key: cord-0332085-5obo3k2t
authors: Cox, Claudia A; Vazquez, Jose A; Wakade, Sushama; Bogacz, Marek; Myntti, Matthew; Manavathu, Elias K
title: Efficacy of Biofilm Disrupters Against Candida auris and Other Candida species
date: 2020-12-03
journal: bioRxiv
DOI: 10.1101/2020.12.02.409250
sha: 81c1c1248f718b94a54fc4fb0183169b229bddd4
doc_id: 332085
cord_uid: 5obo3k2t

Background C. auris has become a globally emerging fungal pathogen, frequently reported to be multi-drug resistant, commonly found with Staphylococcus aureus in polymicrobial nosocomial infections. Although chlorhexidine (CHD) has been shown to be effective, it is associated with serious anaphylaxis reactions. Biofilm disrupters (BDs) are novel agents with a broad spectrum of antimicrobial activity. BDs have been used in the management of chronic wounds and to sterilize environmental surfaces. The goal of this study was to evaluate BDs against polymicrobial biofilms compared to CHD. Methodology We evaluated various BDs (BlastX, Torrent, NSSD) and CHD against Candida spp and S. aureus polymicrobial biofilms by zone of inhibition, biofilm, and time-kill assays. Effectiveness was based on the inhibition zone and the reduction of CFU, respectively, compared to the drug-free control. Results All BDs and CHD inhibited C. auris growth effectively in a concentration-dependent manner. Additionally, CHD and the BDs all showed excellent antimicrobial activity against polymicrobial biofilms. BDs were all highly effective against both C. auris isolates, whereas CHD was only moderately effective against C. auris 0386, suggesting resistance/tolerance. A comparative analysis of the BDs and CHD against C. auris and C. albicans by biofilm kill-curves showed at least 99.999% killing. Conclusions All three BDs and CHD have excellent activity against different Candida species, including C. auris. However, certain isolates of C. auris showed resistance/tolerance to CHD, but not to the BDs. The fungicidal activity of these novel agents will be valuable in eradicating surface colonization of Candida spp, including C. auris.

