AAC Accepted Manuscript Posted Online 1 May 2017

Antimicrob. Agents Chemother. doi:10.1128/AAC.02666-16

Copyright © 2017 American Society for Microbiology. All Rights Reserved.

1 Bacteriophage Lysin CF-301: a Potent Anti-Staphylococcal Biofilm Agent

3 Raymond Schuch,a# Babar K. Khan,a* Assaf Raz,b Jimmy A. Rotolo,a Michael Wittekinda

4 ContraFect Corporation, Yonkers, NY, USAa; Laboratory of Bacterial Pathogenesis and

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

5 Immunology, The Rockefeller University, NY, NY, USAb

6 #Address correspondence to Raymond Schuch, rschuch@contrafect.com.

7 *Present address: Babar K. Khan, KAUST, Thuwal-Jeddah, Saudi Arabia.

8 Running title: Anti-biofilm activity of lysin CF-301

10

11

12

13

14

15

16

1
17 Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of

18 constituent bacteria. Treatments for biofilm-based infections represent a major unmet

19 medical need, requiring novel agents to eradicate mature biofilms. Our objective was to

20 evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus

21 biofilms. We used minimum biofilm eradicating concentration (MBEC) assays on ninety-

22 five S. aureus strains to obtain a MBEC90 value of ≤0.25 μg/ml for CF-301. Mature biofilms

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

23 of coagulase-negative staphylococci, Streptococcus pyogenes, and Streptococcus agalactiae

24 were also sensitive to disruption, with MBEC90 values ranging from 0.25-8 μg/ml. The

25 potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene,

26 glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 hour

27 and killed all released bacteria by 6 hours. Mixed-species biofilms, formed by S. aureus and

28 S. epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms

29 either enriched for small colony variants (SCVs) or grown in human synovial fluid. The

30 antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells

31 in exponential and stationary phase populations. Finally, the anti-biofilm activity of CF-

32 301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when

33 tested against a range of S. aureus strains. In all, the data shows CF-301 is highly effective

34 at disrupting biofilms and killing biofilm bacteria, and, as such, may be an efficient new

35 agent for treating staphylococcal infections with a biofilm component.

36 Over 99.9% of bacteria in the biosphere grow within robust and resilient sessile communities

37 called biofilms (1). Biofilm formation is a precisely controlled developmental process initiated

38 by microbial aggregation on abiotic and biotic surfaces. Importantly, biofilms form in human

39 tissues, facilitating serious chronic infections of the upper respiratory tract, intestinal tract,

2
40 urinary tract, bone, heart valves, middle ear, gingiva and skin that can lead to serious illness and

41 death (2, 3). Indwelling medical devices such as intravenous catheters, stents, prosthetic joints,

42 and pacemakers are frequent sites of biofilm formation and are increasingly associated with

43 morbidity and mortality. In clinical medicine, 65-80% of bacterial infections are biofilm

44 associated, costing the healthcare system billions of dollars each year (4-6).

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

45 Biofilms are densely packed microbial populations held within DNA, polysaccharide and/or

46 proteinaceous matrices (7). In the context of human disease, the biofilm matrix helps to protect

47 bacteria from innate and adaptive immune defenses and enable up to 1000-fold increases in

48 antibiotic tolerance compared to planktonic bacteria. Biofilm microenvironments favor

49 horizontal transfer of antibiotic resistance genes (8) and drive the inactivation of antibiotics by

50 virtue of high metal ion concentrations and low pH (9). Furthermore, biofilms are permeability

51 barriers to some antibiotics (10) and biofilm communities will include subpopulations of semi-

52 dormant persister bacteria (including small colony variants [SCVs]) with phenotypic tolerance to

53 antibiotics (11). The recalcitrance of biofilms to classical antibiotic treatment strategies

54 (including prophylactic, early aggressive, and chronic suppressive therapies), combined with the

55 propensity for human pathogens, such as Staphylococcus aureus, to form and persist in biofilms,

56 have created a medical need for anti-biofilm agents (12).

57

58 There are no approved treatments to specifically target biofilms and, for this reason, research into

59 biofilm prevention, weakening, disruption and direct killing is extensive (3, 13). Several methods

60 are under development to prevent biofilm formation on artificial implants by coating the

61 indwelling surfaces with antibiotics (14, 15), antimicrobial proteins (16), or chemicals (17, 18).

62 Additional treatments to prevent and/or weaken biofilms are based on immunotherapies (19, 20)

3
63 or the use of small-molecule quorum-sensing and c-di-GMP signaling system inhibitors (21, 22),

64 antimicrobial peptides (23, 24), antibiotic cocktails (25, 26), bacteriophage (27) and matrix

65 degrading enzymes like DNase I (28), dispersin B (29), α-amylase (30) and alginate lyase (31).

66 Effective prophylactic treatments are important, since, once formed, mature biofilms are tolerant

67 to most microbiocides and may require mechanical and/or surgical interventions for removal. It

has been suggested that treatments for mature biofilms will require high-dose, long-term

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

68

69 antibiotic regimens combined with agents to weaken or disperse constituent bacteria (3).

70

71 A promising anti-biofilm strategy has recently emerged, based on the use of a family of

72 bacteriophage-encoded peptidoglycan hydrolases, called lysins (32, 33). As purified proteins,

73 lysins exhibit potent bacteriolytic activity against planktonic forms of many Gram-positive

74 pathogens (34-36). Increasingly, anti-biofilm activities have also been reported against biofilms

75 forms formed by S. aureus, Streptococcus pneumoniae and Streptococcus pyogenes (34, 37-43).

76 The use of lysins to rapidly eradicate biofilms and kill constituent bacteria represents a new

77 opportunity in the field of anti-biofilm drug development.

78 We previously described a potent anti-staphylococcal lysin, CF-301, under clinical development

79 for the treatment of S. aureus bacteremia and endocarditis (43). Significantly, we demonstrated

80 the ability of CF-301 to disrupt mature biofilms formed by the methicillin-resistant S. aureus

81 (MRSA) strain BAA-42. In the current study, we provide more detailed analyses of the in vitro

82 and ex vivo anti-biofilm activities of CF-301. The ability of CF-301 to disrupt mature biofilms

83 was demonstrated against a wide range of methicillin-sensitive S. aureus (MSSA) and MRSA

84 isolates, as well as coagulase-negative staphylococci, S. pyogenes and S. agalactiae. The ability

85 of CF-301 to both eradicate biofilm biomass and kill biofilm bacteria was demonstrated on a

4
 86 variety of surfaces, including catheters. Activity was further demonstrated against polymicrobial

87 biofilms, biofilms formed in human blood or synovial fluid, and both planktonic cultures and

88 biofilms enriched for persister forms of S. aureus. Finally, we describe a potent synergistic

89 effect between CF-301 and another cell wall hydrolase, lysostaphin. In all, this work

90 demonstrates the effectiveness of CF-301 as an anti-biofilm agent.

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

91 MATERIALS AND METHODS

92 Bacterial strains, media, antibiotics and growth conditions. Bacterial strains were stored at -

93 80°C. Strains in this study include S. aureus ATCC BAA-42 (American Type Culture Collection

94 [ATCC], Manassas, VA), S. aureus Newman (44), S. aureus MW2 (NRS 123; BEI Resources), S.

95 aureus CFS-1246 (Newman/GFP plasmid pCN57 (45) derived from NRS 623 [BEI Resources]),

96 S. aureus VRS3a (NR-46412; BEI Resources) and S. epidermidis NRS 7 (BEI Resources).

97 Other strains and isolates are described in Tables S2 and S3. The BAA-42 strain was chosen as a

98 model strain in this study based on its strong propensity to form biofilms (43); it is a pediatric

99 MRSA clone, isolated in 1996 from a pneumology ward in Lisbon, Portugal (46).

100 Bacteria were cultivated on BBL™ Trypticase™ soy agar (TSA) with 5% sheep blood

101 (Becton, Dickinson & Company [BD]), Mueller Hinton Broth (MHB; BD), MHB with 25 μg/ml

102 CaCl2 (CA-MHB), Lysogeny Broth (LB; Sigma-Aldrich), and Tryptic Soy Broth (TSB; BD)

103 with 0.2% glucose (TSBg). The following antibiotics (Sigma-Aldrich) were used: vancomycin

104 hydrochloride, daptomycin, linezolid, oxacillin sodium salt, ampicillin sodium salt, carbenicillin

105 disodium salt, ciprofloxacin and erythromycin. Lysostaphin from Staphylococcus simulans was

106 obtained from Sigma-Aldrich.

107 Biofilm survey. Biofilms were examined using a quantitative spectrophotometric

108 microtiter plate assay (47). Colonies from TSA plates were suspended in Phosphate-Buffered

5
109 Saline (PBS; Sigma-Aldrich) to 0.5 McFarland units (~1x108 CFU/ml) and diluted 1:150 in

110 TSBg. Next, 0.15 ml aliquots were added to 96-well, flat-bottom polystyrene microplates (BD

111 Falcon™, Corning Life Sciences) and incubated for 24 hours at 37°C. Plates were washed with

112 PBS and biofilm biomass was stained for 15 minutes with 0.05% crystal violet (Harleco™; EMD

113 Millipore Chemicals). For quantitation, crystal violet was solubilized in 0.2 ml of 33% acetic

114 acid (v/v) and the absorbance at an optical density of 600nm (OD600) was determined using a

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

115 SpectraMax® M3 Multi-Mode Microplate Reader (Molecular Devices, Inc.). Samples were

116 examined in triplicate and TSBg alone was used as the negative control. The OD600 values of

117 TSBg alone only were subtracted from the average OD600 of each strain. Biofilms were classified

118 as strong, moderate, or weak using the guidelines of Stepanovich et al. (47),

119 Minimum biofilm-eradicating concentration (MBEC) assay. The MBEC of CF-301

120 was determined using a broth microdilution (BMD) method, described by the Clinical and

121 Laboratory Standards Institute (48), with modifications. For S. aureus, 95 strong, moderate, and

122 weak biofilm-forming strains were chosen from Table S2. Bacteria were suspended in PBS (0.5

123 McFarland units), diluted 1:100 in TSBg, added as 0.15 ml aliquots to 96-well flat-bottom

124 polystyrene microplates, and incubated 24 hours at 37°C. Biofilms were washed and treated with

125 a 2-fold dilution series of CF-301 or daptomycin in TSBg (containing calcium for daptomycin) at

126 37°C for 24 hours. All samples were examined in triplicate. After treatment, wells were washed,

127 air-dried at 37°C, and stained with 0.05% crystal violet for 10 minutes. The loss of biofilm

128 biomass was assessed visually and crystal violet was quantitated as above. The MBEC of each

129 sample was the minimum drug concentration required to remove >95% of the biofilm biomass

130 assessed by crystal violet quantitation.

