1. Overnight growth of samples E. coli, S. aureus, P. aeruginosa and B. cereus Visual Density check Dilute samples to match turbidity of MacFarlane Standards Plate samples in 96- well Microtiter plates Let separate plates grow for either 0, 6, 24 or 48 hours Staining Procedure Wash biofilms with PBS Stain with Crystal Violet Wash again with PBS Suspend stain with ethanol Read absorbance of Sample Wallac Victor^2 1420 Multilabel Counter Absorbance took at 590nm Statistical Analysis 2 Way ANOVA Tukey Comparison Rate of Biofilm Formation is Influenced by the Bacterial Composition in Mixed Cultures Reichart, Nicholas J.*, Bridgette E. Hagerty, Carolyn F. Mathur Department of Biological Sciences, York College of Pennsylvania In order to study these interactions, we compared the rates of biofilm formation using Pseudomonas aeruginosa (Pa), Escherichia coli (Ec), Staphylococcus aureus (Sa), and Bacillus cereus (Bc). We measured each bacterium alone and in paired combination with each of the other three organisms. Using staining procedures and spectrophotometry, biofilm growth was quantified at 0, 6, 24 and 48 hours. Absorbance (OD) was compared across bacteria and time using a two-way ANOVA followed by Tukey multiple comparisons. Pa was the most robust biofilm producer at 0.46abs/hr. In comparison, Ec biofilm production was 0.11abs /hr., followed by the weakest producers, Sa at 0.04abs/hr. and Bc at 0.03abs/hr. Pa significantly increased the rate of biofilm formation of individual Sa, Bc or Ec cultures when grown with each of them in combination. Sa and Bc significantly decreased the rate of biofilm formation of Ec. Pa rapidly formed biofilms, whether alone or in combination with other bacteria. The latter is probably due to the PA growing faster than the others in the mixed cultures. Explanations for the inhibition of Ec biofilm formation by either Sa or Bc is not so readily apparent and requires further studies. Abstract Grow single and dual cultures of bacteria over various time intervals Use spectrophotometry to quantify biofilm growth at maximum density to get formation rates of each culture Compare the rates of each dual culture to the respective single culture to determine trends of mixed cultures on biofilm growth Objectives Bacteria naturally exist in communities composing biofilms with multiple species. Multiple outcomes can result from mixed cultured biofilms depending on the species present. These interactions can either be adventitious or detrimental to the survival of the bacteria (Burmolle et al. 2006). The ability of bacteria to form a biofilm as a single culture may not accurately portray its survival in mixed cultures due to possible complementation leading to synergistic effects (Lopes et al. 2012). Possible synergistic outcomes seen in biofilms include; improved attachment to surfaces, less of a chance of desiccation, greater possibility for gene transfer, higher virulence potential, and increased resistance to antibacterial compounds and invasion. The relationships among bacteria in mixed biofilms necessitates more research to be done to better understand their associations with one another. Introduction Pa was neither positively or negatively influenced when a second species was added into culture. Due to its high formation rate as a single species, Pa was dominant over the growth trends of the other species. Ec growth rate was increased with Pa but decreased with Sa or Bc. Ec increased growth when added to Bc, but had no significant impact when added to the other poor biofilm former, Sa. Sa and Bc were the slowest biofilm producers of the study. They had no effect on each other when mixed. Conclusion Added Species E. coli S. aureus P. aeruginosa B. cereus Single Species E. coli-Yes (↓)Yes (↑)Yes (↓) S. aureusNo-Yes (↑)No P. aeruginosaNo - B. cereusYes (↑)NoYes (↑)- Figure 5. Example picture of a plate after designated time stained with crystal violet and suspended in ethanol Table 1. Trends of Significant growth difference Burmolle, M., Webb, J.S., Roa, D., Hansen, L.H., Sorensen, S.J., Kjelleberg, S. 2006. Enhanced biofilm formation and increased resistance to antimicrobial agents and bacterial invasion are caused by synergistic interaction in multispecies biofilms. Applied and Environmental Microbiology 72(6): 3916-3922. Lopes, S.P., Ceri, H., Azevedo, N.F., Pereira, M.O. 2012. Antibiotic resistance of mixed biofilms in cystic fibrosis: Impact of emerging microorganisms on treatment of infection. International Journal of Antimicrobial Agents 40(3): 260-263. References Acknowledgements Methods I would like to thank Dr. Mathur for all her support and guidance throughout the whole process of this project. I would also like to thank Dr. Hagerty for her assistance with the statistical analysis. And a big thank you to the York College Biology Department for giving me the opportunity to conduct this research project. Absorbance rate per hour taken from optical growth density at peak time during the experiment. Pa: 0.46 abs/hr Ec: 0.11 abs/hr Sa: 0.04 abs/hr Bc: 0.03 abs/hr Results