusker et al., 1994a,b)
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2.2.3. The incubation and measurements Plates were loosely wrapped in a plastic bag to minimize desiccation but still allowing for efficient aeration. The plates were incubated 4 days at 30 °C when optimizing the method and at 37 °C in the final assay where the invasiveness between strains was compared and correlated with virulence. After incubation, grayscale images of plates were generated with Gel Doc 2000 gel documentation system (Bio-Rad, Italy). Colonies above the agar surface were then washed off with a gentle stream of deionized water, as described before (Roberts and Fink, 1994) and the plates were recorded again. To exclude influences of different brightness (aperture) and focal lengths used during image recording, a square of white paper (1 cm2) was used as a standard for data normalization at every photographed plate. 2.2.4. Calibration The system was first calibrated by milk (3.5% fat) pippetted on parafilm. A scale of volumes from 0.6–30 µl was measured in eight replicates and a calibration curve was made. 2.2.5. Processing of data and quantification A volume tool of Quantity One 4.0.3 (Bio-Rad, Italy) software, originally designed for the analysis of DNA amounts in gels, was used for measuring pre-wash or total (T), post-wash or invasive (I) colony volumes (VT and VI, respectively) and milk drop volumes. Measurements of colony volumes and subtraction of local background intensity were performed according to software instructions, which define the volume as “sum of the intensities of the pixels within the volume boundary × pixel area”. A data output of the software yields adjusted volumes (X) with volume units as “intensity units × mm2”. Normalization was done by division of adjusted volumes of spots (XT,I) with an adjusted volume of standardized areas (XS), which yielded normalized values NT,I = XT,I/XS. Using the equations obtained from the calibration curve, pre-wash (VT) and invasive (VI) colony volumes were calculated. 2.2.6. Invasion and relative invasion Based on different colony sizes due to different relative growth rates, the measurements of absolute invasion (VI) specific for a strain were performed. However, with the intention to compare the invasion ability excluding the parameter of relative growth rate, or in other words, to determine the strain specific tendency to invade rather than grow on the surface, relative invasion was introduced as RI = VI × V−T 1 × 100%. 2.2.7. Validation of the method and validation parameters The measurements were validated indirectly by counting methods. For this purpose, YJM311 was assayed as described, except that the surface colony and the invasive colony were separately suspended in appropriate volume of physiological salt solution and the number of the cells was determined using CFU and haemocytometer (Bürker– Türk) counting methods. Validation parameters describing the assay were also defined. They included: the limit of detection (LOD) and the limit of quantification (LOQ) of the assay, determined statistically and empirically; the assay precision (reproducibility) by calculating relative standard deviations (coefficient of variations, CV) for the 10 S. cerevisiae strains in 7 replicates; and the accuracy/recovery by comparing the measured and the pipetted volumes of milk. 3. Results and discussion When comparing the inoculation techniques, in all three cases we obtained almost the same reproducibility (coefficients of variations, CV): around 10% for VT, and around 20% for VI. However, for various reasons only the inoculation with a loop was satisfactory. Firstly, the inoculation with a micromanipulator was found very time consuming,
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as inoculation of one plate with seven replications lasted 30 min. Secondly, the inoculation from a suspension of cells was also found inappropriate due to numerous disadvantages. A pipetted droplet of a cell suspension characteristically spreads on the surface of agar and forms a much larger spot than in the case of inoculation from colonies with a loop. This is important because our results indicate that an inoculation area has a more significant influence on the final colony volume than an inoculating cell number. Additionally, we noticed that the size and shape of drops are greatly influenced by the agar surface characteristics, which differs between different agar media. As expected, these variations significantly influenced the final colony size. Also, due to the preparation of inocula, the technique was found to be time consuming. By a careful inoculation of strains with a loop, relatively equal colonies were obtained in replicates (Fig. 1A). No significant correlation between the inoculum cell number (100–3000 cells) and the final colony size was observed in practice (data not shown). Rather than a number of cells, a diameter of the inoculum spotted on the agar is important. However, low CV of colony volumes confirm that the precisely defined amounts of inoculums are not needed. Additionally, this inoculating technique was found rapid (one plate with 7 spots per minute) and simple.
