The Dissolvable Bead: a novel in vitro biofilm model for evaluating antimicrobial resistance.

REVISED MANUSCRIPT

Dall GF1,2, Tsang STJ1,2,3, Gwynne PJ1, Wilkinson AJ1, Simpson AHRW2,3, Breusch SJB3, Gallagher MP1

1. School of Biological SciencesUniversity of EdinburghDarwin BuildingKing's BuildingsMayfield RoadEdinburghEH9 3JRUnited Kingdom

2. Department of Orthopaedic surgeryUniversity of EdinburghChancellor’s building49 Little France CrescentOld Dalkeith RoadEdinburghEH16 4SBUnited Kingdom

3. Department of Orthopaedic surgeryRoyal Infirmary of Edinburgh51 Little FranceOld Dalkeith RoadEdinburghEH16 4SAUnited Kingdom

Word count

Text: 2653References: 1336Figures: 161

1

Abstract

In vitro biofilm assays are a vital first step in the assessment of therapeutic effectiveness.

Current biofilm models have been found to be limited by throughput, reproducibility, and

cost. We present a novel in vitro biofilm model, utilising a sodium alginate substratum for

surface biofilm colony formation, which can be readily dissolved for accurate evaluation of

viable organisms. The dissolving bead biofilm assay was evaluated using a range of

clinically relevant strains. The reproducibility and responsiveness of the assay to an

antimicrobial challenge was assessed using standardised methods. Cryo-scanning electron

microscopy was used to image biofilm colonies. Biofilms were grown for 20 hours prior to

testing. The model provides a reproducible and responsive assay to clinically-relevant

antimicrobial challenges, as defined by established guidelines. Moreover cryo-scanning

electron microscopy demonstrates that biofilm formation is localised exclusively to the

alginate bead surface.

Our results suggest that this simple model provides a robust and adaptable assay for

the investigation of bacterial biofilms.

2

Introduction

In vitro biofilm assays are a crucial first step in the assessment of therapeutic

effectiveness (Coenye and Nelis, 2010). The choice of biofilm model is dependent upon

several aspects in the in vitro design: selection of a suitable and uniform platform for

generating and testing the biofilms, selection of appropriate physical state conditions that can

be analysed using the platform, and definition of appropriate end-points. The evaluation of

antimicrobials based on the traditional susceptibility methods, such as the Minimum

Inhibitory Concentration (MIC), is widely recognised to be a poor predictor for microbial

biofilm eradication, commonly resulting in treatment failure (Girard et al., 2010; Olson et al.,

2002). The Minimum Biofilm Eradication Concentration (MBEC) is now widely recognised

and is defined as the lowest concentration of antimicrobial that eradicates 99.9% of the

bacteria in a biofilm state compared to growth controls in the same conditions (Wayne,

1999). This reduction provides a much more robust approximation to the expected in vivo

effect. The MBEC is generally 100-1000 times greater than the MIC (Girard et al., 2010;

Olson et al., 2002).

Several biofilm models have been described, such as the microtiter plate biofilm

assay; constant depth film fermentors; rotating reactors; perfused membrane models; drip

flow biofilm reactors; and flow cells (Coenye and Nelis, 2010; McBain, 2009). These models

are reliable, but can be limited by their throughput (Coenye and Nelis, 2010). Simple agar

plate methods are also described but can be unsuitable in some situations because of their use

of the substratum as the source of nutrition, the exposure of the biofilm to air and the

influence of differing rates of antibiotic diffusion throughout the agar medium (Anderl et al.,

2000; Coenye and Nelis, 2010; Walters et al., 2003). The microtiter plate biofilm assay is a

static biofilm model that utilises crystal violet staining to indirectly quantify biofilm

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

3

formation but does not allow direct cell enumeration (Christensen et al., 1985; Merritt et al.,

2005). The Modified Robbins Device provides a linear array of six ports along a rectangular

cross-section channel that the media flows through, onto which coupons of substrate can be

placed during an experiment for susceptibility testing (McCoy et al., 1981). Although it has

been extensively used for bacterial susceptibility testing, it relies on unidirectional shear and

requires intermittent sterilisation which limits throughput and risks contamination. The multi-

well plate technique described and patented by the Calgary group as the MBECTM device

(Ceri et al., 1999; Harrison et al., 2010) is widely used and capable of high throughput.

