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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
In-situ Microbial Community Assessment
What do you want to know?
Characterization of the microbial community:
1. Viable and Total biomass ( < 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
In-situ Microbial Community Assessment
Classical Plate Count < 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-physiological status, evidence for metabolic activity
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
Detection of Specific genes or rDNA
1. 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
Sampling locations at the Shiprock site, NM
A
C
D
C
C
B
B
E
B
E
E
B
B
D
G
G
C
F
C
D
F
C
B
E
B
F
C
Stds
765
767
770
a
E
B
A
769
774
a
772
780
771
764
763
N(PCR)
Increasing Uranium (VI)* concentration
B
D
B
C
A
DGGE analysis of bacterial communities in
sediment samples. Amplified product was
separated on a gradient of 20%-65%
denaturant
*&
Na+,Mg++,Cl,
SO4--,K+,
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
100
71
79
Umtra DSR group A
90
71
50
Umtra DSR group B
91
Umtra DSR group C
50
84
75
100
94
Umtra DSR group D
92
55
100
75
100
Umtra DSR group E
96
67
100
100
77
100
Umtra DSR group F
97
88
99
100
96
81
100
97
94
100
94
100
100
Umtra DSR group G
100
99
100
75
100
81
100
81
Umtra DSR group H
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
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
Intact Lipids
PLFA
CG/MS
Phospholipids
PG, PE, PC, Cl,
& sn1 sn2 FA
Amino Acid PG
Ornithine lipid
Archea ether lipids
Plamalogens
3. In-situ acidolysis in SFECO2
PHA
Thansesterify &
Derivatize
N-methyl pyridyl
2,6 DPA (Spores)
LPS-Lipid A OH FA
HPLC/ES/MS/MS
Membrane Liability (turnover)
VIABLE
NON-VIABLE
O
O
||
||
H2COC
O H2COC
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
OPolar lipid, ~ PLFA
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
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 i15:0/a15:0 (< 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.
Signature Lipid Biomarker Analysis
Microniche Properties from Lipids
5. Microniches with carbon & terminal electron acceptors with limiting N or
Trace growth factors ~ high ( > 0.2) poly β-hydroxyalkonate (PHA)/PLFA ratios
6. Microniches with suboptimal growth conditions (low water activity,
nutrients or trace components) ~ high ( > 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
Signature Lipid Biomarker Analysis
Phospholipid Fatty Acid [PLFA] Biomarker Analysis =
Single most quantitative, comprehensive insight into insitu microbial community
Why not Universally utilized?
1. Requires 8 hr extraction with ultrapure solvents [emulsions].
2. Ultra clean glassware [incinerated 450oC].
3. Fractionation of Polar Lipids
4. Derivatization [transesterification]
5. GC/MS analysis ~ picomole detection ~ 104 cells LOD
6. Arcane Interpretation [Scattered Literature]
7. 3-4 Days and ~ $250
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
ESI
(cone voltage)
Q-1
ESI/MS/MS
CAD
Q-3
PE-Sciex API 365 HPLC/ESI/MS/MS Functional Sept 29, 2000
Extract with SFECO2
Coupon + Biofilm

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 10:0, 12:0 –Pseudomonas
30H 14:0 -- pathogens & enterics
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
Feasibility of “Flash” Extraction
ASE vs B&D solvent extraction*
Bacteria = B&D, no distortion
Fungal Spores = 2 x B&D
Bacterial Spores = 3 x B&D
Eukaryotic = 3 x polyenoic FA
[2 cycles 80oC, 1200 psi, 20 min]
vs B&D = 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)
Microbial
Insights, Inc.
CEB
Problem: Rapid Detection/Identification of Microbes
Propose a Sequential High Pressure/Temperature
Extractor Delivers Three Analytes to HPLC/ESI/MS/MS
MeOH
MeOH
CHCl3
PO 4-
Pump
CO2
Spe-ed SFE-4
NL
PL
LPS
Fraction Collector
N2 blowdown
Auto
sampler
HPLC/ES/MS/MS
Respiratory Benzoquinone (UQ)
Gram-negative Bacteria with Oxygen as terminal acceptor
LOQ = 580 femtomole/ul, LOD = 200 femtomole/ul ~ 104 E. coli
Q7
Q6
Q10
O
H3OC
CH3
H3OC
O
197 m/z
H
]n
ESI/MS Pyridyl Derivative of Cholesterol
MS/MS LOD should be
~ 100 amoles
pyridyl derivative of cholesterol
chol2 1 (0.620)
Scan ES+
2.13e8
375
100
Unknown
LOD=10 ppb
LOQ=30 ppb
H3C
CH 3
CH 3
H
H3C
CH 3
%
CH 3
H
N
CH 3
CH 3
H
H
O
CH 3
CH 3
CH 3
H
H
479
H
376
480
377
369
0
m/z
210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 510
HPLC/ESI/MS
• Enhanced Sensitivity
• Less Sample
Preparation
• Increased Structural
Information
• Fragmentation highly
specific i.e. no proton
donor/acceptor
fragmentation
processes occurring
O
X O
P
O
CH2
O
O
HC O C R1
O
R2
C O CH2
CEB
Parent product ion MS/MS of synthetic PG
Q-1 1ppm PG scan m/z 110-990
(M –H) -
Sn1 16:0, Sn2 18:2
Q-3 product ion scan of m/z 747 scanned
m/z 110-990
Note 50X > sensitivity
SIM additional 5x > sensitivity ~ 250X
Gram-negative Bacteria  lipid-extracted residue, 
hydrolize [1% Acetic acid ],  extract = Lipid A
Acid sensitive bond
O
[to KDO]
O
O
O P O
OH O
HO
O
HN
O
O
O
Exact Mass: 1765.19
HN
O
OH
O
C93H174N2O24P22-
O
O
P O
O
OH
O
OH
O
O

