Download (MIC) titers Determine mechanism of action of novel antibiotics using

Isolating and Purifying Novel Antibiotics from Soil Bacteria
Heather Fisher, Department of Biological Sciences, York College of Pennsylvania
Expected results
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Column chromatography of
cultures showing ZOI to purify
produced antibiotics
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ZOI test on
C. albicans
plate
MIC of current and newly isolated
antibiotics against E. coli
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ZOI test on
E. coli plate
ZOI test on
S. aureus
plate
Current Antibiotics
MIC titer of
Fractions
n=10
MALDI-TOF mass spectrometry to
determine structure of antibiotics
Image 1. Zones of Inhibition (ZOI) around
various antibiotic discs.
(http://www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/AntibioticDisk
.htm)
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Figure 1. Two newly isolated antibiotics tested for minimal inhibition
concentration against organisms including Gram-positive bacteria,
Gram-negative bacteria, and a yeasts. Antibiotic A shows inhibition
ofGram positive-bacteria, but not much effect on Gram-negative
bacteria or yeast. Antibiotic B shows inhibition of all organisms.
Antibiotic A is narrow spectrum, while Antibiotic B is broad
spectrum.
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•In fact, Streptomyces are responsible for
about 75% of clinically useful antibiotics,
including tetracycline, erythromycin,
streptomycin, neomycin, chloramphenicol,
and lincomycin. (Nelson et. al. 2001)
•Zone of Inhibition (ZOI) and Minimal
Inhibition Concentration (MIC) measures
are commonly used to measure the
efficiency of antibiotics against certain
bacteria. ZOI can also be implemented to
discover the mechanism of the antibiotics.
•The mechanisms of action that antibiotics
use are inhibition of cell wall synthesis,
inhibition of protein synthesis, inhibition of
nucleic acid synthesis, alteration of cell
membranes, or anti-metabolite activity.
Tested organisms
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•Identifying novel antibiotics from various
soil bacteria could prove effective at
inhibiting both Gram-positive and Gramnegative bacteria and providing
effectiveness and potency that is equal to
the antibiotics already in use. This is
possible because many species are able to
produce multiple antibiotics varying in
structure and mechanism of action.
•Streptomyces has been found to produce
many novel antibiotics and continues to be
studied in an attempt to identify more that
are effective against gram-positive and
gram-negative bacteria. (Ceylan et. al.,
2008)
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•Soil bacteria provide an alternative to
existing antibiotics because they produce
antibiotics as secondary metabolites to
improve their survival in competitive
environments.
Make broth
culture of
colonies
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•Antibiotic resistance is constantly on the
rise causing higher rates of morbidity and
mortality and multidrug resistant infections.
•Some strains were found to have the genes
for multiple antibiotics but only produced 1
or 2 in high concentrations at a given time.
This may suggest that antibiotic synthesis
may be influenced and induced by signals
from the environment in addition to the
bacteria’s genetic makeup. (Sarangi et. al.,
2009)
DNA
sequencing of
bacteria via PCR
reaction
10
in
Introduction
•Many Bacillus species are known to
produce antibiotics to control plant diseases
and have been studied using PCR reactions
and MALDI-TOF mass spectrometry to
identify the genes responsible for the
synthesis of specific antibiotics (Sarangi et.
al., 2009)
Antibiotic A
Antibiotic B
ni
ci
ll
•Determine mechanism of action of novel
antibiotics using zone of inhibition.
Dilute soil samples and plate
on soil extract agar
MIC (ug/ml)
•Determine the effectiveness and spectrum of
the antibiotics with zone of inhibition (ZOI)
measures and minimum inhibition
concentration (MIC) titers
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•Purify and identify new antibiotics from
various soil bacteria using column
chromatography and MALDI-TOF mass
spectrometry
•Due to antibiotic producers and resisters
evolving together in the soil, it can be
assumed that a large reservoir of resistance
determinants is present in their genetic
code. (Westman and Wright, 2011)
Research Design
MIC (ug/ml)
Objectives
Review of Literature
MIC of two isolated antibiotics
against six different organisms
Test bacteria against existing
antibiotics to determine antibiotic
mechanism of action
Figure 2. Common antibiotics with various spectrums and a newly isolated
antibiotic tested for minimal inhibition concentration against E. coli. The isolated
antibiotic showing the greatest inhibition followed by sulfacetamide and
ceftriaxone.
Expected conclusions
I expect some antibiotics to exhibit a broad
spectrum and some to only be effective
against Gram-positive bacteria. I also expect to
find some antibiotics that will prove to be more
effective than many of the antibiotics currently
in use.
Literature cited
• Athukorala, Sarangi N.P., W.G. Dilantha Ferando, and Khalid Y. Rashid. "Identification of
antifugal antibiotics of Bacillus species isolated from different microhabitats using
polymerase chain reaction and MALDI-TOF mass spectrometry." Canadian Journal
of Microbiology. 55. (2009): 1021-1032. Web. 28 Jan. 2012.
<http://www.nrcresearchpress.com/doi/abs/10.1139/W09-067>.
•Ceylan O, Okmen G, Ugar A (2008) Isolation of soil Streptomyces as source antibiotics
active against antibiotic-resistant bacteria. EurAsia J BioSci 2, 9, 73-82.
www.ejobios.com/content/2/9/73-82.
•Nelson, Mark, Robert A. Greenwald, and Wolfgang Hillen. Tetracyclines in Biology, Chemistry,
and Medicine. Berlin: Birkhauser, 2001. GoogleBooks. Web. 3 Apr. 2012.
<http://books.google.com/ books?id=kHNW4tFhZD4C&printsec=frontcover&source
=gbs_ge_summary_r&cad=0#v=onepage&q&f=false>.
•Westman, Erin L., and Gerald D. Wright. "The Antibiotic Resistome: Origins, Diversity, and
Future Prospects." Handbook of Molecular Microbial Ecology II: Metagenomics in
Different Habitats. Ed. Frans J. de Bruijn. Hoboken, New Jersey: Wiley-Blackwell,
2011. 165-174. Web. 28 Jan. 2012. <http://books.google.com/books?hl=en&lr
=&id=SJgc8KK52dcC&oi=fnd&pg=PA165&dq =Resistome in
soil&ots=NsKsiNyXHE&sig=DaDHOhyE2TYh4ro24XG_HL2T7Xo
Acknowledgements
I’d like to thank Dr. Singleton for the knowledge and advice
he has provided throughout the duration of this project.