Identification of Biofilm Inhibitors by Screening Combinatorial Libraries

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Biological and Medical Applications of Materials and Interfaces

Identification of Biofilm Inhibitors by Screening Combinatorial Libraries of Metal Oxide Thin Films Michal M. Dykas, Stuti K. Desai, Abhijeet Patra, Mallikarjuna Rao Motapothula, Kingshuk Poddar, Linda J. Kenney, and Thirumalai Venkatesan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02246 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 25, 2018

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ACS Applied Materials & Interfaces

Identification of Biofilm Inhibitors by Screening Combinatorial Libraries of Metal Oxide Thin Films

Michal M. Dykasa,b,#, Stuti K. Desaic,#, Abhijeet Patraa,b,#, Mallikarjuna Rao Motapothulaa, Kingshuk Poddara,b, Linda J. Kenneyc,d,e*, and T. Venkatesana,b,f,g,h* a

NUS Nanoscience & Nanotechnology Institute - NanoCore, 5A Engineering Drive 1,

Singapore 117411, bNUS Graduate School for Integrative Sciences and Engineering, 28 Medical Drive, Singapore 117456, cMechanobiology Institute, National University of Singapore (NUS), 5A Engineering Drive 1, Singapore 117411, dJesse Brown Veterans Affairs Medical Center, 820 S. Damen Avenue, Chicago, IL 60612, USA, eDepartment of Microbiology and Immunology, University of Illinois-Chicago, 1853 West Polk Street, Room 130 CMW, Mail Code 784, Chicago, IL 60612, USA, fDepartment of Electrical Engineering, National University of Singapore, 4 Engineering Drive 3, Singapore 117576 g

Department of Materials Science and Engineering, National University of Singapore, 9

Engineering Drive 1, Singapore 117575, hDepartment of Physics, Faculty of Science, National University of Singapore, 2 Science Drive 3, Singapore 117551. #equal contribution *correspondence: [email protected] and [email protected] Keywords Metal oxides, zinc oxide, thin film deposition, Salmonella enterica, biofilms

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Abstract With the rise in nosocomial infections worldwide, research on materials with an intrinsic ability to inhibit biofilm formation has been generating a great deal of interest. In the present work, we describe how thin film material libraries generated by pulsed laser deposition can be used for simultaneously screening several novel metal oxide mixtures that inhibit biofilm formation in a common human pathogen, Salmonella enterica serovar Typhimurium. We discovered that in a material library constructed using two metal oxides, the net effect on biofilm formation can be modelled as an addition of the activities of the individual oxides weighted to their relative composition at that particular point on the library. In contrast, for similar material libraries constructed using three metal oxides, there was a non-linear relation between the amount of dominant metal oxide and formation of Salmonella biofilms. This non-linearity resulted in several useful metal oxide combinations that were not expected from the weighted average predictions. Our novel application will lead to the discovery of additional alternatives for creating antimicrobial surfaces.

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1. Introduction Chemical or physical modifications of surface properties can affect a range of cellular behaviours. The ability of metals and metal oxides to affect growth, development and differentiation of organisms has been exploited in a variety of medical applications. For example, zirconia coating improves the biocompatibility of implants, while copper and zinc oxides provide anti-microbial surfaces in clinical settings

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. The formation of

biofilms is also regulated by surface chemistry and topography

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. Biofilms are

multicellular aggregates of microbial cells that are encased in a self-secreted matrix. Many genera of bacteria and fungi exist as biofilms in nature which are implicated in biofouling, bioremediation and pathogenesis 6. As nosocomial infections rise sharply worldwide, there is an ever increasing need to harness nanotechnology to improve materials for their use in clinical settings 7. In this regard, pure or mixtures of metal oxides can be useful in controlling such hardy biofilm-mediated infections. Doping of pure metal oxides changes the inherent properties of materials to generate a new range of clinically useful surfaces. For example, addition of a small amount of silver to titanium dioxide inhibited the growth of Escherichia coli to the same extent as in pure silver 8, but at a reduced material cost. Research has also shown that a small amount of doping can drastically change the biological response. A pure coating of zirconium oxide on stainless steel inhibited the growth of E. coli, but upon doping with silver, growth of both E. coli and Staphylococcus aureus was inhibited 9. Therefore, knowledge regarding how surfaces influence biological systems is essential for improving future applications of bioactive surfaces.