Fig. 2. Calibration of the assay. A comparison of yeast colonies (YJM273) incubated on YPD 2, 3 and 4 days at 30 °C (upper row) and corresponding volumes of pipetted milk (lower row). Note that milk volume corresponds to whole colony (surface and invasive part).
In our experiments (YJM311, YPD agar, 30 °C), a significant decrease of the pre-wash colony volumes at colony distances below 25 mm were observed after 4 days and therefore four different geometries were tested. The pre-wash colony volumes on round plates with 9 and on square plates with 16 colonies (the distances between adjacent colonies were 23 and 24 mm, respectively), were considerably smaller (30%) than on round plates with 7 and on square plates with 9 colonies (the distances between adjacent colonies were 27 and 34 mm, respectively). Considering these results, we propose an inoculation of strains in the hexagonal form with a central point using round plates (7 colonies per plate). In the proposed geometry, the distances between all adjacent colonies are equivalent and therefore the error between replications is minimized (Fig. 1A). In order to confirm that post-wash recordings really represent invasive colonies, blocks of such colonies in agar were sliced and photographed laterally (Fig. 1B). When the invasive part of the colonies was checked microscopically, elongated cells and pseudohyphal growth were observed for C. albicans, 56 and YJM311. Other strains showed a pseudohyphal growth to a lesser extent or not at all. The macro-morphology of invasive colonies was strain specific and ranged from a homogenous invasion to the formations of peg-like structures (Scherz et al., 2001). These variations in the colony shape actually do not influence the accuracy of measurement, since the depth of invasion is reflected in pixel intensities — the deeper the invasion, the higher is the intensity (whiteness) of the pixels. Deep invasions result in higher values of signals (area × pixel intensities) in colony area and therefore the calculated volume of invasion is proportionately increased. Milk was found appropriate as a calibrating substance, because it allows accurate pipetting of small volumes and it forms bright spherical drops on parafilm, which well resemble the yeast colony shape (Fig. 2). The average CV of the measured milk drops' volumes ranging from 0.6–30 µl was 5% (data not given). Using “the least squares fit through points”, two calibration curves, one for volumes below 10 µl (Eq. (1)) and the other for volumes above this value (Eq. (2)), sufficiently fit the data, with high R-squared value 0.9999 and 0.9967, respectively. Two calibration curves were used because in this way, more accurate results were obtained. 2 VT;I ðbelow 10 μ1Þ ¼ a NT;I þ b NT;I þ cðc was set to 0Þ
ð1Þ
and VT;I ðabove 10 μlÞ ¼ p NT;I q:
Fig. 1. The Quantitative agar-invasion assay of Saccharomyces cerevisiae YJM273. A: The array of the assay. Strain was assayed on YPD agar plate in 7 replicates and incubated 4 days at 30 °C. A standardized area (1 cm2) of white printing paper was used beside every photographed plate for normalization of processed data. B: The side view of the invasive colony growing into agar.
