Interdisciplinary Perspectives on Infectious Diseases
Volume 2018 (2018), Article ID 3748594, 8 pages
https://doi.org/10.1155/2018/3748594

Research Article

Effects of UVC Irradiation on Growth and Apoptosis of Scedosporium apiospermum and Lomentospora prolificans

Watcharamat Muangkaew, San Suwanmanee, Pantira Singkum, Potjaman Pumeesat, and Natthanej Luplertlop

Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

Correspondence should be addressed to Natthanej Luplertlop; natthanej.lup@mahidol.ac.th

Received 4 August 2018; Accepted 21 November 2018; Published 2 December 2018

Academic Editor: Subhada Prasad Pani

Copyright © 2018 Watcharamat Muangkaew et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Scedosporium apiospermum and Lomentospora prolificans are important fungal species isolated from immunocompromised patients. Previous studies have demonstrated that these filamentous fungi exist as saprophytes in the soil and showed the highest minimum inhibitory concentration to several drugs. We aimed to examine how UVC affects the S. apiospermum and L. prolificans by investigating the role of UVC on growth, induction of apoptosis by ethidium bromide (EB)/acridine orange (AO) staining, and transcriptomic study of caspase recruitment domain family, member 9 (CARD-9) gene. Our studies showed that 15 minutes of exposure to UVC light effectively increased reduction in both organisms and caused changes in colony morphology, color, and hyphal growth pattern. After 15 min of UVC irradiation, apoptotic cells were quantitated by EB/AO staining, and the percentage of apoptosis was 96.06% in S. apiospermum and 28.30% in L. prolificans. CARD-9 gene expression results confirmed that apoptosis was induced in S. apiospermum and L. prolificans after UVC treatment and that S. apiospermum showed a higher expression of apoptosis signaling than L. prolificans. Our study explored the effects of UVC in the inactivation of S. apiospermum and L. prolificans. We hope that our data is useful to other researchers in future studies.

1. Introduction

Scedosporium apiospermum species complex is a group of emerging opportunistic fungal pathogens increasingly found in immunocompromised patients [1]. S. apiospermum and Lomentospora prolificans (former name Scedosporium prolificans) are medically important fungal species that were isolated from patients with Scedosporium infections [2–5]. Previous studies have demonstrated that these filamentous fungi exist as saprophytes in soil, particularly soil from industrial sites, urban playgrounds, agricultural fields, sewers, and polluted water, and are associated with organic matter [6, 7]. In Thailand, infections with S. apiospermum and L. prolificans have been reported [8, 9]. However, the clearance of L. prolificans is more complicated than S. apiospermum because it is more resistant to numerous conventional antifungal drugs [10].

Several studies have evaluated the use of ultraviolet (UV) light as a disinfectant [11, 12]. UV is divided into three wave bands, namely, UVA, UVB, and UVC. UVC (wavelength 100–280 nm) is highly germicidal and commonly used in sterilization [13]. Sullivan et al. determined the effects of UVC in prokaryotic and eukaryotic organisms [14] and showed that 99% of Pseudomonas aeruginosa and Mycobacterium abscessus were killed after 3–5 s of exposure and 99% of Candida albicans and Aspergillus fumigatus were killed after 15–30 s of exposure. Results of this study were supported by Dai et al. who investigated the effects of UVC light on C. albicans after an infection in a mouse with third-degree burns [15]. They demonstrated that UVC treatment carried out on day 0 and day 1 significantly reduced the fungal bioburden in the infected burns.

S. apiospermum and L. prolificans can be opportunistic infection and environmental contamination, especially the soil. Hence, the immunocompromised host could be susceptibility for infection [16]. So, these fungi possibly distribute and contaminate which can highly affect the immunocompromised host.

Studies have mainly focused on the role of UVC as a disinfectant, particularly its fungicidal effects. However, information on the role of UVC on the apoptosis pathway is lacking. The aim of this study was to examine how UVC affects S. apiospermum and L. prolificans by investigating the role of UVC on growth and induction of apoptosis.