6
131 Minimum inhibitory concentration (MIC) assay. CF-301 MICs were determined in

132 MHB as described (43) with the exception that DL–dithiothreitol was not used. Daptomycin

133 MICs were determined in CA-MHB as described (48).

134 Biofilm disruption in 24-well plates. The disruption assay was performed as described

135 (49). Wells of a 24-well, flat-bottom, polystyrene tissue culture plate (Corning Life Sciences)
6
were inoculated with ~1x 10 CFU of S. aureus strain BAA-42 in 2 ml TSBg and incubated at

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

136

137 37°C for either 24 hours or 2 weeks with agitation at 50 rpm. For the 2-week growth, media was

138 aspirated every 3 days and replaced with fresh TSBg. Biofilms were washed with PBS and

139 treated with CF-301 or antibiotic for up to 24 hours. CF-301, vancomycin, and linezolid were

140 used in MHB, while daptomycin was used in CA-MHB. Media-alone controls were included.

141 After treatment, wells were washed, stained for 10 minutes with 0.05% crystal violet, washed

142 again, and photographed. The spectrophotometric measure of crystal violet staining (see above)

143 was used compare CF-301 treatments with buffer treatments according to the following equation:

144 (Absorbance value for CF-301 treatment/Absorbance value for buffer control)(100).

145 Confocal microscopy. An overnight culture of S. aureus strain Newman/pCN57(GFP+)

146 was diluted 1:1000 into 3 ml TSBg with 10 µg/ml erythromycin, in a 35 mm glass-bottom

147 microwell dish (MatTek, Ashland, MA). Dishes were incubated without agitation for 24 hours at

148 37°C, washed, and treated with CF-301 (32 μg/ml) or PBS for 1 hour at 37°C. After treatment,

149 samples were fixed in 2.6% paraformaldehyde, washed and visualized using an inverted TCS

150 SP8 laser scanning confocal microscope (Leica) using a HC PL APO CS2 40X/1.1 water

151 immersion objective. A WLL laser was used at a wavelength of 489 nm, and a HyD detector

152 was used at a range of 500-600 nm. Images were captured using the LAS AF 3.3 software

153 (Leica), and analyzed using Imaris x64 7.7.2 (Bitplane).

7
154 Biofilm disruption on glass chamber slides. Colonies of S. aureus strain BAA-42 from

155 TSA plates were suspended in PBS (0.5 McFarland units), diluted 1:100 in TSBg, and added as

156 1.0 ml aliquots to 4-chambered Nunc® Lab-Tek™ II - CC2™ Chamber Slides (Thermo Scientific).

157 Chambers were incubated for 24 hours at 37°C without agitation, washed and suspended in 1 ml

158 TSBg with or without CF-301 (32 μg/ml) for 1 hour at 37°C. Biofilms were washed, suspended

159 in PBS, and photographed. For microscopy, biofilms were stained with FilmTracer™ biofilm

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

160 stain (Life Technologies Corporation), washed, mounted in ProLong® Gold Antifade Reagent

161 (Life Technologies Corporation), and examined by Differential Interference Contrast (DIC)

162 imaging and fluorescence microscopy (magnification = 400x) using a Nikon Eclipse 80i

163 microscope. Images were captured using a QImaging QIClick digital CCD camera (QImaging

164 Inc.) and processed using NIS-Elements Imaging Software (Version 4.0). Excitation and

165 emission maxima are 494 and 514 nm, respectively. For enumeration, biofilm bacteria and

166 planktonic cells were recovered, diluted in an equivalent volume of 25 mg/ml activated charcoal

167 (Sigma-Aldrich), and plated in quadrants of TSA plates. Activated charcoal is often used for in

168 vitro studies to bind and inactivate residual antibiotic and prevent a “carry-over” effect (50). All

169 samples for CFU determinations in the current study were first diluted into charcoal. The

170 removal of biofilm bacteria (prior to plating) was performed by sonication for 1 minute using a

171 Branson® 3510 ultrasonic cleaner at 40 kHz (51) followed by gentle pipetting. Biofilm removal

172 was confirmed by crystal violet staining as above. The disruption of biofilm particles in

173 suspension, to enable accurate CFU measurements, was confirmed by bright-field microscopy

174 using a Nikon Eclipse 80i microscope (magnification = 2000x).

175 Biofilm disruption in catheters. Biofilms were formed in sterile, 5 cm segments of

® ® ®
176 SmartSite Infusion Set tubing (Alaris SE Pump Signature Edition Infusion System,

8
177 CareFusion Corporation) with a 1 mm internal diameter. The tubing is Poly(Vinyl Chloride)

178 (PVC) with Diethyl Hexyl Phthalate (DEHP) as a softening agent. Segments were inoculated
6
179 with 0.2 ml of TSBg containing 1x10 CFU/ml of S. aureus BAA-42, the ends were sealed, and

180 the tubes was incubated for 3 days at 37°C without agitation. Resulting biofilms were washed

181 and treated for 4 hours at 37°C with CF-301 (32 μg/ml) or daptomycin (1 μg/ml or 1 mg/ml) in

0.225 ml of Lactated Ringer’s (LR) solution (VWR International). The LR was the buffer control

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

182

183 and experiments were in triplicate. For each condition, one set of catheters was drained, washed,

184 air-dried, stained with methylene blue (0.05 wt. %; Sigma-Aldrich) for 15 minutes, washed, air-

185 dried, and photographed. A second set of catheters was used to quantitate biofilm bacteria using

186 the BacTiter-Glo assay described below. A third set of catheters was used for microscopy.

187 Biofilms of strain BAA-42 were also formed on the surface of pre-bisected, 1.25 cm

188 catheter segments suspended in 2 ml TSBg with either 1% human serum (pooled normal human

189 type AB serum, Sigma-Aldrich), 50 % human heat-inactivated plasma (Sigma-Aldrich) or 50%

190 heparinized human whole blood (BioreclamationIVT). Higher concentrations of blood, serum or

191 plasma did not support biofilm formation. After 3 days at 37°C without agitation, catheter

192 segments were then washed and treated for 4 hours with a dilution series of CF-301 suspended in

193 2 ml of either 100% serum, 100 % plasma or 100% whole blood (depending on the condition in

194 which the biofilms were formed). Treatments lacking CF-301 were used as controls. For each

195 condition, one set of catheters was stained with methylene blue (as above) and photographed,

196 while a second set was used to quantitate biomass and a third set was used to quantitate bacteria.

197 Indirect quantitation of catheter biofilm bacteria. Bacterial CFUs were estimated

198 using BacTiter-Glo™ Microbial Cell Viability Assay Kit (Promega). The BacTiter-Glo assay

199 provides a reliable and reproducible method for determining total cell numbers in S. aureus

9
200 biofilms (52). Briefly, treated catheters were washed and loaded with lysis solution (Lactated

201 Ringer’s solution with 100 μg/ml lysostaphin) for 8 minutes at 24°C. The intra-lumenal contents

202 were then recovered, vortexed, and mixed with an equal volume of BacTiter-Glo in solution. A

203 resulting glow-type luminescent signal is proportional to the amount of ATP present, which is

204 directly proportional to the number of cells in culture (based on a standard curve generated as

described using the BacTiter-Glo Kit. Samples were examined in triplicate.

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

205

206 Microscopic analysis of catheter biofilms. Methylene blue-stained segments were

207 bisected longitudinally using surgical scalpels and visualized using a Nikon Eclipse Ti-S inverted

208 microscope (magnification, 400x). Images were captured using a QImaging QIClick digital CCD

209 camera (QImaging Inc.) and processed using NIS-Elements Imaging Software (Version 4.0). For

210 Scanning Electron Microscopy (SEM), bisected segments were fixed in 2.5% glutaraldehyde and

211 0.075M sodium cacodylate (pH 7.4) overnight at 4⁰C, rinsed with MilliQ water, plunged into

212 N2(l), transferred into a Leica EM AFS2 chamber filled with N2(g) and adjusted to room

213 temperature over ~20 hours before being sputter coated with Gold/Palladium using a Denton

214 Vacuum Desk IV and analyzed with a Zeiss Leo 1550 scanning electron microscope.

215 Construction and analysis of GFP-CF-301BD. A 1,116-bp DNA fragment was

216 synthesized at Genewiz (South Plainfield, NJ), encoding an N-terminal poly-histidine tag, a

217 central GFP domain (GFPmut2; GenBank number AAG39983.1), and a C-terminal 126 amino

218 acid fragment of CF-301 encoding the SH3b cell wall binding domain (positions 287-665 in

219 GenBank number KC348602.1). The GFP-CF-301BD construct was cloned into the EcoRI-

220 HindIII sites of vector pBAD24 (53); expression was induced with arabinose and protein was

221 prepared as previously described (54). For binding studies, catheter were bisected and

222 suspended in PBS containing 10 μg/ml GFP-CF-301BD for 30 minutes at 24°C. Segments were

10
223 washed, air-dried, and visualized by bright-field and fluorescence microscopy using a Nikon

224 Eclipse Ti-S inverted microscope (magnification = 400x).

225 Quantitation of planktonic bacteria in treated catheters. Catheters were drained at

226 indicated time points and contents were diluted in activated charcoal, vortexed, serially diluted,

227 and plated for viability on TSA plates for 16 hours at 37°C.

