Fungicidal activity of copper-sputtered flexible surfaces under dark and actinic light against azole-resistant Candida albicans and Candida glabrata

Accepted Manuscript Fungicidal activity of copper-sputtered flexible surfaces under dark and actinic light against azole-resistant Candida albicans and Candida glabrata

Myriam K.S. Ballo, Sami Rtimi, John Kiwi, César Pulgarin, José M. Entenza, Alain Bizzini PII: DOI: Reference:

S1011-1344(16)31086-7 doi: 10.1016/j.jphotobiol.2017.07.030 JPB 10935

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

25 November 2016 25 July 2017 27 July 2017

Please cite this article as: Myriam K.S. Ballo, Sami Rtimi, John Kiwi, César Pulgarin, José M. Entenza, Alain Bizzini , Fungicidal activity of copper-sputtered flexible surfaces under dark and actinic light against azole-resistant Candida albicans and Candida glabrata, Journal of Photochemistry & Photobiology, B: Biology (2017), doi: 10.1016/ j.jphotobiol.2017.07.030

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Fungicidal activity of copper-sputtered flexible surfaces under dark and actinic light against azole-resistant Candida albicans and Candida glabrata

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Myriam K.S. Balloa,b, Sami Rtimia, John Kiwia, César Pulgarina, José M. Entenzab,*,†, Alain Bizzinib,†

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Group of Advanced Oxidation Procedures, Swiss Federal Institute of Technology, EPFL-

SB- ISIC-GPAO, Station 6 CH-1015 Lausanne, Switzerland.

Department of Fundamental Microbiology, University of Lausanne, Quartier Unil-Sorge,

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Biophore Building CH-1015 Lausanne, Switzerland. Equally contributing last authors

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* Corresponding author: Dr. José M. Entenza Department of Fundamental Microbiology, Biophore Building, University of Lausanne, 1015 Lausanne, Switzerland. Phone: +41 21 692 56 12 Fax : + 41 21 692 56 05 E-mail: [email protected] 1

ACCEPTED MANUSCRIPT Abstract Candida spp. are able to survive on hospital surfaces and causes healthcare-associated infections (HCAIs). Since surface cleaning and disinfecting interventions are not totally effective to eliminate Candida spp., new approaches should be devised. Copper (Cu) has widely recognized antifungal activity and the use of Cu-sputtered surfaces has recently been proposed to curb the spread of HCAIs. Moreover, the activity of Cu under the action of actinic light remains underexplored. We investigated the antifungal activity of Cu-sputtered polyester

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surfaces (Cu-PES) against azole-resistant Candida albicans and Candida glabrata under dark and low intensity visible light irradiation (4.65 mW/cm2). The surface properties of CuPES photocatalysts were characterized by diffuse reflectance spectroscopy (DRS) and X-ray fluorescence (XRF). Under dark, Cu-PES showed a fungicidal activity (≥ 3 log10 CFU reduction of the initial inoculum) against both C. albicans DSY296 and C. glabrata DSY565 leading to a reduction of the starting inoculum of 3.1 and 3.0 log10 CFU, respectively, within

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60 min of exposure. Under low intensity visible light irradiation, Cu-PES exhibited an accelerated fungicidal activity against both strains with a reduction of 3.0 and 3.4 log 10 CFU,

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respectively, within 30 min of exposure. This effect was likely due to the semiconductor Cu2O/CuO charge separation. The decrease in cell viability of the two Candida strains under dark and light conditions correlated with the progressive loss of membrane integrity. These results indicate that Cu-PES represent a promising strategy for decreasing the colonization

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of surfaces by yeasts and that actinic light can improve its self-disinfecting activity.

Keywords

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Copper-sputtered polyester; actinic light; antifungal activity; azole-resistant Candida spp.

