Duffy, Brendan

Jaiswal, Swarna

McHale, Patrick

English

Arrow@TUDublin

Technological University Dublin ARROW@TU Dublin Conference Papers Crest: Centre for Research in Engineering Surface Technology 2010-03-22 Rapid Evaluation of Antibacterial Activity by Microtiter Well Coating. Swarna Jaiswal Technological University Dublin, swarna.jaiswal@tudublin.ie Brendan Duffy Technological University Dublin, brendan.duffy@tudublin.ie Patrick McHale Technological University Dublin, patrick.mchale@tudublin.ie Follow this and additional works at: https://arrow.tudublin.ie/cenrescon Recommended Citation Jaiswal S, Duffy B, & McHale P. (2010) Rapid evaluation of antibacterial activity by microtiter well coating. European Conference on Nano Films. March 22nd -24th, 2010 Liège, Belgium. This Conference Paper is brought to you for free and open access by the Crest: Centre for Research in Engineering Surface Technology at ARROW@TU Dublin. It has been accepted for inclusion in Conference Papers by an authorized administrator of ARROW@TU Dublin. For more information, please contact yvonne.desmond@tudublin.ie, arrow.admin@tudublin.ie, brian.widdis@tudublin.ie. This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License RAPID EVALUATION OF ANTIBACTERIAL ACTIVITY BY MICROTITER WELL COATING  Swarna Jaiswal a,b, Brendan Duffy b and Patrick McHale a aSchool of Biological Sciences, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland. bCentre for Research and Engineering Surface Technology (CREST),  FOCAS Institute, Dublin Institute of Technology,  Dublin 8, Ireland.  The colonization of clinical and industrial surfaces with microorganisms, including antimicrobial-resistant strains, has promoted increased research into the development of effective antibacterial and antifouling coatings. In this study the preparation of metal (Ag+) and metal oxide (Cu2+, Zn2+) doped methyltriethoxysilane (MTEOS) coatings and the rapid assessment of their antibacterial activity is described. The wells of polystyrene microtiter plates were coated using various volumes of the sol-gels and cured at different temperatures for varying time periods. Curing parameters were analyzed using by thermogravimetric analysis (TGA) and visual examination. The optimum curing temperature in the microtiter wells was determined to be 50-60⁰ C. when the wells were coated using a sol-gel volume of 200 µl. The coated wells were challenged with cultures of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli. Silver showed the highest antibacterial activity followed by zinc and copper. DLS showed that the size of the silver ions was smaller than that of the zinc and copper. The silver doped sol-gel had broad spectrum antibacterial activity making it potentially useful as a coating for biomaterials. The use of microtiter plates enabled a variety of sol-gel coatings to be screened for their antibacterial activity against a wide range of bacteria in a relatively short time. This route is a pre-screening technique that then applied to the surface of indwelling items (e.g. ear-rings), in which the continuous release of metal and metal oxides has ability to protect skin surfaces from microbial infection.    Rapid Evaluation of Antibacterial Activity by Microtiter Well Coating   Swarna Jaiswala,b, Brendan Duffyb and Patrick McHalea        aSchool of Biological Sciences, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland.      bCentre for Research and Engineering Surface Technology (CREST), FOCAS Institute, Dublin Institute of Technology, Dublin 8, Ireland  Email: bduffy@dit.ie Acknowledgement  The authors wish to acknowledge Dublin Institute of Technology, Dublin, Ireland for funding under the ABBEST scholarship programme  and Enterprise Ireland under the ARE Programme                                                                               Experimental                                                                                                                                           Conclusion The rapid evaluation of metal doped sol gel coatings in microtiter wells has been presented. The technique gave highly reproducible MIC data where the silver doped sol-gel coatings exhibited significant antibacterial activity against all the test organisms, followed by zinc and copper respectively.                                                                               