Yeast Yeast 2015; 32: 533–540 Published online 5 June 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/yea.3077
Research Article
Pulsed light for the inactivation of fungal biofilms of clinically important pathogenic Candida species Mary Garvey1*, Joao Paulo Andrade Fernandes2,3 and Neil Rowan2 1
Department of Life Sciences, Institute of Technology Sligo, Ireland Bioscience Research Institute, Athlone Institute of Technology, Ireland 3 CAPES Foundation, Ministry of Education of Brazil, Universidade Federal do Rio de Janeiro, Brazil 2
*Correspondence to: M. Garvey, Department of Life Sciences, Institute of Technology Sligo, Ash Lane, Sligo, Ireland. E-mail: [email protected]
Received: 21 October 2014 Accepted: 3 May 2015
Abstract Microorganisms are naturally found as biofilm communities more than planktonic free-floating cells; however, planktonic culture remains the current model for microbiological studies, such as disinfection techniques. The presence of fungal biofilms in the clinical setting has a negative impact on patient mortality, as Candida biofilms have proved to be resistant to biocides in numerous in vitro studies; however, there is limited information on the effect of pulsed light on sessile communities. Here we report on the use of pulsed UV light for the effective inactivation of clinically relevant Candida species. Fungal biofilms were grown by use of a CDC reactor on clinically relevant surfaces. Following a maximal 72 h formation period, the densely populated biofilms were exposed to pulsed light at varying fluences to determine biofilm sensitivity to pulsed-light inactivation. The results were then compared to planktonic cell inactivation. High levels of inactivation of C. albicans and C. parapsilosis biofilms were achieved with pulsed light for both 48 and 72 h biofilm structures. The findings suggest that pulsed light has the potential to provide a means of surface decontamination, subsequently reducing the risk of infection to patients. The research described herein deals with an important aspect of disease prevention and public health. Keywords: pathogenic; yeast; UV inactivation; sessile communities; surfaces; Candida
Introduction In the last three decades, fungi have appeared as a major cause of human disease, predominantly among immunocompromised individuals, neonates, burns patients and patients with serious underlying illnesses (Trofa et al., 2008). Candida species are opportunistic eukaryotic fungal pathogens commonly associated with clinical infections resulting in deep tissue infection, high mortality rates and financial burden. Biofilms (sessile communities) are a form of microbial growth where cells grow in a selfproduced protective environment, which allows the cells to escape the dangers of their surrounding location. Biofilms are usually associated with wet or damp surfaces, such as indwelling medical Copyright © 2015 John Wiley & Sons, Ltd.
devices and/or tubing on medical equipment. However, it is now known that microbes can survive for extended periods in a dehydrated state on dry hospital surfaces. Indeed, biofilms have recently been discovered on dry hospital surfaces (Otter et al., 2014). Candida biofilms are composed of yeast cells and filaments that are structurally attached to biotic or abiotic surfaces and embedded in an extracellular matrix (Nailis et al., 2010). Research has shown that such biofilm cell communities are more resistant to chemical disinfection techniques than planktonic (free-floating) cells, due to their extracellular matrix, structural complexity, gene upregulation and metabolic heterogeneity (Fanning and Mitchell, 2012). Biofilm formation by pathogenic microorganisms such as Candida plays a key role in infections resulting
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from intravascular devices in the clinical setting. Indeed, the association between Candida species biofilm formation and continued host infection has become more evident. Once a biofilm forms, it can continuously supply cells which detach from the main structure into the bloodstream, acting as a source of infection. It has been reported by Kumanoto (2002) that conditions with a high flow rate, such as that encountered within the circulatory system, may favour the development of persistent biofilms on devices placed in the bloodstream. Furthermore, Candida species biofilms are quite resistant to antifungals such as fluconazole, amphotericin B, nystatin and voriconazole (Kumanoto, 2002). In recent years there has been a marked increase in non-C. albicans-related bloodstream infections from Candida species, mainly C. parapsilosis, C. krusei and C. tropicalis (Trofa et al., 2008). The resistance of this species to antibiotics and the ability of Candida biofilms to tolerate chemical disinfection suggest the need for alternative methods of removing this pathogen from clinical settings. Prevention of infection is a superior method than infection treatment in terms of cost and patient well-being. An alternative or supplementary means of control is to minimize the extent of exposure of the patient to these fungal pathogens, thereby preventing an infection from occurring. Typical clinical surfaces, such as plastics, have been shown to act as reservoirs for viable pathogenic fungi, such as Candida albicans and C. parapsilosis (Neely and Orloff, 2001). Proper cleaning regimens that include the use of effective surface decontamination techniques can help prevent patient exposure to pathogenic species. The use of ultraviolet (UV) light has proved effective for the inactivation of a range of microbial species; however, there is limited information available on its use for the removal of clinically relevant Candida species or Candida biofilms. UV disinfection occurs following exposure of test species to UV energy at 254 nm, which has a negative effect on genetic material, preventing microbial replication. Standard UV lamps emit UV energy as a continuous wave from either a lowpressure (LP) or a medium-pressure (MP) lamp source at set wavelengths. Ultraviolet C (UVC) has been found to be efficient at sterilizing the inner surface of catheters contaminated with bacterial biofilms and subsequently preventing Copyright © 2015 John Wiley & Sons, Ltd.
M. Garvey et al.
catheter-related infections (Bak et al., 2009). Studies by this research group have focused on the use of an alternative means of delivering UV energy, referred to as ’pulsed UV’ (PUV), which delivers UV light in short-duration pulses of high-intensity light with a broader range of wavelengths in the range 100–1100 nm, enriched with shorter germicidal wavelengths (Farrell et al., 2011). These wavelengths may potentially affect more cellular targets, such as cell membranes and protein structures, than DNA alone, producing irreparable cellular damage. Due to the delivery mechanism of short-duration pulses, PUV also has a better penetration depth than LPUV. Additionally, PUV has proved effective for the inactivation of numerous microbial species, bacterial endospores, biofilms and parasite species in a shorter treatment time than LP- or MP-UV, leading to a reduction in operation costs (Garvey et al., 2014). Considering these important advantages of PUV over LP-UV, the aim of this study was to determine the potential of a PUV light system for the inactivation of clinically important Candida species in both planktonic and biofilm form on surfaces commonly found in the health care setting.