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ACCEPTED MANUSCRIPT 1. Introduction Healthcare-associated infections (HCAIs) are a concern around the globe [1]. The hospital environment is a reservoir of pathogens and plays a major role in the incidence of HCAIs. Indeed, many healthcare-associated pathogens can survive for a long time on inanimate surfaces such as door handles, medical equipment or textiles and possibly be transmitted to a susceptible patient by a simple touch through the hands of healthcare workers or visitors [2-4].

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Candida spp., and among them Candida albicans and Candida glabrata, are commensals of the human mucous membranes and digestive tract but can also be commonly found on abiotic surfaces in the hospital setting, as well as on health care worker’s hands [5, 6]. Candida spp. are the most important fungal agents responsible of HCAIs, especially in immunocompromised hosts [7-9]. The incidence of invasive infections due to Candida spp. in hospitals has increased during the last decades and

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their treatment has been complicated by the rise of resistance to first line agents of the azole or echinocandin class [10, 11]. Surface disinfection and cleaning are recommended by international guidelines as a procedure for preventing HCAIs [12].

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Despite considerable progress, however, efficient Candida spp. elimination from contaminated surfaces remains an unmet clinical problem [13]. Therefore, to counteract HCAIs due to Candida spp., there is a need for development of novel

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antimicrobial materials with antifungal properties [14, 15]. The advanced oxidation processes, the so called photocatalysis, have emerged in

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recent years as a promising approach to be used in bacterial inactivation [16]. Titanium dioxide (TiO2) is the most widely used photocatalyst material with a band gap

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of 3.2 eV, absorbing 4-5% if the visible light above 400 nm and the UV-light under 400 nm. UV < 340 nm is effective for inactivation of microorganisms via cell membrane disruption and DNA damage [17, 18]. Nevertheless, the absorption of TiO 2 mainly in

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the UV-region is a drawback when the utilization of the widely available solar light is desired to inactivate pathogens [19]. Previous studies have shown that TiO 2 surfaces doped with Copper (Cu) can be photoactivated with visible light up to 700 nm [20, 21]. Cu-doped TiO2 (1.7 eV) surfaces have been reported to display bactericidal activity under visible light irradiation against both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus and Escherichia coli [20, 22-24]. Cu and Cu-alloy surfaces have intrinsic antibacterial and antifungal activities [25, 26]. In recent years, the coating-technology known as Direct Current Magnetron Sputtering (DCMS) was used to manufacture Cu-nanoparticulate films on several 3

ACCEPTED MANUSCRIPT fabrics, including polyester. Copper-sputtered polyester (Cu-PES) present a progress in the field of antimicrobial surfaces over previously used coating, such as colloidal coatings on PES.[27] Indeed, Cu-PES presents an adhesive, uniform and stable film on polyester. Furthermore, Cu-PES remains active over time [28, 29]. Since they are mechanically stable, the Cu-coating cannot be removed by contact during the microbial inactivation process [29]. Importantly, rapid bactericidal activity of Cu-PES has been reported against a variety of antibiotic-resistant bacterial pathogens, such as

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methicillin-resistant S. aureus (MRSA) vancomycin-resistant Enterococcus faecalis and extended expectrum β-lactamase producing E. coli [30, 31]. Moreover, Cu-PES are expected to be harmless for medical staff and patients. Indeed, metallic copper is a rare sensitizer and has been shown to not cause skin irritation [32]. In addition, no negative health effects on patients and healthcare workers were reported after exposure to copper surfaces in clinical trials [33] and no skin reaction was observed in rabbits exposed to extracts of copper fabrics [34].

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Cu deposited on the PES is present in the form of CuO (Copper II oxide) and Cu 2O (Copper I oxide) [35]. Previous studies have shown that copper oxide is also a

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semiconductor with lower band gap energy (1.9 eV) than TiO 2 and can absorb visible light [36]. The focus of this study is the inactivation by Cu-PES of Candida spp. both in

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the dark and under actinic light.

