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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, - PowerPoint PPT Presentation
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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 analysisQuantitative, Insight into: viable biomass, community composition,Nutritional-physiological status, evidence for metabolic activity
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 influencedBy 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
Signature Lipid Biomarker Analysis
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
ESCA RP MENT Ba
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Km Boundary
?0730
0725
0836
0846
0848
0837
0847
0843
0833
0841
0839
0815
0813
0827
0832
0603
0731
0728
0838
0835
0812
0826
0814
0828
0816
0844
0923
0928
07340736
0620
0610
0614 0612
0617
0619
0615
06240626
0856
0857
0853
0924
0616
0735
06280630
08550929
0.0025
0.304
0.056
0.0421
0.0519
0.0372
0.0273
0.0303
0.106
0.103
0.388
0.33
0.16
0.814
0.0471
0.0134
0.0355
0.214
0.0314
0.0282
0.123
3.04
0.142
0.315
0.069
0.0426
3.361
3.175
0.09910.392
1.06
1.59
2.26 0.202
0.482
1.61
2.24
1.120.0901
0.16
0.285
0.224
0.372
0.205
0.02210.13
0.178 2.086
0927
0925
09261.216
4.211
3.434.304
1.9260854
U0063300-02
3.5 - 4.5
0.044 - 0.5
0.5 - 1.5
1.5 - 2.5
2.5 - 3.5
<0.044 Uranium (mg/L)
Sample Concentration0.315
# Sample LocationLocation Code0828
$ ESC Location
N
1500 0 1500 Feet
m:\ugw\511\0020\10\u00633\u0063300.apr reynoldm 8/18/1999, 7:56
BackgroundSample location
Proposed backhoe sample location
Sampling locations at the Shiprock site, NM
A A
B
EE
B
D
GG
E
DC
B
B
D
FD
C
CF
B
C
BF
C
E
A
BE
C
BC
B
C
B
N(P
CR
)
763
764
771
780
772
a 774
769
a 770
767
765
Std
s
Increasing Uranium (VI)* concentration
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
Umtra DSR group A
Umtra DSR group B
Umtra DSR group C
Umtra DSR group D
Umtra DSR group E
Umtra DSR group F
Umtra DSR group G
Umtra DSR group H
10071
90 79
71
50
50
91
75
55
75
84
92
94100
100
77
100
8897
100
100 99
96 81
67100
100
96
100
97
100
100
94
94
99 100
100
100
100
81
81
100
75
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
SFECO2 1. Neutral Lipids
UQ isoprenologues
Derivatize –N-methyl pyridyl Diglycerides Sterols Ergostrerol Cholesterol
ESE Chloroform.methanol
2. Polar Lipids
Transesterify
PLFA
CG/MS
Intact Lipids
HPLC/ES/MS/MS
Phospholipids PG, PE, PC, Cl, & sn1 sn2 FAAmino Acid PGOrnithine lipidArchea ether lipidsPlamalogens
PHAThansesterify & Derivatize N-methyl pyridyl
3. In-situ acidolysis in SFECO2
2,6 DPA (Spores)
LPS-Lipid A OH FA
VIABLE NON-VIABLE
O O || ||
H2COC H2COC
| |C O CH C O CH
| |
H2 C O P O CH2CN+ H3
||
|
O
O-
||O
H2 C O H
||O
Polar lipid, ~ PLFA
Neutral lipid, ~DGFA
phospholipase
cell death
Membrane Liability (turnover)
Biofilm Community Composition
Detect viable microbes & Cell-fragment biomarkers : Legionella pneumophila, Francisella tularensis,
Coxellia burnetii, Dienococcus, PLFA oocysts of Cryptosporidium parvum, Fungal spores PLFAActinomycetes Me-br PLFA Mycobacteria Mycocerosic acids, (species and drug resistance)Sphingomonas paucimobilis Sphingolipids Pseudomonas Ornithine lipidsEnterics LPS fragmentsClostridia PlasmalogensBacterial spores Dipicolinic acid Arthropod Frass PLFA, SterolsHuman desquamata PLFA, Sterols
Fungi PLFA, Sterols Algae Sterols, PLFA, Pigments
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 in-situ 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 Lipids4. 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 acceptorDiglyceride ~ cell lysisArchea ~ methanogensLipid ornithine ~ bioavailable phosphateLysyl-phosphatidyl glycerol ~ low pHPoly beta-hydroxy alkanoate ~ unbalanced growth
3. Increased Sensitivity and Specificity ESI/MS/MS
Signature Lipid Biomarker Analysis
ESI (cone voltage) Q-1 CAD Q-3
ESI/MS/MS
PE-Sciex API 365 HPLC/ESI/MS/MS Functional Sept 29, 2000
Coupon + Biofilm Extract with SFECO2
1. Neutral Lipids UQ isoprenologues UQ-8 Enterics, UQ-9 Pseudomonas, UQ-10 ProtozoaDerivatize –N-methyl pyridyl Diglycerides (cell lysis) Sterols, Cholesterol (Protozoa), Ergostrerol (Fungi)
Extract Residue with Chloroform.methanol
2. Polar Lipids
Transesterify, GC/MS . 30H 10:0, 12:0 –Pseudomonas
30H 14:0 -- pathogens & enterics
Lipid Biomarkers
Phospholipids, PC, PE, PG, & sn1 sn2 FAAmino Acid PG, 0rnithine lipids, Plasmalogens
3. LPS OH FA
Acidify, Extract residue with SFECO2
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
*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)
Feasibility of “Flash” Extraction
ASE vs B&D solvent extraction*
Bacteria = B&D, no distortionFungal 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
CEBMicrobial Insights, Inc.
