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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 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 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 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 Sampling locations at the Shiprock site, NM A C D C C B B E 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, 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 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 67 100 100 77 100 Umtra DSR group F 97 88 99 100 96 81 100 97 94 100 94 100 100 Umtra DSR group G 100 99 100 75 100 81 100 81 Umtra DSR group H 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 1. Neutral Lipids SFECO2 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, & sn1 sn2 FA Amino Acid PG Ornithine lipid Archea ether lipids Plamalogens 3. In-situ acidolysis in SFECO2 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 || || H2COC O H2COC 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 OPolar lipid, ~ PLFA 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.~ 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 insitu 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 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 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 ESI (cone voltage) Q-1 ESI/MS/MS CAD Q-3 PE-Sciex API 365 HPLC/ESI/MS/MS Functional Sept 29, 2000 Extract with SFECO2 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, & sn1 sn2 FA Amino Acid PG, 0rnithine lipids, Plasmalogens Acidify, Extract residue with SFECO2 3. LPS OH FA Transesterify, GC/MS . 30H 10:0, 12:0 –Pseudomonas 30H 14:0 -- pathogens & enterics 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 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 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 to HPLC/ESI/MS/MS MeOH MeOH CHCl3 PO 4- Pump CO2 Spe-ed SFE-4 NL PL LPS Fraction Collector N2 blowdown Auto sampler HPLC/ES/MS/MS Respiratory Benzoquinone (UQ) Gram-negative Bacteria with Oxygen as terminal acceptor LOQ = 580 femtomole/ul, LOD = 200 femtomole/ul ~ 104 E. coli Q7 Q6 Q10 O H3OC CH3 H3OC O 197 m/z H ]n ESI/MS Pyridyl Derivative of Cholesterol MS/MS LOD should be ~ 100 amoles pyridyl derivative of cholesterol chol2 1 (0.620) Scan ES+ 2.13e8 375 100 Unknown LOD=10 ppb LOQ=30 ppb H3C CH 3 CH 3 H H3C CH 3 % CH 3 H N CH 3 CH 3 H H O CH 3 CH 3 CH 3 H H 479 H 376 480 377 369 0 m/z 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 510 HPLC/ESI/MS • Enhanced Sensitivity • Less Sample Preparation • Increased Structural Information • Fragmentation highly specific i.e. no proton donor/acceptor fragmentation processes occurring O X O P O CH2 O O HC O C R1 O R2 C O CH2 CEB 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 Gram-negative Bacteria lipid-extracted residue, hydrolize [1% Acetic acid ], extract = Lipid A Acid sensitive bond O [to KDO] O O O P O OH O HO O HN O O O Exact Mass: 1765.19 HN O OH O C93H174N2O24P22- O O P O O OH O OH O O Mol. Wt.: 1766.32 14* 14* E. Coli Lipid A 3 OH 14:0* 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 14:0 3OH 14:0 phthalate siloxane 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. 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 H3OC OCH 3 N O O C9H9NO4 Exact Mass: 195.05 [M+Na]+ 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 NABIR -CEB 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. 104-106 cells/cm2 vs ~ 103-104 /Liter 2. Integrates Over Time 3. 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 Triclosan (Pyridinium derivative) Q1scan +Q1: 181 MCA scans from 0927001.wiff Max. 1.3e9 cps. 101.8 1.3e9 H3C N 1.2e9 1.1e9 380.3 380.3 1.0e9 Cl O O 8.0e8 7.0e8 6.0e8 124.2 Cl 5.0e8 Cl 384.3 74.2 4.0e8 3.0e8 81.3 58.4 110.3 C18H13Cl3NO2+ 80.9 Exact Mass: 380.00 2.0e8 75.2 0.0 60 86.4 80 116.3 100 375.7 Mol. Wt.: 381.66 1.0e8 397.7 165.4 120 140 160 180 200 220 240 260 280 300 m/z, amu 320 340 360 380 400 420 440 +Product (380.3): 181 MCA scans from 0927003.wiff 460 480 500 Max. 9.3e6 cps. 218.1 218.1 9.3e6 9.0e6 Product ion scan 8.5e6 8.0e6 7.5e6 7.0e6 6.5e6 In te n s ity , c p s In te n s ity , c p s 9.0e8 6.0e6 5.5e6 5.0e6 4.5e6 4.0e6 3.5e6 3.0e6 2.5e6 2.0e6 236.1 1.5e6 93.2 1.0e6 0.0 219.1 125.1 5.0e5 79.1110.0 60 80 100 141.0 237.0 112.1 120 140 380.2 204.2 160 180 200 220 240 260 280 300 m/z, amu 320 340 360 380 400 420 440 460 480 500 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 PCA 2 Analysis of Forest Community Soil total PLFA October 2 -1 PCA Analysis Sugar MapleBasswood Black OakWhite Oak Sugar MapleRed Oak 1 -1 PCA 1 August 1 -1 -1 Standard Fore gut Water 817 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 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. Microbial Community in Water (W), Fore Gut (F), Hind Gut (H) 100% 80% 60% Monos Bmonos TBSats MBSats NSats 40% 20% W F H W F H W F H 83101H 83101F 83101 82301H 82301F 82301 81001H 81001F 81001 80301H 80301F 80301 80201H 80201F 80201 0% W F H W F H Microbial Viable Biomass: Water (W), Fore Gut (F), Hind Gut (H) Biomass PLFA Note Log scale 1.00E+08 1.00E+07 1.00E+06 1.00E+04 1.00E+03 1.00E+02 1.00E+01 W F H W F H W F H 83101H 83101F 83101 82301H 82301F 82301 81001H 81001F 81001 80301H 80301F 80301 80201H 80201F 1.00E+00 80201 pmol/g 1.00E+05 W F H W F H 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