228 Biofilm disruption on surgical mesh. For each condition, 4 mm2 segments of sterile

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

229 surgical mesh (1 mm thick silicone elastomer; Goodfellow Cambridge Ltd.) were suspended in 2

230 ml TSBg (containing ~1x 106 CFU of S. aureus BAA-42) in 24-well polystyrene tissue culture

231 plates (Corning Life Sciences). After 3 days at 37°C, media was aspirated and replaced with

232 fresh TSBg for 3 additional days. Treatments consisted of PBS alone and PBS with CF-301 (32

233 μg/ml) for 24 hours at 37°C. Biofilm bacteria were recovered by scraping and vortexing, and

234 CFUs were determined by plating serial dilutions on TSA plates. A spectrophotometric measure

235 of crystal violet staining of mesh biofilms was used as described above.

236 Mixed biofilms. Colonies of S. aureus strain BAA-42 and S. epidermidis strain NRS 7

237 were separately suspended in PBS (each at 0.5 McFarland units) and then diluted 1:100 and 1:50,

238 respectively, into one TSBg aliquot. A precise 2:1 ratio of S. aureus to S. epidermidis was

239 previously established, in preliminary experiments, to be absolutely required for the formation of

240 stable polymicrobial biofilms; other ratios did not support the formation of polymicrobial

241 biofilms. A final mixture of S. aureus to S. epidermidis, in a 2:1 ratio, served as the inoculum to

242 generate biofilms on different surfaces as indicated: for 24-well plates, the mixture was aspirated

243 after 2 days and replaced with fresh TSBg for 1 day, and for catheter and surgical mesh samples,

244 the mixture was aspirated after 3 days and replaced with fresh TSBg for 3 days. Biofilms were

245 washed and treated with CF-301 at 37°C for 4 or 24 hours. Samples were examined in triplicate.

11
246 Single-species biofilms were included as controls. To determine CFUs by quantitative plating,

247 biofilm bacteria on 24-well plates, catheters, and surgical mesh were recovered in PBS, as above,

248 by sonication using a Branson™ 3510 ultrasonic cleaner at 40 kHz followed by gentle pipetting.

249 Biofilm removal was confirmed by crystal violet staining and the disruption of biofilm material

250 in suspension (to enable proper CFU determinations) was confirmed by microscopy. The distinct

251 colony phenotypes of BAA-42 (golden yellow, hemolytic, ~2 mm in diameter) and NRS 7

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

252 (white, non-hemolytic, and ~1 mm in diameter) enabled differential quantitation. A

253 spectrophotometric measure of crystal violet staining of mesh biofilms was also used as

254 described above.

255 Small colony variant (SCV) biofilms. Biofilms enriched for S. aureus SCVs were

256 formed in 24-well polystyrene tissue culture plates (Corning Life Sciences) by inoculating 1x 106

257 CFUs of the biofilm forming strain BAA-42 into 2 ml TSBg with daptomycin (1.0 µg/ml),

258 vancomycin (1.0 µg/ml) oxacillin (0.5 µg/ml). CF-301 was used at 0.1 and 0.05 μg/ml, as higher

259 concentrations prevented biofilm formation. For daptomycin, media was supplemented with

260 CaCl2 to 50 µg/ml. For oxacillin, media was supplemented with 4% NaCl. After 3 days at 37°C,

261 biofilms were washed and treated with CF-301 for 24 hours at 37°C. A spectrophotometric

262 measure of crystal violet staining was used as described above.

263 For examination of the SCV phenotype of biofilm bacteria prior to CF-301 treatment,

264 biofilm biomass was first recovered in the manner described above by sonication using a

265 Branson® 3510 ultrasonic cleaner at 40 kHz followed by gentle pipetting. As above, biofilm

266 removal was confirmed by crystal violet staining of the 24-well plates, and the disruption of

267 biofilm material in suspension (to enable proper CFU determinations) was confirmed by

268 microscopy. The recovered material was then suspended in PBS containing 25 mg/ml of

12
269 activated charcoal, and dilutions thereof were plated on TSA for 24 hours at 37°C. Colonies

270 were enumerated and photographed. The SCVs were non-hemolytic, white, and <0.5 mm in

271 diameter, while the wild-type (or “normal”) colonies were hemolytic, yellow and ~2 mm in

272 diameter. To examine SCV stability, sets of up to 20 SCV colonies from each treatment were

273 subcultured twice on 5% sheep blood agar in absence of antibiotics.

Time kill studies of persister cells treated with antibiotics and CF-301. The

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

274

275 susceptibility of S. aureus persister cells to CF-301 in both stationary and exponential phase

276 cultures was examined in a manner similar to that described (11). As biofilms were not required,

277 the laboratory MRSA strain MW2 was used. For the analysis of stationary phase cultures, CaCl2

278 (50 μg/ml) and daptomycin (100 μg/ml, or 100X MIC) were added to overnight cultures and

279 incubated at 37°C with agitation for 5 hours. CF-301 was then added (to 160 μg/ml, or 5X MIC)

280 for an additional 19 hours. Prior to adding daptomycin (T=0 hours), and at 1, 2, 3, 5, 7, and 24

281 hours thereafter, culture aliquots were removed, washed in 1% NaCl, resuspended in 1% NaCl

282 containing 25 mg/ml activated charcoal, and quantitatively plated on TSA. Colonies were

283 enumerated after incubation for up to 48 hours at 37°C. Experiments were conducted in

284 triplicate. For the analysis of exponential phase bacteria, overnight cultures were diluted 1:100 in

285 MHB and grown at 37°C with agitation to OD600 0.5 before adding ciprofloxacin (3 μg/ml, or 3X

286 MIC). After 4 hours of growth with ciprofloxacin, CF-301 was added (160 μg/ml, or 5X MIC)

287 for 20 additional hours. Bacteria were quantitatively plated as above. For each experiment,

288 control cultures were included to which either no drugs were added (i.e., no antibiotic, no CF-

289 301) or to which CF-301 was added as the 5 hour pretreatment with no antibiotic.

290 CF-301 stability in LR solution. A method was used similar to that described (55). For

291 the 1-, 7-, 14- and 21-day time-points, three 1-ml aliquots of CF-301 (containing either 32, 320

13
292 or 3200 µg/ml in LR) were prepared in triplicate with and without heparin sodium salt (100

293 U/ml; Sigma-Aldrich). Experiments were performed at 37°C and at each time-point, a fresh

294 inoculum of MRSA strain MW2 (0.5 McFarland) was diluted 1:100 into each concentration of

295 CF-301 (with and without heparin, in triplicate) and incubated for 4 hours at 37°C. The CF-301

296 was inactivated with 50 µg/ml Proteinase K (Sigma-Aldrich) for 15 minutes at 24°C, and serial

dilutions were plated on TSA for enumeration. A dilution series of each bacterial inoculum was

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

297

298 plated for viability, to determine the percentage of initial activity.

299 Synergy. Checkerboards were constructed in a manner similar to that described (56).

300 Appropriate dilutions of CF 301 and lysostaphin were combined into 96-well plates containing

301 1-day-old S. aureus biofilms prepared as above. After 4 hours at 37°C, wells were washed and

302 stained with a 0.05% crystal violet solution. The removal of biofilm biomass was assessed

303 visually and by spectrophotometric analysis at OD600 of crystal violet destained in 33% acetic

304 acid. The minimum eradication concentration (MEC) for each drug in combination was

305 determined. The sum of the fractional eradicating concentrations (FECs, which are the

306 equivalents of fractional inhibitory concentrations, or FICs, with the standard broth

307 microdilution method), is calculated. Synergy exists if the sum of the two FECs (ΣFEC =

308 FECCF-301+FECLYSOSTAPHIN) is ≤0.5 (57).

309 Biofilms in human synovial fluid (HSF). Biofilms were formed over 24 hours at 37°C

310 on 0.3 cm segments of SmartSite® Infusion PVC tubing suspended in 0.25 ml HSF

311 (BioreclamationIVT) with 1x106 CFU/ml of S. aureus BAA-42. Resulting biofilms were

312 transferred to 0.25 ml of HSF containing either CF-301 (32 μg/ml), daptomycin (1 μg/ml),

313 lysostaphin (1 μg/ml), CF-301 and lysostaphin (at 4 μg/ml and 0.125 μg/ml, respectively), or

314 buffer control and incubated at 37°C for 4 or 24 hours. Samples were vortexed to dislodge

14
315 adherent bacteria and plated to determine CFUs. To confirm biofilm removal, tubing was

316 stained with methylene blue and examined by phase contrast microscopy. For SEM analyses,

317 biofilms were formed and treated in HSF with CF-301 as above.

318

319 RESULTS

320 Biofilm capacity of S. aureus. Nearly 80% (147/183) of S. aureus strains and isolates tested

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

321 were classified as strong or moderate biofilm producers, with 100% producing some adherent

322 biofilm (Table S1 and Table S2). No notable differences in the distribution of biofilm phenotypes

323 (ie., strong, moderate, or weak) were observed among the different MSSA and MRSA groups

324 analyzed, from the contemporary clinical JMI isolates to the older, well-characterized strains

325 with NRS, ATCC, and HER designations (Table S2).

326 Disruption of mature biofilms with CF-301. The minimum biofilm eradicating

327 concentration (MBEC) of CF-301 was determined using a range of staphylococci and

328 streptococci. Table 1 (and Table S3) shows that all 95 S. aureus strains are susceptible to CF-301

329 in the nanomolar range, with MBEC90 values of 0.125 and 0.25 µg/ml for MSSA and MRSA

330 respectively. There was no correlation between the propensity to form biofilms and high or low

331 MBEC values (Table S2 and Table S3). The MBEC90 value for coagulase-negative staphylococci,

332 including S. epidermidis, was 8 µg/ml, while the MBEC90 values for S. pyogenes and S.

333 agalactiae were 0.25 and 0.5 µg/ml, respectively. These findings contrast with the complete

334 resistance of staphylococcal and streptococcal biofilms to daptomycin (MBEC90 values >1024

335 µg/ml; Table 1 and Table S3).

