1 Title: N-chlorotaurine exhibits fungicidal activity against therapy

1

Title: N-chlorotaurine exhibits fungicidal activity against therapy-refractory Scedosporium 1

species and Lomentospora prolificans 2

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Running title: N-chlorotaurine against Scedosporium 4

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Authors: Michaela Lackner,a Ulrike Binder,a Martin Reindl, Beyhan Gönül, Hannes 6

Fankhauser, Christian Mair, Markus Nagl* 7

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a both authors contributed equally 9

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Department of Hygiene, Microbiology and Social Medicine, Division of Hygiene and Medical 11

Microbiology, Medical University of Innsbruck, Innsbruck, Austria 12

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Keywords: chloramines, antiseptic, fungicidal, mold, Galleria mellonella, Pseudallescheria, 14

Microascales, virulence, germination, Scedosporium, Lomentospora 15

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*Correspondence to: 17

Markus Nagl, MD, Assoc.Prof. 18

Department of Hygiene, Microbiology and Social Medicine 19

Division of Hygiene and Medical Microbiology 20

Medical University of Innsbruck 21

Schöpfstr. 41, 1st floor 22

A-6020 Innsbruck / Austria 23

Tel. +43-(0)512-9003-70708 24

Fax +43-(0)512-9003-73700 25

E-mail: [email protected] 26

AAC Accepted Manuscript Posted Online 3 August 2015Antimicrob. Agents Chemother. doi:10.1128/AAC.00957-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Abstract 27

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N-chlorotaurine (NCT), a well-tolerated endogenous long-lived oxidant that can be applied 29

topically as antiseptic, was tested on its fungicidal activity against Scedosporium and 30

Lomentospora, opportunistic fungi that cause severe infections with limited treatment options 31

mainly in immunocompromised patients. 32

In quantitative killing assays, both hyphae and conidia of Scedosporium apiospermum, 33

Scedosporium boydii, and Lomentospora prolificans (former Scedosporium prolificans) were 34

killed by 55 mM (1.0%) NCT at pH 7.1 and 37 °C with a log10 reduction in CFU of 1 - 4 35

after 4 h and of 4 to > 6 after 24 h. Addition of ammonium chloride to NCT markedly 36

increased this activity. LIVE/DEAD staining of conidia treated with 1.0% NCT for 0.5 to 3 h 37

disclosed increased permeability of the cell wall and membrane. Pre-incubation of the test 38

fungi in 1.0% NCT for 10 - 60 min delayed the time to germination of conidia by 2 h - >12 h 39

and reduced their germination rate by 10.0 - 100.0%. Larvae of Galleria mellonella infected 40

with 1.0 × 10E7 conidia of S. apiospermum and S. boydii died at a rate of 90.0 – 100% after 41

8-12 days. The mortality rate was reduced to 20 - 50.0% if conidia were pre-incubated in 42

1.0% NCT for 0.5 h or if heat-inactivated conidia were used. 43

Our study demonstrates fungicidal activity of NCT against different Scedosporium and 44

Lomentospora species. A postantifungal effect connected with loss of virulence occurs after 45

sublethal incubation times. The augmenting effect of ammonium chloride can be explained by 46

formation of monochloramine. 47

48

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INTRODUCTION 50

51

Fungi associated to the genus Scedosporium (S. apiospermum and S. boydii) and 52

Lomentospora (L. prolificans; former S. prolificans) are opportunists that cause severe 53

infections, mainly in immunocompromised patients (4). The immune status of the patient is 54

the key to the severity and degree of dissemination of scedosporosis (51,52), reaching from 55

mild lymphocutanous infections, osteomyelitis (10,20), otitis, eye infection, endophthalmitis 56

(6), and brain abscess (1) through to life threatening disseminated infections involving the 57

central nervous system (CNS). Invasive pulmonary scedosporiosis typically occurs in 58

predisposed patients (e.g., solid organ transplant patients), and on occasion in healthy humans 59

(e.g., heavy smokers) (28). The therapeutic outcome of invasive and disseminated infections 60

is usually poor and the mortality rate up to 95% in the setting of persistent 61

immunosuppression, as antifungal therapy alone often fails to clear the infection (21,25). 62

