In agar dilution tests EGCG reduced zones of inhibition surrounding Ag NP disĐs ďLJ шϭ ŵŵ agaiŶst all ďaĐterial isolates. Hoǁeǀer, zoŶes of iŶhiďitioŶ produced by Ag NPs are often small, compared to antibiotic discs, and therefore any interaction produced using the agar dilution method was dif- ficult to highlight for significance. Using the disc diffusion method, with discs containing either EGCG or Ag NPs placed adjacently at set distances from each another, an antagonistic interaction was seen between the two compounds (Fig. 1). A reduction in the EGCG zone of inhibition on the side closest the disc containing Ag NPs was observed, when the discs were 10 mm apart. Results and Discussion Correspondence J.betts@qmul.ac.uk Tel: +447716141359 Barts and the London School of Medicine and Dentistry References Marked antagonism was observed on the antibacterial activity of Ag NPs in the presence of EGCG. This may be due to trapping of reactive oxygen species, which are the major mode of bacterial killing by Ag NPs. Although the cytotoxicity of Ag NPs might be im- proved in combination with polyphenols, this is at the expense of antibacterial activity, which is sought after in many medical appli- cations. Future work will seek to monitor Hydrogen peroxide lev- els and use scanning and transmissions electron microscopy to confirm if the reduction of ROS as the mechanism behind the an- tagonism or if another factor, such as nanoparticle agglomeration is the cause. Investigations should also be carried out to discover why Ag NPs can increase the activity of EGCG at sub-inhibitory concentrations against selected bacteria. Further work should be carried out using an in vivo model of infection, to determine whether antagonism is limited to in vitro studies. Conclusions Acknowledgements We would like to gratefully acknowledge Dr Fei Cheng at the Uni- versity of Hull for supplying the Ag NPs and to Unilever, UK for do- nating the EGCG powder. 1 Liu HI, Dai SA, Fu KY, Hsu S. Int. J. Nanomed. 2010; 5: 1017-1028. 2 Pradhu S, Poulose EK. Int. Nano Lett. 2012; 2(32): 1-10. 3 Chen F, Betts JW, Kelly SM, Schaller J, Heinze T. Green. Chem. 2013; 15: 989-998. 4 Xiu Z, Zhang Q, Puppala HL, Colvin VL, Alvarez PJJ. Nano Lett. 2012; 12: 4271-4275 5 Toda M, Okubo S, Hara Y, Shimamura T. Jpn. J. Bacteriol. 1991; 46: 839- 845. 6 Hamilton-Miller JMT, Shah S. J. Antimicrob. Chemother. 2000; 46: 852-853. 7 Gordon NC, Wareham DW. Int. J. Antimicrob. Agents. 2010; 36(2): 129- 131. 8 Betts JW, Wareham DW, Haswell SJ, Kelly SM. J. Microbiol. Biotechnol. 2013; 23(9): 1322-1326. 9 Hu ZQ, Zhao WH, Asano N, Yoda Y, Hara Y, Shimamura T. Antimicrob. Agents. Chemother. 2002; 46(2): 558-560. 10 Özyürek M, Güngör N, Baki S, Güçlü K, Apak R. Anal. Chem. 2012; 84: 8052-8059. 11 Moulton MC, Braydich-Stolle LK, Nadagouda MN, Kunzelman S, Hussain SM, Varma RS. Nanoscale 2010; 2: 763-770. 12 Gordon NC, Png K, Wareham DW. Antimicrob. Agents. Chemother. 2010; 54(12): 5316-5322. 13 Taguri T, Tanaka T, Kouno I. Biol. Pharm. Bull. 2006; 29(11): 226-2235. In Vitro Activity Of Epigallocatechin Gallate (EGCG)-Silver Nanoparticle Combinations Versus Bacteria Associated With Skin And Medical Device Infections J. W. Betts 1 and D. W. Wareham 1,2 Table 1. Minimum inhibitory concentration (MICs) of silver nanoparticles (Ag NPs) and epigallocatechin gal- late (EGCG) and Fractional inhibitory concentration index (FICIs). Strain Ag NPs MIC (µg/mL) EGCG MIC (µg/mL) FICs S. aureus ATCC 25923 11.25 256 4.25 (A) E. coli NCTC 12241 