336 Time course and titration analysis of CF-301 activity on biofilms. We previously

337 demonstrated anti-biofilm activity for CF-301 against S. aureus strain BAA-42 (43). This

15
338 analysis was extended here to show that CF-301 (at the 1X MIC of 32 µg/ml) can disrupt BAA-

339 42 biofilms on 24-well polystyrene plates within 2 hours and prevent regrowth over 24 hours

340 (Fig. 1A). In contrast, daptomycin, vancomycin, and linezolid treatments at 1000X MICs had no

341 anti-biofilm effect by 24 hours. The biofilm biomass remaining after each treatment was

342 quantitated to confirm the relative effectiveness of CF-301 over antibiotics (Fig. 1B).

343 The potency of CF-301 was examined in Fig. 1C. Disruption of 1-day-old BAA-42

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

344 biofilms in 24-well polystyrene plates required as little as 0.5 µg/ml over 24 hours. In a similar

345 titration analysis of 2-week-old biofilms, all biomass was removed by 4 hours using 32 µg/ml

346 CF-301 and by 24 hours using 0.32 µg/ml (Fig. 1D); here, daptomycin was ineffective over a

347 range of concentrations.

348 Microscopic analysis of biofilm disruption. The activity of CF-301 against biofilms

349 formed on glass slides was visualized by microscopy. In one experiment, confocal images are

350 shown for biofilms of MSSA strain Newman (expressing Green Fluorescent Protein [GFP])

351 treated for 1 hour with either buffer or CF-301 at 32 µg/ml (Fig. 2A). While the buffer treatment

352 resulted in an intact three-dimensional biofilm with a thickness of 20-25 μm (left panel), CF-301

353 removed most of the fluorescent signal (right panel). In a second experiment, DIC images and

354 corresponding fluorescence fields are shown for biofilms of MRSA strain BAA-42 treated for 1

355 hour with either buffer or CF-301 at 32 µg/ml (Fig. 2B). Here, the buffer-treated biofilm was

356 packed with bacteria and was highly fluorescent in the presence of calcein green biofilm stain

357 (left column), while the CF-301-treated sample was devoid of bacteria and fluorescence (right

358 column). The removal of BAA-42 biofilms by CF-301 (but not buffer) was confirmed both

359 visually and by determining a ~5-log difference in recoverable CFUs (Fig. 2C).

16
360 Disruption of catheter biofilms. To examine catheter biofilms, MRSA strain BAA-42

361 was grown inside PVC catheters for 3 days prior to treatment with CF-301 or daptomycin for 4

362 hours. Based on staining with methylene blue, CF-301 disrupted all biofilm biomass while

363 daptomycin and buffer had no effect (Fig. 3A). To visualize the anti-biofilm activity, the treated

364 catheters were bisected (as indicated in Fig. 3B) and examined using bright-field microscopy and

365 low-magnification SEM. “Top” views of the buffer- and daptomycin-treated biofilms show

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

366 dense bacterial networks (Fig. 3C and 3E), while “edge” views show extensions protruding from

367 growing bacterial networks (Fig. 3D). For CF-301, regardless of the view, no biofilm biomass

368 was detected (Fig. 3C-E). We confirmed these findings by fluorescence microscopy using

369 buffer- and CF-301-treated catheters stained with a GFP fusion to the cell wall binding domain

370 of CF-301 (GFP-CF-301BD). The buffer control was strongly fluorescent, while the CF-301-

371 treated biofilm (and control lacking biofilm) produced no signal (Fig. S1).

372 Potency of CF-301 against catheter biofilms. A titration analysis was performed to

373 examine the effectiveness of CF-301 against catheter biofilms formed by MRSA strain ATCC

374 BAA-42. In Fig. 4A, CF-301 concentrations ranging from 1X MIC (32 µg/ml) to 0.01X MIC

375 (0.32 µg/ml) removed all observable biofilm biomass within 4 hours. The corresponding drop in

376 biofilm bacteria was >5 log10 CFU/ml. In contrast, daptomycin had little effect on biomass

377 from 5000X MIC (5 mg/ml) to 1X MIC (1 µg/ml) and resulted in only a 1-3 log10 CFU/ml

378 decrease. In an extended analysis of five additional MRSA strains, CF-301 effectively disrupted

379 biofilms down to a concentration of 0.32 μg/ml (Table S4).

380 A series of time-course experiments were performed to further examine the potency of

381 CF-301 against biofilms formed by MRSA strain ATCC BAA-42. Biofilm biomass was

382 completely removed after only 1 hour with CF-301 at 32 µg/ml (Fig. 4B), and the corresponding

17
383 drop in biofilm bacteria was >5 log10 CFU/ml. By SEM, the anti-biofilm effects of CF-301

384 were obvious almost upon contact (Fig. 5). Within 30 seconds after adding CF-301 (32 µg/ml),

385 the biofilm matrix and the majority of bacteria were lost and remaining bacteria appeared

386 “deflated” perhaps because of cytoplasmic leakage. By 15 minutes, only bacterial debris

387 remained. In contrast, the buffer-treated biofilms appeared as robust bacterial networks

388 embedded in an extracellular matrix after 15 minutes (Fig. 5).

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

389 Susceptibility of planktonic bacteria (inside catheters) to CF-301. To address the

390 possibility that CF-301 only disrupted the biofilms on catheters and did not kill the bacterial

391 components, we followed the viability of lumenal planktonic cells after treatment with

392 daptomycin or CF-301. Here, daptomycin resulted in a <1-log10 drop in CFUs over 24 hours,

393 while CF-301 resulted in a >4-log10 drop to the threshold of detection by 6 hours (and out to 24

394 hours) (Fig. S2). These findings, taken with the results from Fig. 4B, support a sequence in

395 which CF-301 removes all biofilm bacteria by 1 hour and kills all lumenal bacteria by 6 hours.

396 Formation and disruption of mixed-species biofilms. To generate stable mixed-species

397 biofilms, an inoculum comprised of S. aureus BAA-42 (CF-301 MIC and MBEC values of 32

398 µg/ml and 0.125 µg/ml, respectively) and S. epidermidis NRS 7 (CF-301 MIC and MBEC

399 values of 256 µg/ml and 8 µg/ml, respectively) in a precise 2:1 ratio were coincubated for 3 days

400 in 24-well dishes and over 6 days in catheters and surgical mesh. The resulting biofilms were

401 consistently comprised of S. aureus and S. epidermidis in ratios of ~10:1 (Fig. 6A).

402 Mixed-species biofilms, formed on either 24-well plate, catheter or surgical mesh

403 surfaces, were susceptible to disruption by CF-301 at concentrations down to 0.032 µg/ml over

404 24 hours (Fig. 6B, D, and E and Fig. S3B). While the susceptibility of single-species S. aureus

405 biofilms was nearly identical to that of mixed-species, the single-species S. epidermidis biofilms

18
406 alone were up to 100 times more resistant. S. epidermidis biofilms were, therefore, more

407 sensitive to CF-301 when formed with S. aureus. This is particularly evident in 4 hour

408 treatments with CF-301 (Fig 6C and Fig. S3A), in which S. epidermidis was resistant to 320

409 µg/ml as a single-species and susceptible to 3.2 µg/ml as a mixed-species.

410 Disruption of biofilms enriched for small colony variants (SCVs). To generate SCVs,

411 biofilms of S. aureus strain BAA-42 were grown for 24-hours with sublethal concentrations of

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

412 antibiotics. Daptomycin, vancomycin, and oxacillin treatments selected for SCV sub-

413 populations ranging from 18.2 to 20.1% (Fig 7A and Table S5). In contrast, biofilms grown

414 either without antibiotics or with CF-301 yielded SCV subpopulations ranging from 0.5 to 1.7%

415 (Table S5).

416 Biofilms formed in either the presence or absence of daptomycin, vancomycin and

417 oxacillin exhibited MBEC values for CF-301 of 0.32 µg/ml (Fig. 7B-D). To confirm a

418 bactericidal effect for CF-301, we determined CFUs in both the adherent and planktonic cell

419 fractions of biofilms grown with daptomycin and then treated with CF-301. As shown in Fig.

420 7E, CF-301 effectively killed both biofilm and planktonic bacteria at concentrations down to the

421 MBEC value of 0.32 µg/ml. The SCVs were, therefore, susceptible to killing by CF-301.

422 Biofilms formed in the presence of sublethal concentrations of CF-301 were also completely

423 susceptible to CF-301, yielding MBEC values of 0.32 µg/ml (data not shown). No resistance to

424 CF-301 was ever observed following treatment with CF-301.

425 Killing planktonic S. aureus persister cells. Because of the importance of persister cells

426 to the antibiotic tolerance of biofilms (11), we confirmed the effectiveness of CF-301 against

427 planktonic persisters of S. aureus strain MW2. In Fig. S4A, stationary phase cultures were

428 treated with 100 μg/ml daptomycin (100X MIC) to yield a biphasic time-kill curve similar to that

19
429 previously described in which the antibiotic treatment selects for highly robust persisters (11,

430 58). Significantly, the addition of 160 µg/ml CF-301 (5X MIC) killed the entire persistent

431 subpopulation within 2 hours and prevented regrowth by 24 hours. In Fig. S4B, exponential

432 phase cultures were treated with 3 μg/ml of ciprofloxacin (3X MIC) to obtain a biphasic kill

433 curve as observed with daptomycin. Here, the addition of 160 µg/ml CF-301 killed all persisters

within 1 hour and prevented regrowth by 24 hours.

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

434

435 CF-301 does not select for persister cells. Treatments of stationary phase and

436 exponential phase S. aureus cultures with CF-301 alone resulted in monophasic time-kill curves,

437 distinct from the biphasic curves observed with antibiotics (Fig. S4A and S4B). The CFU counts

438 of each culture treated with CF-301 (with no antibiotic) rapidly declined to the threshold of

439 detection by 2 hours and remained undetectable to at least 24 hours. Consistent with our

440 findings that CF-301 kills persister cells, the treatment of either stationary phase or exponential

441 phase S. aureus with CF-301 does not select for persistent subpopulations.

442 Disruption of biofilms in human serum, plasma, and whole blood. Biofilms of MRSA

443 strain BAA-42 were formed on catheters in the presence of either 1% HuS, 50% heat-inactivated

444 plasma or 50% whole blood. After formation, the biofilms were then treated with CF-301 (or

445 buffer control) in either 100% HuS, 100% heat-inactivated plasma or 100% whole blood

446 (respectively) for 4 hours. In each case, the CF-301 treatments (but not buffer control) resulted

447 in complete biofilm removal and/or CFU reduction, at concentrations as low as 0.32 µg/ml (Fig.