Problem: Rapid Detection/Identification of Microbes
Propose a Sequential High Pressure/Temperature Extractor Delivers Three Analytes to HPLC/ESI/MS/MS
CO2
Pump
N2 blowdownAutosampler
HPLC/ES/MS/MS
Fraction Collector
Spe-ed SFE-4 NL
PL
LPS
MeOHMeOHCHCl3PO4
-
Q6
Q7Q10
O
O
H3OC
H3OC
CH3
]H
n
197 m/z
Respiratory Benzoquinone (UQ)
Gram-negative Bacteria with Oxygen as terminal acceptor LOQ = 580 femtomole/ul, LOD = 200 femtomole/ul ~ 104 E. coli
pyridyl derivative of cholesterol
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 510m/z0
100
%
chol2 1 (0.620) Scan ES+ 2.13e8375
369
479
376
377
480
ESI/MS Pyridyl Derivative of Cholesterol
CH3
H H
CH3
H3C
H
H
CH3
CH3
O
CH3
H H
CH3
H3C
N
CH3
H
CH3
CH3
Unknown
LOD=10 ppbLOQ=30 ppb
MS/MS LOD should be ~ 100 amoles
HPLC/ESI/MS
CEB
• Enhanced Sensitivity• Less Sample
Preparation• Increased Structural
Information• Fragmentation highly
specific i.e. no proton donor/acceptor fragmentation processes occurring
CH2
HC O C
O
R1
CH2OC
O
R2
OP
O
O
OX
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
OOP
OO
OH O HN
O
OHO
OHN O
P O
OH
O
O
OH
OO
OO
O
O
O
OH
C93H174N2O24P22-
Exact Mass: 1765.19
Mol. Wt.: 1766.32
14*14*
Gram-negative Bacteria lipid-extracted residue, hydrolize [1% Acetic acid ], extract = Lipid A
Acid sensitive bond
[to KDO]
E. Coli Lipid A 3 OH 14:0*
Lipid A from E. coliFatty acids liberated by acid hydrolysis followed by
acid–catalyzed (trans) esterification
14:03OH 14:0
3OH 14:0 TMS
phthalatesiloxane
GC/MS of Methyl esters
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.
[M+H]+
[M+Na]+NOCH3
O
H3OC
O
C9H9NO4Exact Mass: 195.05
Mobile phase: MeOH + 1mM ammonium acetateCone: 40V
ES+
ESI Spectrum of 2, 6-Dimethyl Dipicolinate
LOD ~ 103 spores ~ 0.5 femtomoles/ul
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)18:19c, 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
-CEBNABIR
Rapid Assessment of Subsurface in-situ Microbial Communities by Lipid Biomarkers for Remediation Potential, Monitoring
Effectiveness, and as Rational End-Points
-CEBMicrobial Insights, Inc.
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-offDiatoms Filamentous Algae (pollution) Diatoms
1. 104-106 cells/cm2 vs ~ 103-104 /Liter 2. Integrates Over Time3. Pathogen trap & nurture
(including Cryptosporidum oocysts) 4. Serves as a built in solid phase extractor for
hydrophobic drugs, hormones, bioactive agents5. Convenient to recover & analyze for biomarkers Its not in the water but the slime on the pipe
Biofilms not pelagic in the fluid
Sampling Drinking Water-- Collect Biofilms on Coupons
+Q1: 181 MCA scans from 0927001.wiff Max. 1.3e9 cps.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500m/z, amu
0.0
1.0e8
2.0e8
3.0e8
4.0e8
5.0e8
6.0e8
7.0e8
8.0e8
9.0e8
1.0e9
1.1e9
1.2e9
1.3e9
In
te
ns
ity
, c
ps
101.8
380.3
124.2
384.374.2
81.3 110.3
58.480.9
375.7
116.3397.775.2 165.486.4
Triclosan (Pyridinium derivative) Q1scan
+Product (380.3): 181 MCA scans from 0927003.wiff Max. 9.3e6 cps.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500m/z, amu
0.0
5.0e5
1.0e6
1.5e6
2.0e6
2.5e6
3.0e6
3.5e6
4.0e6
4.5e6
5.0e6
5.5e6
6.0e6
6.5e6
7.0e6
7.5e6
8.0e6
8.5e6
9.0e69.3e6
In
te
ns
ity
, c
ps
218.1
236.1
93.2219.1
380.2125.1204.2141.0110.079.1 237.0112.1
Cl
Cl
O
O
Cl
NH3C
C18H13Cl3NO2+
Exact Mass: 380.00
Mol. Wt.: 381.66
Product ion scan
380.3
218.1
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
October
-1
2
-1
1
August
-1
1
-1
PCA 2 Analysis of Forest Community Soil total PLFAP
CA
1
PCA Analysis Sugar Maple-Basswood Black Oak- White Oak Sugar Maple- Red Oak
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.
Wat
er 8
31
Wat
er 8
17
Sta
ndar
d
For
e gu
t
Hin
d gu
t
Water changed composition between Aug 17 & 31st, much > diversity than shrimp gut, Fore gut less diverse than Hind gut.
Major bands have been RecoveredFor sequencing& Phylogenetic analysis
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
0%
20%
40%
60%
80%
100%
8020
1
8020
1F
8020
1H
8030
1
8030
1F
8030
1H
8100
1
8100
1F
8100
1H
8230
1
8230
1F
8230
1H
8310
1
8310
1F
8310
1H
Monos
Bmonos
TBSats
MBSats
NSats
Microbial Viable Biomass: Water (W), Fore Gut (F), Hind Gut (H)
W F H W F H W F H W F H W F H
Biomass PLFA
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
80201
80201F
80201H
80301
80301F
80301H
81001
81001F
81001H
82301
82301F
82301H
83101
83101F
83101H
pm
ol/g
Note Log scale
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