Methods Test strain culture conditions All Candida test strains used in this study were sourced from the American Type Tissue Culture Collection (ATCC), with the exception of a C. albicans hospital isolate, which was sourced from a blood culture at the National University of Ireland, Galway Hospital. Saccharomyces cerevisiae (ATCC 9763) was also sourced from the ATCC and used for PUV studies as a comparative strain to Candida. Strains were cultured and maintained on malt extract agar (Cruinn Diagnostics, Ireland) at 37 °C following removal from storage in microbank vials (Cruinn Diagnostics, Ireland) at 80 °C. C. parapsilosis (ATCC 22019) was cultured and maintained on Sabouraud agar (Cruinn Diagnostics, Ireland) at 30 °C and incubated at 5 ° C for short-term storage on agar slopes. Strains were identity confirmed by Gram staining and the germ-tube assay, according to Farrell et al. (2011), following a maximal 2 week storage period. For treatment studies, single colonies were Yeast 2015; 32: 533–540. DOI: 10.1002/yea
Inactivation of Candida biofilms using pulsed light
aseptically transferred to 100 ml malt extract broth and incubated at 37 °C under rotary conditions at 125 rpm for S. cerevisiae, C. albicans (ATCC 10231), C. albicans (clinical isolate NUIG 6250), C. krusei (ATCC 14243) and C. tropicalis (ATCC 13803); C. parapsilosis was cultured in 100 ml Sabouraud broth at 30 °C at 125 rpm for 18 h.
CDC reactor biofilm growth All fungal biofilm growth was achieved by the use of a CDC biofilm reactor (Biosurface Technologies Corp., Bozeman, MT, USA). The CDC reactor is a recognized method for the growth of microbial biofilms under high shear and continuous flow (Coenye and Nelis, 2010) and is the standard method in use by the American Society for Testing and Materials (ASTM 2012). The CDC reactor is composed of a glass vessel, which holds the reactor medium. Placed into this vessel is a polyethylene top which holds eight removable polypropylene rods, each of which in turn has three inserts for holding the coupons on which the microbial cells attach. Therefore, each biofilm reactor has the capacity for 24 coupons equivalent to 24 separate biofilms, which makes it an ideal apparatus for inactivation studies. In the centre of the reactor a magnetic stirrer is present, which provides a continuous flow of nutrients over the colonized surface of the coupons. Continuous mixing of the culture liquid is achieved by placing the reactor on a magnetic stir plate (RCT Basic Stir Plate IKA®, Staufen, Germany) at 130 rpm. The reactor vessel was filled with 300 ml malt extract broth for C. albicans (ATCC), hospital strains and C. krusei; 300 ml Sabouraud broth was added for C. parapsilosis; 1 ml of an 18 h culture of the test strain, grown in suitable broth, was then added to the reactor vessel and incubated at 37 °C (30 °C for C. parapsilosis) under rotary conditions at 130 rpm. The reactor was incubated for 48 and 72 h to allow for biofilm growth to a maximal cell density, according to the method of Nailis et al. (2010). Fungal cell counts were performed at inoculation of the reactor broth and at 48 and 72 h of incubation to determine the planktonic cell density of the reactor. The cell density of sessile cells was determined for stainless steel and PVC surfaces by scraping the surface with a sterile cell scraper (Sarstedt, Germany) into 10 ml sterile phosphatebuffered saline (sPBS; Sigma-Aldrich, Ireland), Copyright © 2015 John Wiley & Sons, Ltd.
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according to the ASTM-recommended procedure for biofilm cell density analysis. Colony forming units (cfu/ml) were determined by spreading 100 μl serially diluted sessile cells on suitable agar, followed by incubation for 24 h at 30 °C or 37 °C.
Pulsed UV studies Pulsed-light inactivation was conducted on test strains in planktonic form in suspension and on agar surfaces for comparative studies to that of the biofilm communities on PVC and stainless steel surfaces. The PUV system used throughout this study was the PUV-01 (Samtech Ltd, Glasgow, UK), consisting of two main components, a treatment chamber and a driver circuit. The driver unit consists of the trigger and discharge outputs, frequency control, trigger control and the discharge voltage control. The trigger cable connects the trigger output of the driver unit with the trigger electrode of the flashlamp, while the discharge cable connects the discharge output of the driver unit with the lamp anode and cathode. The treatment chamber consists of a polyvinyl chloride housing containing a xenon light source and a circular treatment table. The light source has an automatic frequency control function that allows it to operate at 1 pulse/s (pps), which was used throughout this study. Petri dishes used in the tests were placed directly below the lamp, which ensured that full coverage of the plate surface occurred and eliminated possible shading effects. In this study, standard treatments involved exposing the test samples to lamp discharge energy of 16.2 J at 8 cm distance from the light source.
PUV treatment of planktonic cells Planktonic cells of all Candida test strains and S. cerevisiae were treated by PUV for analysis compared to the sessile cells. For PUV studies, a single colony of the test strain was aseptically transferred to 100 ml sterile malt extract broth, followed by incubation at 37 °C for 18 h at 125 rpm. For surface treatment, 100 μl of an appropriate dilution was spread onto malt agar surfaces. Sabouraud broth and agar were used as previously described for the growth of C. parapsilosis. Test plates were then exposed to pulses of UV light at 16.2 J at varying fluences at a rate of 1 pulse/s, according to Garvey et al. (2014), up to a PUV Yeast 2015; 32: 533–540. DOI: 10.1002/yea
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fluence of 8 μJ/cm2 (treatment time of 80 s) for surfaces and 11 μJ/cm2 (treatment time of 100 s) for fungal suspensions. PUV studies were also conducted on samples diluted from the 18 h broth in 20 ml final volumes of sterile PBS at 8 cm from the light source, after which 100 μl treated liquid was transferred to suitable agar and incubated at 37 °C for 24 h, or 30 °C for C. parapsilosis.
PUV treatment of biofilm structures PVC and stainless steel coupons were aseptically removed from the reactor, rinsed with sPBS and transferred to a sterile Petri dish. Samples were exposed to pulses of UV light at 16.2 J at 8 cm from the light source at varying UV fluence (μJ/cm2) up to a PUV fluence of 6.48 μJ/cm2 (equivalent to a treatment time of 60 s). Once treated, the coupons were immersed in 10 ml sterile PBS and the surface scraped, using a sterile cell scraper, to remove the treated biofilms and to determine fungal cell viability. The liquid was then transferred to a sterile 20 ml container and centrifuged at 800 × g for 10 min to pellet the cells. The sample was then resuspended and agitated to ensure biofilm dispersion. Serial dilutions were made from the biofilm suspensions and 100 μl spread on triplicate agar plates to determine the cfu/ml of treated samples. This process was repeated for coupons at varying UV fluences to determine the log10 reduction obtained with increasing UV fluence compared to an untreated control. A cell count was also conducted on the medium present in the reactor vessel, using the spread plate technique.