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ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1. Microorganisms and growth conditions C. albicans DSY296 and C. glabrata DSY565 [37, 38] were used in this study. Both strains were stored at -80°C in cryovial bead preservation systems (Microbank; ProLaboratory Diagnostics, Richmond Hill, Canada) and streaked onto blood agar plates before each experiment. Cultures were prepared by inoculating a single colony from the blood agar plate into Sabouraud dextrose broth (Oxoid, Basingstoke, United

2.2. Cu-PES preparation and characterization

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Kingdom) followed by overnight incubation under agitation (180 rpm) at 37°C.

The polyester (PES) was obtained from the Swiss Federal Laboratories for materials Science and Technology (EMPA). Test clothe sample No 407 was a polyester Dacron polyethylene terephthalate, type 54 spun, 130 microns thick, plain weave ISO 105-

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F04. Cu-PES samples were prepared by sputtering Cu for 160 seconds onto the polyester (4 cm2) at temperature < 130°C by DCMS as previously described [30].

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Coating layers of CuO (Copper II oxide) and Cu2O (Copper I oxide) were deposited on PES [35]. The Cu-content of Cu-PES was evaluated by X-ray fluorescence (XRF) using a PW2400 spectrometer (PANalytical; Almelo, The Netherlands). The optical

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absorption of Cu on the sputtered polyesters samples was assessed by diffuse reflectance spectroscopy (DRS) with a Lambda 900 UV-VIS-NIR spectrometer (Perkin

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Elmer; Waltham, MA) within the wavelength range of 200-800 nm.

2.3. Killing activity of Cu-PES in the dark and under actinic light

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The activity of Cu-PES was evaluated by direct transfer of the samples onto agar plates as previously described [30, 31]. Briefly, overnight cultures of C. albicans

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DSY296 and C. glabrata DSY565 were washed 2 times with 0.9% NaCl and serially diluted to the required concentration. Then, unsputtered-PES (control) and Cu-PES were loaded with 20 µl of the tested strains, corresponding to a final inoculum of ca. 107 CFU. Inoculated coupons were then incubated for 15, 30, or 60 min in a humidified chamber at room temperature (20-23oC), in the dark or under actinic light. To measure the activity of PES and Cu-PES under light, a light box equipped with tubular actinic lamps (Master TLD-18W/865, Philips, The Netherlands) with a visible emission spectrum between 400 and 700 nm, generating a light intensity of 4.65 mW/cm2, was used. The temperature inside the light box was maintained at 20°-23°C by a ventilating system. At each time point, agar plates were separated in four 5

ACCEPTED MANUSCRIPT quadrants and the polyester coupons placed in each quadrant with fungi facing the agar. Falcon tubes (BD Biosciences, San Jose, CA) were filled with 50 ml of water and successively applied (0.07 N/cm2 pressure) onto the polyester for 1 min to induce the transfer of fungi on the agar plates. After 48 h of incubation at 37°C, CFU number was determined. The detection limit of this procedure was 1 log10 CFU. Fungicidal activity was defined as a reduction of ≥ 3 log 10 (99.9%) CFU of the initial inoculum. Experiments were performed in duplicate and repeated at least three independent

2.4. Contrast phase and fluorescence microscopy

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The effects of Cu-PES on the Candida structure were assessed by contrast phase and fluorescence microscopy. To that aim, Cu-PES and unsputtered-PES samples were inoculated with 108 CFU of either C. albicans DSY296 or C. glabrata DSY565

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and incubated for 15, 30 and 60 min in a humidified chamber at room temperature in the dark and under actinic light. The yeast cells were subsequently detached from the coupons through vigorous vortexing for 3 min in 1 ml of NaCl. A 10-times higher

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inoculum than in killing experiments were used in order to increase the amount of cells available for staining. Indeed, less yeast cells could be retrieved using the vortexing technique compared to the direct transfer method. Cell staining was performed using

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the LIVE/DEAD® BaclightTM viability kit (Life technologies, Carlsbad, CA). This kit contains SYTO9™ and propidium iodide. SYTO9® is a green nucleic acid fluorescent

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dye which can cross membranes of both live and dead microorganism cells and stains cells green. Propidium iodide is a red nucleic acid fluorescent dye entering the cells

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with damaged membranes and stains cells red. Thus, cells with intact membranes will fluoresce green while cells with damaged membranes will fluoresce red. Cells were observed using an automated inverted microscope (Leica DMI 4000 B, Heerbrugg,

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Switzerland).