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Metal oxides are generally suspended in the growth medium as nanoparticles (NPs) and tested for their bacterial toxicity. Zinc oxide, when used in the form of NPs instead of as a powder, showed enhanced anti-microbial activity 10. The particle size of such zinc oxide NPs was correlated with its anti-microbial effect. Large (>100 nm) zinc oxide NPs did not inhibit growth of S. auereus, but medium sized (12 nm–100 nm) NPs inhibited growth, while small ( 1 nm) across the entire ternary library (Fig. 5b and c). When Salmonella biofilms were analyzed from each of the sub-regions of the ternary library, we discovered metal oxide effects that were unexpected compared to the biofilms formed on pure metal oxide films (Fig. 1). The ternary library resulted in the highest level of biofilm inhibition on the side of the wafer with a higher concentration of ZnO and the lowest level of inhibition for the ZrO2-rich surface (Fig. 6a), as expected. However, there were subregions in the ternary library that exhibited non-linear behavior with ZnO composition and biofilm inhibition (Fig. 6b-d). For example, in well 13, it is evident that ZnO was around 20% (i.e. low), yet in the presence of TiO2 and ZrO2, biofilms were 40% inhibited. When ZnO was the only metal present, 50% inhibition required 97% metal. Interestingly, the library identified metal oxide combinations that inhibited biofilms by close to 40%, in spite of having a low content of ZnO (Fig. 6a). Thus, CCS-PLD libraries enabled the identification of novel metal oxide mixtures with desired properties.

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1µm -2.3 [nm]

Fig. 5. Characterization of a ternary CCS-PLD library. a) ZnO, ZrO2 and TiO2 compositions for each of the 32 wells as measured by RBS. b) Surface roughness at each of the 32 wells was less than 1 nm as measured by AFM. c) Representative AFM images for wells I, II and III as shown in (b).

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a) Zinc oxide rich 50%

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Fig. 6. Lower amounts of ZnO were capable of inhibiting biofilms when present as ternary metal oxide complexes with ZrO2 and TiO2. a) Diagrammatic representation of the percentage of biofilms formed at each of the 32 wells against the respective uncoated control wells as measured by crystal violet staining. Wells used for further analysis were numbered. The amount of biofilms formed in the top row, well numbers 1, 7 and 8, and the bottom row, well numbers 12, 13 and 14 were similar, in spite of having wide differences in ZnO composition (97% compared to 20%, respectively). b) Formation of biofilms in wells 1 to 6 varied with the percentage composition of ZnO (curve with black squares), TiO2 (curve with black triangles) and ZrO2 (curve with black circles) in the ternary mixtures, n = 3, Mean ± SD was plotted. c) Weighted average analysis of the percentage of biofilms in wells 1 to 6 with ZnO content exhibited a non-linear correlation. d) Formation of biofilms in wells 7 to 12 also varied with the percentage composition of ZnO (curve with black squares), TiO2 (curve with black triangles) and ZrO2 (curve with black circles), n = 3; Mean ± SD is plotted. e) Weighted average analysis of the percentage of biofilms in wells 7 to 12 with the ZnO content again showed a non-linear dependence, indicating that in ternary metal oxide mixtures, ZnO was no longer dominant. 3.5. How do coated material libraries affect biofilms? The ability of Salmonella Typhimurium to form biofilms on solid surfaces is dependent on the transcriptional regulator CsgD 19. It was therefore of interest to determine whether the metal oxide mixtures of the binary and ternary libraries affected its expression. We first examined the effect of a specific ZnO-CuO mixture (from the binary library) and a ZnO-TiO2-ZrO2 mixture (from the ternary library) on the promoter activity of a

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Salmonella strain harboring a transcriptional fusion of the csgD promoter with the green fluorescent protein (GFP) (Supporting information 3). Expression of the csgD-gfp transcriptional fusion decreased in the presence of the ZnO-CuO mixture compared to the uncoated control quartz and was lowest when a mixture of ZnO-TiO2-ZrO2 was used (Fig. S2). To confirm the inhibitory effect of metal oxide mixtures on CsgD, we examined the levels of CsgD protein by western blotting using a monoclonal antibody against CsgD. CsgD protein is visible in the uncoated control (Fig. 7), but was not detectible in the presence of the ZnO-CuO and ZnO-TiO2-ZrO2 mixtures. Thus, we established that metal oxide mixtures inhibited the formation of biofilms by a CsgD-dependent mechanism19. To identify whether the inhibition of csgD transcription was due to the release of free metal ions into the growth medium, we used inductively coupled plasma mass spectrometry (ICP-MS) (Supporting information 2). We incubated the ZnO-CuO binary library and ZnO-TiO2-ZrO2 ternary library with the growth medium and after two days determined the levels of Zn+2, Cu+2, Ti+2 and Zr+2 ions. Zn+2 levels were in the range of 5 to 8 ppm, while Cu+2, Ti+2 and Zr+2 ions were less than 0.1 ppm from both the binary and ternary material libraries (Table S1). Free Zn+2 ions in the range of 5 to 8 ppm are much lower than the concentration of zinc known to decrease the in vivo burden of Salmonella in pigs (2,000 to 3,000 ppm) 27. Therefore, we propose that the inhibition of Salmonella biofilms by metal oxide mixtures is primarily due to their chemical composition.

