Transcript Soil-borne antibiotic-producing bacteria and characterization of their

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.
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BDx
IH5x
GC3y
βΔ3d
ϵ2x
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Figure 8. Average ZOI size (mm) of antibiotic extractions against S. simulans lawn indicating that
antibiotics were extracted into solvents and concentrated.
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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
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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.
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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
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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)
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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.