Statistics All the experiments were performed three times, with three plate replicates for each experimental data point, providing a mean result for each experimental
batch. The log reduction was calculated as log10 of the ratio of the concentration (cfu/ml) of the nontreated (N0) and UV-treated (N) samples [log10 (N0/N)]. Linear regression analysis was used to determine the rate of inactivation for each test species under the regime of PUV treatments applied at the 95% significance level. Student’s t-test and oneway ANOVA model (Minitab software release 16; Mintab Inc., State College, PA, USA) were used to determine the sensitivity of each test strain to PUV light.
Results and discussion Candida biofilm formation The CDC reactor proved an effective tool for the formation and growth of fungal biofilms. Sessile colony counts showed that high-density biofilms formed at the 48 and 72 h incubation time points for both surface types in the reactor (Table 1). After 48 h, a 4.7, 4.2 and 4.6 log10 cfu/ml biofilm formed for C. albicans, C. albicans (clinical isolate) and C. parapsilosis, respectively, on PVC surfaces. There was a significant difference (p < 0.05) in the sessile population density on stainless steel surfaces, where a 4.2 log10 cfu/ml biofilm formed for each test species. A similar trend was observed following 72 h of incubation with 4.8, 5.2, 5.1 and 4.3, 4.0 and 5.0 log10 cfu/ml biofilm growths for C. albicans, C. albicans (clinical isolate) and C. parapsilosis on PVC and stainless steel coupons, respectively. These data suggest that both surface types can support the formation of densely populated Candida biofilm structures; however, PVC appears more favourable for growth, evident from the significantly higher sessile cell count than on stainless steel surfaces.
Table 1. Clinically relevant Candida species biofilm cell density after 48 and 72 h of growth in a CDC biofilm reactor (± SD) Biofilms cell density (log10 cfu/ml) 48 h PVC C. albicans C. albicans (clinical) C. parapsilosis
4.7 (±0.2)A 4.2 (±0.1)B 4.2 (±0.05)B
72 h SS
PVC
SS
4.2 (±0.01)B 4.2 (±0.05)B 4.2 (±0.1)B
4.8 (±0.1)A 5.2 (±0.03)F 5.1 (±0.02)E
4.3 (±0.05)D 4.0 (±0.1)C 5.0 (±0.04)E
A–E, significant differences in cfu/ml; SS, stainless steel.
Copyright © 2015 John Wiley & Sons, Ltd.
Yeast 2015; 32: 533–540. DOI: 10.1002/yea
Inactivation of Candida biofilms using pulsed light
Furthermore, it was found that, with further incubation (>72 h), there was no significant increase in the number of sessile cells present. Biofilm cell counts reached a maximum at 72 h, after which there was no increase in cell number on either surface material, a similar trend to that reported by Nailis et al. (2010). Indeed, research by this group concluded that the CDC reactor allows for the formation of more densely populated Candida biofilms than alternative methods, such as microtitre plates (Nailis et al., 2010), and attributed this to the availability of nutrients within the reactor. Studies assessing in vivo models for catheterassociated Candida infections, such as microscopic structure analysis, indicate that in vitro techniques such as the CDC reactor show structured biofilm communities similar to those found in vivo (Lopez-Ribot, 2005). Furthermore, in vitro studies have shown that Candida can survive in the low-iron environment found in the tissues surrounding implanted devices, such as catheters, with the additional factor of its filamentous life cycle making it proficient at colonizing inert surfaces such as PVC (Suci and Tyler, 2002). These studies suggest that in vitro model systems do mimic in vivo events, indicating that the research outputs made are clinically relevant.
Pulsed UV inactivation of planktonic and sessile Candida test strains The Candida and Saccharomyces test strains under study proved sensitive to PUV inactivation, albeit
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to varying extents. Figure 1 details the inactivation of test strains on agar surfaces following exposure to PUV irradiation. Interestingly, the C. albicans clinical isolate proved significantly (p < 0.05) more sensitive to UV disinfection that the reference strain C. albicans (ATCC), with 5.6 and 3.25 log10 cfu/ml inactivation obtained with a PUV fluence of 1.08 μJ/ cm2 for each strain, respectively, on agar surfaces. This trend of an increased sensitivity of the clinical isolate continued for all applied treatment fluences up to 5.39 μJ/cm2 (treatment time of 50 s). A fluence of 5.39 μJ/cm2 was needed to obtain a 5.8 log10 cfu/ml of C. albicans reference strain, with 2.15 μJ/cm2 giving a similar inactivation rate of the clinical isolate (Figure 1). C. albicans (ATCC) and S. cerevisiae showed similar levels of inactivation (ca. 3.2 log10) at 1.08 μJ/cm2 (treatment time of 10 s). This UV fluence resulted in a ca. 5 log10 cfu/ml inactivation of C. albicans (clinical), C. krusei and C. parapsilosis and 4 log10 cfu/ml inactivation of C. tropicalis. A fluence of 5.39 μJ/cm2 resulted in a ca. 5.8 log10 cfu/ml inactivation of S. cerevisiae, C. tropicalis and C. albicans and a ca. 7.5 log10 cfu/ml inactivation of C. parapsilosis, C. krusei and C. albicans (clinical), indicating that levels of sensitivity to treatment varied with the UV fluence on agar surfaces. The order of sensitivity from least to most resistant to PUV at 5.39 μJ/cm2/ pulse on agar surfaces was: C. parapsilosis, C. krusei, C. albicans (clinical), C. tropicalis, S. cerevisiae and C. albicans (ATCC). A similar trend was observed when treated in suspension, where C. albicans (clinical) proved
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Figure 1. Pulsed light inactivation of Candida and Saccharomyces test species on agar surfaces (± SD) Copyright © 2015 John Wiley & Sons, Ltd.
