Rapid Lipid Biomarker Analysis for Quantitative Assessment of Microbial Community Composition and Activity

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Rapid Lipid Biomarker Analysis for Quantitative
Assessment of Microbial Community Composition and
Activity
David C. White, Cory Lytle, Aaron Peacock,
Yun-Juan Chang, Jonas S. Almeida, Ying Dong
Gan, Institute for Applied Microbiology, 10515
Research Drive, Suite 300, Knoxville,
TN,37932-2575,

University of Tennessee
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In-situ Microbial Community Assessment
What do you want to know? Characterization of
the microbial community 1. Viable and Total
biomass ( lt 0.1 culturable VBNC ) 2.
Community Composition General proportions of
clades Specific organisms (? Pathogens)
3. Physiological/Nutritional Status Evidence
for 4 Metabolic Activities (Genes Enzymes
Action) 5.Community Interactions
Communications
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In-situ Microbial Community Assessment
Classical Plate Count lt 1.0 to 0.1 of
community, takes days, lose community
interactions Physiology Two Biomarker
Methods DNA Recover from surface, Amplify
with PCR using rDNA primers , Separate with
denaturing gradient gel electrophoresis (DGGE),
sequence for identification and phylogenetic
relationship. Great specificity Lipids
Extract, concentrate, structural
analysis Quantitative, Insight into viable
biomass, community composition, Nutritional-physio
logical status, evidence for metabolic activity
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Signature Lipid Biomarker Analysis
Cathedral from a Brick Predict impact of Cr
contamination (from 50-200,000 ppm) on soil
microbial community by artificial neural network
(ANN) analysis PLFA (phospholipid fatty acid)
excellent x 102-103 ppm Cr with (PLFA). DNA
is non compressible perfect code not so
influenced By microniche conditions as cell
membranes PLFA is compressible as contains
physiological status input Contains holistic
information responds to perturbations
Predict it is a Cathedral or a Prison DNA a
perfect brick PLFA a non-linear mixture of
bricks and a window

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Detection of Specific genes or rDNA

• Recover DNA from samples (often aqueous of
• lipid extract is best)
• 2. Amplify with PCR using rDNA eubacterial
  primers
• 3. Separate Amplicons with Denaturating
• Gel Gradient Electrophoresis (DGGE)
• 4. Isolate Bands,
• 5. Sequence and match with rDNA database
• 6. Phylogenetic analysis

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Sampling locations at the Shiprock site, NM
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Na,Mg,Cl-, SO4--,K,
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Bands Phylogenetic group Metal Metabolism / Metal Transport Properties Associated with Group
A Arthrobacter Plasmid-borne heavy metal resistance
B Bacillus Known resistance to metals incl. Hg, Cd, Pb, Zn, etc.
C Vibrio Heavy metal resistant (via membrane permeability and transport)
D Shewanella Facultative anaerobes (anaerobic respiration utilizes Fe3, Mn4, U6)
E Pseudomonas Metal efflux genes on both plasmid and chromosome (commonly detected at metal contaminated sites) also ability to reduce metal
F Marinomonas As above (Pseudomonas)
G Pedomicrobium Metal (Fe3, Mn4) oxidizers
Table Identification of sequences derived from
DGGE bands
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(No Transcript)
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LIPID Biomarker Analysis
1. Intact Membranes essential for Earth-based
life 2. Membranes contain Phospholipids 3.
Phospholipids have a rapid turnover from
endogenous phospholipases . 4. Sufficiently
complex to provide biomarkers for viable
biomass, community composition,
nutritional/physiological status 5. Analysis
with extraction provides concentration
purification 6. Structure identifiable by
Electrospray Ionization Mass Spectrometry at
attomoles/uL (near single bacterial cell) 7.
Surface localization, high concentration ideal
for organic SIMS mapping localization
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Lyophilized Soil Fractions, Pipe Biofilm
1. Neutral Lipids
SFECO2
UQ isoprenologues
ESE Chloroform.methanol
Derivatize N-methyl pyridyl Diglycerides
Sterols Ergostrerol Cholesterol
2. Polar Lipids
Transesterify PLFA
Intact Lipids
Phospholipids PG, PE, PC, Cl, sn1
sn2 FA Amino Acid PG Ornithine lipid Archea ether
lipids Plamalogens
3. In-situ acidolysis in SFECO2
CG/MS
PHA Thansesterify Derivatize N-methyl
pyridyl
2,6 DPA (Spores)
LPS-Lipid A OH FA
HPLC/ES/MS/MS
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Membrane Liability (turnover)
VIABLE
NON-VIABLE
O
O