2. Materials and Methods

2.1. Isolates and Culture Conditions

S. apiospermum CBS 117410 and L. prolificans CM324 were provided by Dr. Ana Alastruey Izquierdo (Servicio de Micología, Instituto de Salud Carlos III, Madrid, Spain). Each isolate was incubated on Sabouraud Dextrose Agar (SDA; Difco, USA) slants at 35°C for 7 days. Conidia were collected and suspended in phosphate-buffered saline (PBS, pH 7.4).

2.2. Qualitative Evaluation of UVC on the Growth of S. apiospermum and L. prolificans

To determine the effects of UVC on the growth of S. apiospermum and L. prolificans, 1 ml of the conidia suspension with a concentration of 1 × 10⁶ cells/ml was aliquoted into 6-well plates and exposed to UVC radiation of wavelength 254 nm (CL-1000 Ultraviolet Crosslinker, Canada) 54 mJ cm⁻²[13] for 15 min. Following that 20 μl of suspension was transferred onto SDA plates and incubated at 37°C for 7 days. Colony morphology (diameter, color, and growth rate) was observed by the naked eye on each day until day 7. The remaining conidia suspension was centrifuged at 2,000 rpm for 5 min, and the pellet was resuspended in PBS (pH 7.4) and used apoptosis study by ethidium bromide (EB) and acridine orange (AO) staining, and transcriptomic study of the caspase recruitment domain family, member 9 (CARD-9) gene.

2.3. Scanning Electron Microscopy

The conidia suspension of S. apiospermum and L. prolificans after UVC exposure was transferred onto SDA plates and incubated at 37°C for 4 days; after that the fungal hyphae were collected and placed on a 13 mm circular coverslip. The cell morphology was determined by SEM. Firstly, the samples were fixed with 2.5% glutaraldehyde in sucrose phosphate buffer solution (one time for 1 h (.The fixative solution was aspirated out and the samples were dehydrated by methanol for 1 min and aspirated out until they were dry. After that, the samples were coated with gold and examined by a scanning electron microscope (model JSM-6610Lv, JEOL Ltd., Tokyo, Japan).

2.4. Apoptosis Study

Apoptotic cells were detected by staining with EB/AO as previously reported with minor modifications [17–19]. Briefly, 2 µl of 100 µg/ml EB and 100 µg/ml AO each was added to 20 µl of the sample, the and samples were immediately observed under a fluorescent microscope (Olympus/BX41). Live and apoptotic cells were determined. Live cells showed a green normal nucleus, whereas apoptotic cells showed condensed or fragmented chromatin.

2.5. Transcriptomic Study of the CARD-9 Gene

To investigate apoptosis at a molecular level, transcriptome levels of the apoptosis regulator gene CARD-9, the apoptosis related gene regulation, the expression of IL-1β, and also interaction with BCL10/CLAP were quantitated [20–22]. Total RNA from S. apiospermum CBS 117410 and L. prolificans CM324 after 15 min of UVC exposure and corresponding controls without UVC exposure were isolated using TRIzol® (Invitrogen, USA), according to the manufacturer’s instructions. Approximately 1 × 10⁶ cells were lysed with 750 μl of TRIzol® reagent, using chloroform as the separating phase. Isopropanol was used to precipitate RNA prior to washing with 75% ethanol and DNase treatment. RNase-free water was used to solubilize RNA. The RNA yield was determined with a NanoDrop 2000 spectrophotometer (Thermo Fisher, Wilmington, DE, USA) and the total RNA concentration was adjusted.