Introduction This work presents results for the rapid evaluation of the antibacterial action of metal ions in nanostructured sol gel coatings using microtiter plates. The technique facili-tates multiple assays using standard dilution micro methods, measuring optical densities, determining minimum inhibitory concentrations (MIC) and minimum bacteri-cidal concentrations (MBC).  The antibacterial activities of silver, copper and zinc doped sol-gels against a range of Gram-positive and Gram-negative organisms, includ-ing multi drug resistant (MDR) ones, are determined.  50 µl 100 µl 150 µl 200 µl                                                                    Results and Discussion  Sol-Gel Synthesis: Under continuous stirring, MTEOS was hydrolysed under acidic conditions in the presence of a metal nitrate salt of interest and allowed to stir for 24 hours. All metal concentrations are quoted in w/w%. Characterisation: Thermogravimetric analysis (TGA) of the sols was carried out using a Shimadzu DTG 60 under a nitrogen flow of 40 mL.min-1. Dynamic light spectroscopy was performed using a Malvern nanosizer.  Microtiter Coating: Various sols volumes were dropped into a 96 well microtiter plate and cured at a TGA determined temperature Fig:3. MIC of Ag+ ,Cu2+ and Zn2+ doped MTEOS coatings against (3a) E. coli and (3b) EnterobacterWT6. The results in Fig:3. indicate that silver has the highest antibacterial activity of the metals against both test organisms. The MIC of silver for E. coli was 0.5%, 0.7% for zinc and 1% for copper. For Enterobacter WT6, the MICs were 0.5%, 1% and >1% for silver, zinc and copper respectively. Table:1.. MBC of Ag+ ,Cu2+ and Zn2+ doped MTEOS coatings against all test organisms. Fig:1. Coated microtiter wells with different volume of Ag+, Cu2+ and Zn 2+ doped MTEOS at different concentrations. Fig:4. Flow diagram shows modified imprint method for the determination of MBC of different concentrations of metal doped sol-gel against S. aureus.  Synthesis of metal doped methyltriethoxysilane (MTEOS) sol-gels  Determination of the sizes of metal species by Dynamic Light Spectroscopy (DLS)   Coating curing parameters and analysis: Volume, Temperature and Time  Assessment of antibacterial activity of coated plate The antibacterial activity of metal-doped sol gel coated microtiter well were assessed against Gram-positive (Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis CSF 41498), Gram-negative ( Escherichia coli ATCC 25922) and clinical isolates MDR Enterobacter WT6 and MDR Pseudomonas WTZ using standard dilution micro methods, measuring optical densi-ties and determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) by streak out. Antibacterial Activity: Analysis of curing Parameters  The wells of polystyrene microtiter plates were coated using various volumes of the sol-gels (Fig:1.) and cured at set temperatures for varying time periods. The optimum curing parameters were determined using TGA (Fig:2.) followed by visual examination. Fig:2. Thermogravimetric analysis  (TGA) of  Ag+ , Cu2+and Zn2+ doped  MTEOS of different concen-trations under a nitrogen atmosphere at 50° C The optimum volume for the microtiter well coating was 200 µl and the curing temperature was determined to be 50-60⁰ C for 5 hours. From DLS analysis of different concentrations of metal species in the sols it was observed that the silane species are reproducible at 1-3 nm in diameter, but there was no evidence of metal based nanoparticles for silver or zinc. This was con-firmed by the absence of any yellow colouring as would be expected. Non repro-ducible evidence of copper nanoparticles was observed, however the sols retained a blue colour indicative of Cu2+ chemistry.  Assessment of antibacterial activity  The antibacterial activity of the coating was determined by measuring the optical density at 600 nm of the challenge organisms (100µl of 106 CFU/ml) after 24 hours of incubation at 37 ºC. To avoid deformation of the microtiter plate, temperatures above 70°C were avoided. Low volumes of sols were found to have poor film forming properties with cracking occurring below 100 µl.  S. aureus culture in coated microtiter well after 24 h of incubation.    5µl of the tested samples were transferred from microtiter well and imprinted to agar plate. MBC Samples Culture MBC Ag E. coli 0.5% Zn 0.7% Cu 1% Ag P. aeruginosa WTZ  0.5% Zn 1% Cu >1% Ag Enterobacter WT6  0.5% Zn 1% Cu >1% Ag S. epidermidis 0.7% Zn 1% Cu 1% Ag S. aureus 0.5% Zn 1% Cu >1%  

Rapid Evaluation of Antibacterial Activity by Microtiter Well Coating.

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Rapid Evaluation of Antibacterial Activity by Microtiter Well Coating.

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