H2COC
H2COC
O
O
phospholipase




cell death
C O CH
C O CH

O


H2 C O H
H2 C O P O CH2CN H3

Neutral lipid, DGFA
O-
Polar lipid, PLFA
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Biofilm Community Composition
Detect viable microbes Cell-fragment biomarkers
Legionella pneumophila, Francisella
tularensis, Coxellia burnetii, Dienococcus,
PLFA oocysts of Cryptosporidium parvum,
Fungal spores PLFA Actinomycetes Me-br PLFA
Mycobacteria Mycocerosic acids, (species and
drug resistance) Sphingomonas paucimobilis
Sphingolipids Pseudomonas Ornithine
lipids Enterics LPS fragments Clostridia
Plasmalogens Bacterial spores Dipicolinic acid
Arthropod Frass PLFA, Sterols Human
desquamata PLFA, Sterols Fungi
PLFA, Sterols Algae Sterols,
PLFA, Pigments
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Signature Lipid Biomarker Analysis
Microniche Properties from Lipids 1.
Aerobic microniche/high redox potential. high
respiratory benzoquinone/PLFA ratio, high
proportions of Actinomycetes, and low levels of
i150/a150 (lt 0.1) characteristic of
Gram-positive Micrococci type bacteria,
Sphinganine from Sphingomonas 2. Anaerobic
microniches high plasmalogen/PLFA ratios
(plasmalogens are characteristic Clostridia), the
isoprenoid ether lipids of the methanogenic
Archae. 3. Microeukaryote predation high
proportions of phospholipid polyenoic fatty acids
in phosphatidylcholine (PC) and cardiolipin (CL).
Decrease Viable biomass (total PLFA) 4.
Cell lysis high diglyceride/PLFA ratio.

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Signature Lipid Biomarker Analysis
Microniche Properties from Lipids 5.
Microniches with carbon terminal electron
acceptors with limiting N or Trace growth factors
high ( gt 0.2) poly ß-hydroxyalkonate
(PHA)/PLFA ratios 6. Microniches with
suboptimal growth conditions (low water activity,
nutrients or trace components) high ( gt 1)
cyclopropane to monoenoic fatty acid ratios in
the PG and PE, as well as greater ratios of
cardiolipin (CL) to PG ratios. 7.
Inadequate bioavailable phosphate high lipid
ornithine levels 8. Low pH high lysyl
esters of phosphatidyl glycerol (PG) in
Gram-positive Micrococci. 9. Toxic
exposure high Trans/Cis monoenoic PLFA

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Signature Lipid Biomarker Analysis

• Phospholipid Fatty Acid PLFA Biomarker
  Analysis Single most quantitative,
  comprehensive insight into in-situ microbial
  community
• Why not Universally utilized?
• Requires 8 hr extraction with ultrapure solvents
  emulsions.
• Ultra clean glassware incinerated 450oC.
• Fractionation of Polar Lipids
• Derivatization transesterification
• 5. GC/MS analysis picomole detection 104
  cells LOD
• 6. Arcane Interpretation Scattered Literature
• 7. 3-4 Days and 250

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Signature Lipid Biomarker Analysis
Expand the Lipid Biomarker Analysis
1. Increase speed and recovery of extraction
Flash 2. Include new lipids responsive to
physiological status HPLC (not need
derivatization) Respiratory quinone redox
terminal electron acceptor Diglyceride cell
lysis Archea methanogens Lipid ornithine
bioavailable phosphate Lysyl-phosphatidyl
glycerol low pH Poly beta-hydroxy alkanoate
unbalanced growth 3. Increased Sensitivity
and Specificity ESI/MS/MS

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ESI (cone voltage)
Q-1
CAD
Q-3
ESI/MS/MS
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PE-Sciex API 365 HPLC/ESI/MS/MS Functional Sept
29, 2000
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Coupon Biofilm
Extract with SFECO2
?
1. Neutral Lipids
UQ isoprenologues UQ-8 Enterics, UQ-9
Pseudomonas, UQ-10 Protozoa
Derivatize N-methyl pyridyl Diglycerides (cell
lysis) Sterols, Cholesterol (Protozoa),
Ergostrerol (Fungi)
Extract Residue with Chloroform.methanol
2. Polar Lipids
Lipid Biomarkers
?
Phospholipids, PC, PE, PG, sn1 sn2 FA Amino
Acid PG, 0rnithine lipids, Plasmalogens
Acidify, Extract residue with SFECO2

