Downloaded from //aem.asm.org/content/aem/early/2015/10/19/AEM.02806-15.full.pdf · 49 high concentrations required (10% (v/v)), make it impractical for widespread use [5]. 50

Silver nanoparticles decrease the viability of Cryptosporidium parvum oocysts 1

2

Pamela Cameron2,4*, Birgit K. Gaiser3, Bidha Bhandari1, Paul M. Bartley2, Frank 3

Katzer2 and Helen Bridle1 4 5 1Heriot-Watt University, School of Engineering, Edinburgh, EH14 4AS, UK 6 2Moredun Research Institute, Bush Loan, Penicuik, EH26 0PZ, UK 7 3Heriot-Watt University, School of Life Sciences, Nanosafety Research Group, 8

Edinburgh, EH14 4AS, UK 9 4Novo Science Ltd., 3 Hatton Mains Cottages, Edinburgh, EH27 8EB 10

11

12 *Corresponding author 13

14

Keywords: Cryptosporidium, nanoparticles, water, parasite 15

16

17

AEM Accepted Manuscript Posted Online 23 October 2015Appl. Environ. Microbiol. doi:10.1128/AEM.02806-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

on January 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

http://aem.asm.org/

Abstract 18

19

Oocysts of the waterborne protozoan parasite, Cryptosporidium, are highly resistant 20

to chlorine disinfection. We show here for the first time that both silver nanoparticles 21

(Ag NPs) and silver ions significantly decrease oocyst viability in a dose-dependent 22

manner between concentrations of 0.005 and 500 µg/mL, as assessed by an 23

excystation assay, and shell:sporozoite ratio. For percentage excystation, results are 24

statistically significant at 500 µg/mL of AgNPs, reducing from 83% for the control to 25

33%. For Ag ions the results were statistically significant at 500 and 5000 µg/mL but 26

percentage excystation reduced to only 66 and 62% compared to an 86% control. The 27

sporozoite:shell ratio was affected to a greater extent, following AgNP exposure 28

presumably as sporozoites are destroyed by interaction with the NPs. We also 29

demonstrate via hyperspectral imaging that there is a dual mode of interaction with 30

Ag ions entering the oocyst and destroying the sporozoites while AgNPs interact with 31

the cell wall and at high concentrations are able to fully break the oocyst wall. 32

33


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Introduction 34

35

Contaminated drinking water is one of the most important environmental contributors 36

to the human and livestock disease burden, being responsible for an estimated 1.9 37

million human deaths each year, with protozoan parasites such as Cryptosporidium 38

parvum being responsible for a significant proportion of this number [1]. C. parvum is 39

also of significant concern to farmers, with post mortem data indicating that the 40

majority of calves who had died, at less than one month of age, were infected with 41

this parasite [2]. Tap water is the most common risk factor, in recorded human cases 42

[3], since Cryptosporidium oocysts are robust, long-lived, have a low infectious dose 43

and are highly resistant to chlorination [4]. The parasite is resistant to most 44

commercial disinfectants and whilst filtration can physically remove oocysts, this is 45

not always available or completely effective. Recent research has concentrated on 46

finding a means of inactivating the parasite and C. parvum has previously shown 47

sensitivity to oxidative stress, induced by hydrogen peroxide. However the extremely 48

high concentrations required (10% (v/v)), make it impractical for widespread use [5]. 49

50

Recently, numerous studies have established that silver nanoparticles (Ag NPs) are 51

highly toxic to bacteria and fungi [6-9] and that this toxicity is often associated with 52

ion release and induction of oxidative stress [7]. Consequently, Ag NPs are 53

incorporated into consumer products, primarily due to these antibacterial and 54

antifungal properties, such as clothes made of Ag NP-containing fabric [10] and 55

personal hygiene products [11]. Others are incorporated into wound dressings [12], 56

nano-silver toothpastes and colloidal silver suspensions, designed as nutritional 57

supplements [13]. Since C. parvum has shown sensitivity to oxidative stress 58

previously, we sought to determine the effect of Ag NPs on oocyst viability. At the 59

time of undertaking our study no previous work had investigated the impact of 60

nanoparticles on waterborne protozoan pathogens. A recent article reported some 61

degree of C. parvum oocyst inactivation upon exposure to AgNPs [14]. Nevertheless, 62

our work still represents the first study of nanoparticle action on waterborne 63

protozoan pathogens considering dose-dependance and additionally aims to ascertain 64

whether AgNP have any effect on the viability of C. parvum oocysts and whether this 65

is due to the presence of Ag ions, the nanoparticles or a combination of both. 66


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Materials and Methods 67

Materials 68

C. parvum oocysts were obtained from the Creative Science Company (Penicuik, 69

UK). The oocysts were of the Moredun isolate and were stored in PBS at 4˚C. All 70

experiments were performed within 4 months of oocyst preparations, and untreated 71

control samples of identical age were used as a comparison in every excystsation 72

assay. NM300 Ag NPs were purchased from Mercator GmbH and all other chemicals 73

were from Sigma. 74

75

Particles and particle characterisation 76

NM300 is one of the Representative Manufactured Nanomaterials included in the 77

OECD’s Working Party on Manufactured Nanomaterials Sponsorship Programme, 78

and has therefore been chosen for investigation in an increasing number of projects in 79

order to allow comparison of data across multiple sources. It is a colloidal 10% w/w 80

dispersion of Ag NPs in water containing 4% (w/w) each of polyoxyethylene glycerol 81

