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Soil-borne antibiotic-producing bacteria and characterization of their antibiotics Brandon Crane Dr. Steven Spilatro Introduction Confirming production of antibiotics Concentrating antibiotics 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 animals1,2. 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 organisms1,2. As human pathogens continue to acquire resistance to all classes of current antibiotics, it is necessary to develop novel antibiotic compounds1. 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 pathogens3,4,5. Bacteria of the Bacillus genus occur mainly in soil and produce many widely studied antibiotic compounds6,5. For example, Bacillus subtilus produces more than seventy-five known antibiotics consisting predominantly of small, cyclic peptides, but also including phospholipid, lipopeptide, and aminosugar antibiotics7,8,9,3,5. The objectives of this experiment were to survey for antibiotic-producing bacteria found in soil and isolate and characterize antibiotics produced by soilborne 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. Methods 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. 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 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. 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. 7 BDx IH5x GC3y βΔ3d ϵ2x 5 Figure 8. Average ZOI size (mm) of antibiotic extractions against S. simulans lawn indicating that antibiotics were extracted into solvents and concentrated. 9 4 8 3 2 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). 1 0 0 8 16 24 32 40 Incubation time (hrs) Figure 4. Average ZOI size (mm) of antibiotic-producing bacteria broth filtrates against S. simulans lawn at different incubation times. Isolating putative antibiotic-producing bacteria Supernatant n-But. Broth n-But. Extract EtAc Broth EtAc Extract 7 6 5 4 3 2 1 0 IH5x 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 spotlawn tests. 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. Fourteen putative antibiotic-producing bacteria were isolated. 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 ϵ2x did not produce ZOI. 7 Average ZOI size (mm) Figure 3. Spot-lawn test with inoculated spots of putative antibiotic-producing bacteria on a S. simulans lawn showing ZOI where S. simulans growth is inhibited. 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. 5 4 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). 3 2 Figure 9. Gram-stain of Gram-positive streptobacillus BDx (scale bar is 10 µm). 1 0 IH5x GC3y βΔ3d Figure 10. Endospore-stain of BDx revealing green endospores (scale bar is 10 µm). Figure 6. Average ZOI size (mm) of antibiotic-producing bacteria broth filtrates heated to70°C and 100°C against S. simulans lawn. Antibiotic-producing bacteria 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. Characterizing antibiotic-producing bacteria Control 70ºC 100ºC 6 Figure 2. Cross test of horizontally streaked putatitve antibiotic-producing bacterium GC3y across vertically streaked S. simulans resulting in inhibited S. simulans growth. GC3y Antibiotic-producing bacteria Examining properties of antibiotics Results Results Figure 1. Bacterial colonies from a swabbed soil suspension growing on TSA plate. Average ZOI size (mm) Average ZOI size (mm) 6 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. 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 (ϵ2x) was also shown to inhibit the Gram-negative bacterium E. coli.