However, it has been shown to have a number of limitations which impact reproducibility

(Almshawit et al., 2014; Bridier et al., 2010; Coenye and Nelis, 2010; Coraça-Hubér et al.,

2012; Macia et al., 2014; Monsen et al., 2009; Ren et al., 2014).

These limitations led to the development of an alternative and novel method for

investigating the effects of antibiotics on bacterial biofilms. The method presented here

eliminates these concerns and allows rapid, reproducible growth and enumeration of bacterial

biofilms. Established biofilms are easily liberated from the alginate platform by dissolving

the substrate, thus removing the need to physically disrupt the biofilm. Previous techniques

have scraped off the biofilms (Neut et al., 2006), but this has shown to be inferior to

sonicating the biofilms away from the substratum, resulting in significant assay variance

(Bjerkan et al., 2009). Although low-level sonication does not seem to affect the viability of

staphylococci it has been shown to reduce the viability of Gram negative and anaerobic

bacteria (Monsen et al., 2009). A technique using sodium alginate beads has previously been

used for cellular immobilisation (Takata et al., 1977). It has also been shown that sodium

alginate can be chelated and then dissolved to liberate immobilised bacteria without

compromising their viability (Behrendt et al., 2012; Mater et al., 1995; Pedersen et al., 1990;

Sønderholm et al., 2017). We have combined these principles to grow and quantifiably

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

4

recover surface colonies on the alginate to facilitate the study of the biofilm. Initially, as part

of a proof of principle, the approach has been used to examine the effect of locally delivered

antibiotic combinations in the treatment of staphylococcal biofilms, modelling a prosthetic

joint infection (PJI) (Trampuz et al., 2003; Zimmerli et al., 2004).

In this present study the authors aimed to develop an in vitro biofilm model that was:

1) reproducible, 2) allowed the testing of biofilm-forming isolates with antimicrobials 3)

quantitated antimicrobial effectiveness, 4) used a clinically-relevant outcome, and 5) was cost

effective in terms of materials, equipment, and laboratory time.

Materials and methods

Bacterial strains and Media

The following bacterial strains were used; a methicillin-sensitive Staphylococcus

aureus (MSSA-N) reference strain (ATCC #29213), which has been extensively studied in its

biofilm state (Ceri et al., 1999; Pettit et al., 2009; Zimmerli et al., 1994); a coagulase-negative

Staphylococcus (CNS-J) clinical isolate from an infected hip replacement; a Streptococcus

mutans reference strain (NCTC #10923); an Escherichia coli reference strain (ATCC

#25922); an Enterococcus faecalis clinical isolate from an infected heart valve; a Klebsiella

pneumoniae clinical isolate from a ventilator associated pneumonia; a Pseudomonas

aeruginosa clinical isolate from the sputum of an infective exacerbation of bronchiectasis.

Bacterial strains were cultured aerobically in Luria broth (LB) overnight at 37°C prior to

use. LB used in this study was: Bacto tryptone (Difco) (10 g), Bacto yeast extract (Difco) (5

g) and NaCl (10 g), dissolved up to 1L of dH2O (pH 7.2 prior to autoclaving). LB agar was

solidified by adding 15 g/L agar (Difco) prior to autoclaving.

Antibiotics

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

5

Gentamicin (Cidomycin® 4000mg/L Sanofi Aventis, Guildford GU1 4YS) was used in

this study. It was stored and prepared as per the manufacturer’s instructions prior to use

(Andrews, 2001).

Preparation of alginate beads

Beads were prepared using commercially-available Sodium alginate, extracted from the

brown alga (Fisher Scientific, CAS number 9005-38-3) using a wt/vol ratio of 4.0%. Alginate

is a linear, anionic polysaccharide consisting of two forms of 1, 4-linked hexuronic acid

residues: β-d-mannuronopyranosyl (M) and α-l- guluronopyranosyl (G) residues (Yang et al.,

2011). For 100 mL alginate solution, 4.0 g Sodium alginate was dissolved in 100 ml de-

ionised water with agitation and then autoclaved at 120°C for 20 min. A 100mL alginate

solution and 50µL CaCl2 was sufficient to make around 200 beads. Calcium ions are chelated

by Sodium alginate and induce gelation, in an egg-box arrangement of chain−chain

associations (Morris et al., 1978). Sterile reagents were added, in the order listed, into each of

the 96 U-shaped wells (Greiner) using an 8-channel pipette under sterile conditions: 1) 10µl

of 2M CaCl2; 2) 200µl 4% Sodium Alginate (Back-pipetted to avoid clogging the tips); 3)

20µl of 2M CaCl2. Well plates were sealed, incubated at 60°C for 4 hours, and then stored at

4°C. The mean total surface area was 160.31 ±0.05 mm2 and mean weight was 213.1±2.41 µg

for a single alginate bead.