trioleate and Tween 20 [15]. NM300 and the NP-free dispersant control, NM300-DIS, 82

were obtained from Mercator GmbH, Germany. Characterisation data for these Ag 83

NPs have been previously obtained in the original suspension and in cell culture 84

medium by transmission electron microscopy (TEM), dynamic light scattering (DLS), 85

and inductively coupled plasma optical emission spectrometry (ICP-OES) as reported 86

in Kermanizadeh et al. (2012) [16]. NM300 is explicitly produced as a reference 87

material to allow comparison between studies, and the same batch of material was 88

used in this study. 89

In addition, NM300 NPs were characterised by DLS in sterile water at the exposure 90

concentrations used in this study. For this, NPs were sonicated for 8 minutes in sterile 91

water in a sonicating water bath at a concentration of 5 mg/ml in a glass vial, followed 92

by inversion of the vial and a second sonication cycle. Serial dilutions of the 93

suspensions were then examined by DLS according to the manufacturer’s manual. 94

95

Nanoparticle preparation and exposures 96

For in vitro exposures, particle suspensions were prepared by sonicating in a water 97

bath for 2 x 8 min in deionized water as described above. Oocysts (1 x 106/ml final 98

concentration) were incubated in sterile water (SLS), at room temperature, with 99


.asm.org/

Dow

nloaded from

http://aem.asm.org/

varying concentrations of AgNPs, for a maximum of 30 min. Viability was visually 100

assessed by phase contrast light microscopy for intact oocysts. 101

102

103

Assessment of viability by excystation assay 104

Oocysts were incubated as above, and then immediately assessed for viability using 105

the established excystation assay [17]. Briefly, samples were incubated in the 106

presence of trypsin (1% (w/v), pH 3.0) at 37˚C for 30 min. Samples were then 107

centrifuged and supernatant was discarded before sodium deoxycholate (1% w/v) was 108

added and samples were again incubated at 37˚C for 40 min. Viability was assessed 109

by counting the number of oocysts, sporozoites and number of empty shells, by phase 110

contrast microscopy, expressing the number of empty shells divided by number of 111

oocysts and empty shells as a percentage and thus, a measure of excystation of the 112

parasites. Each shell contrains four sporozoites so the sporozoite:empty shell ratio 113

was also calculated. 114

The percentage of excystation was calculated by using the counted number of oocysts 115

and empty shells at different concentration of silver nanoparticles and is given by: 116

117

% Excystation = [ 𝑛𝑛𝑛𝑛𝑛𝑛 𝑜𝑜 𝑛𝑛𝑒𝑒𝑒 𝑠ℎ𝑛𝑒𝑒𝑠𝑛𝑛𝑛𝑛𝑛𝑛 𝑜𝑜 𝑜𝑜𝑜𝑒𝑠𝑒𝑠+ 𝑛𝑛𝑒𝑒𝑒 𝑠ℎ𝑛𝑒𝑒𝑠

] x 100 118

119

The sporozoite per shell ratio was calculated by using counted number of empty shells 120

and the number of sporozoites and is given by: 121

122

Sporozoite:shell ratio = 𝑛𝑛𝑛𝑛𝑛𝑛 𝑜𝑜 𝑠𝑒𝑜𝑛𝑜𝑠𝑜𝑠𝑒𝑛𝑠𝑛𝑛𝑛𝑛𝑛𝑛 𝑜𝑜 𝑛𝑛𝑒𝑒𝑒 𝑠ℎ𝑛𝑒𝑒𝑠

123

124

125

Hyperspectral imaging 126

Oocyst and AgNP samples were sent to Cytoviva Inc (www.cytoviva.com). Samples 127

were analysed using an enhanced darkfield transmission optical microscope (Olympus 128

BX43) equipped with a 100x objective and a hyperspectral imaging 129

spectrophotometer (CytoViva, Inc., Auburn, AL). The spectrophotometer was used 130

for recording spectra with a low signal-to-noise ratio in visible and near-infrared 131

wavelength (400-1000 nm) at a high spectral resolution of 2.5nm. Ten dark current 132


.asm.org/

Dow

nloaded from

http://aem.asm.org/

images were collected at the beginning of each hyperspectral image acquisition and 133

were subtracted from the hyperspectral image data. A spectral classification algorithm 134

(Spectral Angle Mapper) that uses an n-dimensional angle (equal to the number of 135

wavelengths analyzed) was applied to match pixels on the hyperspectral image to 136

reference libraries acquired by analyzing nanosilver samples dispersed in water. 137

138

139

Statistical analysis 140

All data are expressed as means ± standard error of the mean (SEM). Statistical 141

analysis was performed by students’ t-test (unpaired, two-tailed) using GraphPad 142

Prism software (GraphPad software, Inc) and all data represent the mean of at least 143

three independent experiments. A p<0.05 was deemed to be statistically significant. 144

145

146

147


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Results 148

149

Nanoparticle characterisation 150

151

The results of the characterisation performed in deionised water as part of this study 152

are summarized in Figure 1. At concentrations of 5 µg/ml and higher, the z average, 153

which describes the intensity-weighed harmonic mean sizeof the NPs, is in the “nano-154

range” of 100 nm and smaller, with no significant changes over a 7 day period (Figure 155

1A). At the lowest concentrations, higher z averages are found, which can likely be 156

attributed to the relatively higher contribution of larger dust particles as background 157

noise. 158

159

The polydispersity index (pdi) gives an indication of agglomeration of particles in the 160

suspension. The best-dispersed suspensions are present between 0.05 and 5 µg/ml Ag 161

