Bioprocess Biosyst Eng DOI 10.1007/s00449-015-1414-7

ORIGINAL PAPER

Effective inactivation of Candida albicans biofilms by using supercritical carbon dioxide Hyong Seok Park1 • Jungwoo Yang2 • Hee Jung Choi3 • Kyoung Heon Kim1,2

Received: 23 March 2015 / Accepted: 13 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Present sterilization methods for biofilms in medical devices have limitations. Therefore, an alternative sterilization method using supercritical carbon dioxide (SCCO2) was tested on Candida albicans biofilms. The effect of varying pressure, temperature, and treatment time on the inactivation of C. albicans spores in suspensions and in biofilms was examined. The parameters such as treatment time, pressure, and temperature that led to the complete inactivation of C. albicans biofilms ranged 5–20 min, 100–200 bar, and 35–45 °C, respectively. Notably, treatment of SC-CO2 at either 100 bar and 40 °C or 200 bar and 30 °C induced complete inactivation of spores within 5 min. Furthermore, it was found that wet biofilms (0.4 %, w/w) had higher sensitivity to SC-CO2 than dried biofilms. Finally, spore inactivation was confirmed by confocal laser scanning microscopy. In this study, the use of a low-temperature SC-CO2 sterilization method was proven to be effective in fungal biofilm inactivation, and the moisture content of biofilms was revealed to be the key factor for biofilm inactivation.

H. S. Park and J. Yang contributed equally to this work. & Kyoung Heon Kim [email protected] 1

Department of Biotechnology, Graduate School, Korea University, Seoul 136-713, Republic of Korea

2

BK21 PLUS School of Life Science and Biotechnology, Korea University, Seoul 136-713, Republic of Korea

3

Division of Infectious Diseases, Department of Internal Medicine, Ewha Womans University School of Medicine, Seoul 158-710, Republic of Korea

Keywords Sterilization  Supercritical carbon dioxide  Candida albicans  Biofilm

Introduction Members of the genus Candida are mostly pathogenic fungi associated with yeast infection. In humans, this type of infection is known as candidiasis (or thrush) and can occur at sites such as the mouth, vagina, and intestine [1]. Candida albicans, the major candidiasis-causing pathogen in humans, causes both superficial and systemic diseases, especially in immunocompromised individuals such as infants, the elderly, and immunosuppressed patients [2–4]. C. albicans has a dimorphic nature, existing as yeast and filamentous (hyphal) forms. The hyphal form is capable of adhesion to the epithelium, of epithelial penetration, and of invasion [5]. Prior to this pathogenic state, C. albicans generally forms biofilms to ensure protection from antibiotic treatment and to maintain a source of persistent infection [6]. On the host surface, biofilms consist of heterogeneously spaced microbial colonies and extracellular polymeric substances (EPS) [7]. Microbial cells are embedded within the EPS, and the thickness of these exopolymeric materials determines the range of antibacterial resistance [8, 9]. Thus, cells in a biofilm are undoubtedly much more resistant to antimicrobial reagents than cells in suspension [10]. For instance, in Staphylococcus aureus a 600-fold increase in chlorine concentration was required to kill cells in biofilms in comparison with cells in suspensions [11]. Due to this high level of antimicrobial resistance, biofilms can cause serious problems with indwelling medical devices such as dental implants, catheters, and heart valves, as these devices can act as substrates for biofilm growth

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[11–13]. As a consequence, powerful sterilization strategies are urgently required that can completely inactivate biofilms, thus reducing the social costs incurred due to continuous infections occurring in health care facilities [11, 14]. Currently, several sterilization methods are widely applied to medical devices to control biofilms: thermal, chemical, and radiation sterilizations [15]. However, these physical or chemical methods have limitations. Device geometries result in incomplete inactivation of cells in bioflms, their use can result in thermal or mechanical damages to medical devices, and toxic residues may remain inside the devices [12, 16–18]. Recently, supercritical carbon dioxide (SC-CO2) has been proposed as an alternative non-thermal sterilization agent [19]. Carbon dioxide behaves as a supercritical fluid under relatively mild conditions (critical point, 73.8 bar, and 31.1 °C) and can readily penetrate into the cells. Additionally, SC-CO2 is non-toxic, non-flammable, and nonpolluting [20]. SC-CO2 has a gas-like viscosity and a liquid-like dissolving power [20]. Thus, these unique characteristics of SC-CO2 mean that SC-CO2 can be used effectively to target microorganisms both physically and chemically [19, 21, 22]. SC-CO2 treatment (with or without co-agents, such as alcohol and hydrogen peroxide) has been shown to inactivate vegetative cells [23–25] and spores [26–29] of various microorganisms. However, relatively few studies are available on the use of SC-CO2 treatment for biofilm inactivation, in bacteria such as Bacillus mojavensis [30], Pseudomonas aeruginosa [31], and Bacillus cereus [32]. Furthermore, the inactivation of spores in fungal biofilms by SC-CO2 has yet not been reported. The objectives of this study were: (1) to evaluate the effectiveness of SC-CO2 treatment on C. albicans biofilm by varying operating parameters such as pressure, temperature, and exposure time, and (2) to compare the inactivation performance of SC-CO2 for the cells in biofilms and suspensions.