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Figure 2. Pulsed light inactivation of 20 ml fungal suspensions of Candida and Saccharomyces test strains (± SD)
more sensitive to UV exposure than the reference strain (Figure 2) at all treatment fluences. There was no significant difference between the inactivation of S. cerevisiae and C. albicans (clinical) at treatment fluences of 4.32, 5.39 and 7.56 μJ/cm2 in suspension, with complete inactivation of both strains achieved with 8.64 μJ/cm2 (Figure 2). Additionally, C. krusei and C. parapsilosis showed similar levels of sensitivity to PUV with C. tropicalis proving significantly more resistant than both strains in suspension. Significantly more UV fluence was needed to obtain similar levels of inactivation with fungal suspension compared to surface spread for all test strains. The order of sensitivity from least to most resistant to PUV for fungal suspensions at 7.56 μJ/cm2 was S. cerevisiae, C. albicans (clinical), C. parapsilosis, C. krusei, C. tropicalis and C. albicans (ATCC). Neely and Orloff (2001) have shown that fungal pathogens have the ability to survive on clinical surfaces. Established biofilms on these surfaces pose a difficult challenge to hospital cleaning and disinfection, due to their resistance to biocides and difficulty in removing them by detergent cleaning (Otter et al., 2014). Fungal infections are increasing at a disturbing rate, affecting a growing population of severely ill patients, which poses important challenges for health care professionals. Studies by Chandra et al. (2001) have shown that cellular resistance to biocides increased as the biofilm structure matured, with a 72 h biofilm of C. albicans showing a dramatically increased level of resistance than earlier-stage Copyright © 2015 John Wiley & Sons, Ltd.
biofilm counterparts (Chandra et al., 2001). A means of inactivating planktonic and sessile cells that renders the surface free of pathogenic species is essential to prevent patient infection or the contamination of medical materials. The PUV system used in this study repeatedly inactivated Candida species biofilms on both PVC and stainless steel surfaces. Significant levels of inactivation were obtained for C. albicans (ATCC), C. albicans (clinical) and C. parapsilosis for 48 h (Figure 3) and 72 h (Figure 4) biofilm structures. For both time points, ca. 3.5–4 log10 cfu/ml inactivation of all test strains was achieved with a fluence of 6.68 μJ/cm2. There was no difference in the inactivation rates of C. parapsilosis 48 and 72 h biofilms on PVC surfaces. C. albicans biofilms appear more UV sensitive at 48 h, with a significant (p < 0.05) increase in inactivation obtained for each PUV fluence. The C. albicans clinical isolate showed similar or a slightly decreased level of inactivation at 48 h compared to 72 h biofilm formation; 48 h biofilms on stainless steel surfaces appear more UV-sensitive, with an increase in inactivation achieved for C. albicans up to a UV fluence of 4.32 μJ/cm2 and for all treatment fluences for C. albicans (clinical) and C. parapsilosis. At 48 h biofilm formation, C. parapsilosis appears most resistant to UV pulses, with both C. albicans strains showing similar levels of sensitivity on PVC surfaces. A similar trend was observed for biofilms grown for 48 h on stainless steel surfaces. The order of increasing sensitivity to UV fluence was C. parapsilosis, C. albicans and C. albicans Yeast 2015; 32: 533–540. DOI: 10.1002/yea
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Figure 3. Pulsed-light inactivation of 48 h Candida species biofilms grown on (a) PVC and (b) stainless steel surfaces (± SD)
(clinical) on PVC surfaces and C. albicans (clinical), C. parapsilosis and C. albicans on stainless surfaces for 72 h biofilm structures. In general it was found that planktonic cells are more sensitive to PUV than attached cells on either surface material. The order of sensitivity to UV pulses was the same for fungal suspensions and biofilms on stainless steel surfaces, meaning that planktonic cells treated in suspension had the same level of susceptibility to pulsed light as cells in biofilms on stainless steel surfaces. C. parapsilosis proved the most sensitive test strain on agar surfaces and PVCattached biofilms. Biofilm communities are the natural state of microbial habitat, where they are found attached to biotic or abiotic surfaces, more so than planktonic free-floating cells. Regardless of this, planktonic culture remains the main mechanism for
microbiological studies, such as disinfection (Otter et al., 2014). This study assessing the sensitivity of fungal biofilm structures to pulsed-light treatment provides a direct relationship between treatment fluence and loss of viability in sessile cells. Here we have dealt with an important aspect of clinical disinfection and disease prevention. The findings demonstrate the effective use of pulsed UV light for the effective inactivation of clinically relevant Candida species on surfaces commonly associated with the health care setting. Although these initial studies are promising, further studies are warranted to assess the disinfection of multi-species biofilm communities. Biofilms often develop as a multispecies structure, with a synergistic relationship providing protection from a range of environmental stresses. Additionally, studies are merited to assess the potential of fungal biofilms to harbour
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Figure 4. Pulsed-light inactivation of 72 h Candida species biofilms cells grown on (a) PVC and (b) stainless steel surfaces (± SD) Copyright © 2015 John Wiley & Sons, Ltd.
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other opportunistic pathogens, such as viruses and parasite species. Acknowledgements The authors wish to express their gratitude to Professor Martin Cormican, National University of Ireland, Galway (NUIG Ireland), who kindly donated the clinical isolates for use in this study.
References American Society for Testing and Materials (ASTM). 2012. Standard No. E2562–12. Standard test method for quantification of Pseudomonas aeruginosa biofilm grown with high shear and continuous flow using a CDC biofilm reactor. ASTM: West Conshohocken, PA, USA. Bak J, Ladefoged SD, Tvede M, et al. 2009. Fluence requirements for UVC disinfection of catheter biofilms. Biofouling 25: 289–296. Chandra J, Kuhn MD, Mukherjee KP, et al. 2001. Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol 183: 5385–5394. Coenye T, Nelis JJ. 2010. In vitro and in vivo model systems to study microbial biofilm formation. J Microbiol Methods 83: 89–105.
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M. Garvey et al. Fanning S, Mitchell AP. 2012. Fungal biofilms. PLoS Pathog 8: e1002585. Farrell H, Hayes J, Laffey J, Rowan N. 2011. Studies on the relationship between pulsed UV light irradiation and the simultaneous occurrence of molecular and cellular damage in clinically relevant Candida albicans. J Microbiol Methods 84: 317–326. Garvey M, Thokala N, Rowan N. 2014. A comparative study on the pulsed UV and the low pressure UV inactivation of waterborne microorganisms. J Water Environ Res 86: doi:10.2175/WER-D13-00062.1. Kumanoto AC. 2002. Candida biofilms. Curr Opin Microbiol 5: 608–611. López-Ribot LJ. 2005. Candida albicans Biofilms: more than filamentation. Curr Biol 15: doi:10.1016/j.cub.2005.06.020. Nailis H, Kucharikova S, Ricicova M, et al. 2010. PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression. BMC Microbiol 10: 114. Neely NA, Orloff MM. 2001. Survival of some medically important fungi on hospital fabrics and plastics. J Clin Microbiol 39: 3360–3361. Otter AJ, Vickery K, Walker J, et al. 2014. Surface-attached cells, biofilms and biocide susceptibility: implications for hospital cleaning and disinfection. J Hosp Infect 1: 1–12. Suci AP, Tyler JB. 2002. Action of chlorexidine digluconate against yeast and filamentous forms in an early stage Candida albicans biofilm. Antimicrob Agents Chemother 46: 3522–3530. Trofa D, Ga’cser A, Nosanchuk JD. 2008. Candida parapsilosis, an emerging fungal pathogen. Clin Microbiol Rev 21: 606–625.