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CsgD

GroEL

Fig. 7. Metal oxide mixtures decreased the levels of CsgD protein (the master regulator of biofilms). Immunoblot analysis showing the near complete absence of CsgD in the presence of a ZnO-CuO mixture (similar to well number 6) of the binary library and a ZnO-TiO2-ZrO2 mixture (similar to well number 7) of the ternary material library against that of an uncoated quartz control, using GroEL as a loading control. 4. Conclusion The inherent effects of metals and metal oxides on biological systems have been extensively employed to design materials in industry and the healthcare sector. There is an ever growing need to screen a larger number of materials to create “microbiologically inert” surfaces owing to the rise of antibiotic resistance and nosocomial infections. On the other hand, continuous efforts are also needed to test metal oxides or their mixtures for application of prosthetics and implants that have improved characteristics of biocompatibility, microbial toxicity, physical strength and malleability. In this work, we successfully applied the CCS-PLD technique to create thin film metal oxide libraries for screening biofilm inhibitors.

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A fundamental advantage of our method is in its ability to simultaneously screen a large number (32 or more) of metal oxide compositions for a given biological purpose. We discovered that the formation of Salmonella biofilms was strongly correlated with the varied surface chemistries generated using binary or ternary CCS-PLD libraries. Indeed, differences observed in the anti-biofilm effects of zinc oxide and copper oxide mixtures (the binary library) and zinc oxide, titanium dioxide and zirconium dioxide mixtures (the ternary library), indicated that greater complexities generate a wider range of useful metal oxide surfaces. Since most of the metal oxides are easily available as powders, there is an enormous potential of using modified CCS-PLD, as described here, as a materialscreening tool. This would identify newer materials that are cheaper, stronger, more biocompatible or anti-microbial as required. The ability of microbial pathogens to switch from a free-swimming lifestyle to surface-attached communities is essential for the successful establishment and maintenance of the disease state. Identification of materials that inhibit this lifestyle switch will improve our ability to eliminate infectious bacteria. In Salmonella Typhimurium, the expression of the master regulator of biofilms, csgD, is known to be regulated by pH, nutrition, osmolality and temperature

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. We discovered that certain

metal oxide mixtures also inhibited the activity of the csgD promoter, which led to a decrease in the formation of biofilms. Since the basic mechanisms of biofilm formation are largely conserved, it seems likely that metal oxides and metal oxides mixtures that inhibited Salmonella biofilms would also be applicable for controlling other gramnegative pathogens. Supporting information

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Growth rate of Salmonella in presence of single metal oxide crystals (Fig. S1), presence of ions in culture media (Table S1) and measurement of csgD promoter activity(Fig. S2) has been provided in the supporting information. Conflicts of interest The authors declare no conflicts of interest relevant to this publication. Acknowledgements Supported by 5IOBX-000372 from the VA, funding from the Research Centre of Excellence in Mechanobiology, National University of Singapore, from the Ministry of Education to LJK; NUS Nanoscience and Nanotechnology Institute and Office of the Deputy President (NUS) (R-398-000-084-646) to TV. NUS Graduate School for Integrative Sciences and Engineering fellowship is thankfully acknowledged by MD, AP and KP for supporting their graduate studies. We thank Dr. Aaron White, Vaccine and Infectious Disease Organization, University of Saskatchewan, Canada, for the monoclonal anti-CsgD antibody. The PcsgD-GFP plasmid was a kind gift from Dr. Cagla Tukel, Temple University, Pennsylvania. We also thank Carl Zeiss Imaging laboratory, NUS, for the use of their SEM facility. References 1. Sollazzo, V.; Pezzetti, F.; Scarano, A.; Piattelli, A.; Bignozzi, C. A.; Massari, L.; Brunelli, G.; Carinci, F., Zirconium oxide coating improves implant osseointegration in vivo. Dent Mater 2008, 24 (3), 357-361. 2. Grass, G.; Rensing, C.; Solioz, M., Metallic Copper as an Antimicrobial Surface. Applied and Environmental Microbiology 2011, 77 (5), 1541-1547. 3. Memarzadeh, K.; Sharili, A. S.; Huang, J.; Rawlinson, S. C.; Allaker, R. P., Nanoparticulate zinc oxide as a coating material for orthopedic and dental implants. J Biomed Mater Res A 2015, 103 (3), 981-989. 4. Li, B.; Logan, B. E., Bacterial adhesion to glass and metal-oxide surfaces. Colloids and Surfaces B: Biointerfaces 2004, 36 (2), 81-90.

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Identification of Biofilm Inhibitors by Screening Combinatorial Libraries

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