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Rapid Lipid Biomarker Analysis for Quantitative Assessment of Microbial Community Composition and Activity David 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 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 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 r. DNA 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 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 r. DNA 1. Recover DNA from samples (often aqueous Detection of Specific genes or r. DNA 1. Recover DNA from samples (often aqueous of 2. lipid extract is best) 3. 2. Amplify with PCR using r. DNA eubacterial primers 3. Separate Amplicons with Denaturating Gel Gradient Electrophoresis (DGGE) 4. Isolate Bands, 5. Sequence and match with r. DNA database 6. Phylogenetic analysis

Sampling locations at the Shiprock site, NM Sampling locations at the Shiprock site, NM

A C D C C B B E E B B D G G A C D C C B 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-, SO 4 --, K+,

Bands Phylogenetic group Metal Metabolism / Metal Transport Properties Associated with Group A Arthrobacter 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 Fe 3+, Mn 4+, U 6+) 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 (Fe 3+, Mn 4+) oxidizers Table: Identification of sequences derived from DGGE bands

100 71 79 Umtra DSR group A 90 71 50 Umtra DSR group B 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 77 100 Umtra DSR group F 97 88 99 100 96 81 100 97 94 100 100 Umtra DSR group G 100 99 100 75 100 81 Umtra DSR group H

LIPID Biomarker Analysis 1. Intact Membranes essential for Earth-based life 2. Membranes contain Phospholipids 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/u. L (near single bacterial cell) 7. Surface localization, high concentration ideal for organic SIMS mapping localization

Lyophilized Soil Fractions, Pipe Biofilm 1. Neutral Lipids SFECO 2 UQ isoprenologues ESE Chloroform. Lyophilized Soil Fractions, Pipe Biofilm 1. Neutral Lipids SFECO 2 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, & sn 1 sn 2 FA Amino Acid PG Ornithine lipid Archea ether lipids Plamalogens 3. In-situ acidolysis in SFECO 2 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 || || O H 2 COC phospholipase Membrane Liability (turnover) VIABLE NON-VIABLE O O || || O H 2 COC phospholipase || | cell death C O CH O | | || H 2 C O H H 2 C O P O CH 2 CN+ H 3 | Neutral lipid, ~DGFA OPolar lipid, ~ PLFA

Biofilm Community Composition Detect viable microbes & Cell-fragment biomarkers : Legionella pneumophila, Francisella tularensis, 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. ~ 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 i 15: 0/a 15: 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 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 p. H ~ 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, 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 450 o. C]. 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 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 p. H 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 ESI (cone voltage) Q-1 ESI/MS/MS CAD Q-3

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

Extract with SFECO 2 Coupon + Biofilm 1. Neutral Lipids UQ isoprenologues UQ-8 Enterics, Extract with SFECO 2 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, & sn 1 sn 2 FA Amino Acid PG, 0 rnithine lipids, Plasmalogens Acidify, Extract residue with SFECO 2 3. LPS OH FA Transesterify, GC/MS. 30 H 10: 0, 12: 0 –Pseudomonas 30 H 14: 0 -- pathogens & enterics

Lipid Biomarker Analysis Sequential High Pressure/Temperature Extraction (~ 1 Hour) Supercritical CO 2 + Lipid Biomarker Analysis Sequential High Pressure/Temperature Extraction (~ 1 Hour) Supercritical CO 2 + 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 CO 2 + 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 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 80 o. C, 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 Problem: Rapid Detection/Identification of Microbes Propose a Sequential High Pressure/Temperature Extractor Delivers Three Analytes to HPLC/ESI/MS/MS

Respiratory Benzoquinone (UQ) Gram-negative Bacteria with Oxygen as terminal acceptor LOQ = 580 femtomole/ul, Respiratory Benzoquinone (UQ) Gram-negative Bacteria with Oxygen as terminal acceptor LOQ = 580 femtomole/ul, LOD = 200 femtomole/ul ~ 104 E. coli Q 7 Q 6 Q 10 197 m/z