The change in CARD-9 expression after UVC exposure was determined using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). RT-PCR was carried out using 80 ng/reaction of RNA template and PCR reaction mixture comprising KAPA SYBR® FAST One-Step qRT-PCR Master Mix (2X) kit (KAPA Biosystems, USA), 0.4 μl of each 10 μM forward and reverse primers (CARD-9: forward primer 5′-TCCGACCTGGAAGATGGCTCAC-3′, reverse primer 5′-CAGAGCTGCAAAGGGCTGTTTC-3′) [22], and 0.4 μl of 50X RT Mix. PCR amplification was carried out in a CFX96 TouchTM Real-Time PCR Detection System (Biorad, Germany). A negative control that comprised all the reagents except RNA template was used. All the conditions were carried out in duplicate with the same RNA. The level of gene expression in the test and control samples was analyzed by qRT-PCR. β-Tubulin was used as a reference gene for qRT-PCR normalization using the following primers: β-tubulin forward 5 -GGTAACCAAATCGGTGCTGCTTTC-3′ and β-tubulin Reverse 5′-ACCCTCAGTGTAGTGACCCTTGGC-3′ [23]. RNA quantification was carried out according to the method. CARD-9 expression under UV irradiation was compared with the control conditions.

3. Results

3.1. Exposure Time Dependent Effect of UVC Radiation to S. apiospermum and L. Prolificans

The initial test effect of UVC radiation: the time dependent effect was found to significantly decrease the size of colony which was found at 15-minute exposure, and after 15 min there was no significant decrease. Then we used 15-minute exposure in this study (Figure 1).

Figure 1: The UVC exposure time dependent of S. apiospermum CBS 117410 and L. prolificans CM324.

3.2. Effects of UVC on the Growth of S. apiospermum and L. Prolificans

The growth rate was determined by measuring the diameter of colonies and observing the morphology of colonies. Results showed that, in the controls (without UVC irradiation), the colony diameters of S. apiospermum and L. prolificans were significantly larger than those with UVC exposure (Figures 2–4). The edges of the colonies were circular and smooth in the controls of both S. apiospermum and L. prolificans. In contrast, the colonies of S. apiospermum and L. prolificans with UVC exposure had a serrated edge with a radiating halo; they were not circular in days 2–4, but gradually became circular in days 5–7. The color of S. apiospermum controls (without UVC irradiation) on SDA was white at day 2 before gradually turning gray (completely gray at day 7). The colonies of S. apiospermum with UVC exposure also showed the similar results, but with a pale shade of gray compared with the controls. The colonies of L. prolificans controls had black edges radiating from a center that showed a mix of black and gray hyphae, and colonies with UVC exposure were paler in color than the control.

Figure 2: Colonies of S. apiospermum CBS 117410 and L. prolificans CM324 on SDA after 15 min of UVC exposure from days 2 to 7.

Figure 3: The growth kinetic of S. apiospermum CBS 117410 after 15 min of UVC exposure.

Figure 4: The growth kinetic of L. prolificans CM324after 15 min of UVC exposure.

3.3. Scanning Electron Microscopy(SEM)

Under SEM, there were no differences in the cell morphology of S. apiospermum and L. prolificans, with or without UVC exposure (Figure 5).

(a)

(b)

(c)

(d)

Figure 5: Cell morphology under SEM. (a) S. apiospermum CBS 117410 without UVC exposure; (b) S. apiospermum CBS 117410 after 15 min of UVC exposure; (c) L. prolificans CM324 without UVC exposure; and (d) L. prolificans CM324 after 15 min of UVC exposure.

3.4. Effect of UVC on S. apiospermum and L. prolificans Apoptosis Pathways

UVC induces apoptosis in several fungi [13, 24, 25]. In both S. apiospermum and L. prolificans, UVC exposure induced apoptosis, which was observed by chromatin condensation and orange staining of cells after EB and AO treatment (Figure 6). With UVC exposure, the percentage of apoptotic cells was 96.06% for S. apiospermum and 28.30% for L. prolificans. Without UVC exposure, the percentage of apoptotic cells was 2.38% for S. apiospermum and 2.04% for L. prolificans.

(a)

(b)

(c)

(d)

Figure 6: Apoptotic cells on ethidium bromide/acridine orange staining. Cells were observed using fluorescence microscopy. (a) S. apiospermum CBS 117410 without UVC exposure; (b) S. apiospermum CBS 117410 after 15 min of UVC exposure; (c) L. prolificans CM324 without UVC exposure; and (d) L. prolificans CM324 after 15 min of UVC exposure.

