Rapid Lipid Biomarker Analysis for Quantitative Assessment of Microbial Community Composition and Ac...
This presentation is the property of its rightful owner.
Sponsored Links
1 / 44

David C. White, Cory Lytle, Aaron Peacock, Yun-Juan Chang, Jonas S. Almeida, Ying Dong Gan, PowerPoint PPT Presentation


  • 99 Views
  • Uploaded on
  • Presentation posted in: General

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,

Download Presentation

David C. White, Cory Lytle, Aaron Peacock, Yun-Juan Chang, Jonas S. Almeida, Ying Dong Gan,

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    Sampling locations at the Shiprock site, NM


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    Increasing Uranium (VI)* concentration

    N(PCR)

    763

    764

    771

    780

    772

    a

    774

    769

    a

    770

    767

    765

    Stds

    DGGE analysis of bacterial communities in sediment samples. Amplified product was separated on a gradient of 20%-65% denaturant

    E

    B

    B

    B

    E

    E

    E

    E

    D

    B

    C

    B

    D

    B

    C

    D

    B

    F

    B

    F

    C

    F

    C

    C

    C

    C

    C

    D

    B

    A

    A

    A

    B

    G

    G

    *& Na+,Mg++,Cl-,

    SO4--,K+,


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    Table: Identification of sequences derived from DGGE bands


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    100

    71

    79

    90

    71

    50

    Umtra DSR group A

    91

    Umtra DSR group B

    50

    100

    94

    84

    Umtra DSR group C

    75

    92

    55

    100

    75

    Umtra DSR group D

    100

    96

    67

    100

    Umtra DSR group E

    100

    77

    100

    97

    88

    99

    Umtra DSR group F

    100

    81

    96

    100

    97

    94

    100

    94

    100

    100

    100

    99

    Umtra DSR group G

    100

    75

    100

    81

    100

    81

    Umtra DSR group H


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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 SIMSmapping localization


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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 paucimobilisSphingolipids

    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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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.


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    ESI (cone voltage)

    Q-1

    CAD

    Q-3

    ESI/MS/MS


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    PE-Sciex API 365 HPLC/ESI/MS/MS Functional Sept 29, 2000


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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 residuewithSFECO2

    3. LPS OH FA

    Transesterify, GC/MS .  30H 10:0, 12:0 –Pseudomonas

    30H 14:0 -- pathogens & enterics


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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 frompure culture and environmental samples using pressurized accelerated hot solvent

    extraction. J. Microbial Methods 31: 19-27(1997)

    CEB

    Microbial

    Insights, Inc.


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    Problem: Rapid Detection/Identification of Microbes

    Propose a Sequential High Pressure/Temperature Extractor Delivers Three Analytes to HPLC/ESI/MS/MS


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    Respiratory Benzoquinone (UQ)

    Gram-negative Bacteria with Oxygen as terminal acceptor

    LOQ = 580 femtomole/ul, LOD = 200 femtomole/ul ~ 104E. coli

    Q7

    Q10

    Q6

    197 m/z


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    ESI/MS Pyridyl Derivative of Cholesterol

    MS/MS LOD should be

    ~ 100 amoles

    Unknown

    LOD=10 ppb

    LOQ=30 ppb


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    HPLC/ESI/MS

    • Enhanced Sensitivity

    • Less Sample Preparation

    • Increased Structural Information

    • Fragmentation highly specific i.e. no proton donor/acceptor fragmentation processes occurring

    CEB


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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 747scanned m/z 110-990 Note 50X > sensitivity

    SIM additional 5x > sensitivity ~ 250X


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    Gram-negative Bacteria  lipid-extracted residue,  hydrolize [1% Acetic acid ],  extract = Lipid A

    • Acid sensitive bond [to KDO]

    14*

    14*

    E. Coli Lipid A  3 OH 14:0*


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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

    3OH 14:0

    14:0

    phthalate

    siloxane


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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 feces7 (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.


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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

    [M+Na]+


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    ANN Analysis of CR impacted Soil Microbial Communities

    • Cannelton Tannery Superfund Site, 75 Acres on the Saint Marie River near Sault St. Marie, Upper Peninsula, MI

    • Contaminated with Cr+3and other heavy metals between1900-1958 by the Northwestern Leather Co.

    • Cr+3 background ~10-50 mg/Kg to 200,000 mg/Kg.

    • Contained between ~107-109/g dry wt.viable biomass by PLFA; no correlation with [Cr] (P>0.05)

    • PLFA biomass correlated (P<001) with TOM &TOC but not with viable counts (P=0.5)

    -CEB


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    ANN Analysis of Cr+3 impacted Soil Microbial Communities

    • CONCLUSIONS:

    • 1. Non-Linear ANN >> predictor than LinearPCA(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

    -CEB

    NABIR


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    Sampling Drinking Water-- Collect Biofilms on Coupons

    Biofilmsnot pelagic in the fluid

    • 104-106cells/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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    Triclosan (Pyridinium derivative) Q1scan

    380.3

    218.1

    Product ion scan


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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.


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    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


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    PCA 2 Analysis of Forest Community Soil total PLFA

    PCA Analysis

    Sugar Maple-

    Basswood

    Black Oak-

    White Oak

    Sugar Maple-

    Red Oak

    August

    1

    -1

    -1

    October

    2

    -1

    1

    -1

    PCA 1


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    Hind gut

    Fore gut

    Water 831

    Water 817

    Standard

    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.


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    Microbial Community in Water (W), Fore Gut (F), HindGut(H)

    W F HW F HW F HW F H W F H


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    Microbial Viable Biomass: Water (W), Fore Gut (F), HindGut(H)

    Note Log scale

    W F HW F HW F HW F H W F H


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

    ShrimpIn 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 showsHepatopancreas Mycobacteria, Propionobacteria, SRB

      • and algae (chloroplast > BIOMASS THAN WATER

    • DGGE showsHind Gut Vibrio exclusively less diverse community

    • Gut 2-order of magnitude > viable microbial biomass than water

    • Gut and Water different PLFA from Shrimp food


    David c white cory lytle aaron peacock yun juan chang jonas s almeida ying dong gan

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

    Human Breath sample GC/MS


  • Login