Yeast 2015; 32: 533–540. DOI: 10.1002/yea
Research Article
Pulsed light for the inactivation of fungal biofilms of clinically important pathogenic Candida species Mary Garvey1*, Joao Paulo Andrade Fernandes2,3 and Neil Rowan2 1
Department of Life Sciences, Institute of Technology Sligo, Ireland Bioscience Research Institute, Athlone Institute of Technology, Ireland 3 CAPES Foundation, Ministry of Education of Brazil, Universidade Federal do Rio de Janeiro, Brazil 2
*Correspondence to: M. Garvey, Department of Life Sciences, Institute of Technology Sligo, Ash Lane, Sligo, Ireland. E-mail: [email protected]
Received: 21 October 2014 Accepted: 3 May 2015
Abstract Microorganisms are naturally found as biofilm communities more than planktonic free-floating cells; however, planktonic culture remains the current model for microbiological studies, such as disinfection techniques. The presence of fungal biofilms in the clinical setting has a negative impact on patient mortality, as Candida biofilms have proved to be resistant to biocides in numerous in vitro studies; however, there is limited information on the effect of pulsed light on sessile communities. Here we report on the use of pulsed UV light for the effective inactivation of clinically relevant Candida species. Fungal biofilms were grown by use of a CDC reactor on clinically relevant surfaces. Following a maximal 72 h formation period, the densely populated biofilms were exposed to pulsed light at varying fluences to determine biofilm sensitivity to pulsed-light inactivation. The results were then compared to planktonic cell inactivation. High levels of inactivation of C. albicans and C. parapsilosis biofilms were achieved with pulsed light for both 48 and 72 h biofilm structures. The findings suggest that pulsed light has the potential to provide a means of surface decontamination, subsequently reducing the risk of infection to patients. The research described herein deals with an important aspect of disease prevention and public health. Keywords: pathogenic; yeast; UV inactivation; sessile communities; surfaces; Candida
Introduction In the last three decades, fungi have appeared as a major cause of human disease, predominantly among immunocompromised individuals, neonates, burns patients and patients with serious underlying illnesses (Trofa et al., 2008). Candida species are opportunistic eukaryotic fungal pathogens commonly associated with clinical infections resulting in deep tissue infection, high mortality rates and financial burden. Biofilms (sessile communities) are a form of microbial growth where cells grow in a selfproduced protective environment, which allows the cells to escape the dangers of their surrounding location. Biofilms are usually associated with wet or damp surfaces, such as indwelling medical Copyright © 2015 John Wiley & Sons, Ltd.
devices and/or tubing on medical equipment. However, it is now known that microbes can survive for extended periods in a dehydrated state on dry hospital surfaces. Indeed, biofilms have recently been discovered on dry hospital surfaces (Otter et al., 2014). Candida biofilms are composed of yeast cells and filaments that are structurally attached to biotic or abiotic surfaces and embedded in an extracellular matrix (Nailis et al., 2010). Research has shown that such biofilm cell communities are more resistant to chemical disinfection techniques than planktonic (free-floating) cells, due to their extracellular matrix, structural complexity, gene upregulation and metabolic heterogeneity (Fanning and Mitchell, 2012). Biofilm formation by pathogenic microorganisms such as Candida plays a key role in infections resulting
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from intravascular devices in the clinical setting. Indeed, the association between Candida species biofilm formation and continued host infection has become more evident. Once a biofilm forms, it can continuously supply cells which detach from the main structure into the bloodstream, acting as a source of infection. It has been reported by Kumanoto (2002) that conditions with a high flow rate, such as that encountered within the circulatory system, may favour the development of persistent biofilms on devices placed in the bloodstream. Furthermore, Candida species biofilms are quite resistant to antifungals such as fluconazole, amphotericin B, nystatin and voriconazole (Kumanoto, 2002). In recent years there has been a marked increase in non-C. albicans-related bloodstream infections from Candida species, mainly C. parapsilosis, C. krusei and C. tropicalis (Trofa et al., 2008). The resistance of this species to antibiotics and the ability of Candida biofilms to tolerate chemical disinfection suggest the need for alternative methods of removing this pathogen from clinical settings. Prevention of infection is a superior method than infection treatment in terms of cost and patient well-being. An alternative or supplementary means of control is to minimize the extent of exposure of the patient to these fungal pathogens, thereby preventing an infection from occurring. Typical clinical surfaces, such as plastics, have been shown to act as reservoirs for viable pathogenic fungi, such as Candida albicans and C. parapsilosis (Neely and Orloff, 2001). Proper cleaning regimens that include the use of effective surface decontamination techniques can help prevent patient exposure to pathogenic species. The use of ultraviolet (UV) light has proved effective for the inactivation of a range of microbial species; however, there is limited information available on its use for the removal of clinically relevant Candida species or Candida biofilms. UV disinfection occurs following exposure of test species to UV energy at 254 nm, which has a negative effect on genetic material, preventing microbial replication. Standard UV lamps emit UV energy as a continuous wave from either a lowpressure (LP) or a medium-pressure (MP) lamp source at set wavelengths. Ultraviolet C (UVC) has been found to be efficient at sterilizing the inner surface of catheters contaminated with bacterial biofilms and subsequently preventing Copyright © 2015 John Wiley & Sons, Ltd.
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catheter-related infections (Bak et al., 2009). Studies by this research group have focused on the use of an alternative means of delivering UV energy, referred to as ’pulsed UV’ (PUV), which delivers UV light in short-duration pulses of high-intensity light with a broader range of wavelengths in the range 100–1100 nm, enriched with shorter germicidal wavelengths (Farrell et al., 2011). These wavelengths may potentially affect more cellular targets, such as cell membranes and protein structures, than DNA alone, producing irreparable cellular damage. Due to the delivery mechanism of short-duration pulses, PUV also has a better penetration depth than LPUV. Additionally, PUV has proved effective for the inactivation of numerous microbial species, bacterial endospores, biofilms and parasite species in a shorter treatment time than LP- or MP-UV, leading to a reduction in operation costs (Garvey et al., 2014). Considering these important advantages of PUV over LP-UV, the aim of this study was to determine the potential of a PUV light system for the inactivation of clinically important Candida species in both planktonic and biofilm form on surfaces commonly found in the health care setting.
