Soil-borne antibiotic-producing bacteria and characterization of

							                                                                                                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
[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):    Acknowledgements                                                                                                                              rods of the genus Bacillus.
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                   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.
[4] 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.
[5] 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
[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.                            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
[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           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.