Evaluation of the Impact of E. coli Isolation Method on BST Results

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1 Evaluatin f the Impact f E. cli Islatin Methd n BST Results Emily Martin Sil & Aquatic Micrbilgy Labratry Department f Sil & Crp Sciences Texas A&M University 2012 Land Grant and Sea Grant Natinal Water Cnference Bacterial Detectin and Tracking Sympsium May 22, 2012 Imprving Life thrugh Science and Technlgy.

2 Bacteria #1 Cause f Water Quality Impairment in Texas

3 What is Bacterial Surce Tracking (BST)? Sme members f gut cmmunities can be hst specific. Explit specificity t track fecal cntaminatin back t surce. Target indicatrs because f their link t regulatry standards Varius appraches, but nt ne universally accepted

4 Gentypic BST Methds - Library-Dependent Fingerprinting Enterbacterial repetitive intergenic cnsensus sequence-plymerase chain reactin (ERIC-PCR) RibPrinting (RP)

5 Texas State BST E. cli Library Database f Knwn-Surce Fecal Islates Dual-Fingerprints frm Texas BST watershed prjects (Starting in 2006) Dr. DiGivanni, UT Schl f Public Health Current Versin Islates frm 1285 Different Fecal Samples Knwn-Surce Samples frm each watershed Best-Match Apprach (80% Similarity) t Majr Surce Class Human, Livestck and Pets, and Wildlife

6 Texas State BST E. cli Library Surce Class (6-Way Split) Number f Islates Number f Samples Library Cmpsitin and Expected Randm Rate f Calculated Rate f Crrect Classificatin (RCC) Left Unidentified (unique patterns) Crrect Classificatin Human % 89% 19% Pets % 62% 32% Avian Livestck % 70% 25% Nn-Avian Livestck % 80% 11% Avian Wildlife % 80% 20% Nn-Avian Wildlife % 75% 15% Overall RARCC * = 17% ARCC = 79% 18%

7 EPA Apprved Enumeratin Methds: Cunts vs Eclgy Membrane Filtratin using Chrmgenic Media EPA Methd Mdified Membrane Thermtlerant E. cli (mtec) E. cli nly Texas BST E. cli Library EPA Methd 1604 Ttal Clifrms and E. cli (MI) Defined Substrate Technlgy - MPN Clilert System

8 Enumeratin Methd Cmparisns Hamiltn et al. (2005)

9 Culture-Independent Analysis f Clilert E. cli Psitive Wells - Nn-target taxa made up a substantial percentage f cmmunity, especially in creek water (L). - Klebsiella sp. enriched in sewage sample (R) % false psitive rates fr E. cli in creek water. Sercu et al. (2011)

10 Cmparisn f the Diversity f E. cli Islates using Different Enumeratin Methds BST Implicatins Objective: Evaluate diversity f E. cli cmmunities islated using varius EPA apprved methds and assess implicatins fr BST Hypthesis: Cmmunities frm varius methds will select fr different ppulatins and thus bias BST results Prblem: Very few labs acrss TX accredited fr EPA Methd 1603 Sampling Lcatins Burtn Creek Cllege Statin Attyac Bayu Big Irn Ore Creek Nacgdches Little Brazs River Campbells Creek Hearne Welder Wildlife Refuge Mdy Creek Sintn Plum Creek Lckhart White Oak Bayu Hustn

11 Materials and Methds 3 Media Types EPA Methd 1603 n mtec EPA Methd 1604 n MI Clilert Enumerate and Islate E. cli 45 islates per media type/lcatin Cnfirmed using EPA Methd 1603 verificatin prcedures Fingerprint via ERIC-PCR and RibPrinting Fingerprint patterns analyzed with BiNumerics sftware Bidiversity Indices ID using the Texas BST E.cli Library 3-way Split at 80% Similarity

12 E. cli Enumeratin Fur Lcatins

13 ERIC-RP Cmmunity Diversity A Shannn Weiner Diversity Index mtec MI Clilert Burtn Creek Big Irn Ore Creek Campbells Creek Mdy Creek White Oak Bayu Plum Creek B Shannn-Weiner Diversity Index Highest diversity f islates were btained with mtec (A) and MI Clilert selected fr the lease diverse ppulatins (B)

14 ERIC-RP Similarity Analysis Overall Cmmunity Overlap

15 ERIC-RP Similarity Analysis Overall Cmmunity Overlap Big Irn Ore Creek Mdy Creek White Oak Bayu

16 ERIC-RP Similarity Analysis Overall Cmmunity Overlap Burtn Creek Big Irn Ore Creek Campbells Creek Mdy Creek White Oak Bayu Plum Creek % Ttal Islates (n=45/site) mtec 18% 17% 7% 2% 13% 11% MI 15% 16% 16% 2% 7% 20% Clilert 2% 11% 9% 2% 13% 13% mtec + MI 9% 16% 4% 7% 5% 0% mtec + Clilert 7% 13% 24% 0% 0% 0% MI + Clilert 20% 11% 13% 0% 9% 0% mtec + MI + Clilert 29% 16% 27% 87% 53% 56% Mdy Creek Least Diverse Overall average n all three media at 5 ther sites 36% Lack f verlap = Selectin fr different cmmunities

17 3-Way Split ID acrss all Watersheds 3-way ID split: Humans, Dmesticated Animals, and Wildlife Cmparisn back t mtec Library Standard MI: WILD Clilert: DOM, WILD MI & Clilert: UNID

18 Cmparisn f Surce Classificatins Extreme variatin acrss watersheds N cnsistent trend acrss knwn-surce class r watershed Appears nt pssible t use ther enumeratin techniques (MI and Clilert) as basis fr existing BST librarydependent wrk

19 Cnclusins Enumeratin results were variable acrss sites Islatin media had a substantial impact n E. cli cmmunity cmpsitin Analysis suggests need fr standardizatin amng fecal indicatr enumeratin and islatin techniques t be used in bacterial surce tracking applicatins

20 Questins Emily Martin Texas A&M University Cllege Statin, TX Phne: (979) Funding Prvided by Texas State Sil and Water Cnservatin Bard