Adhesion of Candida spp . and Pichia spp . to Wooden Surfaces

Biofi lms are defi ned as highly structured communities of microorganisms that att ach to surfaces where they grow and produce extracellular polymeric substances (1). They are formed by yeast on diff erent surfaces such as stainless steel, plastic, glass, wood and rubber, and present a great risk in the food industry (2). These are very serious issues because of the potential to cause cross-contamination, which leads to shorter shelf-life, food spoilage, and aff ects the consumer’s health (3,4). Use of wood in industry as a food contact surface has been reduced because of the use of new plastic materials. Although wood as a porous material can entrap organic matt er along with microorganisms (5), it is still in use in the developing and developed countries within food supply chain because it is readily available, cheap and easy to handle, and presents a sustainable resource (6,7). Problems caused by microbial fouling in food systems oft en occur due to increased resistance of sessile organisms to the existing disinfectants and sanitising agents (8).


Introduction
Biofi lms are defi ned as highly structured communities of microorganisms that att ach to surfaces where they grow and produce extracellular polymeric substances (1).They are formed by yeast on diff erent surfaces such as stainless steel, plastic, glass, wood and rubber, and present a great risk in the food industry (2).These are very serious issues because of the potential to cause cross-contamination, which leads to shorter shelf-life, food spoilage, and aff ects the consumer's health (3,4).Use of wood in industry as a food contact surface has been reduced because of the use of new plastic materials.Although wood as a porous material can entrap organic matt er along with microorganisms (5), it is still in use in the developing and developed countries within food supply chain because it is readily available, cheap and easy to handle, and presents a sustainable resource (6,7).Problems caused by micro-bial fouling in food systems oft en occur due to increased resistance of sessile organisms to the existing disinfectants and sanitising agents (8).
Bacterial adherence to various surfaces is regularly studied; however, researchers have paid much less att ention to the adhesion of yeasts, particularly Candida and Pichia species, which are usually contaminants isolated from biofi lms on conveyor belts during canning and bottling in the beverage industry (9)(10)(11).Even though Candida albicans is the most signifi cant and frequently isolated yeast pathogen (12), other species such as Candida krusei, Candida glabrata and Candida parapsilosis are oft en found as contaminants in the food industry (13,14).However, C. albicans is commonly found on human skin and can be transferred during handling by workers to food or food contact surfaces.Since it is frequently found in diff erent food industry sett ings, its implications on sanitation issues in the food industry cannot be disregarded (14,15).Microbial adhesion to an abiotic surface is governed by complex interactions among the microorganisms, the substrate surface and the environmental conditions, involving physical, chemical and biochemical factors.However, the eff ects of all these factors on microbial adhesion have not yet been fully clarifi ed, or they have sometimes been reported inconsistently.For example, opposing results have been reported for the eff ect of surface roughness of stainless steel on adhesion: positive correlation and no relation between microbial adhesion and surface roughness (16,17).Several studies have been conducted on microbial adhesion onto diff erent types of food contact surfaces (18,19), but to our knowledge no information is available on the adhesion of Candida and Pichia species to wooden surfaces.
The purpose of this study is to assess the potential of Candida and Pichia species to adhere to two types of wooden surfaces (smooth and rough), material typical for the food processing industry in some developing countries, and investigate the infl uence of wood surface roughness on the degree of yeast adhesion.

Wooden surfaces
Smooth and rough wooden blocks (beech; length, width and thickness of 15.0, 7.0 and 1.0 mm, respectively) were used in this study.The surface roughness of the blocks was expressed in roughness parameter R a .All wooden blocks were autoclaved at 121 °C for 15 min before use.The R a for smooth and rough blocks were (44.0±1.5) and (5.8±1.5)μm, and for surface contact angle, describing wett ability aft er 10 s, (72±10) and (41±7) μm, respectively.

Strains and growth conditions
A total of eight Candida and three Pichia strains were selected from ZIM Culture Collection of Industrial Microorganisms, Ljubljana, Slovenia (Table 1).All strains were stored in yeast peptone dextrose (YPD) medium (Sigma--Aldrich, St. Louis, MO, USA) supplemented with 40 % glycerol at −80 °C.Prior to experiments, the strains were subcultured on malt extract agar (MEA; Merck KGaA, Darmstadt, Germany) for 24 h at 37 °C (Candida strains) or 27 °C (Pichia strains) for microbiological analyses.
Subsequently, a loop of each yeast biomass was inoculated into 4 mL of malt extract broth (MEB; Merck KGaA) for microbiological analyses and incubated for 18 h at 37 °C (Candida strains) or 27 °C (Pichia strains).Aft er 18 h of incubation, 1 mL of culture was diluted in 9 mL of fresh MEB to achieve the fi nal cell concentration of 10 7 colony forming units (CFU) per mL, and the cell count was determined by plate counting on MEA.These cell suspensions were used immediately for adhesion assay.