ð2Þ
Haemocytometer and CFU methods were used to determine the cell numbers in the pre-wash and post-wash colonies. To enable a comparison with the Quantitative agar-invasion assay, we transformed the cell number values obtained by the counting methods to the colony volumes — this was done by multiplying the pre-wash and post-wash cell numbers obtained with the counting methods by the average volume of the pre-wash and post-wash cells, respectively, determined with a microscope. The results clearly show that the
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methods are comparable when colony volumes are considered and especially when the relative invasion is compared (Fig. 3). The standard deviations of both counting methods are besides their characteristic error, additionally increased on account of the errors assigned to the cell volume measurements with a microscope. It has to be mentioned that invasion cannot be measured with the counting techniques unless cell volumes are measured for each strain at each assay — this would be inevitable because the volume of colonization and invasion is dependent on the invasive morphology. Limits of detection and limits of quantification were determined statistically and empirically. When the post-wash plate of the S288c was used as a negative control, LOD and LOQ were calculated 0.01 and 0.04 mm3, respectively. When LOD and LOQ were determined empirically, higher but more realistic values were obtained. Empirical LOQ was determined as the minor invasive colony volume detected in 7 studied replications that contemplated CV acceptable (below 30%). Among the strains assayed at 30 °C, YHUM272 showed the lowest detectable invasion volume with acceptable CV. All 7 measurements of the replicates were above zero and the average VI was 0.19 ± 0.05 mm3 with CV = 25%. Biological LOD and empirical LOQ were then calculated 0.1 mm3 and 0.2 mm3, respectively. To determine the precision of the method, 10 S. cerevisiae strains and C. albicans ATCC 10261 were assayed at 37 °C in 7 replicates per plate and then the standard deviation was calculated (Fig. 4). The results show that the method is highly reproducible considering this generally variable biological system. The average coefficient of variation for VT values of the replicas was 8%, with the maximum and minimum value 16% (ZIM2273) and 3% (YJM273) respectively. The average CV for VI above LOQ (N0.2 mm3) was 15%, maximum 23% (YJM308) and minimum 7% (YJM311). For the relative invasion, CV values were expectably decreased; for the strains, where VI was above LOQ, the average CV for RI was 14%, with the maximum 24% (YJM308) and minimum 7% (strain 56). Strongly invasive strains turn out to be highly reproducible (CV for VI in three independent repeats with YJM311 at 30 °C was 8%). Recovery was calculated as a measured volume divided by the pipetted volume of milk drops. In a separate experiment, the volumes of milk 0.40, 0.50, 0.75, 1, 2, 4, 6, 10, 15, 20, 25 and 40 µl were measured in 6 replicates. The minimum recovery was 84% (at 0.75 μl), maximum 123% (at 0.5 µl) and the average recovery was 104%. To estimate the optimal incubation time, 9 assays with the incubation times from 1 to 9 days were performed with YJM311 grown in 7 replicates on YPD agar plates at 30 °C. It was observed that CV for VI was decreasing with incubation time and growth of colonies.
Fig. 3. Validation of the Quantitative agar-invasion assay. Colony volumes of strain YJM311 were indirectly determined by CFU method (a) and Haemocytometer method (b) in 2 replicates and compared to colony volumes determined by the Quantitative agar-invasion assay (c) in 7 replicates. The error bars indicate standard deviation values. □ pre-wash (total) colony volume; ■ post-wash (invasive) colony volume; ● Relative invasion.
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Fig. 4. Invasion profile of C. albicans and 10 S. cerevisiae strains incubated on YPD agar plates 4 days at 37 °C. Strains are sorted by invasion volumes (VI) from most invasive to non-invasive. The results are the average of 7 replicates. The error bars indicate standard deviation values. □ pre-wash (total) colony volume; ■ post-wash (invasive) colony volume; ● Relative invasion.