Because of the limitations of the present therapeutic options, new approaches are of 63

interest. For topical treatment of Scedosporium and Lomentospora infections, antiseptics of 64

the class of chloramines are conceivable (13). Particularly N-chlorotaurine, an endogenous 65

long-lived oxidant produced by activated granulocytes and monocytes, comes into question 66

(17). It can be synthesized as crystalline sodium salt (Cl-HN-CH2-CH2-SO3-Na, NCT) and 67

applied as well-tolerated antiseptic to different body sites (17). Because of the oxidative 68

mechanism of action, NCT has the typical broad-spectrum microbicidal activity against 69

bacteria, viruses, fungi, and protozoa (17). Phase II clinical studies in conjunctivitis (54), 70

external otitis (43), crural ulcerations (39), and in the oral cavity (29) have already 71

demonstrated tolerability and therapeutical efficacy, and further applications are investigated, 72

e.g. inhalation (11,48). 73

Regarding Scedosporium, irrigations of skin and subcutaneous lesions, body cavities 74

as well as fungal organ ‘abscesses’, and inhalation of NCT could be of advantage. Fungicidal 75


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activity has already been shown against other fungi (yeasts and molds, (30,35,38)) without 76

resistances in contrast to antifungals (26). Particularly for skin lesions, addition of ammonium 77

chloride (NH4Cl) with formation of monochloramine (NH2Cl), which kills fungi within 78

minutes, has to be considered (34,38). 79

The aim of this study was to demonstrate the activity of NCT against different 80

Scedosporium / Lomentospora species using a panel of different tests. The enhancing effect of 81

ammonium chloride was investigated, too. 82

83

84

MATERIALS AND METHODS 85

86

Chemicals. 87

N-chlorotaurine (NCT, molecular weight 181.57 g/mol) was prepared as crystalline sodium 88

salt in our laboratory in pharmaceutical quality as reported and freshly dissolved in 0.1 M 89

phosphate buffer (pH 7.1) for testing (15). Ammonium chloride (reagent grade) was from 90

Merck (Darmstadt, Germany). 91

92

Fungi. 93

Strains of S. apiospermum (IHEM 21159, IHEM 21168, and IHEM 21170, otitis, Spain), L. 94

prolificans (IHEM 21172, and IHEM 21176, both from a blood culture, Spain), and S. boydii 95

(clinical isolate from cystic fibrosis, internal no. M36 in our laboratory, Austria), which are 96

clinically relevant and have distinct resistances against antifungals (26), were tested 97

separately. They were grown on Oatmeal Agar (1.5% agar and 3% oatmeal - BD, Sparks, 98

USA) at 28°C. Suspensions of hyphae and conidia were gained by scraping an aliquot from 99

the plates and growing it in 5.0 ml tryptic soy broth for 72 hours (46). Then, they were 100

centrifuged at 1800 × g, washed in 5.0 ml 0.9% saline, and treated with IKA Ultra Turrax 101


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Tube Drive (level 9) for 90 sec to cleave large aggregates. Suspensions of hyphae were gained 102

by 5 min ultra sonication of suspensions of hyphae and conidia to detach the latter ones in a 103

Sonorex RK 102 (35 kHz, Bandelin Electronic KG, Berlin, Germany) bath. Hyphae were 104

separated by a following low-speed centrifugation (200 × g, 5 min). The procedure of ultra 105

sonication and low-speed centrifugation was repeated, and the hyphae were centrifuged again 106

for 5 min at 1800 × g on a 40.0 µm filter (cell strainer, Falcon®, Corning Inc., NY) and 107

harvested by washing the filter with 0.9% saline (46). Light microscopy revealed a ratio of 108

hyphae:conidia of 10:1 by this procedure. 109

Suspensions of pure conidia were gained by harvesting them from the agar plates with 5.0 ml 110

0.9% saline plus 0.01% Tween 20 after growth for 8 to 10 weeks followed by 10 µm filtration 111

(CellTrics®, Partec GmbH, Görlitz, Germany) (31). For tests using the Galleria mellonella in 112

vivo model (see below), conidia grown and harvested from Scedosporium selective agar 113

(SceSel+, (44)) were ultra sonicated for 10 min in the Sonorex RK 102 apparatus followed by 114