2.5. Visualization of yeast exposed to Cu-PES by transmission electron microscopy (TEM) To visualize whether contact with Cu-PES induced ultrastructural changes on yeast cell morphology, TEM was performed after 24 h of yeast exposure to PES and CuPES at room temperature in the dark. TEM experiments were not performed under light as the effect of copper on the yeast structure is expected to be the same than under dark, as previously demonstrated with other photocatalyst, i.e., TiO2 [39]. C. glabrata DSY565 was used as a test organism. TEM was performed at 24 h because 6

ACCEPTED MANUSCRIPT in a previous work with bacteria [31] we have observed that, although cells were killed after relatively short exposure (within 60 min) to Cu-PES, the killing process at early times induced limited visible damage to the bacterial cells. In contrast, after 24 h of exposure, structural damages were visible by TEM. Briefly, cells of C. glabrata DSY565 (107 CFU) were loaded on unsputtered- and Cu-PES. After 24 h of exposure at room temperature in the dark, cells were fixed with a glutaraldehyde solution (EMS, Hatfield, PA) in 2.5% Phosphate Buffer (PB) (Sigma-Aldrich, St Louis, MO). Samples

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were rinsed in PB buffer and post-fixed with a solution containing 1% osmium tetroxide (EMS) and 1.5% of potassium ferrocyanide (Sigma-Aldrich). Samples were dehydrated in acetone series (Sigma-Aldrich) and embedded in Epon resin (SigmaAldrich). Ultrathin sections of 50 nm were cut by Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) and deposited on a copper grid (EMS) coated with a polystyrene film (Sigma-Aldrich). Sections were stained with 4% uranyl acetate (Sigma-Aldrich). Micrographs were taken with a FEI CM100 transmission electron

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microscope (FEI, Eindhoven, The Netherlands) at an acceleration voltage of 80kV

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with a TVIPS TemCam-F416 digital camera (TVIPS GmbH, Gauting, Germany).

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ACCEPTED MANUSCRIPT 3. Results 3.1. X-ray fluorescence (XRF) and diffuse reflectance spectroscopy (DRS) of Cu-PES The Cu-content of Cu-PES samples, was determined by XRF, and presented a loading of 0.11 % Cu weight/weight PES. The spectral range for Cu(I)/Cu(II)-species, measured by DRS, ranged between 200 and 800 nm allowing a considerable absorption of the actinic light, emitted between

400 and 700 nm. The optical

absorption between 500 and 600 nm is due to the inter-band transition of Cu(I) and

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the absorption between 600 to 720 nm is attributed to the exciton band and the Cu(II) d-d transition (Figure 1).

3.2. Activity of Cu-PES under dark and under visible light irradiation

Figure 2A shows the Cu-PES inactivation of C. albicans DSY296. Under dark conditions, Cu-PES was fungicidal (reduction of the initial inoculum of 3.1 log10 CFU)

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within 60 min. Under visible light, the fungicidal activity of Cu-PES (3.0 log10 CFU reduction) was already achieved within 30 min.

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Similar results were observed in the case of C. glabrata DSY565 (Figure 2B). Indeed, Cu-PES under dark exhibited fungicidal activity (3.0 log10 CFU reduction) after 60 min. Consistently, the fungicidal activity of Cu-PES was also reached more rapidly under

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visible light, with a loss in the starting inoculum of 3.4 log 10 CFU within 30 min and a reduction of 3.9 log10 CFU at 60 min. Of note, control experiments showed that the survival of Candida cells was not affected under dark or actinic irradiation conditions

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upon exposure to unsputtered-PES

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3.3. Fluorescence microscopy revealing the loss of membrane integrity Contrast phase and fluorescence microscopy analysis of C. albicans DSY296 and C.