Growth and challenge of biofilms

The alginate beads were transferred, under sterile conditions, to a 104 CFU ml-1 culture

in LB. Care was taken to ensure beads were completely submerged and free to circulate

within the culture (6 beads/10mls culture). The beads were incubated at 37°C and 150 rpm

for 20 hours. Beads were submerged in sterile water for 60s to remove non-adherent cells.

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

6

The beads were then transferred to antibiotic challenge before being rinsed for a second time

in sterile water.

Recovery of organisms

A dissolving solution was used to liberate the organisms from the alginate beads.

Each bead was placed in 2ml final dissolving solution, crushed using sterile glass-stirring rod,

and placed on a rotatory suspension mixer until the bead was completely dissolved. After

serial dilutions were performed using phosphate-buffered saline 10µl drops of each dilution

was plated onto LB-agar and incubated at 37°C (Miles et al., 1938). Plates were manually

enumerated after 24 hours (48 hours if no growth was initially seen at 24 hours). The

dissolving solution used was: 0.05M Na2CO3 and 0.02M Citric acid (pH 6.8), dissolved in

dH20, filter sterilised (0.20µm filter), and stored at -20°C.

Microtiter assay

The microtiter plate assay was performed, using the MSSA-N reference strain, as described

previously by O’Toole et al (2011).

Cryo-scanning electron microscopy

Following biofilm formation beads were rapidly frozen using a Gatan ALTO 2500

Cryotransfer module in its native hydrated state to preserve the biofilm architecture. Each

bead was freeze-fractured to expose its internal microstructure and sputter-coated with gold-

palladium to allow higher resolution surface imaging A FEI F20 electron microscope (200

kV, field emission gun) equipped with an 8k x 8k CMOS camera was used. Images were

processed using IMAGIC processing software (Image Science Software GmbH, Gillweg 3,

Berlin, Germany)

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

7

Data handling, graphical illustration and statistical analysis

Colony counts were the average of three biological repeats, each with three beads as

technical replicates. A standard deviation (SD) <0.6 was deemed to be sufficient precision

(Goeres et al., 2005). A one-sample Students t-test was used in the comparison between

control and test conditions to assess responsiveness and reproducibility of response to an

antimicrobial challenge. A p-value <0.05 was deemed to be statistically significant. Data

were analysed using GraphPad Prism 6 for Mac OS X software for statistical analysis and

graphing.

Results

Criteria for assessment

As part of the initial evaluation of the dissolvable system an established system for

analysing the data (Parker et al., 2014) was used in order to facilitate comparisons with other

in vitro biofilm models. The central tenets of the approach were to assess 1) reproducibility of

control data 2) responsiveness to antimicrobial challenges and 3) reproducibility of response

to antimicrobial challenges.

Reproducible growth on beads

Data from all 40 independent unchallenged controls of the CNS-J clinical isolate and

35 independent unchallenged controls of the MSSA-N reference strain experiments were

included and shown in Figure 1. A satisfactory level of reproducibility was found with a

mean cell number (CFU mL-1) Log107.00 ± 0.39. For the MSSA-N reference strain there was

a mean cell number (CFU mL-1) Log107.12±0.27. These levels of variance (CNS-J (SD 0.39)

and MSSA-N (SD 0.27) fall within the recognised parameters for biofilm model

reproducibility (Goeres et al., 2005) indicating that the model protocol was suitably robust.

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

8

The reproducibility across other clinically relevant species was also evaluated. Similar cell

numbers and experimental variation were seen with both the Gram negative and non-

staphylococcal Gram-positive strains used in this study (Fig. 1).