After 4 days of incubation time, CV for VT and VI reached reasonably low value of 12 and 14%, respectively. The lowest variations were observed after 7 days of incubation (CV for VT and VI were 6 and 12%, respectively). However, in order to shorten the assay time and to minimize time-dependent changes in invasiveness due to changes in structure and composition of growth medium (Scherz et al., 2001), the optimal incubation time chosen was 4 days. The experiments were done in addition with fresh (1 day) and old (21 days) colonies of YJM311 to check for the possible influence of the age of pre-grown colonies on the assay. The average VI of 7 replicates for the fresh and the old colonies was 2.6 ± 0.3 mm3 and 2.5 ± 0.4 mm3, respectively, suggesting no significant influence of pre-grown colony age on invasion. However, a significant decrease in invasion volumes and changes in macro-morphology were observed when YJM311 was frequently re-inoculated. The possible influence of media thickness on the assay was considered and tested as well in this work. It was observed that more media poured in the plates resulted generally in increased yeast growth and larger colonies (high VT values). On the contrary, measured invasion (VI) in this regard was found to be non-affected (data not shown). However, the differences of VT and VI values on plates with 30 and 35 ml of media were found statistically insignificant, and therefore approximately constant volume of media of around 30 ml was set as default in our work. The most invasive yeast in the performed experiments was C. albicans ATCC 10261, which was expected because of its known virulence and powerful penetration abilities (Zink et al., 1996). Among S. cerevisiae strains, 56, was determined as the most invasive (VI = 6.9 ± 1.1 mm3). Moreover, it showed the highest relative invasion on the
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YPD agar at 30 °C (RI = 31% ± 2%) among all strains used. Interestingly, the strain was originally isolated from the Danish blue veined cheese. Among clinical strains used in this study, YJM311 was found to be the most invasive at 37 °C, resulting in VI = 6.2 ± 0.5 mm3 (Fig. 4). If the results are compared with the references on the virulence of assayed strains (Byron et al., 1995; McCusker et al., 1994a,b), a high correlation can be observed (Table 1, Fig. 4). All strains used in our study that were previously reported as avirulent or non-invasive, were reliably determined as non-invasive by our assay and vice versa; The group of the moderately virulent strains (YJM308, YHUM272 (Σ1278b derived), YJM273) was in accordance with their moderately expressed invasion. Three strains out of four highly virulent/invasive S. cerevisiae strains showed the strongest invasion. The most virulent, S. cerevisiae strain (YJM311) reported previously, was also determined as the most invasive clinical S. cerevisiae strain used in our study. Moreover, at 42 °C, it showed also the highest invasion among all clinical and non-clinical S. cerevisiae strains (data not shown). Beside virulence, also a high correlation between the invasiveness on YPD at 37 °C and flocculation (Byron et al., 1995) can be observed. These data suggest that the Quantitative agar-invasion assay can be used as a method for quick determination of the virulence/flocculation potential. Low coefficients of variations presented in our assays and a wide variety between strains used are indicating that VT,I and even better RI are good phenotypic markers for the strain-specific invasiveness. To our knowledge, the newly-presented assay format for the first time allows determination of invasiveness quantitatively in exact units. The Quantitative agar-invasion assay represents an alternative approach to determine the virulence potential of yeast strains as well as to determine the effects of environmental/external factors. Due to simplicity and short analytical times of the method, it can be successfully used for screening of clinically isolated and mutant strains of S. cerevisiae, other yeasts and yeast-like fungi. Acknowledgements We thank Lene Jespersen from KVL University, Copenhagen, Denmark and Collection of Industrial Microorganisms (ZIM) at the Biotechnical Faculty, Slovenia for providing us the S. cerevisiae strains. This work was financially supported by the Slovenian Ministry of higher education, science and technology under grant No. 1000-05-310064 and EC financial contribution — EU 6-FP-IP PATHOGENCOMBAT FOOD-CT2005-007081. J.Z. also thanks Jan Mavri and Urška Lešnik for their assistance with the review. References Aucott, J.N., Fayen, J., Grossnicklas, H., Morrissey, A., Lederman, M.M., Salata, R.A., 1990. Invasive infection with Saccharomyces cerevisiae: report of three cases and review. Rev. Infect. Dis. 12, 406–411. Barnett, J.A., Payne, R.W., Yarrow, D., 2000. Yeasts: Characteristics and Identification. Cambridge University Press, Cambridge. Braus, G.H., Grundmann, O., Bruckner, S., Mosch, H.U., 2003. Amino acid starvation and Gcn4p regulate adhesive growth and FLO11 gene expression in Saccharomyces cerevisiae. Mol. Biol. Cell 14, 4272–4284.
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