40 µm (cell strainer, Falcon®, Corning Inc., NY) and then 10 µm (CellTrics®) filtration. 115

Subsequently, they were centrifuged at 800 × g for 5 min and washed in PBS three times. 116

117

Quantitative killing assays. 118

The different suspensions (conidia + hyphae, conidia, or hyphae, 400 µl each) were suspended 119

in 3.6 ml phosphate-buffered NCT and NCT plus NH4Cl solutions, respectively, at 37°C and 120

pH 7.1 (38). Final concentrations of NCT were 1.0% (55 mM, 10 mg/mL), 0.1%, and 0.01% 121

and of the added NH4Cl 0.1% (18.7 mM) and 0.005%. Incubation times were 1 h, 4 h, 8 h, 122

and 24 h for NCT and 3 min, 5 min, 10 min, and 30 min for NCT plus NH4Cl. At the end of 123

the incubation time, aliquots of 50.0 µl were spread on Mueller-Hinton agar plates in 124

duplicate using an automatic spiral plater (model WASP 2, Don Whitley, Shipley, UK). NCT 125

(1.0%) was largely inactivated within 3-5 min on the plates as shown by disappearance of the 126

brown color after addition of potassium iodide in separate experiments. Therefore, in tests 127


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with plain NCT and long incubation times no additional inactivation was performed 128

(detection limit 10 CFU/ml taking into account both plates). In tests with NCT plus NH4Cl, 129

0.1 ml aliquots were added to 0.9 ml of saline containing 0.6% sodium thiosulfate before 130

plating (detection limit 100 CFU/ml). Plates were grown for 48-72 h at 28°C, and the number 131

of CFU was counted. Controls (buffer, taurine, NH4Cl, respectively) were performed without 132

test substances and showed no reduction in CFU. Inactivation controls showed that survival of 133

fungi by addition of test oxidant plus inactivator was warranted. 134

135

Live/dead staining. 136

Since killing of microorganisms by NCT is generally accompanied by increased permeability 137

of the cell coatings (17,37), the induced disorder was investigated by respective staining. 138

Scedosporium conidia and Lomentospora conidia, respectively, each 2 × 106/ml in 500 µl 139

PBS, were mixed with 500 µl of 2.0% NCT in PBS and incubated at 37°C. After 0.5 h, 1 h, 2 140

h, 3 h, and 4 h, 200 µl each were removed and mixed with 100 µl of 6% sodium thiosulfate to 141

immediately inactivate NCT. Subsequently, samples were centrifuged at 2000 × g for 5 min, 142

and the supernatant was discarded. Conidia were stained on ice in the dark for 30 min with 143

100 µl of 1.0% aqua fluorescent reactive dye L34957 in PBS (LIVE/DEAD® Fixable Dead 144

Cell Stain Kit, Invitrogen, Inc., Vienna, Austria). After staining, the samples were washed 145

with PBS and fixed with 3.7% formaldehyde. Negative controls with PBS instead of NCT and 146

positive controls with 70 % ethanol instead of NCT were performed in parallel. For the latter 147

ones, the conidia were centrifuged, the supernatant was discarded and 200 µl 70% ethanol 148

added for 0.5 h. After that, the samples were centrifuged again and subjected to staining after 149

discharge of the supernatant. Analysis was done cytometrically in FACSVerse flow cytometer 150

(BD) (50). 151

As a second method, fluorescence microscopy was applied. Spore suspension (2.5 ml) 152

was mixed with 2.5 ml 2.0% NCT and incubated for 1 h, 2.5 h, and 4 h at 37°C under 153


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continuous rotation. After inactivation and washing, the samples were stained with 0.1 mg/ml 154

FITC in 0.1 M Na2CO3 and 0.005% Tween 20 for 30 min at 37°C under rotation. 155

Subsequently, they were washed twice with 2.0 ml 0.1% Tween 20 in PBS. 50 µl were taken 156

and stained with 10 µl of 50 µg/ml propidium iodide. After 5 min, 5 µl of the stained sample 157

were fixed with 5 µl Mowiol on an object slide and evaluated in the fluorescence microscope 158

(Axioplan,Zeiss, Jena, Germany). Live conidia stain green, dead conidia red. 159

160

Reduced germination of Scedosporium and Lomentospora after sublethal incubation in 161