Mol. Wt.: 1766.32

14*
14*
E. Coli Lipid A  3 OH 14:0*
Lipid A from E. coli
Fatty acids liberated by acid hydrolysis followed by
acid–catalyzed (trans) esterification
3OH 14:0 TMS
GC/MS of
Methyl esters
14:0
3OH 14:0
phthalate
siloxane
WQ1 669 524 94
LIPID A:
Pseudomonas 3 0H 12:0 & 3 0H 10:0 (water organism)
Enteric & Pathogens 30H 14:0 (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 14:0 LPS fatty
acids = 3.8
Human feces 7 (0.6)/19 (4) 3 0H 10 +12/ 3 OH 14:0 in human
feces = 0.37
[*mean(SD)].
Pet safety if access to processed non-potable water.
ESI Spectrum of 2, 6-Dimethyl Dipicolinate
LOD ~ 103 spores ~ 0.5 femtomoles/ul
[M+H]+
ES+
Mobile phase: MeOH + 1mM ammonium acetate
Cone: 40V
H3OC
OCH 3
N
O
O
C9H9NO4
Exact Mass: 195.05
[M+Na]+
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 Cr+3 and other heavy metals
between1900-1958 by the Northwestern Leather Co.
3. Cr+3 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] (P>0.05)
5. PLFA biomass correlated (P<001) with TOM &TOC but
not with viable counts (P=0.5)
-CEB
ANN Analysis of Cr+3 impacted Soil Microbial
Communities
CONCLUSIONS:
1. Non-Linear ANN >> predictor than Linear PCA (principal
Components Analysis)
2. No Direct Correlation (P>0.05) Cr+3 with Biomass (PLFA),
Positive correlation between biomass (PLFA) and TOC,TOM
3. ANN: Sensitivity to Cr+3 Correlates with Microeukaryotes
(Fungi)18:19c, and SRB/Metal reducers (i15:0, i 17:0, 16:1w11,
and 10Me 16:0)
4. SRB & Metal reducers peaked 10,000 mg/Kg Cr+3
5. PLFA of stress > trans/cis monoenoic, > aliphatic saturated
with > Cr+3
NABIR
-CEB
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
Microbial
Insights, Inc.
-CEB
Sampling Drinking Water-- Collect Biofilms on Coupons
Biofilms not pelagic in the fluid
1. 104-106 cells/cm2 vs ~ 103-104 /Liter
2. Integrates Over Time
3. 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
Triclosan (Pyridinium derivative) Q1scan
+Q1: 181 MCA scans from 0927001.wiff
Max. 1.3e9 cps.
101.8
1.3e9
H3C
N
1.2e9
1.1e9
380.3
380.3
1.0e9
Cl
O
O
8.0e8
7.0e8
6.0e8
124.2
Cl
5.0e8
Cl
384.3
74.2
4.0e8
3.0e8
81.3
58.4
110.3
C18H13Cl3NO2+
80.9
Exact Mass: 380.00
2.0e8
75.2
0.0
60
86.4
80
116.3
100
375.7
Mol. Wt.: 381.66
1.0e8
397.7
165.4
120
140
160
180
200
220
240
260
280
300
m/z, amu
320
340
360
380
400
420
440
+Product (380.3): 181 MCA scans from 0927003.wiff
460
480
500
Max. 9.3e6 cps.
218.1
218.1
9.3e6
9.0e6
Product ion scan
8.5e6
8.0e6
7.5e6
7.0e6
6.5e6
In te n s ity , c p s
In te n s ity , c p s
9.0e8
6.0e6
5.5e6
5.0e6
4.5e6
4.0e6
3.5e6
3.0e6
2.5e6
2.0e6
236.1
1.5e6
93.2
1.0e6
0.0
219.1
125.1
5.0e5
79.1110.0
60
80
100
141.0
237.0
112.1
120
140
380.2
204.2
160
180
200
220
240
260
280
300
m/z, amu
320
340
360
380
400
420
440
460
480
500
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.
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
PCA 2 Analysis of Forest Community Soil total PLFA
October
2
-1
PCA Analysis
Sugar MapleBasswood
Black OakWhite Oak
Sugar MapleRed Oak
1
-1
PCA 1
August
1
-1
-1
Standard
Fore gut
Water 817
Water 831
Hind gut
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 >
diversity than shrimp gut, Fore gut less diverse than Hind gut.
Microbial Community in Water (W), Fore Gut (F), Hind Gut (H)
100%
80%
60%
Monos
Bmonos
TBSats
MBSats
NSats
40%
20%
W F H W F H
W F H
83101H
83101F
83101
82301H
82301F
82301
81001H
81001F
81001
80301H
80301F
80301
80201H
80201F
80201
0%
W F H W F H
Microbial Viable Biomass: Water (W), Fore Gut (F), Hind Gut (H)
Biomass PLFA
Note Log scale
1.00E+08
1.00E+07
1.00E+06
1.00E+04
1.00E+03
1.00E+02
1.00E+01
W F H W F H
W F H
83101H
83101F
83101
82301H
82301F
82301
81001H
81001F
81001
80301H
80301F
80301
80201H
80201F
1.00E+00
80201
pmol/g
1.00E+05
W F H W F H
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 biomass~same, 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 > BIOMASS THAN WATER
•
•
DGGE shows Hind Gut Vibrio exclusively less diverse community
Gut 2-order of magnitude > viable microbial biomass than water
•
Gut and Water different PLFA from Shrimp food
Problem: Rapid Non-invasive Detection of Infection or
Metabolic stress for Emergency room Triage
Human Breath sample GC/MS