3.5. mRNA Expression of CARD-9 Gene

Increased CARD-9 mRNA expression was observed in both S. apiospermum and L. prolificans after 15 min of UVC exposure, compared with controls (without UVC irradiation). Interestingly, S. apiospermum showed a higher expression of apoptosis signaling than L. prolificans after UVC exposure, reaching statistical significance (P < 0.05) (Figure 7).

Figure 7: CARD-9 mRNA expression in S. apiospermum CBS 117410 and L. prolificans CM324 after 15 min of UVC exposure compared with controls. Gene expression was calculated using the method. Triplicate experiments were carried out to derive mean ± standard deviation. The CARD-9 expression levels were normalized to β-tubulin gene.

4. Discussion

Scedosporium species (including L. prolificans) are shown to be involved in opportunistic infections, particularly in immunocompromised patients. In Thailand, S. apiospermum has been reportedly found in brain abscesses of near-drowning and renal transplant patients [8, 26] and L. prolificans has been reportedly found in a case of disseminated infection in a patient with acute myeloid leukemia with prolonged febrile neutropenia (Damronglerd et al. 2014). In this patient, L. prolificans was shown to be resistant to antifungal agents such as amphotericin B, voriconazole, and posaconazole (minimal inhibitory concentration > 32 μg/ml) [17]. In environmental investigation studies, we found that the S. apiospermum species complex is widespread in soils across Bangkok and detected predominance of S. apiospermum sensu stricto [27]. As mentioned above, L. prolificans showed high minimal inhibitory concentrations to many conventional antifungal drugs while S. apiospermum appeared to be more susceptible to these drugs [28]. Because of antifungal resistance, numerous studies have investigated new drugs in combination with conventional drugs for synergy in fungicidal activity [29–31].

Numerous studies have demonstrated the role of UV light as a disinfectant, particularly in bacteria. UV light is also used as a disinfectant in areas such as indoor swimming pools [32] and hospital surfaces [33]. UV light is divided into bands of UVA, UVB, and UVC. UVC light is commonly used as a tool for inactivation of microorganism. Lakretz et al. showed that UVC wavelengths between 254 nm and 270 nm were the most effective for inactivation of microorganisms, and wavelengths of 254, 260, or 270 nm were effective in biofilm control [34]. Therefore, the wavelength of 254 nm was selected in our study. To date, no study has explained the role of UVC irradiation on the growth of S. apiospermum and L. prolificans. In our study, we demonstrated the effectiveness of UVC radiation in reducing the growth of both organisms after 15 min of UVC exposure, which was accompanied by changes in the color and morphology of the colonies and hyphal growth pattern. In bacteria, UVC exerts its bactericidal activity through DNA damage [35]. Bak et al. showed that UVC killed Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa in a dose- and time-dependent manner, with no viable counts after 2 min of UVC exposure, while C. albicans was killed after 20 min of UVC exposure [36]. The microbicidal effects of UV irradiation were dependent on the genus and species of target microorganisms. Conner-Kerr et al. found that Enterococcus faecalis was more susceptible to killing by UV light than S. aureus [37] and UVC does not discriminate between antibiotic-resistant strains and susceptible strains [35, 37].

After conducting a review of literature, we were interested in UVC induced programmed cell death. In this study, we showed that apoptosis was induced in 96.06% of S. apiospermum and 28.30% of L. prolificans cells after UVC exposure. Our results show that the S. apiospermum was three times more sensitive to UVC than L. prolificans, adding value to the hypothesis that UVC acts is a genus/species dependent manner. We further explored the effects on apoptosis on a molecular level by analyzing the CARD-9 gene expression. CARD-9 can interact with the CARD domain of BCL10, a positive regulator of apoptosis and NF-κB activation [38]. Gene expression results showed that CARD-9 expression was induced in S. apiospermum and L. prolificans cells after UVC treatment and S. apiospermum showed a higher expression of apoptosis signaling than L. prolificans. However, the morphological analysis through SEM did not show any change or significant point in UVC exposed; it might be from the UVC which did not affect directly morphology but affected the internal signal transduction involved in the growth of fungal and other functions which need to be further investigated.