Figure 1. Reproducibility of growth controls in numerous species. Formation of biofilm after 24 hours

growth using the alginate bead experimental protocol. Gram positive organisms (light coloured) Gram

negative organisms (dark coloured)

Response to antibiotic challenge

In order to assess the responsiveness of the model, the alginate bead biofilms were

exposed to gentamicin for three hours to obtain an eradication curve. The responsiveness of

the model was compared to the crystal violet microtiter plate assay. Each gentamicin

concentration was tested using three biological replicates, with each replicate undergoing

three technical repeats. The mean values of the replicates were plotted for each gentamicin

concentration (Fig. 2). The lowest gentamicin concentration with a log10 reduction of three

was 64 mgL-1 (Fig. 2), which was identified as the MBEC, as per recognised standards

(Wayne, 1999). The precision of the estimated response to a gentamicin challenge was

assessed using the variance between biological replicates. Guidelines suggest that a SD < 0.7

would be a satisfactory level of precision (Tilt and Hamilton, 2002). All concentrations

produced repeatable log10 reductions using this definition. To highlight the responsiveness of

the dissolving bead assay the same gentamicin challenges were performed using the

microtiter biofilm assay. No changes in optical density were seen with the microtiter assay

with varying gentamicin concentrations (Fig. 3).

Figure 2. Eradication curve of gentamicin. MSSA-N exposed to gentamicin for 3 hours. (Error bars:

standard deviation)

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

9

Figure 3. Comparison of assay responsiveness to an antibiotic challenge. MSSA-N exposed to gentamicin

for 3 hours. (Error bars: standard deviation). Dissolving bead data (shown in grey) taken from Figure 2.

Surface localisation of growth

Cryo-scanning electron microscopy was performed to assess the distribution of the

biofilms on the dissolvable beads. It was chosen as it minimised damage to the beads and the

biofilm and also allowed the beads to be fractured without contamination of the interior

surface. In Figure 4 the presence of an extracellular polymeric substance (EPS) in close

association with the S. aureus colonies confirmed that biofilms were growing on the alginate

bead surface. A cross sectional image of the bead did not show the presence of organisms or

EPS penetrating the bead surface (Fig. 4C) suggesting that the biofilm was homogenously

exposed to the test conditions.

Figure 4. Alginate bead and Cryo-scanning electron microscopy images. Fig. 4A. Photograph of an intact

alginate bead (left) and a fractured alginate bead (right); Fig. 4B. Cryo-SEM image taken of the bead surface

showing the extracellular polymeric substance (EPS), secreted by and encasing the colonies of S. aureus. The

undamaged alginate bead substratum is seen in the background; Fig. 4C Cryo-SEM image taken of a fractured

frozen bead. No organisms are seen within the alginate bead core below the bead surface.

Discussion

We present an in vitro biofilm model that reproducibly generates clinically relevant

antibiotic susceptibility data of multiple species in a cost- and time-efficient manner.

Application of such a model is desirable in the field of biofilm research, particularly biofilm-

associated healthcare infections such as PJI. (Girard et al., 2010; Shirtliff and Leid, 2009;

Stoica et al., 2017).

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

10

Assessment of the alginate biofilm model

The method was found to have satisfactory reproducibility of controls (Feldsine et al.,

n.d.; Goeres et al., 2005; No authors, 2008) (Fig. 1) and responsiveness of staphylococcal

biofilms (Fig. 2). The precision of estimated response to a gentamicin challenge was even

satisfactory around the critical levels used to define the MBEC (Fig. 2). Previous methods

using submerged substratum (Cerca et al., 2005; Harrison et al., 2010; Olson et al., 2002) and

in studies that have used a time-kill kinetic assessment method (Baldoni et al., 2010; Moriarty

et al., 2005; Smith et al., 2009) have been found to have declining precision around the

MBEC. This variable response to antimicrobials has previously been attributed to the

physiological heterogeneity of bacterial cells within the biofilm (Stewart and Franklin, 2008).

Uniformity of beads

The cryo-scanning electron microscopy technique preserved the extracellular

polymeric substance and biofilm structure well. This demonstrates that the biofilm appeared

to be exclusively localised to the surface of the bead, thus ensuring homogenous exposures to

nutrients and antimicrobials. The findings are consistent with previous models that found

10% of the silastic substratum in urethral catheters were colonised with a S. aureus biofilm 5-

6 cells thick within 2 hours of exposure (Jones et al., 2001).

Conclusion

The dissolving alginate bead model produces reproducible control data of

staphylococcal and non-staphylococcal biofilms, and reliably quantifies their eradication, as

measured by reduction in cell counts. The alginate biofilm model is inexpensive in terms of

materials, lends itself to a moderate throughput, and is readily adaptable. Further

modifications to the alginate gel might allow it to be used as a dissolvable coating to cover

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

11

the 96-pin microtiter lid of the MBEC device. Such an approach would improve throughput

and enhance recovery of viable organisms of the dissolvable bead assay. It may also be

possible to incorporate antimicrobials into the alginate matrix to mimic impregnated bone

cements and similar delivery systems. The model protocol could also be adapted to

incorporate optical density measurements to evaluate bacterial regrowth (Lindqvist, 2006).