NPs, and at higher concentrations an increase in agglomeration to a pdi of 162

approximately 0.5 was measured (Figure 1B). At high concentrations of NPs, 163

collisions between particles occur more frequently, and therefore agglomeration is 164

encouraged. Similar to the z average, pdi remained stable over the 7 day time course. 165

Despite this increase in polydispersity, the z average at the higher concentrations 166

indicates a good dispersion and small average diameter, which may be partly due to 167

larger agglomerates precipitating. 168

169

In addition to this information, Kermanizadeh reported sizes of NM300 NPs by X-ray 170

diffraction as 7 nm (wet phase) and 14 nm (dried sample), and an average diameter of 171

17.5 nm as measured by TEM, with primary particle sizes ranging from 8 to 45 nm 172

[16]. Particles were mainly euhydral, with some elongated or sub-spherical 173

morphologies present. Ag NP dissolution in water was assessed over 24 h at 1, 16 and 174

128 µg/ml and found to be <0.01 %, 0.78% and 0.59%, respectively [16]. Particles 175

were assessed for endotoxin contamination and were endotoxin-free (data not shown). 176

177

178

The dose dependent effect of silver nanoparticles on C. parvum oocyst integrity 179

180


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Samples containing 1x106 Cryptosporidium oocysts were exposed to silver 181

nanoparticle concentrations ranging from 5ng/mL to 5 mg/mL. For each 182

concentration, three replicate experiments were performed. After 30 minutes of 183

nanoparticle incubation on the sample, the effect of the nanoparticles upon the oocysts 184

was assessed visibly using phase contrast microscopy. A clear AgNP concentration 185

dependence upon oocyst destruction was observed. Figure 2 shows typical images of 186

the samples following nanoparticle exposures at the highest concentration studied. 187

These images indicate that silver nanoparticles induce oocyst death, at high 188

concentrations leading to a break up of the oocyst structure such that only debris was 189

observed in the images. 190

191

The dose dependent effect of silver nanoparticles on the excystation of C. parvum 192

oocysts 193

194

While the above experiments indicated that high concentrations of nanoparticles were 195

capable of destroying the oocyst outer wall, a further research question was whether 196

those oocysts, which remained intact, also remained viable and infectious. Since 197

oocysts cannot be cultured in the lab via traditional microbiological techniques, 198

methods such as the minimum inhibitory concentration (MIC) or minimum 199

bactericidal concentration (MBC) cannot be utilized. Several methods have been 200

developed to assess oocyst viability although the gold standard remains animal 201

models, which are expensive time-consuming and should be used with caution as 202

other assessment techniques could provide adequate data without requiring infectivity 203

studies[17]. An alternative marker for infectivity is the ability of the oocyst to 204

undergo excystation, the process by which oocysts rupture releasing the sporozoites 205

which initiate infection in host cells, as is approved by The Drinking Water 206

Inspectorate. 207

208

Here, an identical set of oocyst nanoparticle exposures was repeated with the impact 209

of the nanoparticles assessed by excystation. Excystation was triggered using a 210

standard protocol of exposure as described in the Materials and Methods. The effect 211

of nanoparticles on the viability of oocysts was analysed through two measures of 212

excystation: percentage of excystation (Figure 3A) and the sporozoite/shell ratio 213

(Figure 3B) (Table 1). 214


.asm.org/

Dow

nloaded from

http://aem.asm.org/

The results indicate very little change in percentage excystation on increasing dose of 215

AgNPs, although the highest AgNP concentration (500µg/mL) caused significantly 216

less sporozoite excystation from 83.3±3% to 33.3±17.5%. In contrast, the 217

sporozoite/shell ratio decreases significantly as the amount of AgNPs added is 218

increased, with significant changes starting at 5 µg/mL (p<0.05), most likely 219

reflecting the toxicity of AgNPs to naked sporozoites. 220

221

222

The dose dependent effect of silver ions on the excystation of C. parvum oocysts 223

224

Samples containing 1x106 Cryptosporidium oocysts were exposed to silver ions 225

concentrations ranging from 0.0005 to 5000 µg/mL. For each concentration, three 226

replicate experiments were performed. As with the silver nanoparticles the impact of 227

silver ions on viability and infectivity was assessed using excystation protocols. 228

Again, percentage excystation (Figure 4A) and the sporozoite/shell ratio (Figure 4B) 229

were employed as a means to determine the extent of excystation (Table 1). As with 230

the AgNPs, significant effects on excystation percentage are only observed at 231

concentrations of 500µg/mL and above. However, the sporozoite/shell ratio is not 232

impacted at lower concentrations, with the 5 µg/mL result not being significantly 233

different from the positive control. At higher concentrations, 500µg/mL and above, it 234

is clear that silver ions do reduce the sporozoite shell ratio (p<0.05). 235

236

Hyperspectral Imaging 237

238

Enhanced Darkfield Hyperspectral Microscopy is a technique which allows for the 239 optical visualization and spectral characterization of nanosized objects. As a result, 240 the location of silver nanoparticles can be determined [18-20]. Enhanced darkfield 241 microscopy enables detection of the scatter from silver nanoparticles at sizes below 242 the optical microscopy resolution limit. Hyperspectral imaging of these samples 243 enables spectral characterization of silver nanoparticles based on their unique spectral 244 characteristics. Figure 5 shows images of oocysts comparing the negative control 245 sample with one exposed to AgNPs, with the latter clearly showing signs of obvious 246 morphological changes with such damage likely to mediate loss of viability. Figure 6 247 shows the response of the reference AgNP sample as well as an image of an oocyst 248 with the presence of AgNPs. Figure 6B shows a NP interacting with the oocyst wall 249 whereas internalization of AgNPs was not observed. 250