A study by El-Azizi et al. demonstrated the effectiveness of UVC in combination with anti-staphylococcal antibiotics in the disinfection of catheter biofilms of methicillin-susceptible and methicillin-resistant staphylococcal strains [39]. Further studies could be carried out to ascertain the effectiveness of UVC light in combination with antifungal drugs on S. apiospermum and L. prolificans. Overall, our study observed the effects of UVC on inactivation of S. apiospermum and L. prolificans. Therefore, this knowledge could be applied for therapeutic approach such as UVC topical application of mycoses in burns, dermatophytes infection, and other superficial and subcutaneous fungal infections. However, further evaluations are required in terms of safety and efficacy. We hope that our data is useful to other researchers in future studies as well.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Disclosure

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

We are grateful to Medical Mycology and Virology Laboratory, Faculty of Tropical Medicine, Mahidol University, for providing the laboratory room and some required machines and we would like to thank Dr. Yuvadee Mahakunkijcharoen for general consultation and suggestion. This work was supported by the Tropical Medicine Grants 2015 from Faculty of Tropical Medicine, Mahidol University to Watcharamat Muangkaew, Health System for Research Institute (HSRI) 2016 and National Research Council of Thailand (NRCT) 2016 to Natthanej Luplertlop.

References

P. Parize, S. Billaud, A. L. Bienvenu et al., “Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis,” Journal of Cystic Fibrosis, vol. 13, no. 6, pp. 667–673, 2014.

E. Bibashi, G. S. De Hoog, E. Kostopoulou, M. Tsivitanidou, J. Sevastidou, and P. Geleris, “Invasive infection caused by Pseudallescheria boydii in an immunocompetent patient,” Hippokratia, vol. 13, no. 3, pp. 184–186, 2009.

E. L. Campagnaro, K. J. Woodside, M. G. Early et al., “Disseminated Pseudallescheria boydii (Scedosporium apiospermum) infection in a renal transplant patient,” Transplant Infectious Disease, vol. 4, no. 4, pp. 207–211, 2002.

S. Bhagavatula, L. Vale, J. Evans, C. Carpenter, and R. A. Barnes, “Scedosporium prolificans osteomyelitis following penetrating injury: a case report,” Medical Mycology Case Reports, vol. 4, no. 1, pp. 26–29, 2014.

J. L. Rodriguez-Tudela, J. Berenguer, J. Guarro et al., “Epidemiology and outcome of Scedosporium prolificans infection, a review of 162 cases,” Medical Mycology, vol. 47, no. 4, pp. 359–370, 2009.

A. Harun, F. Gilgado, S. C.-A. Chen, and W. Meyer, “Abundance of Pseudallescheria/Scedosporium species in the Australian urban environment suggests a possible source for scedosporiosis including the colonization of airways in cystic fibrosis,” Medical Mycology, vol. 48, no. 1, pp. S70–S76, 2010.

J. Kaltseis, J. Rainer, and G. S. De Hoog, “Ecology of Pseudallescheria and Scedosporium species in human-dominated and natural environments and their distribution in clinical samples,” Medical Mycology, vol. 47, no. 4, pp. 398–405, 2009.

N. Larbcharoensub, P. Chongtrakool, C. Wirojtananugoon et al., “Treatment of a brain abscess caused by scedosporium apiospermum and phaeoacremonium parasiticum in a renal transplant recipient,” Southeast Asian Journal of Tropical Medicine and Public Health, vol. 44, no. 3, pp. 484–489, 2013.

B. Satirapoj, P. Ruangkanchanasetr, S. Treewatchareekorn, O. Supasyndh, L. Luesutthiviboon, and T. Supaporn, “Pseudallescheria boydii Brain Abscess in a Renal Transplant Recipient: First Case Report in Southeast Asia,” Transplantation Proceedings, vol. 40, no. 7, pp. 2425–2427, 2008.

M. Cuenca-Estrella, B. Ruiz-Díez, J. V. Martínez-Suárez, A. Monzón, and J. L. Rodríguez-Tudela, “Comparative in-vitro activity of voriconazole (UK-109,496) and six other antifungal agents against clinical isolates of Scedosporium prolificans and Scedosporium apiospermum,” Journal of Antimicrobial Chemotherapy, vol. 43, no. 1, pp. 149–151, 1999.