?
3. LPS OH FA
Transesterify, GC/MS . ? 30H 100, 120
Pseudomonas 30H 140 --
pathogens enterics

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Lipid Biomarker Analysis
Sequential High Pressure/Temperature Extraction
( 1 Hour) Supercritical CO2 Methanol enhancer
Neutral Lipids, (Sterols, Diglycerides,
Ubiquinones) Lyses Cells
Facilitates DNA Recovery and Adenine
Nucleotides for Adenylate Energy Charge (for
off-line analysis) 2. Polar solvent Extraction
Phospholipids CID detect negative ions
Plasmalogens Archeal Ethers 3).
In-situ Derivatize Extract Supercritical CO2
Methanol enhancer 2,6 Dipicolinic acid
Bacterial Spores Ester-Linked Hydroxy
Fatty acids Gram-negative LPS Three
Fractions for HPLC/ES/MS/MS Analysis
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Feasibility of Flash Extraction
ASE vs BD solvent extraction Bacteria BD,
no distortion Fungal Spores 2 x BD Bacterial
Spores 3 x BD Eukaryotic 3 x polyenoic
FA 2 cycles 80oC, 1200 psi, 20 min vs BD
8 -14 Hours
Macnaughton, S. J., T. L. Jenkins, M. H. Wimpee,
M. R. Cormier, and D. C. White. 1997. Rapid
extraction of lipid biomarkers from pure culture
and environmental samples using pressurized
accelerated hot solvent extraction. J.
Microbial Methods 31 19-27(1997)
CEB
Microbial Insights, Inc.
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Problem Rapid Detection/Identification of
Microbes
Propose a Sequential High Pressure/Temperature
Extractor Delivers Three Analytes to
HPLC/ESI/MS/MS
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Respiratory Benzoquinone (UQ)
Gram-negative Bacteria with Oxygen as terminal
acceptor LOQ 580 femtomole/ul, LOD 200
femtomole/ul 104 E. coli
Q7
Q10
Q6
197 m/z
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ESI/MS Pyridyl Derivative of Cholesterol
MS/MS LOD should be 100 amoles
Unknown
LOD10 ppb LOQ30 ppb
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HPLC/ESI/MS

• Enhanced Sensitivity
• Less Sample Preparation
• Increased Structural Information
• Fragmentation highly specific i.e. no proton
  donor/acceptor fragmentation processes occurring

CEB
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Parent product ion MS/MS of synthetic PG
Q-1 1ppm PG scan m/z 110-990
(M H) -
Sn1 160, Sn2 182
Q-3 product ion scan of m/z 747 scanned m/z
110-990 Note 50X gt sensitivity
SIM additional 5x gt sensitivity 250X
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Gram-negative Bacteria ? lipid-extracted
residue, ? hydrolize 1 Acetic acid , ? extract
Lipid A

• Acid sensitive bond
  to KDO

?
?
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E. Coli Lipid A ? 3 OH 140
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Lipid A from E. coli Fatty acids liberated by
acid hydrolysis followed by acidcatalyzed
(trans) esterification
3OH 140 TMS
GC/MS of Methyl esters
3OH 140
140
phthalate siloxane
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WQ1 669 524 94
LIPID A     Pseudomonas 3 0H 120 3 0H 100
(water organism) Enteric Pathogens 30H 140
(fecal potential pathogen) Toilet bowl biofilms
High flush vs Low flush rate ? Higher
monoenoic, lower cyclopropane PLFA
Gram-negative more actively growing bacteria
mol ratios of 72 (30)/19 (4) of 3 0H 10 12/
3 OH 140 LPS fatty acids 3.8 Human feces
7 (0.6)/19 (4) 3 0H 10 12/ 3 OH 140 in human
feces 0.37 mean(SD).
Pet safety if access to processed non-potable
water.
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ESI Spectrum of 2, 6-Dimethyl Dipicolinate
LOD 103 spores 0.5 femtomoles/ul
MH
ES
Mobile phase MeOH 1mM ammonium acetate Cone
40V
MNa
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ANN Analysis of CR impacted Soil Microbial
Communities

1. Cannelton Tannery Superfund Site, 75 Acres on the
   Saint Marie River near Sault St. Marie, Upper
   Peninsula, MI
2. Contaminated with Cr3 and other heavy metals
   between1900-1958 by the Northwestern Leather Co.
3. Cr3 background 10-50 mg/Kg to 200,000 mg/Kg.
4. Contained between 107-109/g dry wt. viable
   biomass by PLFA no correlation with Cr
   (Pgt0.05)
5. PLFA biomass correlated (Plt001) with TOM TOC but
   not with viable counts (P0.5)