251


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Discussion 252 253

Recently, there has been considerable interest in the use of nanotechnology in 254

water purification. In particular, the use of nanomaterials in small-scale, point-of-use 255

or emergency response treatment systems has been proposed and investigated. For 256

example, silver embedded ceramic filters have been trialled in several developing 257

countries and bactericidal silver nanoparticle paper was recently reported in 258

Environmental Science and Technology [21]. However, while it is known that certain 259

nanoparticles exhibit antibacterial activity, only one other study has investigated the 260

impact of nanoparticles on protozoa [14], also concentrating on Cryptosporidium, 261

even though this pathogen is a leading cause of waterborne disease [1]. The results 262

presented here confirm that silver nanoparticles and silver ions are toxic to the 263

waterborne protozoan pathogen, Cryptosporidium and are the first quantification of 264

the dose-dependance of this effect. 265

266

Whether silver nanoparticle toxicity is due to particles, ions or a combination 267

has been hotly debated over the last decade, and many mechanisms of action have 268

been proposed (e.g. [22, 23]). It is well-known that AgNPs can be oxidized in 269

aqueous solutions leading to release of silver ions and recent publications suggest 270

AgNP toxicity is in the case of controlled aqueous laboratory media due to silver ion 271

release into the exposure medium [9, 24, 25]. However, this may vary in water 272

containing high concentrations of chlorides or organic matter, which could form 273

largely insoluble complexes with Ag ions and therefore reduce their toxicity [26].. 274

A particular advantage of studying Cryptosporidium is that this pathogen 275

offers a unique opportunity to gather deeper insight into the mechanisms of 276

nanoparticle interaction with biological samples; in most cellular suspensions the 277

halogen-containing culture medium precludes the accurate evaluation of silver ion 278

toxicity, as silver halides tend to precipitate at low concentrations. Furthermore, 279

nanoparticle properties such as agglomeration and aggregation, surface charge and 280

adsorption of proteins are generally highly dependent on their surrounding media (e.g. 281

[27, 28]). The robustness of Cryptosporidium oocysts enabled experiments to be 282

conducted in water, so that both the comparison of nanoparticles versus ions and the 283


.asm.org/

Dow

nloaded from

http://aem.asm.org/

mechanisms of action of the particles as close to their native state as possible in a 284

solution could be investigated. 285

Recent work by Xiu et al concluded that there was negligible particle-specific 286

antibacterial activity of silver nanoparticles, though note that organism-specific 287

responses could lead to different observations in other biological systems [9]. They 288

found that antibacterial activity as a function of released silver ions from AgNPs was 289

indistinguishable from the equivalent concentration of silver ions (from silver nitrate). 290

However, we observe greater toxicity with AgNPs at equivalent concentrations, 291

suggesting the oocyst response to AgNPs differs from bacteria. 292

Over a range of concentrations, comparable to those observed for bacterial 293

toxicity, both silver nanoparticles and silver ions were observed to either trigger 294

destruction of the oocyst or render oocysts non-viable (Table 1). At the same 295

microbial density, the MBC for E. coli and S. aureus with AgNPs were reported to be 296

20 and 40µg/mL, respectively [6]. This is a factor of ten lower than observed for 297

Cryptosporidium by considering excystation percentage though a factor of ten higher 298

when using the sporozoite/shell ratio as a measure of viability. The same study found 299

the MBC for these bacteria with silver ions to be 7.5µg/mL [6], a factor of one 300

hundred less than observed for the Cryptosporidium, with either measure of 301

excystation. The result with silver ions is as expected, since Cryptosporidium oocysts 302

have a protective outer wall which for example resists their disinfection by chlorine, 303

to which bacteria are particularly susceptible. Given this robust outer wall it is 304

surprising to observe similar toxicity of AgNPs as for bacteria. 305

Additionally, while the bacteria study found silver ions to be more cytotoxic 306

than AgNPs [6], our results indicate that AgNPs appear to be slightly more toxic to 307

oocysts than silver ions. For example the impact on excystation percentage becomes 308

significant at the same concentration (of 500µg/mL), with AgNPs leading to greater 309

reduction in excystation percentage (down to 33% with AgNPs compared to 62% 310

with ions). Previous characterization of the NPs employed revealed that less than 1% 311

of the AgNPs dissolve in water [16], suggesting that the equivalent silver ion 312

concentration of 500µg/mL AgNPs is 5µg/mL of silver ions, further confirming our 313

hypothesis that AgNPs are more toxic as there is a clear impact of 500µg/mL AgNPs 314

whereas no changes are observed at 5µg/mL of silver ions. Additionally, when using 315


.asm.org/

Dow

nloaded from

http://aem.asm.org/

the sporozoite/shell ratio as a measure, AgNP toxicity is noted at much lower 316

concentrations than silver ions, most likely because sporozoites are more suspectible 317

to NPs than oocysts and therefore they are more easily destroyed reducing the 318

observed ratio. Greater AgNP toxicity has been previously reported through the 319

impact of the medium on silver ion bioavailability. 320

321

322

One previous study of oocyst exposures to AgNPs and ions was conducted by 323

Su et al, who utilized 100µg/mL of AgNPs and the equivalent of 63.5µg/mL of Ag 324

ions (100µg/mL of AgNO3), with four hour exposures [14]. These concentrations 325

were at the upper range of what we investigated though our exposures took place over 326

just 30 mins. Su et al reported a 42.7% excystation percentage for oocysts exposed to 327

