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 Methods Methods the growth of or destroy bacteria and other microorganisms, and are frequently Five of the putative antibiotic-producing bacteria were cultured in tryptic soy Supernatants of GC3y and IH5x culture broth were each extracted separately used to combat bacterial infections in humans and animals1,2. Antibiotics are broth (TSB) at 37°C for 40 hours. Every 8 hours, a small volume of the with n-butanol and ethyl acetate. The n-butanol extracts were evaporated to routinely added to livestock feed which, along with intensive broad-spectrum supernatant of each culture broth was filtered with 0.2 µm pore membranes. This dryness with air while the ethyl acetate extracts were rotary-evaporated. The antibiotic use for the treatment of human diseases, has led to the emergence of broth filtrate was transferred to wells bored into the media of TSA plates that had extraction products were resuspended in a small volume of water and well-lawn antibiotic-resistant organisms1,2. As human pathogens continue to acquire been swabbed with S. simulans (well-lawn test). Plates were incubated at 37°C tests were performed. The culture broth supernatants with which the extractions resistance to all classes of current antibiotics, it is necessary to develop novel and ZOI were measured from the edge of each well to the perimeter of each ZOI. were performed were also applied to well-lawn tests. antibiotic compounds1. New antibiotic-producing strains of bacteria isolated from soil samples around Results Results the world are continually being discovered and patented, and the effectiveness of Average ZOI size for all five antibiotic-producing bacteria increased as Ethyl acetate and n-butanol extracted the IH5x antibiotic from broth while only the antibiotics produced by these strains is being tested against a wide range of incubation time increased except for bacterium βΔ3d, for which average ZOI size ethyl acetate effectively extracted the GC3y antibiotic from broth. The n-butanol human and animal pathogens3,4,5. Bacteria of the Bacillus genus occur mainly in increased until 32 hours and then decreased. extraction product produced a slightly smaller average ZOI than the GC3y soil and produce many widely studied antibiotic compounds6,5. For example, 7 supernatant, indicating that n-butanol either partially inactivated the antibiotic or Bacillus subtilus produces more than seventy-five known antibiotics consisting was ineffective at extracting GC3y antibiotic from broth. BDx predominantly of small, cyclic peptides, but also including phospholipid, 6 IH5x Figure 8. Average ZOI size (mm) of antibiotic extractions against S. simulans lawn indicating that lipopeptide, and aminosugar antibiotics7,8,9,3,5. GC3y Average ZOI size (mm) 5 βΔ3d antibiotics were extracted into solvents and concentrated. The objectives of this experiment were to survey for antibiotic-producing ϵ2x 9 bacteria found in soil and isolate and characterize antibiotics produced by soil- 4 borne bacteria. Putative antibiotic-producing bacteria were isolated from soil and 8 Supernatant antibiotic production was confirmed. The culture broth filtrates were treated to 3 Average ZOI size (mm) 7 n-But. Broth determine whether the antibiotics were peptides or non-peptides. Antibiotics n-But. Extract 2 6 EtAc Broth were extracted and concentrated from culture broth and the antibiotic-producing EtAc Extract Figure 5. ZOI produced by broth filtrates bacteria were characterized by Gram- and endospore-staining. 1 (incubated for 40 hours) diffusing into 5 It was hypothesized that antibiotic-producing bacteria would be of the genera TSA from well and inhibiting growth of 4 Bacillus or Streptomyces and that the antibiotics would be peptides that inhibited 0 S. simulans (TSB served as control). 0 8 16 24 32 40 the growth of Gram-positive bacteria. 3 Incubation time (hrs) 2 Figure 4. Average ZOI size (mm) of antibiotic-producing bacteria broth filtrates against S. simulans lawn at different incubation times. 