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

Summary

Yeast adhesion to and biofilm formation on surfaces is present in many different environments. In food industry, biofilms may be a source of contaminations, causing food spoilage and reducing quality of products. Candida and Pichia are two common yeast genera involved in the spoilage of some food products. The aim of this study is to assess the potential of Candida and Pichia species to adhere to two types of wooden surfaces (smooth and rough), one of the materials typical for the food processing industry, and investigate the influence of surface roughness of wood on the degree of yeast adhesion. The adhesion of the cells to the wooden surfaces was determined by rinsing them from the surface, followed by methylene blue staining, and quantification after imaging under microscope by automatic counting of viable cells. The results showed that all Candida and Pichia strains were able to adhere to the wooden surfaces in a species- and strain-dependent manner. On the other hand, our data indicated that adhesion by these yeasts was not significantly affected by the roughness of the wood surfaces.

Introduction

Biofilms are defined as highly structured communities of microorganisms that attach to surfaces where they grow and produce extracellular polymeric substances (1). They are formed by yeast on different 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 affects 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 matter 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 often 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 attention to the adhesion of yeasts, particularly Candida and Pichia species, which are usually contaminants isolated from biofilms on conveyor belts during canning and bottling in the beverage industry (9–11). Even though Candida albicans is the most significant and frequently isolated yeast pathogen (12), other species such as Candida krusei, Candida glabrata and Candida parapsilosis are often 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 different food industry settings, 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 effects of all these factors on microbial adhesion have not yet been fully clarified, or they have sometimes been reported inconsistently. For example, opposing results have been reported for the effect 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 different 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 influence of wood surface roughness on the degree of yeast adhesion.

Materials and Methods

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 wettability after 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.

Table 1. Yeast strains used in the study and their origin.

Species (strain)	Origin
Candida albicans (ATCC 10261) Candida glabrata (ZIM 2367) Candida glabrata (ZIM 2369) Candida glabrata (ZIM 2382) Candida parapsilosis (ATCC 22019) Candida parapsilosis (ZIM 2014) Candida parapsilosis (ZIM 2234) Candida krusei (ATCC 6258) Pichia pijperi (ZIM 1368) Pichia membranifaciens (ZIM 2302) Pichia membranifaciens (ZIM 2417)	human fingernail infection: paronychia tracheal aspiration bronchoalveolar lavage (BAL) urine from indwelling catheter human: sprue sputum fruit juice concentrate human: bronchial secretion Refošk must spoiled wine white cheese from cow’s milk

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). After 18 h of incubation, 1 mL of culture was diluted in 9 mL of fresh MEB to achieve the final cell concentration of 10⁷ 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 bottom of Petri dishes (30 mm in diameter). For each strain, 2 mL of cell suspension (10⁷ CFU/mL) prepared as above were pipetted 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. After incubation period, non-adherent cells were removed by washing three times with phosphate-buffered 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 quantification after imaging under microscope using Leica Application Suite software v. 3.7.0 (Leica Microsystems AG, Heerbrugg, Switzerland) by automatic counting of viable cells (20). Briefly, 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 filled with 20 µL of the stained suspension. After adjusting the settings of the microscope for counting only viable cells, images were analysed using ImageJ, v. 1.43u, image processing software (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 significant 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 different 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 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, 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 reflects inherent physiological differences, and may have significance 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.

Fig. 1.

Adhesion of Candida spp. to smooth and rough wooden surfaces: a) C. albicans, b) C. glabrata, c) C. parapsilosis and d) C. krusei. Each bar represents the mean value±standard deviation (S.D.)

Additionally, under the conditions of our study all assayed Pichia strains were able to adhere to wooden surfaces, as shown in Fig. 2. P. pijperi ZIM 1368 and P. membranifaciens ZIM 2302 had a better 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 different food industry settings because these microorganisms can be a source of food contamination.

Fig. 2.

Adhesion of Pichia spp. to smooth and rough wooden surfaces. Each bar represents the mean value± standard deviation (S.D.)

Our study has confirmed previous findings that Candida species are capable of adhering to abiotic surfaces (23, 24). Nevertheless, to the best of our knowledge, this is the first time the adhesion of Candida and Pichia species to wooden surfaces has been shown. Considerable differences in adhesion ability among Candida species were observed. C. albicans and C. glabrata strains adhered better than C. parapsilosis and C. krusei (Fig. 1). These results confirm previous findings, which showed that C. albicans had good biofilm growth on the surface of PVC catheter discs (25) and on silicone elastomer discs (26). Furthermore, Shin et al. (27) observed that biofilm 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 findings could be the fact that microbial adhesion is also influenced by the properties of the different substrates, contact medium and methods used to quantify adhesion.

It is well known that the surface properties of materials, such as surface roughness, can significantly influence the quality and quantity of fungal adhesion. Evaluation of C. albicans adhesion to denture base resin with different 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 difficulties in surface cleaning (31), resulting in rapid regrowth of a biofilm. In contrast, in our study the roughness of wooden surfaces did not have significant influence 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 influenced by surface roughness of different materials (32, 33). The effect of surface roughness on adhesion could be attributed to species- and strain-specific 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 better 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 significantly affected by the roughness of the wooden surfaces. Therefore, as yeast adhesion is the first step of biofilm 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 biofilm formation in food processing environment.