2.1 Design
This is an experimental semi-quantitative study that has been carried out entirely at the Medical Mycology Laboratory of the University Miguel Hernández (UMH. Alicante. Spain).
2.2 Yeast isolates
A set of 10 species of yeast previously described as members of the Low Respiratory Tract[4] were used in the study. Candida albicans, Candida parapsilosis, Nakaseomyces glabrata (formerly Candida glabrata), Meyerozyma guillermondii (formerly Candida guilliermondii), Cryptococcus neoformans, Cryptococcus deneoformans, Cryptococcus deuterogattii and three Malassezia strains: Malassezia arunalokei, Malassezia pachydermatis and Malassezia restricta. M. restricta was acquired from the type-culture collection of CBS-KNAW (Westerdijk Fungal Biodiversity Institute, Utrecht. The Netherlands). The others were obtained from our own culture collection and were isolated from clinical strains consisting of human BAL samples (C. parapsilosis and M. guilliermondii), human oral cavity (Nakaseomyces glabrata, and C. albicans), human blood (C. deneoformans), human cerebrospinal fluid (C. neoformans and C. deuterogattii), human ear canal (M. arunalokei) and dog ear (M. pachydermatis). The origin of the isolates is listed in Table 1.
Table 1. Yeast strains used in the study: Abbreviation, name of the species, identification code and origin of the isolates. CLA: Collection of yeasts of Alicante (Spain); CCA: Collection of Cryptococcus of Alicante (Spain);CBS-KNAW: VI-KNAW Culture Collection. Utrecht (The Netherlands). *Name of species according to to Hagen F, et al26, and Kidd SE, et al27. **Two isolates of the same strain of Malassezia restricta were assayed.
The strains were kept in skimmed milk at -80ºC. To obtain fresh cultures, all strains were first grown on SDA (for Candida, Nakaseomyces, Meyerozyma and Cryptococcus) and MLNA (for the three Malassezia species). These two were considered as “standard media”. All isolates were incubated at 30º C (optimal temperature for M. restricta) for periods of 24 or 48 hours, until 28 days for M. restricta. Once growth was obtained, the macroscopic and microscopic morphology was checked and verified as the typical morphology of each microorganism. Additionally, the catalase test was used to assess the presence of M. restricta (catalase negative) and the avoidance of contamination from the other two Malassezia species (catalase positive) during the experimental procedure. The aspect of the growth of the isolates recovered on SDA and MLNA, was used to assess and semi-quantify the ability to grow on other lipidic media, comparing the macroscopic aspect of both plates and applying the following criterium: Value 0: similar growth to the one on standard medium; Value 0,5: better growth than on the standard medium; Value -0.5: scarce growth, less than on the standard medium; Value -1: no growth at all.
2.3. In vitro growth assays on different lipidic sources
2.3.1. Inoculum preparation. The inoculum of each isolate was prepared from fresh cultured cells that were suspended in sterile distilled water. Its concentration was adjusted by counting in a Neubauer chamber, and according to the McFarland scale and was of 106 UFC/mL. Incubation was performed at 30º C degrees in an air incubator (Heidolph Inkubator 1000).
2.3.2. Culture media used. Nine culture media with different lipidic content were assayed, including the standard media for lipophilic yeasts (Modified Leeming and Notman agar medium -MLNA)[28]. One new medium (NM1) was created substituting the glycerol monostearate (GME) by DPPC, and another 7 more media were designed, consisting of a common nutrient base (Base Medium plus glycerol - MBG) to which 7 different lipids were added separately: Glycerol Monostearate (GME), 4 Tween components (T20, T40, T60 and T80), purified DPPC (Sigma-Aldrich®) and pig surfactant extract (Curosurf®, Chiesi Laboratories). Table 2 shows the detailed composition of these media. SDA was used also as a culture medium, as mentioned above, as standard medium for non- lipophilic yeasts.
Table 2- Detailed composition of the culture media used for testing the ability of yeast to grow on different lipidic sources.
2.3.3 Culture methods. For all the yeasts tested, three different culture methods were assayed and performed in triplicate: growth on surface of solid medium; growth in depth (dug wells) in solid medium, and growth in liquid medium in a microtiter plate.
- Growth on surface of solid medium:
Plates were prepared with each of the culture media described before and were divided into numbered sections. Each isolate was inoculated in its corresponding section by a 5 µL drop of inoculum. The plates were left at rest and were incubated at 30º C. Growth checked at 24 hours, 5 days, and every 3 days until 28.
- Deep growth in wells in solid medium
A new set of plates of the designed culture media was also divided into numbered sections. Wells of 3-5 mm depth were performed by a sterile punch of 5 mm of diameter in each of the sections. Each well was inoculated by 50 µL of the yeast cells suspensions. They were left to stand at 30°C and the results were read at 24 hours, 5 days, and every 3 days until 28.
- Growth in liquid media (microtiter plates)
Eight different liquid media were prepared with the components described for Tw20, Tw40, Tw60, Tw80, GME, DPPC, SURF and SDA, excluding the agar-agar in all of them (Table 2). After sterilization, the media were disposed in the columns of three 96-well microtiter plates. Each well was filled with 150 μL of medium. Rows from A to I were inoculated by 50 μL of yeast cell suspension and row H was kept sterile as contamination control. Three sets of two plates (A and B) were used to test all the strains distributed as follows: plate A: yeasts of the genus Candida, Nakaseomyces, Meyerozyma and Cryptococcus; and plate B: the tree species of Malassezia (for M. restricta two rows per plate were used). The growth of the strains was assessed by serial readings, starting at 24 hours for plate A, and every 3 days until 21 days for plate B, since the generation times of each of the species are different. The reading was facilitated by the addition of 2.5 μL of a 5 µg/mL of alamar blue solution (Resazurine. Sigma-Aldrich®) to the wells. Growth was indicated by a colour change from blue to red-pink[29].