L. M. Li, T. Wong, E. Rose, G. Wickham, and E. Bryce, “Evaluation of an ultraviolet C light–emitting device for disinfection of electronic devices,” American Journal of Infection Control, vol. 44, no. 12, pp. 1554–1557, 2016.

N. A. Napolitano, T. Mahapatra, and W. Tang, “The effectiveness of UV-C radiation for facility-wide environmental disinfection to reduce health care-acquired infections,” American Journal of Infection Control, vol. 43, no. 12, pp. 1342–1346, 2015.

A. Valero, M. Begum, S. L. Leong et al., “Effect of germicidal UVC light on fungi isolated from grapes and raisins,” Letters in Applied Microbiology, vol. 45, no. 3, pp. 238–243, 2007.

P. K. Sullivan and T. A. Conner-Kerr, “A comparative study of the effects of UVC irradiation on select procaryotic and eucaryotic wound pathogens.,” Ostomy Wound Management, vol. 46, no. 10, pp. 28–34, 2000.

T. Dai, G. B. Kharkwal, J. Zhao et al., “Ultraviolet-C light for treatment of Candida albicans burn infection in mice,” Photochemistry and Photobiology, vol. 87, no. 2, pp. 342–349, 2011.

N. Luplertlop, P. Pumeesat, W. Muangkaew, T. Wongsuk, and A. Alastruey-Izquierdo, “Environmental screening for the Scedosporium apiospermum species complex in public parks in Bangkok, Thailand,” PLoS ONE, vol. 11, no. 7, 2016.

S. Kasibhatla, G. Amarante-Mendes, D. Finucane, T. Brunner, and E. Bossy-Wetzel, “Acridine orange/ethidium bromide (AO/EB) staining to detect apoptosis,” Cold Spring Harbor Protocols, vol. 2006, no. 3, p. 4493, 2006.

K. Liu, P.-C. Liu, R. Liu, and W. Xing, “Dual AO/EB staining to detect apoptosis in osteosarcoma cells compared with flow cytometry,” Medical Science Monitor Basic Research, vol. 21, pp. 15–20, 2015.

C. Ciniglia, G. Pinto, C. Sansone, and A. Pollio, “Acridine orange/Ethidium bromide double staining test: A simple In-vitro assay to detect apoptosis induced by phenolic compounds in plant cells,” Allelopathy Journal, vol. 26, no. 2, pp. 301–308, 2010.

L. Bouchier-Hayes and S. J. Martin, “CARD games in apoptosis and immunity,” EMBO Reports, vol. 3, no. 7, pp. 616–621, 2002.

J. Bertin, Y. Guo, L. Wang et al., “CARD9 Is a Novel Caspase Recruitment Domain-containing Protein That Interacts With BCL10/CLAP and Activates NF-κB,” The Journal of Biological Chemistry, vol. 275, no. 52, pp. 41082–41086, 2000.

L. Mao, L. Zhang, and H. Li, “Pathogenic fungus Microsporum canis activates the NLRP3 inflammasome,” Infection and Immunity, vol. 82, no. 2, pp. 882–892, 2014.

R. B. Raggam, H. J. F. Salzer, E. Marth, B. Heiling, A. H. Paulitsch, and W. Buzina, “Molecular detection and characterisation of fungal heat shock protein 60,” Mycoses, vol. 54, no. 5, pp. e394–e399, 2011.

J. H. Yang, U. I. Wu, H. M. Tai, and W. H. Sheng, “Effectiveness of an ultraviolet-C disinfection system for rduction of healthcare-associated pathogens Journal of Microbiology,” Immunology and Infection, pp. S1684–S1182, 2017.

T. Dai, G. P. Tegos, G. Rolz-Cruz, W. E. Cumbie, and M. R. Hamblin, “Ultraviolet C inactivation of dermatophytes: Implications for treatment of onychomycosis,” British Journal of Dermatology, vol. 158, no. 6, pp. 1239–1246, 2008.