-CEB
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ANN Analysis of Cr3 impacted Soil Microbial
Communities
CONCLUSIONS 1. Non-Linear ANN gtgt predictor than
Linear PCA (principal Components Analysis) 2. No
Direct Correlation (Pgt0.05) Cr3 with Biomass
(PLFA), Positive correlation between biomass
(PLFA) and TOC,TOM 3. ANN Sensitivity to Cr3
Correlates with Microeukaryotes (Fungi)181?9c,
and SRB/Metal reducers (i150, i 170, 161w11,
and 10Me 160) 4. SRB Metal reducers peaked
10,000 mg/Kg Cr3 5. PLFA of stress gt trans/cis
monoenoic, gt aliphatic saturated with gt Cr3
-CEB
NABIR
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Rapid Assessment of Subsurface in-situ Microbial
Communities by Lipid Biomarkers for Remediation
Potential, Monitoring Effectiveness, and as
Rational End-Points
Rational (Defensible) End Point Multi species,
multiple tropic level assessments vs single
species toxicity assessment How Clean is
Clean Quantitatively Monitor Microbial
Community Composition When uncontaminated
subsurface sediment has same, or is approaching
the same type of community composition as
treated sediment Biofilms are Very
satisfactory for surface water run-off Diatoms ?
Filamentous Algae (pollution) ? Diatoms

-CEB
Microbial Insights, Inc.
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Sampling Drinking Water-- Collect Biofilms on
Coupons
Biofilms not pelagic in the fluid

• 104-106 cells/cm2 vs 103-104 /Liter
• Integrates Over Time
• Pathogen trap nurture
• (including Cryptosporidum oocysts)
• 4. Serves as a built in solid phase extractor for
  hydrophobic drugs, hormones, bioactive agents
• 5. Convenient to recover analyze for
  biomarkers
• Its not in the water but the slime on the pipe

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Triclosan (Pyridinium derivative) Q1scan
380.3
218.1
Product ion scan
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Toxicity Biomarkers

• Hypochlorite, peroxide exposure induces
• 1. Formation of oxirane (epoxy) fatty acids from
  phospholipid ester-linked unsaturated fatty
  acids
• 2. Oxirane fatty acid formation correlates with
  inability
• to culture in rescue media. Viability?
  
• 3. Oxirane fatty acid formation correlates
  with
• cell lysis indicated by diglyceride formation
  and loss of phospholipids.

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WQ1 669 524 94
Goal      Provide a Rapid (minutes)
Quantitative Automated Analytical System that can
analyze coupons from water systems to 1).)
Monitor for Chlorine-resistant pathogens
Legionella, Mycobacteria, Spores 2). Provide
indicators for specific tests (Sterols for
Cryptosporidium, LPS OH-FA for enteric bacteria
3). Monitor hydrophobic drugs bioactive
molecules ? Establish Monitored Reprocessed
Waste Water as safer than the wild type
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PCA 2 Analysis of Forest Community Soil total
PLFA
PCA 1
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Major bands have been Recovered For
sequencing Phylogenetic analysis
Figure 1. DGGE analysis bacterial community in
water and shrimp gut samples. Amplified 16S rDNAs
were separated on a gradient of 20 to 65
denaturant.
Water changed composition between Aug 17 31st,
much gt diversity than shrimp gut, Fore gut less
diverse than Hind gut.
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Microbial Community in Water (W), Fore Gut (F),
Hind Gut (H)

W F H W F H W F H W F
H W F H
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Microbial Viable Biomass Water (W), Fore Gut
(F), Hind Gut (H)
Note Log scale

W F H W F H W F H
W F H W F H
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Shrimp In Mariculture Water Gut Microbial
Community

• Shifts Gut Water Microbiota in 52 days of
  growth
• pathogen-controlled shrimp outgrowth in a
  closed system, can be solar heated
• Water microbial biomasssame, Algal and
  Microeukaryotes decrease
• Desulfobacter increase Desulfovibrio slight
  decrease
• Gram-negative bacteria increase then decrease
• Gut Community very different from water
• DGGE shows Hepatopancreas Mycobacteria,
  Propionobacteria, SRB
• and algae (chloroplast gt BIOMASS THAN WATER
• DGGE shows Hind Gut Vibrio exclusively less
  diverse community
• Gut 2-order of magnitude gt viable microbial
  biomass than water
• Gut and Water different PLFA from Shrimp food

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Problem Rapid Non-invasive Detection of
Infection or Metabolic stress for Emergency room
Triage

Human Breath sample GC/MS