1. Introduction Antibiotics are substances produced by various microorganisms that inhibit the growth of or destroy bacteria and other microorganisms, and are frequently used to combat bacterial infections in humans and animals 1,2. Antibiotics are routinely added to livestock feed which, along with intensive broad-spectrum antibiotic use for the treatment of human diseases, has led to the emergence of antibiotic-resistant organisms 1,2. As human pathogens continue to acquire resistance to all classes of current antibiotics, it is necessary to develop novel antibiotic compounds 1. New antibiotic-producing strains of bacteria isolated from soil samples around the world are continually being discovered and patented, and the effectiveness of the antibiotics produced by these strains is being tested against a wide range of human and animal pathogens 3,4,5. Bacteria of the Bacillus genus occur mainly in soil and produce many widely studied antibiotic compounds 6,5. For example, Bacillus subtilus produces more than seventy-five known antibiotics consisting predominantly of small, cyclic peptides, but also including phospholipid, lipopeptide, and aminosugar antibiotics 7,8,9,3,5. The objectives of this experiment were to survey for antibiotic-producing bacteria found in soil and isolate and characterize antibiotics produced by soil- borne bacteria. Putative antibiotic-producing bacteria were isolated from soil and antibiotic production was confirmed. The culture broth filtrates were treated to determine whether the antibiotics were peptides or non-peptides. Antibiotics were extracted and concentrated from culture broth and the antibiotic-producing bacteria were characterized by Gram- and endospore-staining. It was hypothesized that antibiotic-producing bacteria would be of the genera Bacillus or Streptomyces and that the antibiotics would be peptides that inhibited the growth of Gram-positive bacteria. Isolating putative antibiotic-producing bacteria Methods Soil samples collected from Ohio, Georgia, Florida, and Alaska were suspended in water and swabbed on plates of tryptic soy agar (TSA). Zones of inhibition (ZOI) were identified on the plates and the inhibitory capabilities of putative antibiotic-producing bacteria were confirmed with cross tests and spot- lawn tests. Results Fourteen putative antibiotic-producing bacteria were isolated. Figure 1. Bacterial colonies Figure 2. Cross test of Figure 3. Spot-lawn test with from a swabbed soil suspension horizontally streaked putatitve inoculated spots of putative growing on TSA plate. antibiotic-producing bacterium antibiotic-producing bacteria GC3y across vertically streaked on a S. simulans lawn showing S. simulans resulting in inhibited ZOI where S. simulans growth S. simulans growth. is inhibited. Literature Cited [1] Gould IM. 2008. The epidemiology of antibiotic resistance. International Journal of Antimicrobial Agents 32S: S2-S9. [2] Iovine NM and Blaser MJ. 2004. Antibiotics in animal feed and spread of resistant Campylobacter from poultry to humans. Emerging Infectious Diseases 10(6): 1158-1159. [3] Rosado AS and Seldin L. 1993. Production of a potentially novel anti-microbial substance by Bacillus polymyxa. World Journal of Microbiology and Biotechnology, 9: 521-528. [4] Woolford MK. 1972. The semi large-scale production, extraction, purification and properties of an antibiotic produced by Bacillus licheniformis strain 2725. Journal of Applied Bacteriology, 35: 227-331. [5] Mannanov RN and Sattarova RK. 2001. Antibiotics produced by Bacillus bacteria. Chemistry of Natural Compounds, 37(2): 117-123. [6] Al-Janabi AAHS. 2006. Identification of Bacitracin produced by local isolate of Bacillus licheniformis. African Journal of Biotechnology, 5(18): 1600-1601. [7] Stein T. 2005. Bacillus subtilus antibiotics: structures, syntheses and specific functions. Molecular Microbiology, 56(4): 845-857. [8] Katz E and Demain AL. 1977. The peptide antibiotics of Bacillus: Chemistry, biogenesis, and possible functions. Bacteriological Reviews, 41(2): 449-474. [9] Tamehiro N, Okamoto-Hosoya Y, Okamoto S, Ubukata M, Hamada M, Naganawa H, and Ochi K. 2001. Bacilysocin, a novel phospholipid antibiotic produced by Bacillus