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Title: N-chlorotaurine exhibits fungicidal activity against therapy-refractory Scedosporium 1
species and Lomentospora prolificans 2
3
Running title: N-chlorotaurine against Scedosporium 4
5
Authors: Michaela Lackner,a Ulrike Binder,a Martin Reindl, Beyhan Gönül, Hannes 6
Fankhauser, Christian Mair, Markus Nagl* 7
8
a both authors contributed equally 9
10
Department of Hygiene, Microbiology and Social Medicine, Division of Hygiene and Medical 11
Microbiology, Medical University of Innsbruck, Innsbruck, Austria 12
13
Keywords: chloramines, antiseptic, fungicidal, mold, Galleria mellonella, Pseudallescheria, 14
Microascales, virulence, germination, Scedosporium, Lomentospora 15
16
*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
28
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
49
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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
254
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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
279
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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
357
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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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REFERENCES 365
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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
620
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FITC Propidium iodide
Negative control
NCT 1h
NCT 2.5h
NCT 4h
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