NCT. 162

In previous studies, bacteria and yeasts treated for sublethal incubation times with NCT 163

showed a lag of regrowth (postantibiotic effect) and a loss of virulence (35-37). This may be 164

important for a possible future in-vivo application of NCT against Scedosporium, too. 165

A lag of regrowth was tested by monitoring the germination from NCT-pretreated 166

conidia by time lapse microscopy (live cell imaging Biostation®, Nikon, Tokyo, Japan), 167

which allows an exact documentation of fungal outgrowth (27). Conidia (1 × 105/ml) were 168

incubated in 0.5 ml 1.0% NCT in PBS at 37°C under rotation for 1 min, 10 min, 30 min, and 169

60 min (controls without NCT for 60 min). After inactivation with sodium thiosulfate, the 170

samples were centrifuged and the supernatant replaced by 1.0 ml of Sabouraud broth (BD, 171

Sparks, USA) containing 2% glucose. Germination was monitored for 20 h at 37°C in the 172

Biostation® with photodocumentation every 20 min. Both the rate of germination and the 173

time to germination were evaluated. 174

175

Loss of virulence of Scedosporium after sublethal incubation in NCT in the Galleria 176

mellonella in-vivo model. 177

Furthermore, we investigated if a lag of germination would influence the virulence of 178

Scedosporium in vivo, as it had been shown previously for Staphylococcus aureus and 179


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Streptococcus pyogenes (36,37). An invertebrate model system, Galleria mellonella (3), was 180

applied. Conidia (final concentration 5 × 108/ml) of S. apiospermum or S. boydii were 181

incubated in 0.5 ml 1.0% NCT in PBS at 37°C for 0.5 h, 1 h, 4 h, and 24 h. Negative controls 182

were performed with plain PBS and with heat-inactivated (autoclaved) conidia in PBS. 183

Subsequent to incubation and inactivation with 6% sodium thiosulfate, an aliquot of 10 µl was 184

removed and subjected to quantitative cultures performed with the spiral plater (model WASP 185

2). The remaining part of the samples was centrifuged at 15000 × g for 2 min and the 186

supernatant replaced by 450 µl of 10% glycerol in insect physiological saline (8.76 g NaCl, 187

0.36 g KCl, 15.76 g TrisHCl, 3.72 g EDTA, in 1000 ml water). The samples were 188

immediately frozen in liquid nitrogen and stored at minus 20°C until injection into larvae. 189

The G. mellonella virulence testing was carried out as published previously (9). Sixth-190

instar larvae of G. mellonella (Kurt Pechmann, Langenzersdorf, Austria) were stored in the 191

dark at 18°C prior to use. Larvae weighing between 0.3 g and 0.4 g were used, each (n = 20) 192

infected with 1×107 conidia in 20 µl insect physiological saline by injection into the hemocoel 193

via the hind pro-leg. Untreated larvae and larvae injected with insect physiological saline 194

served as controls. Further controls consisted of larvae injected with heat-inactivated conidia 195

(negative control) and with PBS-treated conidia (positive control). Larvae were incubated at 196

30°C in the dark and monitored daily up to 9-12 days. 197

198

Statistics. 199

Data are presented as mean values and standard deviations (SD). Student’s unpaired t test in 200

case of two groups, or one-way ANOVA and Bonferroni’s and Dunnett’s multiple 201

comparison test in case of more than two groups were used to test for a difference between the 202

test and control group. Significance of mortality rate data (G. mellonella) was evaluated by 203

using Kaplan-Meier survival curves and Mantel-Cox log rank test. P < 0.05 was considered 204


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significant for all tests. Calculations were done with GraphPad Prism 6.01 software 205

(GraphPad Inc., La Jolla, CA, USA). 206

207

208

RESULTS 209

210

Quantitative killing assays. 211

All tested Scedosporium spp. were killed by 1.0% (55 mM) NCT. In Fig. 1A, the condensed 212

mean killing curve of mixtures of hyphae and conidia of all tested strains is shown. A highly 213

significant reduction in CFU by >99.0% occurred after an incubation time of 4 h. The 214

susceptibilities of S. apiospermum and L. prolificans were higher than that of S. boydii after 215