AgNPs, 71.4% for those treated with silver nitrate and 89.5% for untreated oocysts, 328

comparable to our results. Su et al concluded that Ag ions were ineffective at 329

inactivation of oocysts, implying that the impact of AgNP action was in someway 330

related to an intrinsic property of the NP itself. This is also in agreement with our 331

observation that AgNPs are more toxic than silver ions to oocysts. However, Su et al 332

did note that AgNP action was often attributed to the release of silver ions, though 333

offered no explanation for why in their results little effect was observed with Ag. We 334

have compared the influence of particles and ions by performing viability dose-335

response assays for each state of the material. Our findings indicate that while both 336

AgNP and Ag ions are capable of inactivation of C. parvum oocysts there is a strong 337

dose-dependence, with AgNPs exhibiting greater toxicity. 338

339

The greater toxicity of AgNPs was more apparent depending upon the 340

excystation measure employed. The ratio of number of oocysts to number of shells 341

(excystation percentage) might not reveal the influence of silver upon oocysts if the 342

inactivation occurs through sporozoite destruction as empty oocysts might still excyst. 343

However, by considering the sporozoite/shell ratio this factor can be accounted for. 344

Our results clearly indicated the decrease in sporozoite/shell ratio with increasing 345

silver doses, showing that exposure reduces oocyst viability through sporozoite 346

destruction. This is in agreement with the findings of Su et al, who observed 347

sporozoite destruction in phase contrast imaging and dielectrophoretic response [14]. 348


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Sporozoite destruction would suggest internalization of silver nanoparticles or ions. 349

Su et al observed sporozoite destruction within intact oocysts, confirming the wall 350

was not destroyed via propidium iodide staining. At high concentrations we have 351

observed destruction of the oocyst wall, at which point it is reasonable to infer 352

sporozoite destruction as they are much less robust than the oocyst itself, and indeed 353

are packaged within the oocyst structure to protect them from the environment. Our 354

results show sporozoite/shell ratio decreases at much lower AgNP concentrations than 355

with silver ions (statistically significant effects noted at 5μg/mL versus 500μg/mL). 356

One explanation is that the presence of AgNPs contributes to greater silver 357

internalization, potentially mediated by local low pH enhancing silver release 358

following AgNP binding to the membrane, in an enhanced Trojan Horse effect which 359

has previously been suggested as a mechanism for nanoparticle toxicology after 360

uptake into lysosomes[29]. An alternative is that bound AgNPs are not removed 361

before the excystation assay and thus remain a source of silver ions as sporozoites are 362

excysted, although the Su results suggest sporozoite destruction before the oocyst 363

wall is broken. 364

365

Hyperspectral imaging with enhanced darkfield microscopy offers 366

significantly higher signal to noise ratio and therefore better scatter detection of 367

particles than conventional optical methods and the spectral profiles of NPs can be 368

used to detect the NPs in hyperspectral imaging; the method has been increasingly 369

used for environmental samples [18, 19, 30], including the semi-quantitative uptake 370

of NPs by protozoa [19] and to study disinfection of water samples [31]. We 371

employed hyperspectral imaging to study both whether nanoparticles interact with the 372

membrane, and the oocyst morphology after exposure. These experiments have 373

enabled preliminary conclusions on the mode of nanoparticle action. Our results 374

supported by hyperspectral imaging clearly highlight the interaction of silver 375

nanoparticles with the oocyst wall. Previous work has noted the accumulation of 376

AgNPs on cell surfaces and the formation of ‘pits’ [8]. Taken together with our other 377

findings it appears that interaction of AgNPs with the oocyst wall is a critical step in 378

mediation of the toxic effects observed, resulting in greater toxicity than Ag ions 379

alone. 380

381


.asm.org/

Dow

nloaded from

http://aem.asm.org/

In conclusion, we have provided the first detailed characterization of silver 382

nanoparticle and ion toxicity on protozoan pathogens such as Cryptosporidium. 383

Furthermore, dose-response experiments have shown that high concentrations of 384

nanoparticles and ions, respectively, cause oocyst destruction with lower 385

concentrations impacting viability. Comparison of excystation percentage and 386

sporozoite/shell ratios has indicated greater sensitivity of the sporozoites to silver, 387

with AgNPs leading to greater sporozoite destruction. The work has revealed that for 388

oocysts there is a particle-specific mechanism, imparting greater toxicity for 389

nanoparticles versus ions. Additionally, the use of hyperspectral imaging allowed us 390

to confirm the interactions of AgNPs with the oocyst membrane and observe the 391

subsequent oocyst disruption. 392

393

Protozoan pathogens are a major contributor to the waterborne disease burden and 394

this dose-dependent analysis of silver nanoparticle and silver ion response will be 395

highly useful in assisting the application of these materials for oocyst disinfection. 396

While, improvements in efficacy are also required to achieve a truly effective 397

disinfectant, this can perhaps be achieved by combination with other 398

materials/reagents. However, as noted above the presence of e.g. other ions or organic 399

components in a sample can impact upon toxicity and further investigations in 400

finished and raw waters are required. 401

402

Funding Information/Acknowledgements: 403

404

PC, BG and HB would like to acknowledge the Heriot-Watt Crucible training 405

programme, which inspired the initiation of this project and also provided financial 406

support through a grant from the Heriot-Watt Crucible fund, and the EU FP7 project 407

