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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,

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

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  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. Sampling locations at the Shiprock site, NM

  7. 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+,

  8. Table: Identification of sequences derived from DGGE bands

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  18. ESI (cone voltage) Q-1 CAD Q-3 ESI/MS/MS

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

  20. 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

  21. 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

  22. 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.

  23. Problem: Rapid Detection/Identification of Microbes Propose a Sequential High Pressure/Temperature Extractor Delivers Three Analytes to HPLC/ESI/MS/MS

  24. 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

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

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

  27. 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

  28. 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*

  29. 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

  30. 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.

  31. 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]+

  32. 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

  33. 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

  34. 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

  35. 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

  36. Triclosan (Pyridinium derivative) Q1scan 380.3 218.1 Product ion scan

  37. 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.

  38. 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

  39. 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

  40. 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.

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

  42. 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

  43. 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

  44. Problem: Rapid Non-invasive Detection of Infection or Metabolic stress for Emergency room Triage Human Breath sample GC/MS

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