subtilis 168. Antimicrobial Agents and Chemotherapy, 46(2): 315-320. Confirming production of antibiotics Methods Five of the putative antibiotic-producing bacteria were cultured in tryptic soy broth (TSB) at 37°C for 40 hours. Every 8 hours, a small volume of the supernatant of each culture broth was filtered with 0.2 µm pore membranes. This broth filtrate was transferred to wells bored into the media of TSA plates that had been swabbed with S. simulans (well-lawn test). Plates were incubated at 37°C and ZOI were measured from the edge of each well to the perimeter of each ZOI. Results Average ZOI size for all five antibiotic-producing bacteria increased as incubation time increased except for bacterium βΔ3d, for which average ZOI size increased until 32 hours and then decreased. Figure 5. ZOI produced by broth filtrates (incubated for 40 hours) diffusing into TSA from well and inhibiting growth of S. simulans (TSB served as control). Figure 4. Average ZOI size (mm) of antibiotic-producing bacteria broth filtrates against S. simulans lawn at different incubation times. Examining properties of antibiotics Methods Well-lawn tests were performed for antibiotic-producing bacteria broth filtrates that had been heated to 70°C and 100°C for five minutes. Results The 100°C treatment caused average ZOI size for two antibiotic-producing bacteria (GC3y and βΔ3d) to decrease, while average ZOI size of the bacterium IH5x was unaffected by the 100°C treatment. Broth filtrates of bacteria BDx and 2x did not produce ZOI. Figure 7. ZOI produced by the following GC3y broth filtrates against a S. simulans lawn: untreated (left), heated to 70 °C (middle) and heated to 100°C (right). Figure 6. Average ZOI size (mm) of antibiotic-producing bacteria broth filtrates heated to70°C and 100°C against S. simulans lawn. Characterizing antibiotic-producing bacteria Methods The antibiotic-producing bacteria were Gram-stained and endospore-stained using standard staining procedures. Results All five antibiotic-producing bacteria were endospore-forming Gram-positive rods. Figure 9. Gram-stain of Gram-positive Figure 10. Endospore-stain of BDx revealing streptobacillus BDx (scale bar is 10 µm). green endospores (scale bar is 10 µm). Conclusions All five antibiotic-producing bacteria were endospore-forming Gram-positive rods of the genus Bacillus. It was not confirmed whether the antibiotics were peptides or non-peptides, however it is possible that the antibiotics that were peptides were those that were sensitive to heating, began producing antibiotics later, and had smaller ZOI. All five antibiotics inhibited the Gram-positive bacteria S. simulans and B. subtilis and one (2x) was also shown to inhibit the Gram-negative bacterium E. coli. Concentrating antibiotics Methods Supernatants of GC3y and IH5x culture broth were each extracted separately with n-butanol and ethyl acetate. The n-butanol extracts were evaporated to dryness with air while the ethyl acetate extracts were rotary-evaporated. The extraction products were resuspended in a small volume of water and well-lawn tests were performed. The culture broth supernatants with which the extractions were performed were also applied to well-lawn tests. Results Ethyl acetate and n-butanol extracted the IH5x antibiotic from broth while only ethyl acetate effectively extracted the GC3y antibiotic from broth. The n-butanol extraction product produced a slightly smaller average ZOI than the GC3y supernatant, indicating that n-butanol either partially inactivated the antibiotic or was ineffective at extracting GC3y antibiotic from broth. Figure 8. Average ZOI size (mm) of antibiotic extractions against S. simulans lawn indicating that antibiotics were extracted into solvents and concentrated. Acknowledgements I would like to thank Dr. McShaffrey for teaching me how to obtain good scientific images, Dr. Brown for giving me advice (and GC3y), Dr. Lustofin for helpful criticism, the 2008-2009 capstone class for their help and support, Dr. Spilatro for guiding me throughout this project and being my academic advisor, and my family. Soil-borne antibiotic-producing bacteria and characterization of their antibiotics Brandon CraneDr. Steven Spilatro