InLiveTox (Grant agreement NMP4-SL-2009-228789) for providing the NM300 408

silver particles. PB, PC and FK would also like to acknowledge Scottish Government 409

Funding. HB would also like to acknowledge the Royal Academy of 410

Engineering/EPSRC for her research fellowship. The authors would also like to thank 411

Prof. David Smith (Moredun Research Institute) and Prof. Vicki Stone (Heriot-Watt 412

University) for the use of laboratory space and consumables. We would also like to 413

acknowledge Mr Byron Cheatham and CytoViva (www.cytoviva.com) for the 414

technical assistance and undertaking of the hyperspectral imaging experiments. 415


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Figure Legends 416

417

Fig. 1 Z average and polydispersity index of Ag nanoparticles suspended in sterile 418

water at the concentration range used in this study and stability of suspensions over 419

time (1, 3 and 7 days). Values are the mean of three individual experiments + SEM 420

421

Fig. 2 The effect of silver nanoparticles on C. parvum viability, as assessed by phase-422

contrast light microscopy (x 400 magnification). Viable intact oocysts (panel A) and 423

non-viable oocysts/cell debris in the presence of AgNP (500 mg/ml). 424

425

Fig. 3 The effect of silver nanoparticles on C. parvum viability, as assessed by % 426

excystation (panel A) and sporozoite:shell ratio (panel B). Values are the mean of 427

three individual experiments ± SEM * p<0.05 428

429

Table 1. Summary table of all the results for the excystation protocols by both 430

measures of excystation and with both AgNP and Ag ions. Bold font and * indicate 431

statistical significance (p<0.05). 432

433

Fig. 4 The effect of silver nitrate on C. parvum viability, as assessed by % excystation 434

(top panel) and sporozoite:shell ratio (bottom panel). Values are the mean of three 435

individual experiments ± SEM * p<0.05 436

437

Fig. 5 Hyperspectral imaging. 438

Top panel: visualisation of AgNPs via spectral imaging.The NPs vary significantly in 439

colour from blue (smallest) to red (largest) when illuminated with full spectrum light 440

due to the plasmon resonance effect. The left image shows the microscope view of the 441

slide whereas the right image shows the spectral profile of different particles. 442

Bottom panel: observation of NPs interacting with the oocyst wall. Again the left 443

image is the microscope view – overlayed bright field and spectral image to show 444

both the oocyst and the AgNPs. The right image is the spectral profile of the 445

highlighted green NP interacting with the oocyst wall. 446

447

Fig. 6 448

Hyperspectral imaging depicting the effect of silver NPs on C. parvum 449


.asm.org/

Dow

nloaded from

http://aem.asm.org/

A: Control, intact oocyst 450

B: NPs added to the oocyst 451

C: Oocyst after 2 hours of exposure to NPs, observable impact on cell wall and 452

interior 453

All scale bars are 20μm. 454

455

456

457

458


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Fig. 1 Z average and polydispersity index of Ag nanoparticles suspended in sterile water at the concentration range used in this study and stability of suspensions over time (1, 3 and 7 days).Values are the mean of three individual experiments + SEM

A B

459


.asm.org/

Dow

nloaded from

http://aem.asm.org/

A

B

Fig. 2 The effect of silver nanoparticles on C. parvum viability, as assessed by phase-contrast light microscopy (x 400 magnification). Viable intact oocysts (panel A) and non-viable oocysts/cell debris in the presence of AgNP (500 µg/ml).

25 µm

25 µm

460


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Fig. 3 The effect of silver nanoparticles on C. parvum viability, as assessed by % excystation (panel A) and sporozoite:shellratio (panel B). Values are the mean of three individual experiments ± SEM * p<0.05

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Positive 0.005 0.05 0.5 5 50 500

Spor

ozoi

te:S

hell r

atio

Concentration (µg/ml)

B *

*

*

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

Positive 0.005 0.05 0.5 5 50 500

% E

xcys

tatio

n

Concentration (µg/ml)

A*

461


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Fig. 4 The effect of silver nitrate on C. parvum viability, as assessed by % excystation (panel A) and sporozoite:shell ratio (panel B). Values are the mean of three individual experiments ± SEM * p<0.05

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

Positive 0.0005 0.005 0.05 0.5 5 50 500 5000

% E

xcys

tatio

n

Concentration (ug/ml)

* *

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Positive 0.0005 0.005 0.05 0.5 5 50 500 5000

Spor

ozoi

te:S

hell

ratio

Concentration (ug/ml)

**

462 463


.asm.org/

Dow

nloaded from

http://aem.asm.org/

AgNP % Excystation Sporozoite/Shell ratio Control 83.33 ± 3.3 1.93 ± 0.03 0.005 79 ± 9.5 1.2 ± 0.46 0.05 88 ± 2.5 2.03 ± 0.58 0.5 62.33 ± 8.65 0.87 ± 0.52 5 53 ± 16.8 0.77 ± 0.38 * 50 71 ± 12.3 0.38 ± 0.3 * 500 33.33 ± 17.5 * 0.13 ± 0.07 * 464 Ag ions % Excystation Sporozoite/Shell ratio Control 85.67 ± 3.93 2 ± 0.4 0.0005 82.67 ± 3 1.2 ± 0.46 0.005 72.33 ± 13.5 1.37 ± 0.57 0.05 78 ± 9 1.97 ± 0.47 0.5 80 ± 8.5 1.55 ± 0.35 5 79.67 ± 7 1.47 ± 0.09 50 83.33 ± 2 1.5 ± 0.35 500 66.33 ± 0.5 * 0.37 ± 0 * 5000 62 ± 2.5 * 0.07 ± 0.03 * 465 Table 1. Summary table of all the results for the excystation protocols by both 466