stock culture was preserved in yeast extract peptone dextrose (YPD) medium (Difco, Detroit, MI, USA) containing 10 % (v/v) glycerol (Junsei Chemical, Tokyo, Japan) and was stored at -80 °C until used. Preparation of spore suspensions One loop (ca. 10 lL) of stock culture of C. albicans was streaked on Sabouraud’s dextrose agar (SDA; Difco) and incubated at 37 °C for 24 h. A single colony was then transferred into 25 mL of YPD medium and incubated, with agitation at 150 rpm, at 35 °C for 18 h. Spores were then harvested by centrifugation (CR-21G II; Hitachi, Tokyo, Japan) at 67609g at 4 °C for 10 min. The resulting spore pellets were washed twice using 25 mL of sterile phosphate-buffered saline (PBS; KH2PO4 0.24 g/L, KCl 0.2 g/L, NaCl 8 g/L, Na2HPO4 1.44 g/L; pH 7.4; Sigma, St. Louis, MO, USA). Finally, washed spores were suspended in 25 mL of PBS, and the initial spore densities were measured as approximately 8.4 9 108 colony forming units (CFU)/mL. Meanwhile, the formation of spores in suspensions and in biofilms was visually confirmed by confocal microscopy as shown in Fig. 4. Development of biofilms on stainless steel plates For the development of C. albicans biofilms, we have followed the previous method [33] with minor modification as follows. Briefly, stainless steel plates (2 cm 9 5 cm 9 1 mm, SUS 304) were obtained from POSCO (Pohang, Korea) and autoclaved before use. The stainless steel plates were first immersed in 25 mL of freshly prepared spore suspension and incubated at 37 °C for 3 h to facilitate cell attachment. Then, the plates were transferred into 25 mL of fresh YPD medium and incubated at 37 °C for 4 days to facilitate biofilm development. The YPD medium was changed at 24 h intervals during the 4-day incubation. The initial spore densities in biofilms were measured as approximately 7.5 9 107 CFU/mL. Preparation of spores in dried biofilms

Materials and methods Strain C. albicans strain was isolated from a vaginal candidiasis patient at Ewha Womans University Hospital in Seoul, Korea. After the isolation of single colony, the colony was subjected to the morphological analysis and biochemical assay. Then, 18S rRNA gene, internal transcribed spacers, and the 50 end of the 28S rRNA of genomic DNA from the colony were PCR-amplified, sequenced, and analyzed. A

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To investigate the effect of biofilm moisture content on cell viability, biofilms on stainless steel plates were dried using a vacuum-drying oven (SH-45S, Biofree, Seoul, Korea) at room temperature for 4 h. The moisture contents of biofilms [wet (not dried) vs. vacuum-dried] were determined by drying biofilms in a drying oven at 105 °C for 24 h. The moisture content of wet and vacuum-dried biofilms was measured to be 0.4 and 0 % (w/w), respectively. For the case of vacuum-dried biofilms, the CFU of spores in biofilms did not change even after vacuum drying.