Methods Test strain culture conditions All Candida test strains used in this study were sourced from the American Type Tissue Culture Collection (ATCC), with the exception of a C. albicans hospital isolate, which was sourced from a blood culture at the National University of Ireland, Galway Hospital. Saccharomyces cerevisiae (ATCC 9763) was also sourced from the ATCC and used for PUV studies as a comparative strain to Candida. Strains were cultured and maintained on malt extract agar (Cruinn Diagnostics, Ireland) at 37 °C following removal from storage in microbank vials (Cruinn Diagnostics, Ireland) at 80 °C. C. parapsilosis (ATCC 22019) was cultured and maintained on Sabouraud agar (Cruinn Diagnostics, Ireland) at 30 °C and incubated at 5 ° C for short-term storage on agar slopes. Strains were identity confirmed by Gram staining and the germ-tube assay, according to Farrell et al. (2011), following a maximal 2 week storage period. For treatment studies, single colonies were Yeast 2015; 32: 533–540. DOI: 10.1002/yea
Inactivation of Candida biofilms using pulsed light
aseptically transferred to 100 ml malt extract broth and incubated at 37 °C under rotary conditions at 125 rpm for S. cerevisiae, C. albicans (ATCC 10231), C. albicans (clinical isolate NUIG 6250), C. krusei (ATCC 14243) and C. tropicalis (ATCC 13803); C. parapsilosis was cultured in 100 ml Sabouraud broth at 30 °C at 125 rpm for 18 h.
CDC reactor biofilm growth All fungal biofilm growth was achieved by the use of a CDC biofilm reactor (Biosurface Technologies Corp., Bozeman, MT, USA). The CDC reactor is a recognized method for the growth of microbial biofilms under high shear and continuous flow (Coenye and Nelis, 2010) and is the standard method in use by the American Society for Testing and Materials (ASTM 2012). The CDC reactor is composed of a glass vessel, which holds the reactor medium. Placed into this vessel is a polyethylene top which holds eight removable polypropylene rods, each of which in turn has three inserts for holding the coupons on which the microbial cells attach. Therefore, each biofilm reactor has the capacity for 24 coupons equivalent to 24 separate biofilms, which makes it an ideal apparatus for inactivation studies. In the centre of the reactor a magnetic stirrer is present, which provides a continuous flow of nutrients over the colonized surface of the coupons. Continuous mixing of the culture liquid is achieved by placing the reactor on a magnetic stir plate (RCT Basic Stir Plate IKA®, Staufen, Germany) at 130 rpm. The reactor vessel was filled with 300 ml malt extract broth for C. albicans (ATCC), hospital strains and C. krusei; 300 ml Sabouraud broth was added for C. parapsilosis; 1 ml of an 18 h culture of the test strain, grown in suitable broth, was then added to the reactor vessel and incubated at 37 °C (30 °C for C. parapsilosis) under rotary conditions at 130 rpm. The reactor was incubated for 48 and 72 h to allow for biofilm growth to a maximal cell density, according to the method of Nailis et al. (2010). Fungal cell counts were performed at inoculation of the reactor broth and at 48 and 72 h of incubation to determine the planktonic cell density of the reactor. The cell density of sessile cells was determined for stainless steel and PVC surfaces by scraping the surface with a sterile cell scraper (Sarstedt, Germany) into 10 ml sterile phosphatebuffered saline (sPBS; Sigma-Aldrich, Ireland), Copyright © 2015 John Wiley & Sons, Ltd.
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according to the ASTM-recommended procedure for biofilm cell density analysis. Colony forming units (cfu/ml) were determined by spreading 100 μl serially diluted sessile cells on suitable agar, followed by incubation for 24 h at 30 °C or 37 °C.
Pulsed UV studies Pulsed-light inactivation was conducted on test strains in planktonic form in suspension and on agar surfaces for comparative studies to that of the biofilm communities on PVC and stainless steel surfaces. The PUV system used throughout this study was the PUV-01 (Samtech Ltd, Glasgow, UK), consisting of two main components, a treatment chamber and a driver circuit. The driver unit consists of the trigger and discharge outputs, frequency control, trigger control and the discharge voltage control. The trigger cable connects the trigger output of the driver unit with the trigger electrode of the flashlamp, while the discharge cable connects the discharge output of the driver unit with the lamp anode and cathode. The treatment chamber consists of a polyvinyl chloride housing containing a xenon light source and a circular treatment table. The light source has an automatic frequency control function that allows it to operate at 1 pulse/s (pps), which was used throughout this study. Petri dishes used in the tests were placed directly below the lamp, which ensured that full coverage of the plate surface occurred and eliminated possible shading effects. In this study, standard treatments involved exposing the test samples to lamp discharge energy of 16.2 J at 8 cm distance from the light source.
PUV treatment of planktonic cells Planktonic cells of all Candida test strains and S. cerevisiae were treated by PUV for analysis compared to the sessile cells. For PUV studies, a single colony of the test strain was aseptically transferred to 100 ml sterile malt extract broth, followed by incubation at 37 °C for 18 h at 125 rpm. For surface treatment, 100 μl of an appropriate dilution was spread onto malt agar surfaces. Sabouraud broth and agar were used as previously described for the growth of C. parapsilosis. Test plates were then exposed to pulses of UV light at 16.2 J at varying fluences at a rate of 1 pulse/s, according to Garvey et al. (2014), up to a PUV Yeast 2015; 32: 533–540. DOI: 10.1002/yea
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fluence of 8 μJ/cm2 (treatment time of 80 s) for surfaces and 11 μJ/cm2 (treatment time of 100 s) for fungal suspensions. PUV studies were also conducted on samples diluted from the 18 h broth in 20 ml final volumes of sterile PBS at 8 cm from the light source, after which 100 μl treated liquid was transferred to suitable agar and incubated at 37 °C for 24 h, or 30 °C for C. parapsilosis.
PUV treatment of biofilm structures PVC and stainless steel coupons were aseptically removed from the reactor, rinsed with sPBS and transferred to a sterile Petri dish. Samples were exposed to pulses of UV light at 16.2 J at 8 cm from the light source at varying UV fluence (μJ/cm2) up to a PUV fluence of 6.48 μJ/cm2 (equivalent to a treatment time of 60 s). Once treated, the coupons were immersed in 10 ml sterile PBS and the surface scraped, using a sterile cell scraper, to remove the treated biofilms and to determine fungal cell viability. The liquid was then transferred to a sterile 20 ml container and centrifuged at 800 × g for 10 min to pellet the cells. The sample was then resuspended and agitated to ensure biofilm dispersion. Serial dilutions were made from the biofilm suspensions and 100 μl spread on triplicate agar plates to determine the cfu/ml of treated samples. This process was repeated for coupons at varying UV fluences to determine the log10 reduction obtained with increasing UV fluence compared to an untreated control. A cell count was also conducted on the medium present in the reactor vessel, using the spread plate technique.