measures of excystation and with both AgNP and Ag ions. Bold font and * indicate 467

statistical significance (p<0.05). 468

469 470


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Fig. 5 Hyperspectral imaging.Top panel: visualisation of AgNPs via spectral imaging.The NPs vary significantly in colour from blue (smallest) to red (largest) when illuminated with full spectrum light due to the plasmonresonance effect. The left image shows the microscope view of the slide whereas the right image shows the spectral profile of different particles. Bottom panel: observation of NPs interacting with the oocystwall. Again the left image is the microscope view – overlayedbright field and spectral image to show both the oocyst and the AgNPs. The right image is the spectral profile of the highlighted green NP interacting with the oocyst wall.

471 472


.asm.org/

Dow

nloaded from

http://aem.asm.org/

473

474 Fig. 6 475

Hyperspectral imaging depicting the effect of silver NPs on C. parvum 476

A: Control, intact oocyst 477

B: NPs added to the oocyst 478

C: Oocyst after 2 hours of exposure to NPs, observable impact on cell wall and 479

interior 480

All scale bars are 20μm. 481

References 482

483

1. Striepen, B., 2013. Time to tackle cryptosporidiosis. Nature. 503: p. 189-484 191. 485

2. Thomson, S., Cryptosporidios in farmed livestock, 2015, University of 486 Glasgow. 487

3. Tam, C. C., L. C. Rodrigues, L. Viviani, J. P. Dodds, M. R. Evan, P. R. 488 Hunter, J. J. Gray, L. H. Letley, G. Rait, D. S. Tompkins, and S. J. O'Brien. 489 2011. Longitudinal study of infectious intestinal disease in the UK (IID2 490 study): incidence in the community and presenting to general practice 491 Gut Published on line doi:10.1136/gut.2011.238386. 492

4. Rose, J.B., Huffman, D.E., Gennaccaro A., 2002. Risk and control of 493 waterborne cryptosporidiosis. FEMS Microbiol Rev. 26: p. 113-23. 494

5. Barbee S.L., Weber D.J., M. D. Sobsey, and W. A. Rutala. 1999. 495 Inactivation of Cryptosporidium parvum oocyst infectivity by disinfection 496 and sterilization processes. Gastrointest Endosc. 49:605-611. 497

6. Greulich, C., Braun, D., Peetsch, A., Diendorf, J., Siebers, B., Epple, M. 498 and Koller, M., 2012. The toxic effect of silver ions and silver 499 nanoparticles towards bacteria and human cells occurs in the same 500 concentration range. RSC Advances. 2: p. 6981-6987. 501

7. Johnston, H.J., Hutchison, G., Christensen, F. M., Peters, S., Hankin, S. 502 and Stone, V., 2010. A review of the in vivo and in vitro toxicity of silver 503 and gold particulates: Particle attributes and biological mechanisms 504 responsible for the observed toxicity. Critical Reviews in Toxicology. 40: 505 p. 328-346. 506

8. Marambio-Jones, C. and E. V. Hoek 2010. A review of the antibacterial 507 effects of silver nanomaterials and potential implications for human 508


.asm.org/

Dow

nloaded from

http://aem.asm.org/

health and the environment. Journal of Nanoparticle Research. 12(5): 509 1531-1551. 510

9. Xiu, Z.-M., Zhang, Q-B., Puppala, H.L., Colvin, V.L., Alvarez, P.J.J. . 2012. 511 Negligible particle-specific antibacterial activity of silver nanoparticles. 512 NanoLetters 12:4271-4275. 513

10. Kulthong, K., Srisung, S., Boonpavanitchakul, K., 514 Kangwansupamonkon, W. and Maniratanachote, R. . 2010. 515 Determination of silver nanoparticle release from antibacterial fabrics 516 into artificial sweat. Part Fibre Toxicol 7:1743-8977. 517

11. Chao, J.B., Liu, J. F., Yu, S. J., Feng, Y. D., Tan, Z. Q., Liu, R. and Yin, Y. G., 518 2011. Speciation analysis of silver nanoparticles and silver ions in 519 antibacterial products and environmental waters via cloud point 520 extraction-based separation. Anal Chem. 83: p. 6875-6882. 521

12. Silver, S., Phung le, T. and Silver, G., 2006. Silver as biocides in burn and 522 wound dressings and bacterial resistance to silver compounds. J Ind 523 Microbiol Biotechnol. 33: p. 627-634. 524

13. Nowack, B., Krug, H. and Height, M. 2011. 120 Years of Nanosilver 525 History: Implications for Policy Makers (vol 45, pg 1177, 2011). 526

14. Su, Y.-H., Tsegaye, M., Varhue, W., Liao, K-T., Abebe, L.S., Smith, J.A., 527 Guerrant, R.L. and Swami, N.S., 2014. Quantitative dielectrophoretic 528 tracking for characterization and separation of persistent subpopulations 529 of Cryptosporidium parvum. Analyst. 139: p. 66-73. 530