4h, as shown in Fig. 1B-D. S. apiospermum IHEM 21170 was the most susceptible strain with 216

a reduction below the detection limit (10 CFU/ml) after 4 h. 217

Against conidia, where higher inocula could be achieved, the killing curves were 218

similar, without marked differences between the test strains (Fig. 2). Reduction in CFU 219

reached approximately 2 log10 after 4 h and 5 log10 after 24 h incubation time. Inocula with 220

prevailing hyphae (ration hyphae:conidia equaled 10:1) needed a 24 h incubation for a highly 221

significant killing despite the low CFU count (Fig. 3). This might be explained by formation 222

of aggregates, which are more difficult to kill. NCT at a lower concentration of 0.1% (5.5 223

mM) reduced the number of CFU of S. apiospermum IHEM 21170 conidia by 2.19 - 2.66 224

log10 after 24 hours, while the reduction was 5.31 – 5.47 log10 for S. boydii after the same time 225

(starting value 6.21 - 6.23 log10 CFU). A concentration of 0.01% NCT caused no more 226

significant killing. Because of long incubation times needed with lower NCT concentrations, 227

which are usually irrelevant in practice, we performed the further investigations (live/dead 228

staining, germination, virulence) with the clinically applied 1% NCT. 229


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By addition of 0.1% (18.7 mM) ammonium chloride to NCT, the antifungal activity 230

could be enhanced significantly (P < 0.01 versus NCT alone), resulting in killing times of few 231

minutes (Fig. 4). This was true against hyphae and conidia. The activity of 1.0% and 0.1% 232

NCT plus 0.1% NH4Cl each was similar. If sodium thiosulfate was applied for inactivation, 233

the detection limit increased to 100 CFU/ml, and the CFU count was zero after 5 min. Special 234

inactivation controls, where a low count of hyphae and conidia (8.0 × 102 – 2.3 × 103 235

CFU/ml) was mixed with NCT plus ammonium chloride previously inactivated with sodium 236

thiosulfate, showed no decrease of the CFU count. The same was true for controls with 1% 237

taurine. 238

239

Live/dead staining. 240

Increased permeability after incubation in 1.0% NCT was detected by uptake of the 241

LIVE/DEAD® fluorescent reactive dye L34957 using conidia of S. apiospermum IHEM 242

21170, L. prolificans IHEM 21172, and S. boydii clinical isolate. Cytometry revealed uptake 243

of the stain for all species with increasing incubation time (Fig. 5). Uptake was slowest with 244

L. prolificans. After a longer incubation time in 1.0% NCT (24 h), the percentage of dead 245

fungal cells resembled that of 70.0% ethanol, i.e. almost 100.0%. 246

Uptake of propidium iodide by the same fungal strains occurred more rapidly, i.e. 247

already after 1.0 h of incubation in 1.0% NCT, and the uptake of FITC was more pronounced 248

compared with the controls. Representative exemplary slides of fluorescence microscopy of 249

L. prolificans IHEM 21172 are shown in Fig. 6. With increasing previous incubation time in 250

NCT, the appearance of propidium iodide uptake became less planar, but more focused within 251

the fungal cells. There were no differences between the single test strains (3 independent 252

experiments per strain). 253

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Reduced germination of Scedosporium and Lomentospora after sublethal incubation in 255

NCT. 256

Strains showing a good germination within 10 h of monitoring in the live cell imaging 257

Biostation® after pre-incubation in PBS for 1 h (negative controls) were used, i.e. S. 258

apiospermum IHEM 21170, L. prolificans IHEM 21176, and S. boydii. Remarkably, L. 259

prolificans strain IHEM 21172 did not grow under these conditions. After pre-incubation in 260

1.0% NCT in PBS at 37°C for 10 min, 30 min, and 60 min, both the rate of germination and 261

the time to germination of Scedosporium spp. were reduced compared to the controls (Fig. 7). 262