2.8 2048 4.05 (A) S. maltophilia NCTC 10258 11.25 1024 4.25 (A) A. baumannii ATCC 19606 22.5 1024 4.5 (A) P. aeruginosa ATCC 27853 2.8 2048 8.31 (A) S. epidermidis SE-51 2.8 128 20.1 (A) MRSA - Clinical isolate 1 5.6 256 10.04 (A) MRSA - Clinical isolate 2 5.6 128 12.04 (A) S = Synergy, I = Intermediate effect, A = Antagonism Background Silver nanoparticles (Ag NPs) have a range of medical and indus- trial uses, most notably as antimicrobials in bandages/ catheters/intravenous lines. Antimicrobial activity has been demonstrated against many species of bacteria including methi- cillin-resistant S. aureus (MRSA) and E. coli 1-3 . This is reported to be size dependant and linked to the generation of reactive oxygen species (ROS) on the release of Ag + ions 4 . Although this is beneficial for antimicrobial activity, this is also cytotoxic to human cells. Like Ag NPs, polyphenols are a group of com- pounds that have been shown to produce significant antimicro- bial activity against a variety of organisms 5-8 and can enhance the activity of antibiotics 9 . Although antioxidants, they can also act as pro-oxidants via the formation of hydrogen peroxide, which can damage cell membranes. Tea polyphenols have been shown to be useful in the green synthesis of Ag NPs by antioxi- dant reduction of Ag + ions in the presence of silver seeds 10 . Pol- yphenols act as reducing and capping agents this process known as seed mediated growth. Ag NPs produced in this pro- cess have shown reduced cytotoxicity to human cell lines com- pared to non-polyphenol synthesized Ag NPs 11 . The proposed mechanism for this is that polyphenol residues on the surface of the Ag NPs protects the cell lines from ROS damage. The ob- jective of this study was to discover if Ag NPs in combination with the polyphenol EGCG results in a beneficial or antagonistic antimicrobial activity against bacteria associated with skin and medical device infections. Materials and Methods Five antibiotic susceptible bacterial type strains including Esch- erichia coli NCTC 12241, Acinetobacter baumannii ATCC 19606, Stenotrophomonas maltophilia NCTC 10258, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and clinical isolates of S. epidermidis and MRSA were tested. EGCG powder (>95 % purity) was donated by Unilever PLC and Ag NPs (#12) with a diameter of 4-12 nm were supplied by the Univer- sity of Hull, synthesized by the method previously published (1). All media was purchased from Oxoid (Basingstoke, UK) and autoclave before use. Agar dilution assays were performed us- ing ISO-Sensitest agar supplemented with/without EGCG (1 and 10 mg/L). Discs containing 0.72, 1.04 or 1.46 mg of Ag NPs and antibiotic control discs of gentamicin (10 µg) for Gram-negative isolates and vancomycin (5 µg) for Gram-positive isolates were also added to each plate. Zones of inhibition were measured and recorded after incubation at 37°C for 18-20 h. A second phenotypic screen for interaction was also conducted using EGCG/Ag NP discs, containing 1 mg/1.04mg respectively, placed at decreasing distances (30-10 mm) from each other. Plates were inoculated with a 0.5 McFarland standard of an overnight culture of each bacterial isolate. Interactions between discs set up adjacently were recorded. The minimal inhibitory concentra- tions (MICs) of EGCG and Ag NPs alone and in combination were determined in ISO-Sensitest broth in microtitre plates in checkerboard style. P0319 1 Antimicrobial Research Group, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, UK 