Download The Development of a Method for the Characterization Of

The Development of a Method for the Characterization Of
Non-point Sources of Escherichia Coli
Using Protein Biomarkers
Presenters: Dr. Joe Emily; Dr. Mahtabuddin U. Ahmed; Dr.
Fernande Mofo and Mr. Ronald Whitmore
Research Site: BioAnalytical Support Laboratory (BASL), PO
Box 7351, Department of Biological and Physical Sciences,
South Carolina State University, Orangeburg, South Carolina
29117
Introduction
Non-point source (NPS) microbial contamination is well
recognized as a major threat to the safety of watersheds
utilized for recreation and as sources of drinking water. The
ability to identify the source of microbial pollution,
Microbial source tracking (MST), is essential to any efforts
directed toward eliminating NPS pollution. Although
Coliform bacteria are commonly recognized indicators of
fecal contamination of surface drinking water sources, it is
very difficult to distinguish between contamination from
human versus animal sources. Presently used methods of
microbial source tracking (MST) such as antibiotic
resistance, phenotype number ratios, DNA sequencing and
ribotyping all have certain limitations such as cost, speed,
accuracy and precision. Most of these techniques depend on
the belief that certain strains of E. Coli tend to inhabit the
gut of specific species of warm blooded mammals.
Theory
Although DNA and RNA store and transport messages
from the genetic code, proteins are the actual agents
which mediate the actions which are mandated by this
nucleic acid message. Therefore if DNA and RNA
typing can be used to distinguish between individuals of
the same species then surely proteins can be utilized to
identify different strains within a species. And if it is
true that certain strains of E. Coli tend to inhabit the gut
of specific host mammals species then there may exist
proteins or peptide fragments within E. Coli strains that
can be used to identify their host animal of origin.
Objectives
We propose to develop a method using electrospray
ionization-liquid chromatography/mass spectrometry
(ESI-LC/MS) which is capable of identifying the
specific host sources of microbial contamination based
on peptide biomarkers within the various strains of E.
coli bacteria. By comparing the mass spectra of proteins
and peptides extracted from different E. coli strains
isolated from the intestines of humans, and various wild
and domestic animals we hope to develop a method
capable of accurately linking strains recovered from
suspected contaminated water sources to their host
animal source of origin.
Design of Experiment
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#1 isolate E. Coli strains from several different animal sources
(hogs, cows, chickens, humans, horses)
#2 extract proteins from lysed cells
#3 enzymatically digesting these proteins
#4 separate peptides via multidimensional nanospray liquid
chromatography
#5 optimize the measurement of the peptide fragments via
electrospray MS.
#6 search mass spectra for peptide fragments which are likely
candidates to be used as strain specific biomarkers
#7 test the efficiency of these biomarkers at identifying the specific
animal host source of coliform contaminated waters
Procedure/Method
Water Sample
• pour 100 ml of sample through .45 um filter
• dilute samples with high bacteria counts if necessary
Membrane Filter
• add 2.0 ml of M-FC media to sterilepetri dishes
• place filter pads in petri dishes
Selective Media
(M-FC)
• Place petri dishes in waterproof plastic
whirlpack bags
• seal, invert and submerge bags in water bath
• incubate for 24 hours at 44.5 C
Fecal Coliform Colonies
• Pick individual colonies and transfer each colony
to culture tubes containing LT Broth
• each colony should represents a single coliform
bacteria strain from a potential animal host
Lauryl Tryptose Broth
• Incubate at 35 C for 24 hours
•If gas (+) produced transfer to EC media
• If no gas (-) produced wait another 24 hrs and re-evaluate
EC Broth
• Incubate at 35 C for 24 hours
• If gas (+) produced in 24 hrs then transfer to LB broth
• If no gas (-) produced then wait another 24 hours
• and re-evaluate
LB Broth
(Mid-Log Phase Growth)
• Shake flask at 225 rpm for 24 h at 37 C
Harvested Whole Cells
• centrifuge at 8,000 g for 10 min
• wash 3x with water or 8.5g/L NaCl
Reconstituted Cells
• Resuspend (5mg/ml) in 50mN ammonium
bicarbonate containing 8M urea
• add internal std (bovine insulin, ubiquitin,
equine cytochrome C)
Lyse Cells
• Sonicate for 2 min (1 pulse/s w/
0.75 duration)
Supernatant
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Separate supernatant from cell debris
Determine Protein Concentration
• Determine Protein Concentration
Tryptic Digest
2.
3.
4.
reduce w/ 1mM Dithiothreitol (DTT)
45 min at 37 C
alkylate w/ 10 mN idoacetamide for
1 hr at room temp
digest w/ trypsin (1:50 w/w ratio)
Desalt Peptide Mixture
• Acidify digest to pH 3.0 w/ formic acid
• desalt and concentrate with C18 Solid phase
extraction columns
• elute peptides from column with 75% acetonitrile, 0.1%
formic acid
Peptide Mixture
2D-Liquid Chromatograh
Mass Spectrometer
Peptide MS Data
Intensity
mass
1.Discriminant Analysis
2. Cluster Analysis
Determine the Source of Coliform Contamination
Data
The initial phases of these experiments are
currently in progress.
Conclusion
Developing a method for Microbial Source
Tracking (MST) using protein biomarkers is an
ambitious project which is very challenging. If
successful, MST-Proteomics will prove to be a
very useful tool in watershed management in the
future.
References
1. Meays, Cynthia L,; Broersma, Klass, Source tracking fecal bacteria in water: a
critcal review, J. Of Env. Man., 73, 71-79, 2004
2. Simpson, Joyce; Santo Domingo, Jorge, Microbial Source Tracking: State of the
Science, Env. Sci. & Tech, 36, 5279-5288, 2002
3. Whitlock, John E.; Jones, David T.; Identification of the sources of fecal
coliforms in an urban watershed using antibiotic resistance analysis, Water
Research, 36, 4273-4282, 2002
4. Albert, John M.; Munakaata-Marr, Junko; Statistical Evaluation of Baacterial
Source Tracking Data Obtained by rep-PCR DNA Fingerpringting of
Escherichia coli, Env. Sci & Tech, 37, 4554-4560, 2003
5. Xiang, Fan, Anderson, Anderson, Gordon A.; Smith, Richard D.,
Characterization of Microorganisms and Biomarkers Development from Global
ESI-MS/MS Analyses of Cell Lysates, Anal. Chem., 72, 2475-2481
6. Ho, Yen-Pen; Hsu, Po-Hsi, Investigating the effects of protein patterns on
microorganism identification by high performance liquid chromatography-mass
spectrometry and protein database searches, J. Chrom. A., 976, 103-111
Acknowledgements
Extramural Associates Program (NIH/NICHD)
South Carolina Alliance for Minority Participation (NSF)