Rapid Quantitative Detection & Speciation of Contamination in the Urban Watershed

David C. White, Cory Lytle, Ying Dong Gan, Aaron M. Peacock, Kimberly Salome, Yevette M. Picenco,

Institute for Applied Microbiology, University of Tennessee, Knoxville, TNMicrobial Insights, Inc., Rockford, TN,

-IAM

Continuation of WQ1 669 524 94

Microbial Insights, Inc.

N W R I

WQ1 669 524 94

Objectives:      1). Rapid (< 1 hr) Quantitative sensitive method for CONTAMINATION*

Detection to monitor multiple sites in the watershed

    2). Allows for” regionalized” intensive treatment in an integrated watershed system

CONTAMINATION*

Drugs, hormones & other pharmaceutically active compounds -ppb

Differentiate Pathogenic Bacteria including especially VBNC pathogens ~ Enterics, Legionella, Mycobacteria, indicators for virus

Protozoa Cryptosporidium, Giardia , Microsporidium, Algae

Indicators pf pathogen infectivity    

1. Strategically placed coupons at multiple sites for biofilm formation

2. Biofilms are readily invaded by Pathogens and Nurtured

3. Lipids in biofilms serve 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 coupon

Sampling Urban Watershed --- Scheme #1

6. Coupon sequentially extracted high pressure/temperature

Supercritical Carbon Dioxide Neutral Lipids

(residue) Chloroform/methanol Polar lipids Analysis by HPLC/ES/MS/MS

(residue) Mild Acid , SFE Lipopolysaccharide OH FA Analysis by GC/MS

7. Analysis on GIS Basis by automatons neural network ANN

8. Feedback to purification interdiction systems

Sampling Urban Watershed --- Scheme # 2

1. Add from continuous culture vessels:Pseudomonas Spp.Acetovorax spp.Bacillus spp.

2. Seed with trace surrogate/pathogen E. coli (GFP), Mycobacterium pflei (GFP), Legionella bosmanii , Sphingomonas

In the Drinking Water Biofilm

Reproducibly Generate a Drinking Water Biofilm:

Biofilm Test System

Detecting specific pollution: Drugs, hormones bioactive compounds

Recover from biofilms: Triculture biofilm + E. coli (GFP) established 3 days in presence of 1000

ppb drug Extract EI/MS/MS

Beads Beads +Biofilm Waste Caffeine 4.7% 50 ppb 3.7% 37 ppb ~97% Triclosan* 3.7% 37 ppb 15% 160 ppb ~ 84% Monensin** ~ 0 % 0.18 1.8 ppb ~99%Finasterdine*** ~ 0% 0.4 % 4.2 ppb ~98 % Caffeine LOD ~ 2 ppb*disinfectant widely used in toiletries LOD ~25 ppb ***macrolide antibiotic used as antiparastic in cattle & chickens factories

LOD~2 ppb *****(Proscar) inhibitor of testosterone hydroxylase LOD ~ 300 ppt *

Biofilms concentrate bioactives compared to sterile surface

ESI (cone voltage) Q-1 CAD Q-3

ESI/MS/MS

+Q1: 0.118 to 0.480 min from Sample 1 (finasterdine) of 0928002.wiff Max. 7.4e6 cps.

200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400m/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

7.4e6

In

te

ns

ity

, c

ps

373.7

374.7

395.6

365.3

397.7366.5202.4 343.4253.5231.2 371.7

+Product (373.7): 119 MCA scans from Sample 2 (finasterdine) of 0928002.wiff Max. 1.6e8 cps.

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, amu

0.0

1.0e7

2.0e7

3.0e7

4.0e7

5.0e7

6.0e7

7.0e7

8.0e7

9.0e7

1.0e8

1.1e8

1.2e8

1.3e8

1.4e8

1.5e8

1.6e8

In

te

ns

ity

, c

ps

373.3

305.4

374.4

317.2175.1121.3 189.3147.4 255.4220.3107.2 161.3 215.4 355.3249.3 272.4

NH

O

CH3

CH3

NH

O

CH3

CH3

H3C

H C23H36N2O2Exact Mass: 372.28

Mol. Wt.: 372.54

Finasterdine Q1 scan

Product ion scan305.4

373.3

373.3

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

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

Q6

Q7Q10

O

O

H3OC

H3OC

CH3

]H

n

197 m/z

Respiratory Ubiquinone (UQ)

LOD = 3 ppb (3.7 fmol/uL) ~ 104 Bacteria , UQ-8 E. coli & Enterics,

UQ-9 Pseudomonas, UQ-10 Protozoa, Algae, UQ-12,13 Legionella

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