The effect increased with the incubation time in NCT and was largely similar for all tested 263

species. 264

265

Loss of virulence of Scedosporium after sublethal incubation in NCT in the Galleria 266

mellonella in-vivo model. 267

With conidia of S. apiospermum IHEM 21170, such a loss of virulence could be clearly 268

demonstrated (Fig. 8A). Conidia pre-treated with 1.0% NCT for 0.5 h, 1 h, 4 h or 24 h caused 269

a death rate of larvae similar to that of autoclaved conidia and significantly lower compared to 270

that of untreated conidia (P < 0.01). Untreated and PBS-treated larvae survived 12 days at a 271

level of 90-100%. The clinical isolate of S. boydii showed similar results (Fig. 8B). Although 272

heat-inactivated conidia (negative control) reduced the number of larvae by approximately 30 273

- 60% after 7-9 days, this value was significantly different from untreated, living conidia (90-274

100% reduction, P < 0.05). 275

276

277

DISCUSSION 278

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As a representative of the active chlorine compounds, NCT exerts the broad-spectrum 280

microbicidal activity against all reigns of pathogens (13,17), which is typical for this class of 281

antiseptics. Due to its low reactivity, which is the reason for its good tolerability in sensitive 282

body regions (17), its killing activity against microorganisms is slower compared with highly 283

reactive compounds like hypochlorous acid (7,13). Killing of different Candida species and 284

molds needed incubation times of one or only few hours in previous studies (35,38,45), and 285

that against Scedosporium spp. turned out to be similar in the present study. As with other 286

molds (38), there were no marked differences in the susceptibility of the test strains of this 287

fungus in our study. Moreover, the growth stage of Scedosporium, hyphae (vegetative 288

propagating stage) or conidia (resting stage), did not play a decisive role. The somewhat 289

higher resistance of hyphae can be explained by larger aggregates compared to conidia. This 290

may be supported by the small, but significant decrease of the CFU count of the controls in 291

these tests with hyphae (Fig. 3). 292

The remarkable enhancement of killing of molds (as well as bacteria and protozoa) by 293

addition of NH4Cl to NCT known from previous studies (12,34,38), was confirmed to a 294

similar extent against Scedosporium, too. Due to the more rapid killing of microorganisms in 295

body fluids and exudates and due to the successful therapeutic application of NCT in humans 296

and animals (34,38-40,43,47), we are convinced that this effect plays a role in vivo. It is 297

explained by formation of monochloramine (NH2Cl) in equilibrium with NCT (12,18). 298

Because of its higher lipophilicity, NH2Cl penetrates and kills microorganisms more rapidly 299

than NCT (12,13,22). 300

The importance of penetration of NCT for inactivation of the microorganism has once 301

more been confirmed with Scedosporium. LIVE/DEAD® staining clearly demonstrated an 302

increased permeability of the fungal wall and cell membrane after incubation in NCT. It is 303

true that the kinetics of uptake of the stains do not exactly resemble those of the CFU in the 304

killing tests. Fluorescence microscopy rather indicates an increased permeability for 305


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propidium iodide before a reduction in CFU starts, while FACS analysis using another dye 306

(L34957) revealed a more delayed effect. The continuous rotation during incubation in NCT 307

in the tests applying propidium iodide may contribute to explain these differences, and 308

probably a different permeability of the stains used. Nevertheless, we proved earlier that the 309

rapidly occurring chlorination of the surface of bacteria and fungi by NCT does not kill them 310

(16). Because of this fact and changes seen in electron microscopy and proteomics of bacteria, 311

oxidation and chlorination of the inside of pathogens must be assumed to be essential for their 312

irreversible inactivation (2,13,37). 313

However, already short, sublethal incubation times in NCT caused a lag of regrowth of 314

bacteria and fungi (35,36,41), and a reduction of production of secretory aspartyl proteinases 315

in Candida albicans (35). Moreover, this was connected with a loss of virulence in vivo in a 316

staphylococcal and streptococcal mouse peritonitis model (36,41). A lag of germination and a 317

decrease of the percentage of germination of a fungus after sublethal pre-treatment with NCT 318

could be described for the first time in the present study. Obviously, germination is a sensitive 319

parameter since the impact of NCT was highly significant already after incubation times of 10 320

min to 60 min. A further challenge was to demonstrate if sublethal incubation times of NCT 321

in vitro had an influence on the virulence of Scedosporium in an in vivo model. The virulence 322

of Aspergillus and yeasts in the Galleria mellonella model was shown previously (3,5,8,49), 323

so that we chose it as a practicable one. The model turned out to be suitable for Scedosporium 324

apiospermum (IHEM 21170) and S. boydii and a loss of virulence could be proved 325

unambiguously already after incubation in sublethal concentrations of NCT for 30 min. The 326

moderate killing of larvae by heat inactivated and NCT-treated S. boydii conidia is probably 327

caused by the high inoculum dose and either the physical effect of, or the respective immune 328

response to this high number of foreign particles. 329

The fungicidal activity of NCT against Scedosporium, connected with a loss of 330

virulence after short incubation, render the substance an interesting antiseptic for topical 331


