1. Isolating and Purifying Novel Antibiotics from Soil Bacteria Heather Fisher, Department of Biological Sciences, York College of Pennsylvania Introduction Antibiotic resistance is constantly on the rise causing higher rates of morbidity and mortality and multidrug resistant infections. Soil bacteria provide an alternative to existing antibiotics because they produce antibiotics as secondary metabolites to improve their survival in competitive environments. Identifying novel antibiotics from various soil bacteria could prove effective at inhibiting both Gram-positive and Gram- negative bacteria and providing effectiveness and potency that is equal to the antibiotics already in use. This is possible because many species are able to produce multiple antibiotics varying in structure and mechanism of action. Zone of Inhibition (ZOI) and Minimal Inhibition Concentration (MIC) measures are commonly used to measure the efficiency of antibiotics against certain bacteria. ZOI can also be implemented to discover the mechanism of the antibiotics. The mechanisms of action that antibiotics use are inhibition of cell wall synthesis, inhibition of protein synthesis, inhibition of nucleic acid synthesis, alteration of cell membranes, or anti-metabolite activity. Objectives Purify and identify new antibiotics from various soil bacteria using column chromatography and MALDI-TOF mass spectrometry Determine the effectiveness and spectrum of the antibiotics with zone of inhibition (ZOI) measures and minimum inhibition concentration (MIC) titers Determine mechanism of action of novel antibiotics using zone of inhibition. Research Design Expected results Literature cited Athukorala, Sarangi N.P., W.G. Dilantha Ferando, and Khalid Y. Rashid. "Identification of antifugal antibiotics of Bacillus species isolated from different microhabitats using polymerase chain reaction and MALDI-TOF mass spectrometry." Canadian Journal of Microbiology. 55. (2009): 1021-1032. Web. 28 Jan. 2012.. Ceylan O, Okmen G, Ugar A (2008) Isolation of soil Streptomyces as source antibiotics active against antibiotic-resistant bacteria. EurAsia J BioSci 2, 9, 73-82. www.ejobios.com/content/2/9/73-82. Nelson, Mark, Robert A. Greenwald, and Wolfgang Hillen. Tetracyclines in Biology, Chemistry, and Medicine. Berlin: Birkhauser, 2001. GoogleBooks. Web. 3 Apr. 2012.. Westman, Erin L., and Gerald D. Wright. "The Antibiotic Resistome: Origins, Diversity, and Future Prospects." Handbook of Molecular Microbial Ecology II: Metagenomics in Different Habitats. Ed. Frans J. de Bruijn. Hoboken, New Jersey: Wiley-Blackwell, 2011. 165-174. Web. 28 Jan. 2012. <http://books.google.com/books?hl=en&lr =&id=SJgc8KK52dcC&oi=fnd&pg=PA165&dq =Resistome in soil&ots=NsKsiNyXHE&sig=DaDHOhyE2TYh4ro24XG_HL2T7Xo Acknowledgements I’d like to thank Dr. Singleton for the knowledge and advice he has provided throughout the duration of this project. Expected conclusions I expect some antibiotics to exhibit a broad spectrum and some to only be effective against Gram-positive bacteria. I also expect to find some antibiotics that will prove to be more effective than many of the antibiotics currently in use. Review of Literature Due to antibiotic producers and resisters evolving together in the soil, it can be assumed that a large reservoir of resistance determinants is present in their genetic code. (Westman and Wright, 2011) Many Bacillus species are known to produce antibiotics to control plant diseases and have been studied using PCR reactions and MALDI-TOF mass spectrometry to identify the genes responsible for the synthesis of specific antibiotics (Sarangi et. al., 2009) Some strains were found to have the genes for multiple antibiotics but only produced 1 or 2 in high concentrations at a given time. This may suggest that antibiotic synthesis may be influenced and induced by signals from the environment in addition to the bacteria’s genetic makeup. (Sarangi et. al., 2009) Streptomyces has been found to produce many novel antibiotics and continues to be studied in an attempt to identify more that are effective against gram-positive and gram-negative bacteria. (Ceylan et. al., 2008) In fact, Streptomyces are responsible for about 75% of clinically useful antibiotics, including tetracycline, erythromycin, streptomycin, neomycin, chloramphenicol, and lincomycin. (Nelson et. al. 2001) Dilute soil samples and plate on soil extract agar ZOI test on E. coli plate ZOI test on S. aureus plate ZOI test on C. albicans plate MALDI-TOF mass spectrometry to determine structure of antibiotics DNA sequencing of bacteria via PCR reaction MIC titer of Fractions n=10 Make broth culture of colonies Image 1. Zones of Inhibition (ZOI) around various antibiotic discs. (http://www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/AntibioticDisk.htm) Column chromatography of cultures showing ZOI to purify produced antibiotics Test bacteria against existing antibiotics to determine antibiotic mechanism of action