15. Comero, S., Klein, C., Stahlmecke, B., Romazanov, J., Kuhlbusch, T., 531 van Doren, E., Wick, P., Locoro, G., Koerdel, W., Gawlik, B., Mast, J., Krug, H.F., 532 Hund-Rinke, K., Friedrichs, S., Maier, G., Werner, J., Linsinger, T. 2011. NM-300 533 silver characterisation, stability, homogeneity. JRC Publication No. JRC60709 534 EUR 24693 EN. Publications Office of the European Union. DOI: 10.2788/23079 535 16. Kermanizadeh, A., Pojana, G., Gaiser, B. K., Birkedal, R., Bilanicova, 536 D., Wallin, H., Jensen, K. A., Sellergren, B., Hutchison, G. R., Marcomini, A. 537 and Stone, V., 2012. In vitro assessment of engineered nanomaterials using a 538 hepatocyte cell line: cytotoxicity, pro-inflammatory cytokines and functional 539 markers. Nanotoxicology. 201. 540 17. Robertson, L.J. and B.K. Gjerde, 2007. Cryptosporidium oocysts: 541

challenging adversaries? Trends in Parasitology. 23(8): p. 344-347. 542 18. Beach, J., 2009. Hyperspectral imaging. BioOptics World. 3: p. 2-4. 543 19. Mortimer, M., A. Gogos, N. Bartolomé, A. Kahru, T. D. Bucheli, and V. I. 544

Slaveykova. 2014. Potential of Hyperspectral Imaging Microscopy for 545 Semi-quantitative Analysis of Nanoparticle Uptake by Protozoa. 546 Environmental Science & Technology 48:8760-8767. 547

20. Pratsinis, A., P. Hervella, J.-C. Leroux, S. E. Pratsinis, and G. A. 548 Sotiriou. 2013. Toxicity of Silver Nanoparticles in Macrophages. Small 549 9:2576-2584. 550

21. Dankovich, T.A. and D.G. Gray, 2011. Bactericidal Paper Impregnated 551 with Silver Nanoparticles for Point-of-Use Water Treatment. 552 Environmental Science & Technology. 45(5): p. 1992-1998. 553

22. Ivask A, E.A., Kaweeteerawat C, Boren D, Fischer H, Ji Z, Chang CH, Liu 554 R, Tolaymat T, Telesca D, Zink JI, Cohen Y, Holden PA, Godwin HA., 555 2014. Toxicity mechanisms in Excherichia coli vary for silver 556 nanoparticles and differ from ionic silver. ACS Nano. 28(8): p. 374-86. 557


.asm.org/

Dow

nloaded from

http://aem.asm.org/

23. Yang, X., Gondikas, A.P., Marinakos, S.M., Auffan, M., Liu, J., Hsu-Kim, 558 H., Meyer, J.N., 2012. Mechanism of silver nanoparticle toxicity is 559 dependent on dissolved silver and surface coating in Caernorhabditis 560 elegans. Environ Sci Technol. 46(2): p. 1119-27. 561

24. Gomes, S.I.L., Soares, A. M. V. M., Scott-Fordsmand, J. J., & Amorim, M. 562 J. B. , 2013. Mechanisms of response to silver nanoparticles on 563 Enchytraeus albidus (Oligochaeta): Survival, reproduction and gene 564 expression profile. Journal of Hazardous Materials. 254: p. 336-344. 565

25. Hoheisel, S.M., Diamond, S., & Mount, D., 2012. Comparison of 566 nanosilver and ionic silver toxicity in Daphnia magna and Pimephales 567 promelas. Environmental Toxicology and Chemistry. 31(11): p. 2557-568 2563. 569

26. Fabrega J, L.S., Tyler CR, Galloway TS, Lead JR., 2011. Silver nanoparticles: 570 behaviour and effects in the aquatic environment. Environ Int.. 37(2): p. 571 517-31. 572

27. Behra, R., Sigg, L., Clift, M.J., Herzog, F., Minghetti, M., Johnston, B., Petri-573 Fink, A., Rothen-Rutishauser, B., 2013. Bioavailability of silver 574 nanoparticles and ions: from a chemical and biochemical perspective. J R 575 Soc Interface. 10(87): p. 20130396. 576

28. Topuz, E., Sigg, L., Talinli, I., 2014. A systematic evaluation of 577 agglomeration of Ag and TiO2 nanoparticles under freshwater relevant 578 conditions. Environ Pollut. 193: p. 37-44. 579

29. Sabella, S., Carney, R.P., Brunetti, V., Malvindi, M.A., Al-Juffali, N., Vecchio, 580 G., Janes, S.M., Bakr, O.M., Cingolani, R., Stellacci, F., Pompa, P.P., 2014. A 581 general mechanism for intracellular toxicity of metal-containing 582 nanoparticles. Nanoscale. 6: p. 7052-61. 583

30. Badireddy, A.R., M.R. Wiesner, and J. Liu, 2012. Detection, 584 Characterization, and Abundance of Engineered Nanoparticles in Complex 585 Waters by Hyperspectral Imagery with Enhanced Darkfield Microscopy. 586 Environmental Science & Technology. 46(18): p. 10081-10088. 587

31. Sankar, M. U., S. Aigal, S. M. Maliyekkal, A. Chaudhary, Anshup, A. A. 588 Kumar, K. Chaudhari, and T. Pradeep. 2013. Biopolymer-reinforced 589 synthetic granular nanocomposites for affordable point-of-use water 590 purification. Proceedings of the National Academy of Sciences 110:8459-591 8464. 592

593 594 595


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Documents

Downloaded from //aem.asm.org/content/aem/early/2015/10/19/AEM.02806-15.full.pdf · 49 high concentrations required (10% (v/v)), make it impractical for widespread use [5]. 50