(BlastX, Torrent, NSSD) and CHD against Candida spp and S. aureus polymicrobial biofilms by 23 zone of inhibition, biofilm, and time-kill assays. Effectiveness was based on the inhibition zone 24 and the reduction of CFU, respectively, compared to the drug-free control. Results: All BDs and 25 CHD inhibited C. auris growth effectively in a concentration-dependent manner. Additionally, 26 CHD and the BDs all showed excellent antimicrobial activity against polymicrobial biofilms. BDs 27 were all highly effective against both C. auris isolates, whereas CHD was only moderately 28 effective against C. auris 0386, suggesting resistance/tolerance. A comparative analysis of the 29 BDs and CHD against C. auris and C. albicans by biofilm kill-curves showed at least 99.999% 30 killing. Conclusions: All three BDs and CHD have excellent activity against different Candida 31 species, including C. auris. However, certain isolates of C. auris showed resistance/tolerance to 32 CHD, but not to the BDs. The fungicidal activity of these novel agents will be valuable in 33 eradicating surface colonization of Candida spp, including C. auris. (6) (7) (8) (9) , though this fungal pathogen has now been isolated from several body sites ranging from 44 asymptomatic cutaneous colonization to bloodstream infections (6) . In fact, in the first C. auris 45 outbreak which took place over the course of 16 months in a healthcare setting in Europe, of 46 the 50 patients who became colonized, 44% required antifungal treatment and 18% developed 47 bloodstream infections despite infection prevention measures implemented after the first few 48 cases had been identified (10). 49 C. auris infections largely originate from healthcare facilities (2-4), but are known to be 50 transmitted from person-to-person due to the ability of C. auris to colonize the skin (6, 11, 12) . 51 In this way, infection prevention and control must be administered much in the way of 52 methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant 53 Enterobacteriacea (CRE) (6) . This is in stark contrast to other Candida species, where cases are 54 attributed to a patient's own microbiome leading to autoinfection (6) . This novel yeast poses a 55 serious threat because of its level of multidrug resistance (MDR) and how readily this Candida 56 spp. spreads in the hospital setting (1-4, 11). In fact, once it has established itself in the hospital 57 environment, it has been difficult to eradicate C. auris from hospital surfaces (1-3). Elimination 58 of this MDR pathogen from a London care center that involved 34 patients in 2016 brought with 59 it a cost of over GB£1 million (approximately US$1.3 million) for initial control and GB£58,000 60 per month (approximately US$83,000) the following year (13). 61 In addition, it is now well described that C. auris generates a very significant biofilm (1-4) 62 that increases resistance to antifungal agents and decreases the efficacy of surface agent (3) Further, Candida colonizes several sites on the human body also inhabited by 84 Staphylococcus aureus, a virulent, pathogenic, gram-positive bacteria (26). Co-infections with 85 these microbes have been associated with higher mortality rates than those infections 86 consisting of only one (26, 27) . This is due, in part, to a synergistic relationship between the two 87 microorganisms. This type of microbial relationship has created a significant need for broad-88 spectrum antimicrobial agents. 89 Chlorhexadine (CHD) has been used as a skin antiseptic for over half a century, and is 90 effective against a wide range of microbes: gram-positive and gram-negative bacteria, yeast, 91 and even enveloped viruses like HIV (28). However, CHD has also been linked to hypersensitivity 92 reactions, up to and including anaphylaxis (29), and while anaphylactic reactions are rare there 93 has been a rise in reported cases (30). Because of its wide spectrum of antimicrobial activity and 94 low cost, it is unknowingly omnipresent in products extending beyond its disinfectant properties 95 serving to possibly increase sensitization leading to a possible rise in allergic reactions to the 96 product (29). To that end, it is necessary to find non-toxic alternative disinfectants. 97 The most commonly used antifungal agents are used systemically to treat patients with 98 disseminated or invasive fungal infections. Unfortunately, C. auris is MDR, and thus is tolerant or 99 resistant to many conventional antifungal agents. Moreover, environmental surfaces are 100 generally cleaned with surface disinfectants such as CHD and other quaternary ammonium 101 compounds. Recently, a non-toxic novel topical agent has been developed that has a unique 102 mechanism of action. This new product is a high-osmolarity surfactant solution that has shown 103 in vitro activity against gram-positive cocci, gram-negative bacilli, fungi and yeast (31, 32). 104 Products that use this technology are also known as biofilm disrupting agents (BDs). These 105 products are composed of a surfactant (benzalkonium chloride) and a high osmolarity buffer at 106 a pH of 4.0 (33). In essence, the highly concentrated acid component destroys the extracellular 107 polymeric substance (EPS) or biofilm by removing ionic metal bonds between EPS polymers and 108 allowing for the penetration of the high-osmolarity solution which destroys bacterial and fungal 109 cells. In addition, the pH is maintained for a prolonged period of time ( ~ 5 days) and also 110 affects the persister cells so they can't re-develop new biofilm. 111 The primary objective of this study is to evaluate the efficacy of three novel BDs (1, 5, 6, 12, 14, 197 21, 26) . This highlights the need for a non-specific antimicrobial disinfectant. Moreover, it has 198 been suggested that repeated use of CHD at sub-inhibitory concentrations can lead to reduced 199 activity against several oral pathogens. This may be due to the development of multidrug efflux 200 pumps which can be expressed in both gram-positive and gram-negative bacteria (36). 201 Though highly effective against several species of Candida (37), the broad-spectrum 202 antiseptic CHD has been shown to be less effective against C. auris, which can persist even after 203 twice a day treatment with the antiseptic (38). One study showed that in clinically-relevant 204 doses, mature biofilms were actually resistant to CHD treatment when compared to early 205 biofilms and in a dose-dependent manner (39). To wit, there is a necessity for products 206 designed to disrupt the extracellular polymeric substance of the biofilm, but is also effective 207 against persister cells. The microbicide-treated biofilms were washed with sterile distilled water three times, 424 resuspended in sterile water and the fungal and bacterial CFUs were determined. 425 

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Candida auris sp. 269 nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a 270 Japanese hospital

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FIG 3 Effects of experimental and commercially available microbicides on candidal and 387 bacterial monomicrobial biofilms. C. albicans 90028, C. auris 0381, C. auris 0386 and S. aureus 388 43300 were used in this study. Monomicrobial biofilms were developed in 96-well microtiter 389 plates for 24 h at 37 o C by incubating 0.1 ml cell suspension containing

Sabouraud's dextrose broth (Candida) or Brain Heart Infusion broth (bacteria) as previously 391 described. The biofilms were washed and exposed to full-strength experimental and 392 commercial microbicides (except BlastX, 50%) for 24 h. The microbicide-treated biofilms were 393 washed and their effectiveness was assessed by biofilm CFU assay. The control represents 394 biofilm treated identically with PBS. The threshold of detection for the CFU assay was a 395 minimum of 10. The experiment was repeated once and similar results were obtained

FIG 4 Time kill assays showing the effect of Next Science surface disinfectant and 4% 399 chlorhexidine solution. C. albicans 90028 (A, D), C. glabrata

Cells were washed, pelleted, and resuspended in 20% solution (in SD 401 broth) or full strength NSSD or CHD to a suspension of 1x 10 7 cells/ml. At 1, 5, 10, 30, and 60 402 minute time intervals, cells were removed and washed, at which point serial dilutions were 403 made and plated on SD agar plates

FIG 5 Twenty-four vs forty-eight hour Candida and Staphylococcus aureus polymicrobial biofilm

After cell adhesion, 411 the plates were gently shaken on a gyratory shaker for 5 min and the unbound cells were 412 removed with a pipette. The attached Candida cells were then incubated with 0.2 ml SA43300 413 cell suspension (1 x 10 7 cells/ml) in SD broth for 24 or 48 h for biofilm development

) TC plates for 24 h at 37°C. The biofilms were washed with sterile distilled 421 water two times and then treated with various microbicidal agents prepared in SD broth at a 422 concentration of 50% (A-C) or full strength