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SC-CO2 treatment SC-CO2 was delivered into a vessel containing spores in suspensions or biofilms; these were treated at various pressures and temperatures for different exposure times as previously described [32]. Briefly, when the temperature of the SC-CO2 treatment vessel reached a predetermined level, a biofilm plate or spore suspension (2 mL) was loaded into the treatment vessel and the vessel was tightly closed. Liquid CO2 (99.5 % purity; Daehan Specialty Gases, Seoul, Korea) was then pumped into the treatment vessel from a cylinder through a siphoned system. The CO2 temperature was regulated using a heating bath connected to a computer-based controller. The flow of pressurized CO2 was kept at a constant rate by the CO2 pump, and the vessel was fully pressurized within 3 min. After treatment for the desired time, the CO2 vent valve was immediately opened to stop further reactions in the vessel. All experiments were performed in triplicate. Determination of viable spores in biofilms To quantify the total viable spores in biofilms, 3 g of glass beads (425–600 lm; Sigma-Aldrich) were transferred into a tube containing a stainless plate of biofilms and 25 mL of PBS. The mixture of the biofilm plate and PBS was then vigorously vortexed for 1 min to detach the spores from the biofilm on the surface of the steel plate. The suspension of detached spores were then incubated on SDA medium after tenfold serial dilution using PBS, and the number of spores was determined by viable colony counting after 24 h. The spore viability after SC-CO2 treatment was expressed in log reduction of CFU of spores, which was calculated by using the following equation:  log reduction of CFU = log10

 CFU from not sterilized spores : CFU from sterilized spores

Confocal laser scanning microscopy (CLSM) The fluorescent probes of SYTO 9 and propidium iodide (PI) (Invitrogen, Eugene, OR, USA) were used to differentiate between viable and dead spores in biofilms and in suspensions. SYTO 9 is a membrane-permeable green fluorescent dye, which stains all cells regardless of cell viability, whereas PI is a membrane-impermeable red dye, which stains only dead cells [34]. Staining procedures were performed according to the manufacturer’s instructions. To prepare staining solution, fluorescent dyes were diluted at 3:1000 using sterilized water. Staining solution and spore suspension were mixed at 1:1. The mixture was then incubated in a dark room at 37 °C for 30 min. After

incubation, stained spore suspension was centrifuged at 67609g at 4 °C for 2 min. The spore pellet was washed twice with PBS and suspended in an equal volume of PBS. Next, 100 lL of each spore suspension was transferred onto a cover slide and dried in a dark room at 37 °C. Finally, a drop of mounting oil was loaded onto each specimen and covered with a cover glass. For staining of spores in biofilms, the entire steel plates were stained with 0.2 mL of the staining solution and incubated in a dark room at room temperature for 30 min. The specimens were then rinsed gently with sterilized water to remove excess staining. Finally, the stained spore specimens of biofilms or suspensions were analyzed by CLSM (LSM 5 Exciter, Zeiss, Jena, Germany). The microscope images were analyzed through a 409 lens. The observing conditions of specimens were as follows: for green channel (SYTO 9), an excitation wavelength at 488 nm using argon laser and an emission filter with a band path of 505–530 nm; for red channel (PI), an excitation wavelength at 543 nm using HeNe-laser and an emission filter with a long path of 560 nm.

Results and discussion Effect of SC-CO2 pressure on spore viability Generally, increasing temperature increases the diffusivity of CO2 and the permeability of cell membranes, resulting in increased cell death [19]. At a fixed temperature of 35 °C, we tested the effect of varying SC-CO2 pressure on the viability of C. albicans spores in suspensions and biofilms. At all the pressures tested, spores in suspension were rarely inactivated by SC-CO2 treatment for up to 60 min. The log reduction of CFU of C. albicans spores in suspensions was less than 1.0 (Fig. 1a) in all cases. However, spores in biofilms were completely inactivated within 20 min at all pressures (Fig. 1b). Furthermore, 5-min treatment by SC-CO2 either at 150 bar or at 200 bar was sufficient to achieve complete cell death. These results indicate that spores of C. albicans in suspension are more difficult to inactivate using SC-CO2 than spores in biofilms. This may be due to the presence of the abundant aqueous phase in the spore suspensions lessening the effect of SCCO2, such as simply dispersing CO2 more widely. Since longer treatment time or higher pressure did not affect the inactivation of spores in suspension (Fig. 1a), increasing temperature was investigated. Effect of temperature on spore viability Since the increase of pressure from 100 to 200 or 300 bar did not result in any significant increase in spore

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inactivation at 35 °C (Fig. 1a), treatments at higher temperatures were attempted by fixing the pressure of SCCO2 at 100 bar (Fig. 2). As a control, a spore suspension was treated with 100 bar of SC-CO2 at 35 °C for 60 min, and its log reduction of CFU was less than 1.0, as previously shown in Fig. 1a. For spores in suspension, when the temperature was increased to 40 and 45 °C, log reduction significantly increased to 4.0 and 8.5, respectively (Fig. 2a). In the biofilms, viable spores were completely inactivated within 20 min at all temperature conditions (Fig. 2b), and within 5 min at 40 or 45 °C. Overall, increasing the temperature of SC-CO2 at 100 bar enhanced the inactivation of C. albicans spores both in suspensions and in biofilms. Thus, we have shown that pressure and temperature are both important factors in SC-CO2-mediated inactivation of fungal biofilms as well as bacterial biofilms [30–32] (Figs. 1 and 2).