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glabrata DSY565 were performed to investigate whether the fungicidal activity of CuPES under light and dark conditions was associated with changes in yeast cell morphology and cell membrane integrity. These observations are reported in Figure 3. Contrast phase microscopy images showed that cells of both Candida strains in contact with unsputtered-PES samples preserved their initial membrane integrity. In contrast, cells being in contact with Cu-PES presented damages of the cell membrane and have a tendency to agglomerate to each other. Figure 3A and Figure 3B show that for both Candida isolates, the number of yeast stained red (i.e., being dead) after Cu-PES contact increased within time, indicating progressive cell membrane damage.

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ACCEPTED MANUSCRIPT 3.4. Visualization of ultrastructure changes after Cu-PES exposure TEM was used to visualize the effect of the Cu-PES contact with C. glabrata DSY565 at room temperature in the dark. The results are shown in Figure 4. The C. glabrata DSY565 exposed to unsputtered-PES exhibited normal morphology, with intact and uniform cell wall and inner membrane (Figure 4A). In contrast, after 24 h of exposure to Cu-PES, yeasts showed visible damage with severe morphologic modifications, such as a rough surface, irregular shape, marked invagination of the plasma

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membrane and loss of cytoplasmic structures (Figure 4B).

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ACCEPTED MANUSCRIPT 4. Discussion Over the last decade metallic Cu on objects such as door handles, bed rails, or toilet seats, [25, 40] and Cu-impregnated flexible materials, such as hospital gowns, bed sheets or clothing [15, 26, 30, 34], have been demonstrated to efficiently decrease the bioburden of microorganisms in healthcare facilities. Cu-coated surfaces, however, require significant amounts of Cu, present low uniformity, little adhesion and are not mechanically stable over time [41]. Thus, the existing Cu-coating technology needs to

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be improved. Moreover, there is a need of Cu-coated surfaces active against a broad range of microorganisms including bacteria and yeasts. For instance, particular problems are encountered with surfaces contaminated with fungi that might cause difficult to treat infections [42, 43]. Here we investigated the activity of Cu-impregnated flexible surfaces against Candida spp. and the effect of visible light on its antimicrobial activity. Several important findings were observed.

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First, we showed that Cu-PES possess fungicidal activity against Candida spp. This result extends our previous findings showing that Cu-PES possess antibacterial activity against a wide panel of multidrug-resistant Gram-positive and Gram-negative

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bacteria under dark conditions [30, 31]. However, in comparison with the substantial (>5 log10 CFU reduction) and rapid (15-30 min) bactericidal activity of Cu-PES under dark reported previously by our laboratory [31], the fungicidal activity against Candida

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was reduced (ca. 3 log10 CFU) and slower (within 60 min). This difference might be attributed to the fact that the cell envelope surrounding the eukaryotic yeast cells is

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structurally more complex (composed by 1,3-β glucan and chitin) and thicker than that found in bacteria (70-100 nm vs 20-80 nm, respectively) [44] [45].

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Several reports have been published recently, indicating that the microbiocidal activity of Cu and Cu-coated surfaces was due to the disruption of the cell membrane function [31, 46-48]. In microorganisms the cell envelope is typically composed by a

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cell wall surrounding the plasma membrane. Accordingly, a thicker cell wall would protect the plasma membrane of the yeast against the biocidal effect of copper. Consistently, the lower biocidal activity against yeast as compared with bacteria has also been observed with silver (Ag)-nanoparticles [49]. Then, we showed that the fungi reduction due to Cu-PES against both C. albicans and C. glabrata isolates was accelerated under visible light (>400nm) compared to dark experiments, in agreement with a previous work [20]. The rupture of the cell membrane has also been established to be the mechanism for the antimicrobial activity of semiconductor catalysts [24]. This effect is likely related to an increased production of CuO. Before cell contact, the majority of Cu exists as Cu 2O. After 10