448 S5A and S5B). CF-301 was, therefore, active in human blood and fractions thereof.

449 Stability of CF-301 in LR solution. Antibiotic lock therapies (ALTs) for catheters often

450 use LR solution as a vehicle (55). To explore the use of CF-301 as an ALT to maintain the

451 sterility of catheters, we examined long-term stability for CF-301 in LR. At concentrations of

20
452 320 µg/ml and 3.2 mg/ml, with or without heparin, CF-301 remained 99.9% active over 3 weeks

453 at 37°C (Table S6). At 32 µg/ml, CF-301 was 99% active after 1 day and 28.4% active by 3

454 weeks. For every condition, no changes in solution clarity, color, particulate matter, turbidity, or

455 pH was observed over 3 weeks (data not shown).

456 Synergistic anti-biofilm activity. Several different lysin pairs have been combined for

synergistic killing of planktonic bacteria (59-61). Here, synergy between CF-301 and the lysin-

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

457

458 like molecule, lysostaphin (62), was examined against strong and moderate biofilms formed by

459 ten MSSA, MRSA, VISA, and VRSA strains. Checkerboard assays were used to evaluate the

460 synergy between CF-301 and lysostaphin. Resulting isobolograms yielded concave curves and

461 ΣFECmin values of <0.5 (Fig. S6 and Table S7), indicating potent synergy. Combinations using

462 as little as 1/8th the MBECs CF-301 and lysostaphin were effective anti-biofilm agents.

463 Ex vivo analysis of biofilms formed in human synovial fluid (HSF). HSF is known to

464 support the formation of strong, antibiotic-resistant biofilms comprised of host factors

465 (including fibrin) and bacterial proteins (including fibronectin-and fibrinogen-binding proteins)

466 (63). We showed that biofilms formed by MRSA strain BAA-42 in HSF were susceptible to

467 removal and killing by CF-301 between 4 and 24 hours (Fig. S7A). Lysostaphin alone and in

468 combination with CF-301 also effectively killed biofilm bacteria, however, daptomycin alone

469 had no effect. Scanning electron micrographs of buffer-treated HSF biofilms reveal a thick

470 adherent matrix containing densely packed bacterial cells (Fig. S7B and C). After treatment with

471 CF-301, SEMs reveal a convoluted scaffolding devoid of bacteria and interspersed with

472 bacterial debris (Fig. S7D-F).

473

474 DISCUSSION

21
475 Antimicrobial agents for treating bacterial biofilms represent a major unmet medical need (3,

476 64). Aggressive antibiotic therapies can reduce biofilms and control dispersed planktonic

477 bacteria, however, eradication is difficult because of the high antibiotic concentrations that must

478 be sustained (63, 65-67). To avoid the limitations of antibiotics, a promising new strategy was

479 proposed based on the emerging understanding of bacteriophage lysins as anti-biofilm agents

480 (32, 43). Lysins can potentially destroy biofilms, kill constituent bacteria, and resensitize

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

481 released planktonic bacteria to antibiotics and immune surveillance, and, in doing so, provide

482 effective stand-alone or adjunctive therapies to treat biofilm infections.

483 The potent (and rapid) anti-biofilm activity of CF-301 was first demonstrated using an

484 initial test concentration of 32 μg/ml (1.2 μM; 1x MIC) to eliminate biofilms formed on a range

485 of surfaces, including polystyrene (i.e., 24- and 96-well microtiter plates), glass (i.e., chamber

486 slides), and PVC (i.e., catheter tubing). The sensitivity of catheter biofilms was particularly

487 notable, with the surface-removal of all biofilm bacteria and matrix elements with 15-60 minutes

488 and the complete sterilization of the intra-lumenal environment by 6 hours (Fig. 4 and Fig. S2).

489 The efficient removal and killing of biofilm bacteria by CF-301 are particularly ideal features for

490 catheter lock solutions. Catheter lock solutions are often comprised of heparin and high

491 concentrations of antibiotics (in LR solution) for instillation into the catheter lumen to sterilize,

492 treat catheter-related bacteremia, minimize complications, and avoid catheter removal (70). In

493 support of such a use, we demonstrated potent CF-301 activity in LR solution, with and without

494 heparin, over a 3 week period at 37°C (Table S7).

495 To extend our understanding of CF-301 as an anti-biofilm agent and its potential as a

496 therapeutic tool, a larger-scale study of the breadth and potency of CF-301 activity was also

497 undertaken. Toward this end, the MBEC assay was used to show the effectiveness of very low,

22
498 sub-microgram, concentrations of CF-301 in disrupting biofilms formed by a range of 95 clinical

499 and laboratory S. aureus strains (with MBEC90 values of 0.125 and 0.25 μg/ml against MSSA

500 and MRSA, respectively) (Table 1). In addition to the MBEC assay, we further demonstrated a

501 potent (and rapid) anti-biofilm activity for CF-301 at concentrations as low as 0.32 and 0.032

502 μg/ml against biofilms formed on 24 well plates (Fig. 1), catheters (Fig. 4), and silicone

503 elastomer mesh surfaces (Fig. 6). These findings suggest that the 32 μg/ml concentration of CF-

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

504 301 used in initial activity studies was in vast excess of that needed to effectively remove

505 biofilms. The potency of CF-301 may reflect an anti-biofilm mechanism involving both

506 bacteriolysis within the biofilm and cleavage of peptidoglycan in the structural framework. .

507 The effectiveness of CF-301 was further evaluated against S. aureus biofilms formed in

508 association with S. epidermidis. Multiple studies describe both cooperative and antagonistic

509 behaviors for S. aureus and other pathogens, including S. epidermidis, in the formation of mono-

510 species and mixed-species biofilms (68). While S. epidermidis deploys strategies to inhibit

511 biofilm formation by S. aureus, mixed biofilms do occur clinically with serious consequences

512 (69, 70). In the current report, we examined the sensitivity of mixed biofilms containing S.

513 aureus and S. epidermidis on polystyrene, PVC catheters, and surgical mesh. Both the mixed-

514 species biofilms and S. aureus mono-species biofilms were highly sensitive to removal with CF-

515 301 at concentrations down to 0.032 μg/ml (Fig. 6). These findings show that single and mixed-

516 species biofilms formed by the two most common pathogens that cause medical device

517 colonizations (and subsequent bacteremia) (71) can be disrupted by CF-301.

518 A hallmark of S. aureus pathogenesis is chronic persistence despite intense antibiotic therapy

519 (72). This is attributed not only to biofilms, but to SCVs and persister cells that exist as

520 subpopulations of non- or slow-growing staphylococci, comprised of phenotypic and genotypic

23
521 variants, tolerant of antibiotics (11, 72, 73). An increased understanding of such antibiotic-

522 tolerant variants in chronic disease has, in turn, led to an understanding of the need for a new

523 bactericidal agents to target them (3). In the current study, CF-301 activity was examined against

524 both SCVs and persister cells. First, biofilms were grown in either the presence of DAP, VAN or

525 OXA (to enrich for SCVs) or the absence of antibiotics and demonstrated to be equivalently

526 sensitive to CF-301 down to 0.32 μg/ml (Fig. 7). Second, persister subpopulations of both

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

527 stationary and exponential phase cultures (selected for by exposure to high concentrations of

528 DAP and CIP, respectively), were completely eliminated by CF-301. In every condition tested,

529 persister-enriched populations were killed by CF-301. Importantly, CF-301 alone did not select

530 for persistent subpopulations or SCVs, nor did it select for biofilms resistant to the anti-bioflm

531 activity of CF-301 (Fig. Table S5 and Fig. S4).

532 Our proof-of-concept studies suggest the importance of testing CF-301 in animal models

533 of biofilm-based disease (i.e., infective endocarditis and osteomyelitis). We do expect in vivo

534 biofilms to pose a significant challenge, however, as biofilms formed in host tissues will include

535 matrix proteins like fibrinogen and fibronectin that may block CF-301 (74, 75). Physical access

536 to in vivo biofilms may also be limited, in particular for heart valves, deep-seated bone or joint

537 infections, and dense fibrin-platelet thrombi in subcutaneous tissue, fascia, or muscle.

538 A series of ex vivo studies were performed to examine the potential for CF-301 against in

539 vivo biofilms. In the first study, MRSA biofilms were formed in either 1% human serum, 50%

540 heat-inactivated plasma or 50% whole human blood before treatment with CF-301 in 100%

541 serum, 100% plasma or 100% blood, respectively. In each case, CF-301 effectively disrupted

542 the biofilm biomass and killed all biofilm and planktonic CFUs at concentrations down to 0.32

543 μg/ml (Fig. S5). Neither the electrolytes and proteins found in serum, nor the host matrix

24
544 proteins found in heat-inactivated plasma or heparinized whole blood (and possibly incorporated

545 into biofilms) could inhibit CF-301 activity. The structural framework of S. aureus biofilms

546 formed in vitro in the presence of heat-inactivate plasma have, in particular, been shown to

547 incorporate high levels of host fibrin (76). These findings suggest biofilm-based infections

548 exposed to the bloodstream, like infective endocarditis, may be susceptible to CF-301.

549 In a second set of ex vivo studies, the anti-biofilm effect of CF-301 was tested in human

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

550 synovial fluid to understand the potential for treating joint infections (i.e., septic arthritis). Upon

551 introduction into synovial fluid, S. aureus is known to rapidly form biofilms comprised of large,

552 adherent aggregates enriched with fibrin and resistant to antibiotics (63, 74). In our study,

553 staphylococcal biofilms formed readily in synovial fluid and were highly susceptible to killing

554 by CF-301 (Fig. S7). After CF-301 treatment, large structural frameworks, devoid of bacterial

555 cells, were still observed. It is likely that the remaining frameworks were predominantly

556 comprised of host-derived matrix proteins, since similar treatments of biofilms formed in TSBg

557 yielded no remaining matrix after treatment with CF-301 (Fig. 5). Together, these findings show

558 CF-301 is potent in synovial fluid against biofilms containing host-derived matrix molecules.

559 In addition to demonstrating the potent anti-biofilm activity of CF-301 alone, we also showed

560 the potential for synergy with a second cell wall hydrolase, lysostaphin, to further enhance

561 activity. This finding has important practical implications, as we can envision combinations of

562 lysins providing enhanced treatment options. Additional studies to also assess synergistic anti-

563 biofilm activities between CF-301 and antibiotics are planned.