1 Isolating putative antibiotic-producing bacteria 0 IH5x GC3y Methods Antibiotic-producing bacteria Soil samples collected from Ohio, Georgia, Florida, and Alaska were Examining properties of antibiotics suspended in water and swabbed on plates of tryptic soy agar (TSA). Zones of Methods inhibition (ZOI) were identified on the plates and the inhibitory capabilities of putative antibiotic-producing bacteria were confirmed with cross tests and spot- Well-lawn tests were performed for antibiotic-producing bacteria broth filtrates Characterizing antibiotic-producing bacteria that had been heated to 70°C and 100°C for five minutes. lawn tests. Methods Results Results The antibiotic-producing bacteria were Gram-stained and endospore-stained The 100°C treatment caused average ZOI size for two antibiotic-producing using standard staining procedures. Fourteen putative antibiotic-producing bacteria were isolated. 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 Results ϵ2x did not produce ZOI. All five antibiotic-producing bacteria were endospore-forming Gram-positive 7 rods. Control 6 70ºC 100ºC Average ZOI size (mm) 5 4 Figure 7. ZOI produced by the following GC3y broth 3 filtrates against a S. simulans lawn: untreated (left), heated to 70 °C (middle) and heated to 100°C (right). Figure 1. Bacterial colonies Figure 2. Cross test of Figure 3. Spot-lawn test with 2 from a swabbed soil suspension horizontally streaked putatitve inoculated spots of putative Figure 9. Gram-stain of Gram-positive Figure 10. Endospore-stain of BDx revealing growing on TSA plate. antibiotic-producing bacterium antibiotic-producing bacteria 1 streptobacillus BDx (scale bar is 10 µm). green endospores (scale bar is 10 µm). GC3y across vertically streaked on a S. simulans lawn showing Figure 6. Average ZOI size (mm) of antibiotic-producing S. simulans resulting in inhibited ZOI where S. simulans growth bacteria broth filtrates heated to70°C and 100°C against 0 S. simulans growth. is inhibited. IH5x GC3y βΔ3d S. simulans lawn. Antibiotic-producing bacteria Conclusions Literature Cited All five antibiotic-producing bacteria were endospore-forming Gram-positive  Gould IM. 2008. The epidemiology of antibiotic resistance. International Journal of Antimicrobial Agents 32S: S2-S9.  Iovine NM and Blaser MJ. 2004. Antibiotics in animal feed and spread of resistant Campylobacter from poultry to humans. Emerging Infectious Diseases 10(6): Acknowledgements rods of the genus Bacillus. 1158-1159.  Rosado AS and Seldin L. 1993. Production of a potentially novel anti-microbial substance by Bacillus polymyxa. World Journal of Microbiology and I would like to thank Dr. McShaffrey for teaching me how to obtain good It was not confirmed whether the antibiotics were peptides or non-peptides, Biotechnology, 9: 521-528.  Woolford MK. 1972. The semi large-scale production, extraction, purification and properties of an antibiotic produced by Bacillus licheniformis strain 2725. scientific images, Dr. Brown for giving me advice (and GC3y), Dr. Lustofin for however it is possible that the antibiotics that were peptides were those that were Journal of Applied Bacteriology, 35: 227-331.  Mannanov RN and Sattarova RK. 2001. Antibiotics produced by Bacillus bacteria. Chemistry of Natural Compounds, 37(2): 117-123. helpful criticism, the 2008-2009 capstone class for their help and support, Dr. sensitive to heating, began producing antibiotics later, and had smaller ZOI. All  Al-Janabi AAHS. 2006. Identification of Bacitracin produced by local isolate of Bacillus licheniformis. African Journal of Biotechnology, 5(18): 1600-1601.  Stein T. 2005. Bacillus subtilus antibiotics: structures, syntheses and specific functions. Molecular Microbiology, 56(4): 845-857. Spilatro for guiding me throughout this project and being my academic advisor, five antibiotics inhibited the Gram-positive bacteria S. simulans and B. subtilis and  Katz E and Demain AL. 1977. The peptide antibiotics of Bacillus: Chemistry, biogenesis, and possible functions. Bacteriological Reviews, 41(2): 449-474.  Tamehiro N, Okamoto-Hosoya Y, Okamoto S, Ubukata M, Hamada M, Naganawa H, and Ochi K. 2001. Bacilysocin, a novel phospholipid antibiotic produced by and my family. one (ϵ2x) was also shown to inhibit the Gram-negative bacterium E. coli. Bacillus subtilis 168. Antimicrobial Agents and Chemotherapy, 46(2): 315-320.
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