M. Leechawengwongs, S. Milindankura, A. Liengudom, K. Chanakul, K. Viranuvatti, and P. Clongsusuek, “Multiple Scedosporium apiospermum brain abscesses after near-drowning successfully treated with surgery and long-term voriconazole: A case report,” Mycoses, vol. 50, no. 6, pp. 512–516, 2007.

P. Damronglerd, A. Phuphuakrat, P. Santanirand, and S. Sungkanuparph, “Disseminated Scedosporium prolificans infection in a patient with acute myeloid leukemia and prolonged febrile neutropenia,” Journal of Infectious Diseases and Antimicrob Agents, vol. 31, pp. 101–105, 2014.

J. Meletiadis, J. F. G. M. Meis, J. W. Mouton et al., “In vitro activities of new and conventional antifungal agents against clinical Scedosporium isolates,” Antimicrobial Agents and Chemotherapy, vol. 46, no. 1, pp. 62–68, 2002.

M. Homa, L. Galgóczy, E. Tóth et al., “In vitro susceptibility of Scedosporium isolates to N-acetyl-L-cysteine alone and in combination with conventional antifungal agents,” Medical Mycology, vol. 54, no. 7, pp. 776–779, 2016.

C. Farina, G. Marchesi, M. Passera, C. Diliberto, and G. Russello, “Comparative study of the in vitro activity of various antifungal drugs against Scedosporium spp. in aerobic and hyperbaric atmosphere versus normal atmosphere,” Journal de Mycologie Médicale, vol. 22, no. 2, pp. 142–148, 2012.

H. Schemuth, S. Dittmer, M. Lackner et al., “In vitro activity of colistin as single agent and in combination with antifungals against filamentous fungi occurring in patients with cystic fibrosis,” Mycoses, vol. 56, no. 3, pp. 297–303, 2013.

M. Zare Afifi and E. R. Blatchley, “Effects of UV-based treatment on volatile disinfection byproducts in a chlorinated, indoor swimming pool,” Water Research, vol. 105, pp. 167–177, 2016.

J. M. Boyce, “Modern technologies for improving cleaning and disinfection of environmental surfaces in hospitals,” Antimicrobial Resistance and Infection Control, vol. 5, no. 5, pp. 1–10, 2016.

A. Lakretz, E. Z. Ron, and H. Mamane, “Biofouling control in water by various UVC wavelengths and doses,” Biofouling, vol. 26, no. 3, pp. 257–267, 2010.

M. Dujowich, J. B. Case, G. Ellison, and J. F. X. Wellehan, “Evaluation of Low-Dose Ultraviolet Light C for Reduction of Select ESKAPE Pathogens in a Canine Skin and Muscle Model,” Photomedicine and Laser Surgery, vol. 34, no. 8, pp. 363–370, 2016.

J. Bak, T. Begovic, T. Bjarnsholt, and A. Nielsen, “A UVC device for intra-luminal disinfection of catheters: In vitro tests on soft polymer tubes contaminated with Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Candida albicans,” Photochemistry and Photobiology, vol. 87, no. 5, pp. 1123–1128, 2011.

T. A. Conner-Kerr, P. K. Sullivan, J. Gaillard, M. E. Franklin, and R. M. Jones, “The effects of ultraviolet radiation on antibiotic-resistant bacteria in vitro.,” Ostomy Wound Management, vol. 44, no. 10, pp. 50–56, 1998.

J. Bertin, Y. Guo, L. Wang et al., “CARD9 is a novel caspase recruitment domain-containing protein that interacts with BCL10/CLAP and activates NF-κB,” The Journal of Biological Chemistry, vol. 275, no. 52, pp. 41082–41086, 2000.

M. El-Azizi and N. Khardori, “Efficacy of ultraviolet C light at sublethal dose in combination with antistaphylococcal antibiotics to disinfect catheter biofilms of methicillin-susceptible and methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis in vitro,” Infection and Drug Resistance, vol. 9, pp. 181–189, 2016.