564 In conclusion, we provide evidence that CF-301 is a potent anti-staphylococcal biofilm agent.

565 Our findings support a potential role for CF-301 in treating both catheter-based and joint

566 infections, and strongly suggest the importance of examining efficacy in animal models for

25
567 biofilm-based infections, like infective endocarditis, osteomyelitis, and pneumonia (77). We

568 currently envision the anti-biofilm activity of CF-301 as a complement to antibiotic therapy in

569 treating biofilm-based infections. With CF-301 acting to degrade and disperse staphylococcal

570 biofilms and antibiotics (and CF-301) to kill the released planktonic forms and prevent

571 metastasis, this novel treatment paradigm could provide the first efficacious anti-biofilm therapy.

572

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

573 ACKNOWLEDGEMENTS

574 We acknowledge David B. Huang and Vincent A. Fischetti for helpful discussions; Chiara

575 Indiani, Cara Cassino, Paul Boni, and Gladys Moreira-Olsen for reviewing the manuscript;

576 Alfredo Barea and Nicholas Caliendo for technical assistance; Nadine Soplop and Kunihiro Uryo

577 (Electron Microscopy Resource Center at The Rockefeller University) for electron microscopy;

578 and Bernard Beall (Centers for Disease Control and Prevention) and Mary Windels (The

579 Rockefeller University) for providing streptococcal strains. The work of Raymond Schuch,

580 Babar K. Khan, Jimmy A. Rotolo and Michael Wittekind was supported by ContraFect

581 Corporation. The work of Assaf Raz was supported by U.S. Public Health Service Grant

582 AI11822 (to Vincent A. Fischetti).

583

584 REFERENCES

585

586 1. Hall-Stoodley L, Costerton JW, Stoodley P. 2004. Bacterial biofilms: from the natural
587 environment to infectious diseases. Nat Rev Microbiol 2:95-108.
588 2. Costerton JW, Stewart PS, Greenberg EP. 1999. Bacterial biofilms: a common cause
589 of persistent infections. Science 284:1318-1322.
590 3. Bjarnsholt T, Ciofu O, Molin S, Givskov M, Hoiby N. 2013. Applying insights from
591 biofilm biology to drug development - can a new approach be developed? Nat Rev Drug
592 Discov 12:791-808.

26
593 4. Davies D. 2003. Understanding biofilm resistance to antibacterial agents. Nat Rev Drug
594 Discov 2:114-122.
595 5. Potera C. 1999. Forging a link between biofilms and disease. Science 283:1837, 1839.
596 6. Lebeaux D, Chauhan A, Rendueles O, Beloin C. 2013. From in vitro to in vivo Models
597 of Bacterial Biofilm-Related Infections. Pathogens 2:288-356.
598 7. Flemming HC, Wingender J. 2010. The biofilm matrix. Nat Rev Microbiol 8:623-633.
599 8. Savage VJ, Chopra I, O'Neill AJ. 2013. Staphylococcus aureus biofilms promote
600 horizontal transfer of antibiotic resistance. Antimicrob Agents Chemother 57:1968-1970.
601 9. Hoiby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O. 2010. Antibiotic resistance of
602 bacterial biofilms. Int J Antimicrob Agents 35:322-332.
603 10. Tseng BS, Zhang W, Harrison JJ, Quach TP, Song JL, Penterman J, Singh PK,

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

604 Chopp DL, Packman AI, Parsek MR. 2013. The extracellular matrix protects
605 Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin. Environ
606 Microbiol 15:2865-2878.
607 11. Lechner S, Lewis K, Bertram R. 2012. Staphylococcus aureus persisters tolerant to
608 bactericidal antibiotics. J Mol Microbiol Biotechnol 22:235-244.
609 12. Kiedrowski MR, Horswill AR. 2011. New approaches for treating staphylococcal
610 biofilm infections. Ann N Y Acad Sci 1241:104-121.
611 13. Taylor PK, Yeung AT, Hancock RE. 2014. Antibiotic resistance in Pseudomonas
612 aeruginosa biofilms: Towards the development of novel anti-biofilm therapies. J
613 Biotechnol 191:121-130.
614 14. Meije Y, Almirante B, Del Pozo JL, Martin MT, Fernandez-Hidalgo N, Shan A,
615 Basas J, Pahissa A, Gavalda J. 2014. Daptomycin is effective as antibiotic-lock therapy
616 in a model of Staphylococcus aureus catheter-related infection. J Infect 68:548-552.
617 15. Darouiche RO, Mansouri MD, Zakarevicz D, Alsharif A, Landon GC. 2007. In vivo
618 efficacy of antimicrobial-coated devices. J Bone Joint Surg Am 89:792-797.
619 16. Belyansky I, Tsirline VB, Montero PN, Satishkumar R, Martin TR, Lincourt AE,
620 Shipp JI, Vertegel A, Heniford BT. 2011. Lysostaphin-coated mesh prevents
621 staphylococcal infection and significantly improves survival in a contaminated surgical
622 field. Am Surg 77:1025-1031.
623 17. Ketonis C, Parvizi J, Jones LC. 2012. Evolving strategies to prevent implant-associated
624 infections. J Am Acad Orthop Surg 20:478-480.
625 18. Berra L, Kolobow T, Laquerriere P, Pitts B, Bramati S, Pohlmann J, Marelli C,
626 Panzeri M, Brambillasca P, Villa F, Baccarelli A, Bouthors S, Stelfox HT, Bigatello
627 LM, Moss J, Pesenti A. 2008. Internally coated endotracheal tubes with silver
628 sulfadiazine in polyurethane to prevent bacterial colonization: a clinical trial. Intensive
629 Care Med 34:1030-1037.
630 19. Flores-Mireles AL, Pinkner JS, Caparon MG, Hultgren SJ. 2014. EbpA vaccine
631 antibodies block binding of Enterococcus faecalis to fibrinogen to prevent catheter-
632 associated bladder infection in mice. Sci Transl Med 6:254ra127.
633 20. Brady RA, O'May GA, Leid JG, Prior ML, Costerton JW, Shirtliff ME. 2011.
634 Resolution of Staphylococcus aureus biofilm infection using vaccination and antibiotic
635 treatment. Infect Immun 79:1797-1803.
636 21. O'Loughlin CT, Miller LC, Siryaporn A, Drescher K, Semmelhack MF, Bassler BL.
637 2013. A quorum-sensing inhibitor blocks Pseudomonas aeruginosa virulence and biofilm
638 formation. Proc Natl Acad Sci U S A 110:17981-17986.

27
639 22. Sambanthamoorthy K, Sloup RE, Parashar V, Smith JM, Kim EE, Semmelhack
640 MF, Neiditch MB, Waters CM. 2012. Identification of small molecules that antagonize
641 diguanylate cyclase enzymes to inhibit biofilm formation. Antimicrob Agents Chemother
642 56:5202-5211.
643 23. de la Fuente-Nunez C, Reffuveille F, Haney EF, Straus SK, Hancock RE. 2014.
644 Broad-spectrum anti-biofilm peptide that targets a cellular stress response. PLoS Pathog
645 10:e1004152.
646 24. de la Fuente-Nunez C, Korolik V, Bains M, Nguyen U, Breidenstein EB, Horsman S,
647 Lewenza S, Burrows L, Hancock RE. 2012. Inhibition of bacterial biofilm formation
648 and swarming motility by a small synthetic cationic peptide. Antimicrob Agents
649 Chemother 56:2696-2704.

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

650 25. Leite B, Gomes F, Teixeira P, Souza C, Pizzolitto E, Oliveira R. 2011. In vitro activity
651 of daptomycin, linezolid and rifampicin on Staphylococcus epidermidis biofilms. Curr
652 Microbiol 63:313-317.
653 26. Luther MK, Arvanitis M, Mylonakis E, LaPlante KL. 2014. Activity of daptomycin
654 or linezolid in combination with rifampin or gentamicin against biofilm-forming
655 Enterococcus faecalis or E. faecium in an in vitro pharmacodynamic model using
656 simulated endocardial vegetations and an in vivo survival assay using Galleria mellonella
657 larvae. Antimicrob Agents Chemother 58:4612-4620.
658 27. Alves DR, Gaudion A, Bean JE, Perez Esteban P, Arnot TC, Harper DR, Kot W,
659 Hansen LH, Enright MC, Jenkins AT. 2014. Combined use of bacteriophage K and a
660 novel bacteriophage to reduce Staphylococcus aureus biofilm formation. Appl Environ
661 Microbiol 80:6694-6703.
662 28. Kaplan JB, LoVetri K, Cardona ST, Madhyastha S, Sadovskaya I, Jabbouri S,
663 Izano EA. 2012. Recombinant human DNase I decreases biofilm and increases
664 antimicrobial susceptibility in staphylococci. J Antibiot (Tokyo) 65:73-77.
665 29. Kaplan JB. 2009. Therapeutic potential of biofilm-dispersing enzymes. Int J Artif
666 Organs 32:545-554.
667 30. Craigen B, Dashiff A, Kadouri DE. 2011. The Use of Commercially Available Alpha-
668 Amylase Compounds to Inhibit and Remove Staphylococcus aureus Biofilms. Open
669 Microbiol J 5:21-31.
670 31. Lamppa JW, Griswold KE. 2013. Alginate lyase exhibits catalysis-independent biofilm
671 dispersion and antibiotic synergy. Antimicrob Agents Chemother 57:137-145.
672 32. Fischetti VA, Nelson D, Schuch R. 2006. Reinventing phage therapy: are the parts
673 greater than the sum? Nat Biotechnol 24:1508-1511.
674 33. Entenza JM, Loeffler JM, Grandgirard D, Fischetti VA, Moreillon P. 2005.
675 Therapeutic effects of bacteriophage Cpl-1 lysin against Streptococcus pneumoniae
676 endocarditis in rats. Antimicrob Agents Chemother 49:4789-4792.
677 34. Schmelcher M, Donovan DM, Loessner MJ. 2012. Bacteriophage endolysins as novel
678 antimicrobials. Future Microbiol 7:1147-1171.
679 35. Pastagia M, Schuch R, Fischetti VA, Huang DB. 2013. Lysins: the arrival of pathogen-
680 directed anti-infectives. J Med Microbiol 62:1506-1516.
681 36. Roach DR, Donovan DM. 2015. Antimicrobial bacteriophage-derived proteins and
682 therapeutic applications. Bacteriophage 5:e1062590.