Statistics All the experiments were performed three times, with three plate replicates for each experimental data point, providing a mean result for each experimental
batch. The log reduction was calculated as log10 of the ratio of the concentration (cfu/ml) of the nontreated (N0) and UV-treated (N) samples [log10 (N0/N)]. Linear regression analysis was used to determine the rate of inactivation for each test species under the regime of PUV treatments applied at the 95% significance level. Student’s t-test and oneway ANOVA model (Minitab software release 16; Mintab Inc., State College, PA, USA) were used to determine the sensitivity of each test strain to PUV light.
Results and discussion Candida biofilm formation The CDC reactor proved an effective tool for the formation and growth of fungal biofilms. Sessile colony counts showed that high-density biofilms formed at the 48 and 72 h incubation time points for both surface types in the reactor (Table 1). After 48 h, a 4.7, 4.2 and 4.6 log10 cfu/ml biofilm formed for C. albicans, C. albicans (clinical isolate) and C. parapsilosis, respectively, on PVC surfaces. There was a significant difference (p < 0.05) in the sessile population density on stainless steel surfaces, where a 4.2 log10 cfu/ml biofilm formed for each test species. A similar trend was observed following 72 h of incubation with 4.8, 5.2, 5.1 and 4.3, 4.0 and 5.0 log10 cfu/ml biofilm growths for C. albicans, C. albicans (clinical isolate) and C. parapsilosis on PVC and stainless steel coupons, respectively. These data suggest that both surface types can support the formation of densely populated Candida biofilm structures; however, PVC appears more favourable for growth, evident from the significantly higher sessile cell count than on stainless steel surfaces.
Table 1. Clinically relevant Candida species biofilm cell density after 48 and 72 h of growth in a CDC biofilm reactor (± SD) Biofilms cell density (log10 cfu/ml) 48 h PVC C. albicans C. albicans (clinical) C. parapsilosis
4.7 (±0.2)A 4.2 (±0.1)B 4.2 (±0.05)B
72 h SS
PVC
SS
4.2 (±0.01)B 4.2 (±0.05)B 4.2 (±0.1)B
4.8 (±0.1)A 5.2 (±0.03)F 5.1 (±0.02)E
4.3 (±0.05)D 4.0 (±0.1)C 5.0 (±0.04)E
A–E, significant differences in cfu/ml; SS, stainless steel.
Copyright © 2015 John Wiley & Sons, Ltd.
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Furthermore, it was found that, with further incubation (>72 h), there was no significant increase in the number of sessile cells present. Biofilm cell counts reached a maximum at 72 h, after which there was no increase in cell number on either surface material, a similar trend to that reported by Nailis et al. (2010). Indeed, research by this group concluded that the CDC reactor allows for the formation of more densely populated Candida biofilms than alternative methods, such as microtitre plates (Nailis et al., 2010), and attributed this to the availability of nutrients within the reactor. Studies assessing in vivo models for catheterassociated Candida infections, such as microscopic structure analysis, indicate that in vitro techniques such as the CDC reactor show structured biofilm communities similar to those found in vivo (Lopez-Ribot, 2005). Furthermore, in vitro studies have shown that Candida can survive in the low-iron environment found in the tissues surrounding implanted devices, such as catheters, with the additional factor of its filamentous life cycle making it proficient at colonizing inert surfaces such as PVC (Suci and Tyler, 2002). These studies suggest that in vitro model systems do mimic in vivo events, indicating that the research outputs made are clinically relevant.
Pulsed UV inactivation of planktonic and sessile Candida test strains The Candida and Saccharomyces test strains under study proved sensitive to PUV inactivation, albeit
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to varying extents. Figure 1 details the inactivation of test strains on agar surfaces following exposure to PUV irradiation. Interestingly, the C. albicans clinical isolate proved significantly (p < 0.05) more sensitive to UV disinfection that the reference strain C. albicans (ATCC), with 5.6 and 3.25 log10 cfu/ml inactivation obtained with a PUV fluence of 1.08 μJ/ cm2 for each strain, respectively, on agar surfaces. This trend of an increased sensitivity of the clinical isolate continued for all applied treatment fluences up to 5.39 μJ/cm2 (treatment time of 50 s). A fluence of 5.39 μJ/cm2 was needed to obtain a 5.8 log10 cfu/ml of C. albicans reference strain, with 2.15 μJ/cm2 giving a similar inactivation rate of the clinical isolate (Figure 1). C. albicans (ATCC) and S. cerevisiae showed similar levels of inactivation (ca. 3.2 log10) at 1.08 μJ/cm2 (treatment time of 10 s). This UV fluence resulted in a ca. 5 log10 cfu/ml inactivation of C. albicans (clinical), C. krusei and C. parapsilosis and 4 log10 cfu/ml inactivation of C. tropicalis. A fluence of 5.39 μJ/cm2 resulted in a ca. 5.8 log10 cfu/ml inactivation of S. cerevisiae, C. tropicalis and C. albicans and a ca. 7.5 log10 cfu/ml inactivation of C. parapsilosis, C. krusei and C. albicans (clinical), indicating that levels of sensitivity to treatment varied with the UV fluence on agar surfaces. The order of sensitivity from least to most resistant to PUV at 5.39 μJ/cm2/ pulse on agar surfaces was: C. parapsilosis, C. krusei, C. albicans (clinical), C. tropicalis, S. cerevisiae and C. albicans (ATCC). A similar trend was observed when treated in suspension, where C. albicans (clinical) proved
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Figure 1. Pulsed light inactivation of Candida and Saccharomyces test species on agar surfaces (± SD) Copyright © 2015 John Wiley & Sons, Ltd.
