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

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0837

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0833

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0839

0815

0813

0827

0832

0603

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0835

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0826

0814

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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