28
683 37. Sass P, Bierbaum G. 2007. Lytic activity of recombinant bacteriophage phi11 and phi12
684 endolysins on whole cells and biofilms of Staphylococcus aureus. Appl Environ
685 Microbiol 73:347-352.
686 38. Domenech M, Garcia E, Moscoso M. 2011. In vitro destruction of Streptococcus
687 pneumoniae biofilms with bacterial and phage peptidoglycan hydrolases. Antimicrob
688 Agents Chemother 55:4144-4148.
689 39. Linden SB, Zhang H, Heselpoth RD, Shen Y, Schmelcher M, Eichenseher F, Nelson
690 DC. 2014. Biochemical and biophysical characterization of PlyGRCS, a bacteriophage
691 endolysin active against methicillin-resistant Staphylococcus aureus. Appl Microbiol
692 Biotechnol 99:741-752.
693 40. Shen Y, Koller T, Kreikemeyer B, Nelson DC. 2013. Rapid degradation of

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

694 Streptococcus pyogenes biofilms by PlyC, a bacteriophage-encoded endolysin. J
695 Antimicrob Chemother 68:1818-1824.
696 41. Gutierrez D, Ruas-Madiedo P, Martinez B, Rodriguez A, Garcia P. 2014. Effective
697 removal of staphylococcal biofilms by the endolysin LysH5. PLoS One 9:e107307.
698 42. Meng X, Shi Y, Ji W, Meng X, Zhang J, Wang H, Lu C, Sun J, Yan Y. 2011.
699 Application of a bacteriophage lysin to disrupt biofilms formed by the animal pathogen
700 Streptococcus suis. Appl Environ Microbiol 77:8272-8279.
701 43. Schuch R, Lee HM, Schneider BC, Sauve KL, Law C, Khan BK, Rotolo JA,
702 Horiuchi Y, Couto DE, Raz A, Fischetti VA, Huang DB, Nowinski RC, Wittekind
703 M. 2014. Combination therapy with lysin CF-301 and antibiotic is superior to antibiotic
704 alone for treating methicillin-resistant Staphylococcus aureus-induced murine
705 bacteremia. J Infect Dis 209:1469-1478.
706 44. Baba T, Bae T, Schneewind O, Takeuchi F, Hiramatsu K. 2008. Genome sequence of
707 Staphylococcus aureus strain Newman and comparative analysis of staphylococcal
708 genomes: polymorphism and evolution of two major pathogenicity islands. J Bacteriol
709 190:300-310.
710 45. Charpentier E, Anton AI, Barry P, Alfonso B, Fang Y, Novick RP. 2004. Novel
711 cassette-based shuttle vector system for Gram-positive bacteria. Appl Environ Microbiol
712 70:6076-6085.
713 46. Sa-Leao R, Santos Sanches I, Dias D, Peres I, Barros RM, de Lencastre H. 1999.
714 Detection of an archaic clone of Staphylococcus aureus with low-level resistance to
715 methicillin in a pediatric hospital in Portugal and in international samples: relics of a
716 formerly widely disseminated strain? J Clin Microbiol 37:1913-1920.
717 47. Stepanovic S, Vukovic D, Hola V, Di Bonaventura G, Djukic S, Cirkovic I, Ruzicka
718 F. 2007. Quantification of biofilm in microtiter plates: overview of testing conditions and
719 practical recommendations for assessment of biofilm production by staphylococci.
720 APMIS 115:891-899.
721 48. Institute CLS. 2012. Methods for dilution antimicrobial susceptibility tests for bacteria
722 that grow aerobically; approved standard, 9th ed. Clinical & Laboratory Standards
723 Institute, Wayne, PA. http://antimicrobianos.com.ar/ATB/wp-
724 content/uploads/2012/11/03-CLSI-M07-A9-2012.pdf.
725 49. Izano EA, Sadovskaya I, Wang H, Vinogradov E, Ragunath C, Ramasubbu N,
726 Jabbouri S, Perry MB, Kaplan JB. 2008. Poly-N-acetylglucosamine mediates biofilm
727 formation and detergent resistance in Aggregatibacter actinomycetemcomitans. Microb
728 Pathog 44:52-60.

29
729 50. Grohs P, Fantin B, Lefort A, Wolff M, Gutmann L, Mainardi JL. 2011. Differences
730 in daptomycin and vancomycin ex vivo behaviour can lead to false interpretation of
731 negative blood cultures. Clin Microbiol Infect 17:1264-1267.
732 51. Fernandez-Barat L, Ferrer M, Sierra JM, Soy D, Guerrero L, Vila J, Li Bassi G,
733 Cortadellas N, Martinez-Olondris P, Rigol M, Esperatti M, Luque N, Saucedo LM,
734 Agusti C, Torres A. 2012. Linezolid limits burden of methicillin-resistant
735 Staphylococcus aureus in biofilm of tracheal tubes. Crit Care Med 40:2385-2389.
736 52. Stiefel P, Rosenberg U, Schneider J, Mauerhofer S, Maniura-Weber K, Ren Q.
737 2016. Is biofilm removal properly assessed? Comparison of different quantification
738 methods in a 96-well plate system. Appl Microbiol Biotechnol 100:4135-4145.
739 53. Guzman LM, Belin D, Carson MJ, Beckwith J. 1995. Tight regulation, modulation,

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

740 and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol
741 177:4121-4130.
742 54. Schuch R, Fischetti VA. 2006. Detailed genomic analysis of the Wβ and γ phages
743 infecting Bacillus anthracis: implications for evolution of environmental fitness and
744 antibiotic resistance. J Bacteriol 188:3037-3051.
745 55. Van Praagh AD, Li T, Zhang S, Arya A, Chen L, Zhang XX, Bertolami S, Mortin
746 LI. 2011. Daptomycin antibiotic lock therapy in a rat model of staphylococcal central
747 venous catheter biofilm infections. Antimicrob Agents Chemother 55:4081-4089.
748 56. Moody J. 2010. Synergy testing: broth microdilution checkerboard and broth
749 macrodilution methods, p 5.12.11-15.12.23. In Garcia LS (ed), Clinical Microbiology
750 Procedures Handbook, vol 2. ASM Press, Washington, D.C.
751 57. Tallarida RJ. 2012. Revisiting the isobole and related quantitative methods for assessing
752 drug synergism. J Pharmacol Exp Ther 342:2-8.
753 58. Mascio CT, Alder JD, Silverman JA. 2007. Bactericidal action of daptomycin against
754 stationary-phase and nondividing Staphylococcus aureus cells. Antimicrob Agents
755 Chemother 51:4255-4260.
756 59. Schmelcher M, Powell AM, Becker SC, Camp MJ, Donovan DM. 2012. Chimeric
757 phage lysins act synergistically with lysostaphin to kill mastitis-causing Staphylococcus
758 aureus in murine mammary glands. Appl Environ Microbiol 78:2297-2305.
759 60. Becker SC, Foster-Frey J, Donovan DM. 2008. The phage K lytic enzyme LysK and
760 lysostaphin act synergistically to kill MRSA. FEMS Microbiol Lett 287:185-191.
761 61. Loeffler JM, Fischetti VA. 2003. Synergistic lethal effect of a combination of phage
762 lytic enzymes with different activities on penicillin-sensitive and -resistant Streptococcus
763 pneumoniae strains. Antimicrob Agents Chemother 47:375-377.
764 62. Donovan DM. 2007. Bacteriophage and peptidoglycan degrading enzymes with
765 antimicrobial applications. Recent Pat Biotechnol 1:113-122.
766 63. Dastgheyb S, Hammoud S, Ketonis C, Liu AY, Fitzgerald K, Parvizi J, Purtill J,
767 Ciccotti M, Shapiro IM, Otto M, Hickok NJ. 2015. Staphylococcal persistence due to
768 biofilm formation in synovial fluid containing prophylactic cefazolin. Antimicrob Agents
769 Chemother 59:2122-2128.
770 64. Wu H, Moser C, Wang HZ, Hoiby N, Song ZJ. 2014. Strategies for combating
771 bacterial biofilm infections. Int J Oral Sci 7:1-7.
772 65. Hall Snyder AD, Vidaillac C, Rose W, McRoberts JP, Rybak MJ. 2014. Evaluation
773 of High-Dose Daptomycin Versus Vancomycin Alone or Combined with Clarithromycin

30
774 or Rifampin Against Staphylococcus aureus and S. epidermidis in a Novel In Vitro
775 PK/PD Model of Bacterial Biofilm. Infect Dis Ther 4:51-65.
776 66. Baldoni D, Furustrand Tafin U, Aeppli S, Angevaare E, Oliva A, Haschke M,
777 Zimmerli W, Trampuz A. 2013. Activity of dalbavancin, alone and in combination with
778 rifampicin, against meticillin-resistant Staphylococcus aureus in a foreign-body infection
779 model. Int J Antimicrob Agents 42:220-225.
780 67. Hengzhuang W, Wu H, Ciofu O, Song Z, Hoiby N. 2011.
781 Pharmacokinetics/pharmacodynamics of colistin and imipenem on mucoid and
782 nonmucoid Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 55:4469-
783 4474.
784 68. Nair N, Biswas R, Gotz F, Biswas L. 2014. Impact of Staphylococcus aureus on