Our results suggest that this approach is reliable and relatively inexpensive.

Funding

This work was supported by the British Hip Society (Grant to G.D), London, United

Kingdom.

Conflicts of interest

None to declare

References

Almshawit, H., Macreadie, I., Grando, D., 2014. A simple and inexpensive device for biofilm

analysis. J Microbiol Methods 98, 59–63. doi:10.1016/j.mimet.2013.12.020

Anderl, J.N., Franklin, M.J., Stewart, P.S., 2000. Role of antibiotic penetration limitation in

Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob

Agents Chemother 44, 1818–24.

Andrews, J.M., 2001. Determination of minimum inhibitory concentrations. J Antimicrob

Chemother 48, 5–16. doi:10.1093/jac/48.suppl_1.5

Baldoni, D., Steinhuber, A., Zimmerli, W., Trampuz, A., 2010. In vitro activity of gallium

maltolate against staphylococci in logarithmic, stationary, and biofilm growth phases:

Comparison of conventional and calorimetric susceptibility testing methods. Antimicrob

Agents Chemother 54, 157–163. doi:10.1128/AAC.00700-09

229

230

231

232

233

234

235

236

237

238

239

240

241

242243244245

246

247

248

249

250

251

252

253

254

255

12

Behrendt, L., Schrameyer, V., Qvortrup, K., Lundin, L., Sørensen, S.J., Larkum, A.W.D.,

Kühl, M., 2012. Biofilm growth and near-infrared radiation-driven photosynthesis of the

chlorophyll d-containing cyanobacterium Acaryochloris marina. Appl Environ

Microbiol 78, 3896–904. doi:10.1128/AEM.00397-12

Bjerkan, G., Witsø, E., Bergh, K., 2009. Sonication is superior to scraping for retrieval of

bacteria in biofilm on titanium and steel surfaces in vitro. Acta Orthop 80, 245–250.

doi:10.3109/17453670902947457

Bridier, A., Dubois-Brissonnet, F., Boubetra, A., Thomas, V., Briandet, R., 2010. The biofilm

architecture of sixty opportunistic pathogens deciphered using a high throughput CLSM

method. J Microbiol Methods 82, 64–70. doi:10.1016/j.mimet.2010.04.006

Cerca, N., Martins, S., Cerca, F., Jefferson, K.K., Pier, G.B., Oliveira, R., Azeredo, J., 2005.

Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci

in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT

colorimetry. J Antimicrob Chemother 56, 331–336. doi:10.1093/jac/dki217

Ceri, H., Olson, M.E., Stremick, C., Read, R.R., Morck, D., Buret, A.A., 1999. The Calgary

Biofilm Device: New Technology for Rapid Determination of Antibiotic Susceptibilities

of Bacterial Biofilms. J Clin Microbiol 37, 1771–1776.

Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett, F.F., Melton,

D.M., Beachey, E.H., 1985. Adherence of coagulase-negative staphylococci to plastic

tissue culture plates: a quantitative model for the adherence of staphylococci to medical

devices. J Clin Microbiol 22, 996–1006.

Coenye, T., Nelis, H.J., 2010. In vitro and in vivo model systems to study microbial biofilm

formation. J Microbiol Methods 83, 89–105. doi:10.1016/j.mimet.2010.08.018

Coraça-Hubér, D.C., Fille, M., Hausdorfer, J., Pfaller, K., Nogler, M., 2012. Evaluation of

MBECTM-HTP biofilm model for studies of implant associated infections. J Orthop Res

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

13

30, 1176–1180. doi:10.1002/jor.22065

Feldsine, P., Abeyta, C., Andrews, W.H., AOAC International Methods Committee, n.d.

AOAC International methods committee guidelines for validation of qualitative and

quantitative food microbiological official methods of analysis. J AOAC Int 85, 1187–

200.