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7 6 5 4 C. albicans (ATCC) C. albicans (clinical) C. krusei C. tropicalis C. parapsilosis S. cerevisiae
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Figure 2. Pulsed light inactivation of 20 ml fungal suspensions of Candida and Saccharomyces test strains (± SD)
more sensitive to UV exposure than the reference strain (Figure 2) at all treatment fluences. There was no significant difference between the inactivation of S. cerevisiae and C. albicans (clinical) at treatment fluences of 4.32, 5.39 and 7.56 μJ/cm2 in suspension, with complete inactivation of both strains achieved with 8.64 μJ/cm2 (Figure 2). Additionally, C. krusei and C. parapsilosis showed similar levels of sensitivity to PUV with C. tropicalis proving significantly more resistant than both strains in suspension. Significantly more UV fluence was needed to obtain similar levels of inactivation with fungal suspension compared to surface spread for all test strains. The order of sensitivity from least to most resistant to PUV for fungal suspensions at 7.56 μJ/cm2 was S. cerevisiae, C. albicans (clinical), C. parapsilosis, C. krusei, C. tropicalis and C. albicans (ATCC). Neely and Orloff (2001) have shown that fungal pathogens have the ability to survive on clinical surfaces. Established biofilms on these surfaces pose a difficult challenge to hospital cleaning and disinfection, due to their resistance to biocides and difficulty in removing them by detergent cleaning (Otter et al., 2014). Fungal infections are increasing at a disturbing rate, affecting a growing population of severely ill patients, which poses important challenges for health care professionals. Studies by Chandra et al. (2001) have shown that cellular resistance to biocides increased as the biofilm structure matured, with a 72 h biofilm of C. albicans showing a dramatically increased level of resistance than earlier-stage Copyright © 2015 John Wiley & Sons, Ltd.
biofilm counterparts (Chandra et al., 2001). A means of inactivating planktonic and sessile cells that renders the surface free of pathogenic species is essential to prevent patient infection or the contamination of medical materials. The PUV system used in this study repeatedly inactivated Candida species biofilms on both PVC and stainless steel surfaces. Significant levels of inactivation were obtained for C. albicans (ATCC), C. albicans (clinical) and C. parapsilosis for 48 h (Figure 3) and 72 h (Figure 4) biofilm structures. For both time points, ca. 3.5–4 log10 cfu/ml inactivation of all test strains was achieved with a fluence of 6.68 μJ/cm2. There was no difference in the inactivation rates of C. parapsilosis 48 and 72 h biofilms on PVC surfaces. C. albicans biofilms appear more UV sensitive at 48 h, with a significant (p < 0.05) increase in inactivation obtained for each PUV fluence. The C. albicans clinical isolate showed similar or a slightly decreased level of inactivation at 48 h compared to 72 h biofilm formation; 48 h biofilms on stainless steel surfaces appear more UV-sensitive, with an increase in inactivation achieved for C. albicans up to a UV fluence of 4.32 μJ/cm2 and for all treatment fluences for C. albicans (clinical) and C. parapsilosis. At 48 h biofilm formation, C. parapsilosis appears most resistant to UV pulses, with both C. albicans strains showing similar levels of sensitivity on PVC surfaces. A similar trend was observed for biofilms grown for 48 h on stainless steel surfaces. The order of increasing sensitivity to UV fluence was C. parapsilosis, C. albicans and C. albicans Yeast 2015; 32: 533–540. DOI: 10.1002/yea
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Figure 3. Pulsed-light inactivation of 48 h Candida species biofilms grown on (a) PVC and (b) stainless steel surfaces (± SD)
(clinical) on PVC surfaces and C. albicans (clinical), C. parapsilosis and C. albicans on stainless surfaces for 72 h biofilm structures. In general it was found that planktonic cells are more sensitive to PUV than attached cells on either surface material. The order of sensitivity to UV pulses was the same for fungal suspensions and biofilms on stainless steel surfaces, meaning that planktonic cells treated in suspension had the same level of susceptibility to pulsed light as cells in biofilms on stainless steel surfaces. C. parapsilosis proved the most sensitive test strain on agar surfaces and PVCattached biofilms. Biofilm communities are the natural state of microbial habitat, where they are found attached to biotic or abiotic surfaces, more so than planktonic free-floating cells. Regardless of this, planktonic culture remains the main mechanism for
microbiological studies, such as disinfection (Otter et al., 2014). This study assessing the sensitivity of fungal biofilm structures to pulsed-light treatment provides a direct relationship between treatment fluence and loss of viability in sessile cells. Here we have dealt with an important aspect of clinical disinfection and disease prevention. The findings demonstrate the effective use of pulsed UV light for the effective inactivation of clinically relevant Candida species on surfaces commonly associated with the health care setting. Although these initial studies are promising, further studies are warranted to assess the disinfection of multi-species biofilm communities. Biofilms often develop as a multispecies structure, with a synergistic relationship providing protection from a range of environmental stresses. Additionally, studies are merited to assess the potential of fungal biofilms to harbour
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Figure 4. Pulsed-light inactivation of 72 h Candida species biofilms cells grown on (a) PVC and (b) stainless steel surfaces (± SD) Copyright © 2015 John Wiley & Sons, Ltd.
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other opportunistic pathogens, such as viruses and parasite species. Acknowledgements The authors wish to express their gratitude to Professor Martin Cormican, National University of Ireland, Galway (NUIG Ireland), who kindly donated the clinical isolates for use in this study.
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M. Garvey et al. Fanning S, Mitchell AP. 2012. Fungal biofilms. PLoS Pathog 8: e1002585. Farrell H, Hayes J, Laffey J, Rowan N. 2011. Studies on the relationship between pulsed UV light irradiation and the simultaneous occurrence of molecular and cellular damage in clinically relevant Candida albicans. J Microbiol Methods 84: 317–326. Garvey M, Thokala N, Rowan N. 2014. A comparative study on the pulsed UV and the low pressure UV inactivation of waterborne microorganisms. J Water Environ Res 86: doi:10.2175/WER-D13-00062.1. Kumanoto AC. 2002. Candida biofilms. Curr Opin Microbiol 5: 608–611. López-Ribot LJ. 2005. Candida albicans Biofilms: more than filamentation. Curr Biol 15: doi:10.1016/j.cub.2005.06.020. Nailis H, Kucharikova S, Ricicova M, et al. 2010. PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression. BMC Microbiol 10: 114. Neely NA, Orloff MM. 2001. Survival of some medically important fungi on hospital fabrics and plastics. J Clin Microbiol 39: 3360–3361. Otter AJ, Vickery K, Walker J, et al. 2014. Surface-attached cells, biofilms and biocide susceptibility: implications for hospital cleaning and disinfection. J Hosp Infect 1: 1–12. Suci AP, Tyler JB. 2002. Action of chlorexidine digluconate against yeast and filamentous forms in an early stage Candida albicans biofilm. Antimicrob Agents Chemother 46: 3522–3530. Trofa D, Ga’cser A, Nosanchuk JD. 2008. Candida parapsilosis, an emerging fungal pathogen. Clin Microbiol Rev 21: 606–625.
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