ESI/MS Pyridyl Derivative of Cholesterol MS/MS LOD should be ~ 100 amoles Unknown LOD=10 ESI/MS Pyridyl Derivative of Cholesterol MS/MS LOD should be ~ 100 amoles Unknown LOD=10 ppb LOQ=30 ppb

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

Parent product ion MS/MS of synthetic PG Q-1 1 ppm PG scan m/z 110 Parent product ion MS/MS of synthetic PG Q-1 1 ppm PG scan m/z 110 -990 Sn 1 16: 0, (M –H) - Sn 2 18: 2 Q-3 product ion scan of m/z 747 scanned m/z 110 -990 Note 50 X > sensitivity SIM additional 5 x > sensitivity ~ 250 X

Gram-negative Bacteria lipid-extracted residue, hydrolize [1% Acetic acid ], extract = Lipid A êAcid Gram-negative Bacteria lipid-extracted residue, hydrolize [1% Acetic acid ], extract = Lipid A êAcid sensitive bond [to KDO] 14* E. Coli Lipid A 3 OH 14: 0*

Lipid A from E. coli Fatty acids liberated by acid hydrolysis followed by acid–catalyzed Lipid A from E. coli Fatty acids liberated by acid hydrolysis followed by acid–catalyzed (trans) esterification 3 OH 14: 0 TMS GC/MS of Methyl esters 14: 0 3 OH 14: 0 phthalate siloxane

WQ 1 669 524 94 LIPID A: Pseudomonas 3 0 H 12: 0 & WQ 1 669 524 94 LIPID A: Pseudomonas 3 0 H 12: 0 & 3 0 H 10: 0 (water organism) Enteric & Pathogens 30 H 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 0 H 10 +12/ 3 OH 14: 0 LPS fatty acids = 3. 8 Human feces 7 (0. 6)/19 (4) 3 0 H 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 ESI Spectrum of 2, 6 -Dimethyl Dipicolinate LOD ~ 103 spores ~ 0. 5 femtomoles/ul [M+H]+ ES+ Mobile phase: Me. OH + 1 m. M ammonium acetate Cone: 40 V [M+Na]+

ANN Analysis of CR impacted Soil Microbial Communities 1. Cannelton Tannery Superfund Site, 75 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 between 1900 -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 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 9 c, and SRB/Metal reducers (i 15: 0, i 17: 0, 16: 1 w 11, and 10 Me 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 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. Sampling Drinking Water-- Collect Biofilms on Coupons Biofilms not pelagic in the fluid 1. 104 -106 cells/cm 2 vs ~ 103 -104 /Liter 2. Integrates Over Time 3. Pathogen trap & nurture 4. (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) Q 1 scan 380. 3 218. 1 Product ion scan Triclosan (Pyridinium derivative) Q 1 scan 380. 3 218. 1 Product ion scan

Toxicity Biomarkers Hypochlorite, peroxide exposure induces: 1. Formation of oxirane (epoxy) fatty acids from 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.

WQ 1 669 524 94 Goal: Provide a Rapid (minutes) Quantitative Automated Analytical System WQ 1 669 524 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 PCA 2 Analysis of Forest Community Soil total PLFA October 2 -1 PCA Analysis Sugar Maple. Basswood Black Oak. White Oak Sugar Maple. Red Oak 1 -1 PCA 1 August 1 -1 -1

Water 817 Standard Fore gut Water 831 Hind gut Major bands have been Recovered Water 817 Standard Fore gut 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 16 S r. DNAs were separated on a gradient of 20% to 65% denaturant. Water changed composition between Aug 17 & 31 st, much > diversity than shrimp gut, Fore gut less diverse than Hind gut.

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

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

Shrimp In Mariculture Water & Gut Microbial Community Shifts Gut & Water Microbiota in 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 Problem: Rapid Non-invasive Detection of Infection or Metabolic stress for Emergency room Triage Human Breath sample GC/MS