Girard, L.P., Ceri, H., Gibb, A.P., Olson, M., Sepandj, F., 2010. MIC Versus MBEC to

Determine the Antibiotic Sensitivity of Staphylococcus aureus in Peritoneal Dialysis

Peritonitis. Perit Dial Int 30, 652–656. doi:10.3747/pdi.2010.00010

Goeres, D.M., Loetterle, L.R., Hamilton, M.A., Murga, R., Kirby, D.W., Donlan, R.M., 2005.

Statistical assessment of a laboratory method for growing biofilms. Microbiology 151,

757–762. doi:10.1099/mic.0.27709-0

Harrison, J.J., Stremick, C.A., Turner, R.J., Allan, N.D., Olson, M.E., Ceri, H., 2010.

Microtiter susceptibility testing of microbes growing on peg lids: a miniaturized biofilm

model for high-throughput screening. Nat Protoc 5, 1236–54. doi:10.1038/nprot.2010.71

Jones, S.M., Morgan, M., Humphrey, T.J., Lappin-Scott, H., 2001. Effect of vancomycin and

rifampicin on meticillin-resistant Staphylococcus aureus biofilms. Lancet 357, 40–41.

doi:10.1016/S0140-6736(00)03572-8

Lindqvist, R., 2006. Estimation of Staphylococcus aureus growth parameters from turbidity

data: characterization of strain variation and comparison of methods. Appl Environ

Microbiol 72, 4862–70. doi:10.1128/AEM.00251-06

Macia, M.D., Rojo-Molinero, E., Oliver, A., 2014. Antimicrobial susceptibility testing in

biofilm-growing bacteria. Clin Microbiol Infect 20, 981–990. doi:10.1111/1469-

0691.12651

Mater, D.D.G., Jean-Noel, B., Jose Edmundo, N.S., Nicole, T., Daniel, T., 1995. Effect of

gelation temperature and gel-dissolving solution on cell viability and recovery of two

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

14

Pseudomonas putida strains co-immobilized within calcium alginate or kappa-

carrageenan gel beads. Biotechnol Tech 9, 747–752. doi:10.1007/BF00159242

McBain, A., 2009. In vitro biofilm models: an overview, in: Advances in Applied

Microbiology. pp. 99–132.

McCoy, W.F., Bryers, J.D., Robbins, J., Costerton, J.W., 1981. Observations of fouling

biofilm formation. Can J Microbiol 27, 910–7.

Merritt, J.H., Kadouri, D.E., O ’toole, G.A., 2005. Growing and Analyzing Static Biofilms.

Curr Protoc Microbiol 1. doi:10.1002/9780471729259.mc01b01s00

Miles, A., Misra, S., Irwin, J., 1938. The Estimation of the Bactericidal Power of the Blood. J

Hyg (Lond) 6, 732–749.

Monsen, T., Lövgren, E., Widerström, M., Wallinder, L., 2009. In vitro effect of ultrasound

on bacteria and suggested protocol for sonication and diagnosis of prosthetic infections.

J Clin Microbiol 47, 2496–2501. doi:10.1128/JCM.02316-08

Moriarty, F., Elborn, S., Tunney, M., 2005. Development of a rapid colorimetric time-kill

assay for determining the in vitro activity of ceftazidime and tobramycin in combination

against Pseudomonas aeruginosa. J Microbiol Methods 61, 171–179.

doi:10.1016/j.mimet.2004.11.010

Morris, E.R., Rees, D.A., Thom, D., Boyd, J., 1978. Chiroptical and stoichiometric evidence

of a specific, primary dimerisation process in alginate gelation. Carbohydr Res 66, 145–

154. doi:10.1016/S0008-6215(00)83247-4

Neut, D., Hendriks, J.G.E., van Horn, J.R., Kowalski, R.S.Z., van der Mei, H.C., Busscher,

H.J., 2006. Antimicrobial efficacy of gentamicin-loaded acrylic bone cements with

fusidic acid or clindamycin added. J Orthop Res 24, 291–299. doi:10.1002/jor.20058

No authors, 2008. Guide to the expression of uncertainty in measurement.

O’Toole, G.A., 2011. Microtiter dish biofilm formation assay. J Vis Exp. doi:10.3791/2437

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

15

Olson, M.E., Ceri, H., Morck, D.W., Buret, A.G., Read, R.R., 2002. Biofilm bacteria:

Formation and comparative susceptibility to antibiotics. Can J Vet Res 66, 86–92.

Parker, A.E.E., Walker, D.K.K., Goeres, D.M.M., Allan, N., Olson, M.E.E., Omar, A., 2014.