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treatment of infections with this pathogen. The marked enhancement of activity in the 332

presence of ammonium supports this intention. In human body fluids and exudates, 333

ammonium is present and increases the killing activity exactly at the location of treatment 334

(13,14,19,34,38-40). At the same time, the tolerability of NCT is high, therefore it is of 335

particular advantage for sensitive body regions, including body cavities (17). Regarding 336

Scedosporium, topical treatment of infections of the skin and subcutaneous tissue (39), of the 337

eye (47,54), of the ear (43), of the paranasal sinuses (42), and even organ abscesses by 338

irrigations via catheters are well conceivable. In case of locations with low exudation, 339

addition of ammonium chloride can be considered, for instance in the eye (55) or on the skin 340

(Nagl M, unpublished). According to recent findings, also the lower respiratory system can be 341

accessed with NCT by inhalation due to its outstanding tolerability compared to other 342

antiseptics (11,33,48). The latter application would be of particular interest for the 343

decontamination of cystic fibrosis patients suffering from allergic bronchopulmonary 344

aspergillosis or invasive bonchopulmonary scedosporiosis and in general for eradication of 345

pathogens from the lower respiratory tract. The contraindication of lung transplantation is 346

discussed for patients with a Scedosporium-colonized lower respiratory tract, as it was 347

reported in cases of disseminated scedosporiosis (32,53). The role of fungi in exacerbations of 348

chronic bronchitis, lung infections, and as allergic agents in the respiratory tract of cystic 349

fibrosis patients was highlighted recently (23,24). 350

As a conclusion, NCT has broad-spectrum fungicidal activity against Scedosporium 351

spp., and L. prolificans including hyphae and conidia. Inhibition of germination and loss of 352

virulence are early phenomena, followed by increased permeability and killing of the fungus. 353

Addition of ammonium chloride to NCT leads to a marked acceleration of killing. Clinical 354

application of NCT for topical treatment of infections by Scedosporium spp. and L. 355

prolificans should be investigated. 356

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ACKNOWLEDGEMENTS 358

We are grateful to Bettina Sartori, Andrea Windisch, and Magdalena Hagleitner for excellent 359

technical assistance. 360

This study was supported by the Division of Hygiene and Medical Microbiology of the 361

Medical University of Innsbruck. 362

The authors have no conflict of interest to declare. 363

364


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544

545


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Figure legends 546

547

Fig 1 548

Activity of 1.0% NCT (-■-) against hyphae and conidia of Scedosporium spp. and L. 549

prolificans at pH 7.1 and 37°C. Controls in phosphate buffer without NCT (-●-). Mean values 550

± SD. Detection limit 1 log10. * P < 0.05 and ** P < 0.01 versus control by Student’s unpaired 551

t test. 552

(A) summary of all 6 test strains; n = 18. 553

(B) S. apiospermum (strains IHEM 21159, 21168, and 21170); n = 9. 554

(C) L. prolificans (strains IHEM 21172 and 21176); n = 6. 555

(D) S. boydii (clinical isolate); n = 3. 556

557

Fig 2 558

Activity of 1.0% NCT (-■-) against conidia of Scedosporium spp. and L. prolificans at pH 7.1 559

and 37°C. Controls in phosphate buffer without NCT (-●-). Mean values ± SD of all six test 560

strains used in Fig. 1, n = 18. Detection limit 1 log10. * P < 0.05 and ** P < 0.01 versus 561

control by Student’s unpaired t test. 562

563

Fig 3 564

Activity of 1.0% NCT (-■-) against hyphae of Scedosporium spp. and L. prolificans at pH 7.1 565

and 37°C. Controls in phosphate buffer without NCT (-●-). Mean values ± SD of S. 566

apiospermum (IHEM 21168), L. prolificans (IHEM 21172) and S. boydii (clinical isolate), n = 567