Adhesion assay
Adhesion assays were performed on the two types of wooden blocks, smooth and rough.Three blocks of each type were placed on the bott om of Petri dishes (30 mm in diameter).For each strain, 2 mL of cell suspension (10 7 CFU/mL) prepared as above were pipett ed into each plate, covering the discs.The plates were incubated for 24 h at 37 °C (Candida strains) or 27 °C (Pichia strains).In control plates, the wooden blocks were inoculated with 2 mL of yeast-free MEB.Aft er incubation period, non-adherent cells were removed by washing three times with phosphate-buff ered saline (PBS; Oxoid, Hampshire, UK), and the blocks were then transferred to 15-mL Falcon tubes with 2 mL of PBS.Wood samples were centrifuged at 1500×g for 3 min to detach the adhering cells.The remaining adhering cells were determined by rinsing, followed by methylene blue staining, and their quantifi cation aft er imaging under microscope using Leica Application Suite soft ware v. 3.7.0(Leica Microsystems AG, Heerbrugg, Switzerland) by automatic counting of viable cells (20).Briefl y, 50 μL of cell suspension were diluted with methylene blue at a ratio of 1:1 (by volume), and Bürker−Türk haemocytometer (100 μm depth; Brand, Wertheim, Germany) was then fi lled with 20 μL of the stained suspension.Aft er adjusting the sett ings of the microscope for counting only viable cells, images were analysed using ImageJ, v. 1.43u, image processing soft ware (National Institutes of Health (NIH), Bethesda, MD, USA) as previously described (20).

Statistical analysis
All quantitative data are presented as mean values with error bars representing standard deviation (S.D.) from two independent experiments with three replicates.The analysis of variance (ANOVA) was used for statistical analysis.The results are considered signifi cant at p<0.05.

Results and Discussion
Evaluation of adhesion of Candida spp. in the present study revealed that these yeasts possess the ability to adhere to wooden surfaces, although to diff erent extents depending on the species and strains.Fig. 1 shows the number of cells (mean value±S.D.) of Candida strains adhered to the smooth and rough surfaces of wood.Statistical analysis showed that C. albicans ATCC 10261 and C. glabrata (ZIM 2367, ZIM 2369 and ZIM 2382) strains exhibited a much greater propensity for adherence to both types of wooden surfaces than C. parapsilosis (ATCC 22019, ZIM  C. glabrata strains adhered in equivalent amount to wooden surfaces (p>0.05).Some previous studies also showed strain variation among C. parapsilosis regarding adherence to abiotic surfaces (21,22).This variation among species and strains refl ects inherent physiological diff erences, and may have signifi cance in relation to the pathogenic potential, since it is known that majority of pathogenic events start with adherence to the relevant surface.Nevertheless, we should mention that only a few strains were used in our study and other strains of the same species can have a greater potential for adhesion.
Additionally, under the conditions of our study all assayed Pichia strains were able to adhere to wooden surfaces, as shown in Fig. 2. P. pĳ peri ZIM 1368 and P. membranifaciens ZIM 2302 had a bett er ability to adhere to the smooth surfaces than P. membranifaciens ZIM 2417 (p<0.05).Therefore, the ability of Candida spp.and Pichia spp. to adhere to wooden surfaces is important in diff erent food industry sett ings because these microorganisms can be a source of food contamination.
Our study has confi rmed previous fi ndings that Candida species are capable of adhering to abiotic surfaces (23,24).Nevertheless, to the best of our knowledge, this is the fi rst time the adhesion of Candida and Pichia species to wooden surfaces has been shown.Considerable diff erences in adhesion ability among Candida species were observed.C. albicans and C. glabrata strains adhered bett er than C. parapsilosis and C. krusei (Fig. 1).These results confi rm previous fi ndings, which showed that C. albicans had good biofi lm growth on the surface of PVC catheter discs (25) and on silicone elastomer discs (26).Furthermore, Shin et al. (27) observed that biofi lm formation by isolates of C. parapsilosis (73 %) followed by C. glabrata (28 %) and C. albicans (8 %) on polystyrene was most frequent.The reason for these contradictory fi ndings could be the fact that microbial adhesion is also infl uenced by the properties of the diff erent substrates, contact medium and methods used to quantify adhesion.
It is well known that the surface properties of materials, such as surface roughness, can signifi cantly infl uence the quality and quantity of fungal adhesion.Evaluation of C. albicans adhesion to denture base resin with diff erent surface roughness has revealed greater adhesion to rough surfaces than to smooth ones (28,29).This phenomenon is understandable since a rough surface is irregular, has an extended surface area, and likely to possess more binding sites for adhering microorganisms (30).The promoting effect of surface roughness on microbial adhesion may also be related to the diffi culties in surface cleaning (31), resulting in rapid regrowth of a biofi lm.In contrast, in our study the roughness of wooden surfaces did not have signifi cant infl uence on the adhesion of Candida and Pichia strains (p>0.05), as shown in Figs. 1 and 2. Such result is in agreement with previous studies which showed that bacterial adhesion was not infl uenced by surface roughness of diff erent materials (32,33).The eff ect of surface roughness on adhesion could be att ributed to species-and strain-specifi c cell surface characteristics.

Conclusions
The present study indicates that Candida and Pichia strains readily adhered to smooth and rough surfaces of wood, which are nowadays frequently used in food processing environments in some countries.C. albicans and C. glabrata adhered bett er to wooden surfaces than C. parapsilosis and C. krusei, while all tested Pichia strains adhered in a strain-dependent manner.Additionally, adhesion of these yeasts was not signifi cantly aff ected by the roughness of the wooden surfaces.Therefore, as yeast adhesion is the fi rst step of biofi lm formation, which may be responsible for contamination and adulteration of food products, the potential ability of contaminant yeast to adhere to wood must be taken into account to prevent undesirable biofi lm formation in food processing environment.

Table 1 .
Yeast strains used in the study and their origin white cheese from cow's milk 2014 and ZIM 2234) strains and C. krusei ATCC 6258 (p<0.05).Considerable intraspecies variation was found for C. parapsilosis.C. parapsilosis ATCC 22019 has weaker capacity to adhere to wood (p<0.05)than C. parapsilosis ZIM 2014 and C. parapsilosis ZIM 2234.On the other hand,