Ruggedness and reproducibility of the MBEC biofilm disinfectant efficacy test. J

Microbiol Methods 102, 55–64. doi:10.1016/j.mimet.2014.04.013

Pedersen, S.S., Shand, G.H., Hansen, B.L., Hansen, G.N., 1990. Induction of experimental

chronic Pseudomonas aeruginosa lung infection with P. aeruginosa entrapped in

alginate microspheres. APMIS 98, 203–211. doi:10.1111/j.1699-0463.1990.tb01023.x

Pettit, R.K., Weber, C.A., Pettit, G.R., 2009. Application of a high throughput Alamar blue

biofilm susceptibility assay to Staphylococcus aureus biofilms. Ann Clin Microbiol

Antimicrob 8, 1–7. doi:10.1186/1476-0711-8-28

Ren, D., Madsen, J.S., de la Cruz-Perera, C.I., Bergmark, L., Sørensen, S.J., Burmølle, M.,

2014. High-Throughput Screening of Multispecies Biofilm Formation and Quantitative

PCR-Based Assessment of Individual Species Proportions, Useful for Exploring

Interspecific Bacterial Interactions. Microb Ecol 68, 146–154. doi:10.1007/s00248-013-

0315-z

Shirtliff, M., Leid, J., 2009. The role of biofilms in device-related infections. Heidelberg.

Smith, K., Perez, A., Ramage, G., Gemmell, C.G., Lang, S., 2009. Comparison of biofilm-

associated cell survival following in vitro exposure of meticillin-resistant

Staphylococcus aureus biofilms to the antibiotics clindamycin, daptomycin, linezolid,

tigecycline and vancomycin. Int J Antimicrob Agents 33, 374–8.

doi:10.1016/j.ijantimicag.2008.08.029

Sønderholm, M., Kragh, K.N., Koren, K., Jakobsen, T.H., Darch, S.E., Alhede, M., Jensen,

P.Ø., Whiteley, M., Kühl, M., Bjarnsholt, T., 2017. Pseudomonas aeruginosa Aggregate

Formation in an Alginate Bead Model System Exhibits In Vivo-Like Characteristics.

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

16

Appl Environ Microbiol 83, e00113-17. doi:10.1128/AEM.00113-17

Stewart, P.S., Franklin, M.J., 2008. Physiological heterogeneity in biofilms. Nat Rev

Microbiol 6, 199–210. doi:10.1038/nrmicro1838

Stoica, P., Chifiriuc, M.C., Rapa, M., Lazăr, V., 2017. 1 – Overview of biofilm-related

problems in medical devices, in: Biofilms and Implantable Medical Devices. pp. 3–23.

doi:10.1016/B978-0-08-100382-4.00001-0

Takata, I., Tosa, T., Chibata, I., 1977. Screening of matrix suitable for immobilization of

microbial cells. J Solid-Phase Biochem 2, 225–236. doi:10.1007/BF02996744

Tilt, N., Hamilton, M.A., 2002. Repeatability and reproducibility of germicide tests: a

literature review. J AOAC Int 82, 384–9.

Trampuz, A., Steckelberg, J.M., Osmon, D.R., Cockerill Iii, F.R., Hanssen, A.D., Patel, R.,

2003. Advances in the laboratory diagnosis of prosthetic joint infection. Rev Med

Microbiol 14, 1–14.

Walters, M.C., Roe, F., Bugnicourt, A., Franklin, M.J., Stewart, P.S., Stewart, P.S., 2003.

Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to

tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin.

Antimicrob Agents Chemother 47, 317–23. doi:10.1128/aac.47.1.317-323.2003

Wayne, P., 1999. Methods for Determining Bactericidal Activity of Antimicrobial Agents;

Approved Guidelines:NCCLS.

Yang, J.-S., Xie, Y.-J., He, W., 2011. Research progress on chemical modification of

alginate: A review. Carbohydr Polym 84, 33–39. doi:10.1016/j.carbpol.2010.11.048

Zimmerli, W., Frei, R., Widmer, A.F., Rajacic, Z., 1994. Microbiological tests to predict

treatment outcome in experimental device-related infections due to Staphylococcus

aureus. J Antimicrob Chemother 33, 959–967.

Zimmerli, W., Trampuz, A., Ochsner, P.E., 2004. Prosthetic-joint infections. N Engl J Med

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

17

351, 1645–54. doi:10.1056/NEJMra040181381

382

18