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Fig. 1 Effect of supercritical carbon dioxide pressure on inactivation of C. albicans spores both in a suspensions and b biofilms at 35 °C

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Fig. 4 Confocal laser scanning microscopy images of C. albicans spores. a Untreated spores in suspensions stained with SYTO 9; b spores in suspensions treated with supercritical carbon dioxide (SC-

CO2) at 100 bar and 45 °C for 30 min stained with PI; c untreated spores in biofilms stained with SYTO 9; and d spores in biofilms treated with SC-CO2 at 100 bar and 45 °C for 20 min stained with PI

Effect of moisture content on spore viability

important from a different viewpoint because biofilms inherently contain water as channels for the distribution of nutrients and signaling molecules [7]. Therefore, the moisture content can either negatively or positively affect the survival of spores in biofilms under SC-CO2 treatment. To investigate the effect of biofilm moisture content on SC-CO2 inactivation, spores in biofilms with two different moisture contents [0.4 % (w/w) for wet biofilms (not dried) and 0 % (w/w) for vacuum-dried biofilms] were treated by SC-CO2 at 100 bar and 35 °C for 5–60 min (Fig. 3). Spores in the wet biofilms were completely inactivated within 20 min,

The lethal effect of SC-CO2 may be complex and still arguable. However, one of possible physicochemical effects can be simply explained by the penetration of SC-CO2 molecules into cells because of the increased fluidity and permeability of phospholipid bilayers in cell membranes under SC-CO2 conditions [35]. As a result, the release of DNA and proteins leads to cell death. However, this lethal effect of SC-CO2 seems to be lessened when samples are in dried form [24, 30]. Meanwhile, the moisture content is

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whereas spores in the dried biofilms were rarely inactivated even after 60 min. The average reductions of log CFU were 7.5 and 0.8 in the wet and dried biofilms, respectively. These results suggest that fully dried biofilms do not seem to allow CO2 molecules to invade the biofilm. Similar phenomena were previously observed in a bacterium, Listeria monocytogenes [24]. When the SC-CO2 inactivation of vegetative cells was tested, reductions in log CFU were 3–8 times lower for dried L. monocytogenes (2–10 %, w/w) than for wet cells (80–100 %, w/w). Conversely, increasing moisture content in bacterial biofilms has been reported to be inversely proportional to spore inactivation [31]. Specifically, the reduction in log CFU of spores in waterimmersed biofilms (7 % of moisture, v/v) was maximally 1.5, whereas in control biofilms (\0.1 % moisture, v/v) the reduction in log CFU reached 6.0 within 7 min. Taken together, these results suggest that effective inactivation of spores in biofilms requires determination of the optimal ranges of moisture for each inactivation condition. Analysis of biofilms by CLSM Untreated spore samples both in suspensions (Fig. 4a) and in biofilms (Fig. 4c) were mostly stained green by SYTO9, indicating the presence of live cells. Conversely, treated samples were strained red by PI, indicating disruption of spore membrane integrity (Fig. 4b and 4d). These CLSM images indicate that the membrane integrity of spores in both suspensions and biofilms was disrupted by the SCCO2 treatment. The observation of disrupted membranes in the spores is consistent with results from the inactivation of vegetative cells [23].

Conclusions In this study, SC-CO2 treatment at a moderate temperature is demonstrated to be an effective method for the inactivation of C. albicans biofilms. In addition to SC-CO2 pressure and temperature, biofilm moisture content was also shown to influence the effectiveness of SC-CO2 treatment. Furthermore, CLSM analysis revealed that SCCO2 treatment disrupted spore membrane integrity. These results will contribute to the development of strategies for effective inactivation of biofilms in locations that are difficult to reach. Acknowledgments This work was supported by grants from the Advanced Biomass R&D Center of Korea (2011-0031353) funded through the Korean Government (MSIP) and also from the Ministry of Trade, Industry and Energy (10047873). This research was performed at the Korea University Food Safety Hall for the Institute of Biomedical Science and Food Safety.

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Effective inactivation of Candida albicans biofilms by using supercritical carbon dioxide.

Present sterilization methods for biofilms in medical devices have limitations. Therefore, an alternative sterilization method using supercritical car...
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