ACCEPTED MANUSCRIPT contact with the microorganisms, there is a significant oxidation of Cu 2O to CuO, as investigated by X-ray photoelectron spectroscopy (XPS) [29, 35, 50]. The variation from Cu+ to Cu2+ is indicative of redox catalysis and, as previously demonstrated, Cu2+ plays a major role in the antimicrobial activity [51]. While the primary target of Cu-PES under dark conditions seems to be the plasma membrane, similar results were found when Cu-PES was photo-activated. Indeed, Live/Dead® staining showed that the contact of Cu-PES with yeast under light also

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leads to the disruption of the membrane integrity. Similar to this study, Mitoraj et al. have shown that the structure and the thickness of the cell wall might help the microorganisms to resist more efficiently to the stress induced by oxidative semiconductor photocalysts [52].

Finally, as reported previously for bacteria like MRSA and E. coli [20, 31], we showed using TEM that exposure for 24 h to Cu-PES altered the ultrastructure of the yeast, and induce profound changes in the yeast cell wall and the cytoplasmic

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membrane, as well as disappearance of the nucleus and other cytoplasmic structures. As the reduction mediated by Cu-PES seems to be related with the structural

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composition of the microorganism envelope [53], further investigations are required to assess whether Cu-PES might also be able to inactivate filamentous fungi (e.g., Aspergillus spp.) or spore-forming (e.g. Clostridium spp.) microbes which are also a

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problem in healthcare facilities.

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5. Conclusions

This study focused on the activity of Cu-impregnated flexible surfaces against

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Candida spp. and the effect of visible light on its antimicrobial activity. Stable, uniform and adhesive film of Cu on PES samples have been shown to induce fungicidal activity against Candida spp within a short period of time under actinic light.

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These results further support the potential of Cu-PES as a valid surface to prevent microbial infection in hospital environments.

Acknowledgements We thank Dominique Sanglard (Institute of Microbiology, University of Lausanne) for the gift of the Candida strains, and Bruno Humbel and Jean Daraspe (Electron Microscopy Facility, University of Lausanne) for the help with the electron microscopy experiments. We thank the COST Action 1106 for interactive discussions during the course of this study. 11

ACCEPTED MANUSCRIPT Funding This work was supported by the Swiss National Science Foundation [grant number

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200021_143283/1].

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ACCEPTED MANUSCRIPT Figure legends

Fig. 1. Diffuse reflectance spectroscopy (DRS) of copper-sputtered polyesters (CuPES). Cu was sputtered by Direct Current Magnetron Sputtering (DCMS) on PES for 160 sec. Fig. 2. Loss of viability induced by unsputtered- (dashed lines) or copper-sputtered

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polyesters (solid lines) against C. albicans DSY296 (A) and C. glabrata DSY565 (B) in dark (closed symbols) and visible light (open symbols) conditions. The horizontal dot lines represent a 3 log10 CFU reduction of the initial inoculum sizes (fungicidal activities). Data represent the mean ± standard deviation of three independent experiments.

Fig. 3. Fluorescence microscopy images of C. albicans (A) and C. glabrata (B) cells

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exposed to unsputtered-polyesters (PES) or copper-sputtered polyesters (Cu-PES) under

dead cells. For other details see text.

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light irradiation. Green fluorescence indicates live yeast cells and red fluorescence shows

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Fig. 4. TEM images of Candida glabrata DSY565 within 24 h of contact with unsputteredpolyesters (PES; sample A) or with copper-sputtered PES (Cu-PES; sample B). Changes in

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the cell envelope and cytoplasm can be observed. The plain arrow indicates an abnormal

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and irregular cell wall, and the dashed arrow the invagination of the membrane.

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- Candida spp. can survive on hospital surfaces and are important causes of infections - Copper-sputtered polyesters (Cu-PES) showed a rapid fungicidal activity under dark - The antifungal activity of Cu-PES was faster upon visible light irradiation

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- The yeast envelop was damaged after Cu-Pes exposure

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- Self-disinfecting Cu-sputtered surfaces could be implemented in healthcare facilities

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