9. Detection limit 1 log10. * P < 0.05 and ** P < 0.01 versus control by Student’s unpaired t 568

test. 569

P < 0.01 between time zero and 24 h for controls by Student’s paired t test. 570

571


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Fig 4 572

Activity of 1.0 % NCT plus 0.1 % NH4Cl (-■-, full line) and 0.1 % NCT plus 0.1 % NH4Cl (-573

▲-, dotted line) against hyphae and conidia of Scedosporium spp. and L. prolificans at pH 7.1 574

and 20°C. Controls in phosphate buffer without NCT (-●-). Mean values ± SD of S. 575

apiospermum (IHEM 21168, 21168, and 21170) and S. boydii (clinical isolate), n = 4. 576

Detection limit 1 log10. ** P < 0.01 versus control by one-way ANOVA and Dunnett’s 577

multiple comparison test. 578

579

Fig 5 580

Cytometric analysis of LIVE/DEAD® staining of conidia of S. apiospermum IHEM 21170 (-581

■-), L. prolificans IHEM 21172 (-▼-), and S. boydii clinical isolate (-▲-) after incubation in 582

1.0% NCT at pH 7.1 and 37°C for 0.5 h - 4 h. Positive controls with 70% ethanol instead of 583

NCT (-♦-) and negative controls in PBS instead of NCT (-●-). Fungal cells with uptake of 584

stain were quoted as dead cells. Mean values ± SEM of n = 3, (n = 9 for controls). * P < 0.05 585

and ** P < 0.01 versus control by one-way ANOVA and Dunnett’s multiple comparison test. 586

a P < 0.01 versus NCT. 587

588

Fig 6 589

Uptake of FITC (green, 525 nm) and propidium iodide (red, 620 nm) by conidia of L. 590

prolificans IHEM 21172 after incubation in 1.0% NCT at 37°C and pH 7.1 for 1 h, 2.5 h, and 591

4 h. Control in PBS. Representative slides of fluorescence microscopy from one of three 592

independent experiments. Magnification 400×; bar 50 µm. 593

594

Fig 7 595

Time to germination (left panels) and percentage of germination (right panels) of S. 596

apiospermum IHEM 21170, L. prolificans IHEM 21176, and S. boydii clinical isolate after 597


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pre-incubation in 1.0% NCT in PBS at 37°C and pH 7.1 for 10 min, 30 min, and 60 min. 598

Controls were pre-incubated in plain PBS for 60 min. Mean values ± SD of n =5. 599

* P < 0.05 and ** P < 0.01 versus control by one-way ANOVA and Dunnett’s multiple 600

comparison test. 601

602

Fig 8 603

Kaplan-Meier survival curves of Galleria mellonella infected with 1 × 107 conidia/larva of S. 604

apiospermum IHEM 21170 (A) and S. boydii (B). Loss of virulence of conidia pre-treated 605

with 1.0% NCT (dotted lines) at 37°C and pH 7.1 for 0.5 h (--), 1 h (--), 4 h (--), and 24 606

h (--). Controls consisted of untouched larvae (-■-), larvae injected with 20 µl of insect 607

physiological saline (--), with heat-inactivated (autoclaved) conidia (-▲-), and with 608

untreated living conidia (-▼-). Mean values of three independent experiments. 609

** P < 0.01 of living conidia versus all other groups (Mantel-Cox log rank test) in (A); 610

*,** P < 0.01 of living conidia versus 4 h and 24 h NCT and P < 0.05 versus 0.5 h and 1 h 611

NCT in (B). 612

P > 0.05 between NCT-treated and heat-inactivated conidia in (A) and (B). 613

P > 0.05 in (A) and * P < 0.05 in (B) between untouched or saline-treated larvae and larvae 614

challenged with NCT-treated or heat-inactivated conidia. 615

616

617

618

619

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50 µm

FITC Propidium iodide

Negative control

NCT 1h

NCT 2.5h

NCT 4h


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Documents

1 Title: N-chlorotaurine exhibits fungicidal activity against therapy