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

785 pathogenesis in polymicrobial infections. Infect Immun 82:2162-2169.
786 69. Vandecandelaere I, Matthijs N, Nelis HJ, Depuydt P, Coenye T. 2013. The presence
787 of antibiotic-resistant nosocomial pathogens in endotracheal tube biofilms and
788 corresponding surveillance cultures. Pathog Dis 69:142-148.
789 70. Stoodley P, Conti SF, DeMeo PJ, Nistico L, Melton-Kreft R, Johnson S, Darabi A,
790 Ehrlich GD, Costerton JW, Kathju S. 2011. Characterization of a mixed
791 MRSA/MRSE biofilm in an explanted total ankle arthroplasty. FEMS Immunol Med
792 Microbiol 62:66-74.
793 71. Otto M. 2009. Staphylococcus epidermidis--the 'accidental' pathogen. Nat Rev Microbiol
794 7:555-567.
795 72. Conlon BP. 2014. Staphylococcus aureus chronic and relapsing infections: Evidence of a
796 role for persister cells: An investigation of persister cells, their formation and their role in
797 S. aureus disease. Bioessays 36:991-996.
798 73. Conlon BP, Rowe SE, Lewis K. 2015. Persister cells in biofilm associated infections.
799 Adv Exp Med Biol 831:1-9.
800 74. Dastgheyb S, Parvizi J, Shapiro IM, Hickok NJ, Otto M. 2015. Effect of biofilms on
801 recalcitrance of staphylococcal joint infection to antibiotic treatment. J Infect Dis
802 211:641-650.
803 75. Bjarnsholt T, Alhede M, Alhede M, Eickhardt-Sorensen SR, Moser C, Kuhl M,
804 Jensen PO, Hoiby N. 2013. The in vivo biofilm. Trends Microbiol 21:466-474.
805 76. Kwiecinski J, Kahlmeter G, Jin T. 2015. Biofilm formation by Staphylococcus aureus
806 isolates from skin and soft tissue infections. Curr Microbiol 70:698-703.
807 77. Schuch R, Sauve K, Ramirez RA, Raz A, Rotolo J, Wittekind W. 2015. Lysin CF-301
808 Activates Daptomycin in Pulmonary Surfactant in vitro and Rescues Mice from Lethal
809 Staphylococcal Pneumonia, abstr presented at the 55th Interscience Conference on
810 Antimicrobial Agents and Chemotherapy, San Diego, California,
811

812

813

814

31
815 TABLE 1 Activity of CF-301 and daptomycin (DAP) against mature biofilms

[CF-301] (μg/ml) [DAP] (μg/ml)

Organism n MBEC90 Range MBEC90 Range

MSSA 40 0.125 0.125-1 >1024 512- >1024

MRSA 55 0.25 0.125-0.5 >1024 >1024

CoNSa 46 8 0.125-32 >1024 256- >1024

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

S. pyogenes (Group A) 27 0.25 0.03-1 >1024 256- >1024

S. agalactiae (Group B) 20 0.5 0.03-1 >1024 512- >1024

a
816 Coagulase-negative staphylococci examined in this study include the following (number of

817 isolates in parentheses): S. epidermidis (21), S. warnerii (9), S. hominis (5), S. capitis (2), S.

818 saprophyticus (2), S. cohnii (1), S. hyicus (1), S. lugdunensis (2), S. sciuri (2) and S. simulans (1).

819

820

821

822

823

824

825

826

827

828

829

830

32
831 FIGURE LEGENDS

832 FIG 1 Disruption of S. aureus biofilm biomass. Biofilms of MRSA strain ATCC BAA-42 were

833 grown over 24 hours (A-C) or 2 weeks (D) in 24-well polystyrene plates and treated with either

834 CF-301 (1X MIC = 32 μg/ml) or antibiotics (1000X MIC = 1 mg/ml) in MHB or CA-MHB.

835 Media-alone controls were included. (A) Crystal violet staining of biofilms treated for 24 hours.

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

836 (B) Quantitation of crystal violet staining over a 24 hour time course. Assays were performed in

837 triplicate and OD600 data is the mean ± standard deviation. (C) Biofilms treated with a 2-fold

838 dilution series of CF-301 for 4 hours prior to crystal violet staining and quantitation. (D)

839 Biofilms treated with a 10-fold dilution series of CF-301 or DAP for 4- or 24-hours prior to

840 staining with crystal violet.

841 FIG 2 Microscopic analysis of 24 hour-old biofilms on glass surfaces. (A) Confocal analysis of

842 GFP-expressing MSSA strain Newman (CFS-1246) treated for 1 hour with buffer or CF-301 (32

843 μg/ml). The box size is 290 µm x 290 µm x 25 µm and voxel dimensions are 0.284 µm x 0.284

844 µm x 0.420 µm (Scale bars = 40.0 µm). (B) Biofilms of ATCC BAA-42 on glass chamber

845 slides, treated for 1 hour with buffer or CF-301 (32 μg/ml), and stained with FilmTracer™

846 Calcein Green Biofilm Stain. DIC images are shown with and without fluorescence

847 (magnification=400x). (C) Unmagnified biofilms of MRSA strain ATCC BAA-42 on glass

848 chamber slides, treated for 1 hour with buffer or CF-301 (32 μg/ml). After treatment, biofilm

849 CFU were determined.

850 FIG 3 Disruption of catheter biofilms. Biofilms of MRSA strain ATCC BAA-42 were grown for

851 3 days in the catheter lumen (covering half of the internal diameter) before treatment for 4 hours

852 with buffer, Daptomycin (DAP; 1 μg/ml and 1 mg/ml), or CF-301 (32 μg/ml). (A) Methylene

33
853 blue-stained biofilm biomass remaining after treatment. (B) Representation of catheter biofilms,

854 cut longitudinally to expose the internal biomass. “Top-down” and “leading-edge” views are

855 indicated. (C) Top-down view of methylene blue-stained biofilm biomass. Bright-field

856 microscopic images are shown at 400x magnification. (D) Leading-edge view of methylene

857 blue-stained biofilms. Darkly stained material represents the biofilm. Bright-field microscopic

858 images are shown at 400x magnification. (E) SEMs of catheter biofilms remaining after the

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

859 indicated treatments. The following magnifications were used (in parentheses): buffer (500x),

860 DAP (7,500x), CF-301 (7,500x) and no biofilm (7,500x).

861 FIG 4 Titration and time-course analysis of CF-301-mediated biofilm biomass disruption and

862 associated CFU reduction. Biofilms of MRSA strain ATCC BAA-42 were grown for 3 days in

863 the catheter lumen before treatment. (A) Biofilms treated for 4 hours with buffer, DAP or CF-

864 301. Drug concentrations are based MIC values; 1X MIC concentrations of DAP and CF-301

865 are 1 μg/ml and 32 µg/ml, respectively. Methylene blue-stained biofilms are shown with a

866 matched analysis of biofilm CFUs remaining after each treatment. Asterisks denote the limit of

867 detection. (B) Biofilms treated with CF-301 (32 μg/ml) over a 4 hour time course. At the

868 indicated time points, catheters were washed and biofilms were either strained with methylene

869 blue or quantitated to determine CFUs. Asterisks denote the limit of detection.

870 FIG 5 SEMs of catheter biofilms treated with CF-301. Biofilms of MRSA strain ATCC BAA-42

871 were grown for 3 days in the catheter lumen prior to either a 30 second or 15 minute treatment

872 with CF-301 (32 μg/ml). A 15 minute buffer treatment was included as a control and all

873 magnifications are indicated.

34
874 FIG 6 Formation of mixed-species biofilms and analysis of CF-301 susceptibility. Biofilms

875 containing S. aureus (strain BAA-42) and/or S. epidermidis (strain NRS-7) were grown either on

876 24-well polystyrene plates for 3 days or on catheters and surgical mesh for 6 days before

877 treatment with a 10-fold dilution series of CF-301. (A) Quantitation of S. aureus and S.

878 epidermidis CFUs in mixed biofilms prior to CF-301 treatment. Experiments were performed in

879 triplicate and CFUs represent the mean ± standard deviation. (B) Crystal violet staining of

Downloaded from http://aac.asm.org/ on May 4, 2017 by guest

880 single- and mixed-species biofilms on 24-well plates treated for 24 hours with CF-301 (relative

881 to untreated control). (C) Catheter biofilms treated for 4 hours. (D) Catheter biofilms treated for

882 24 hours. (E) Surgical mesh biofilms treated for 24 hours.

883 FIG 7 Disruption of S. aureus biofilms enriched for SCVs. To select for SCVs, biofilms of

884 MRSA strain BAA-42 were formed on 24-well plates over 3 days in the presence of daptomycin

885 (DAP; 1 µg/ml), vancomycin (VAN; 1 µg/ml), or oxacillin (OXA; 0.5 µg/ml). (A) Appearance

886 of SCVs among bacteria recovered from biofilms grown with and without DAP. SCVs are

887 indicated by arrows. (B) Crystal violet staining of biofilms after growth in the presence and

888 absence of DAP and treatment with CF-301 for 24 hours. Duplicate experiments are shown. (C)

889 Quantitation of crystal violet staining of biofilms grown in the presence and absence of DAP and

890 treated with CF-301 for 24 hours. Values were determined as a percentage of the buffer-treated

891 (i.e., no CF-301) control. (D) Quantitation of crystal violet staining of biofilms grown in the

892 presence and absence of VAN or OXA and treated with a CF-301 for 24 hours. Values were

893 determined as a percentage of the buffer-treated (i.e., no CF-301) control. (E) CF-301-mediated

894 killing of biofilm bacteria grown in the presence and absence of DAP. After 24-hour treatment

895 with CF-301, both the biofilm and planktonic cells were quantitated. Asterisks denote the limit of

896 detection.

35
Downloaded from http://aac.asm.org/ on May 4, 2017 by guest
Downloaded from http://aac.asm.org/ on May 4, 2017 by guest
Downloaded from http://aac.asm.org/ on May 4, 2017 by guest
Downloaded from http://aac.asm.org/ on May 4, 2017 by guest
Downloaded from http://aac.asm.org/ on May 4, 2017 by guest
Downloaded from http://aac.asm.org/ on May 4, 2017 by guest
Downloaded from http://aac.asm.org/ on May 4, 2017 by guest