2 Division of Infection, Barts and the London NHS Trust, London, UK Fig. 1. Disc diffusion assay showing zones of inhibition for EGCG (Left) and Ag NPs (Right) against S. aureus ATCC 25923 with antagonism pro- duced as discs are place closer to each other B A C D Fig. 2. Time-kill graphs showing antagonism between Ag NPs and EGCG against E. coli (A+B) and S. aureus (C+D) over 24h. Checkerboard assays confirmed the initial phenotypic observations. The MICs of Gram-positive isolates ranged from 2.8 to 11.25 mg/L for Ag NPs and 128 and 256 mg/L for EGCG. Against Gram-negative isolates the MICs raŶged ďetǁeeŶ Ϯ.8 aŶd ϮϮ.ϱ ŵg/L aŶd ш ϭϬϮϰ ŵg/L respeĐtiǀelLJ. As edžpeĐted, the MICs for EGCG ǁere higher iŶ the Gram-negative isolates, consistent with previous research 13 . In checkerboard assays the addition of EGCG se- verely reduced the antibacterial effect of Ag NPs with MICs increasing significantly to 45 mg/L for Gram-positive bacteria and between 11.25 and 90 mg/L for Gram-negative isolates. Similarly EGCG MICs were increased in the presence of Ag NPs with MICs against the Gram-positive bacteria up to fold higher than without Ag NPs. However, the MICs of EGCG for the Gram-negative isolates were reduced in the presence of Ag NPs. Although when the FICs were calculated, values of between 4.25 and 20.1 were generated, indicating overall antagonism. Time-kill data supported checkerboard and disc diffusion assay findings, with the antibacterial action of Ag NPs being inhibited by sub-inhibitory concentrations of EGCG against E. coli (Fig 2a) and the antibacterial activity of EGCG being fractionally increased in the presence of sub-inhibitory concentrations of Ag NPs (Fig. 2b). Against S. aureus both combinations resulted in a reduced antimicrobial activity, with the secondary antimicrobial at a sub- inhibitory concentration promoting antagonism with the CFU/mL being significantly higher than with the com- pounds alone. Fractional inhibitory concentration indices (FICIs) were calculated as previ- ously described 12 , to determine a synergistic, additive or antagonistic effect, ǁhere a FICI ǀalue of ч Ϭ.ϱ ǁas defiŶed as sLJŶergLJ, a ǀalue that ǁas >Ϭ.ϱ aŶd ч ϰ.Ϭ ǁas defiŶed as iŶterŵediate/additiǀe aŶd a ǀalue > ϰ.Ϭ ǁas deter- mined to be antagonistic. Time-Kill assays were performed against S. aureus and E. coli, as two species representing Gram-positive and Gram-negative species. A 1/1000 dilution of an overnight culture, equating to approximately 10 6 CFU/mL was used as the starting inoculum. Falcon tubes containing 10 mL of Iso-Sensitest broth were inoculated with S. aureus/E. coli and supplemented with Ag NPs (0.5/ x1 MIC), EGCG (0.5/x1 MIC), and Ag NPs-EGCG (x1 MIC-0.5 MIC) or EGCG-Ag NPs (x1 MIC-0.5 MIC) and were incubated and continuously agitated at 35° C. At 0, 2, 4, 6 and 24 h, 100 µL aliquots were taken from each tube and seri- al dilutions plated onto Iso-Sensitest agar for viable counts. Colonies on all plates were counted after incubation at 35°C for 20 h. The mechanism behind the antagonism is possibly due to EGCG acting as an antioxidant, neutralising ROS produced by the Ag NPs 11 , the suggested basis of their antimicrobial action. However, other mechanisms such as agglomeration of nanoparticles and fur- ther seed-mediated nanoparticle growth in the presence of EGCG could also contribute to the reduced antimicrobial activity. It has been previously shown that the antibacterial action of Ag NPs de- creases as their size increases 3 .