A tool box strategy using Bacteroides genetic markers to differentiate human from non-human sources of fecal contamination in natural water

Leila Kabiri, Absar Alum, Channah Rock, Jean E. McLain, Morteza Abbaszadegan

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Bacteroides genetic markers have been widely used to identify fecal pollution of water originating from human and animal sources. Many of the assays currently used for detecting human-specific Bacteroides produce false positive results. The focus of this study was to develop a microbial source tracking (MST) tool box strategy for differentiating Bacteroides from human and animal sources. Bacteroides 16S rRNA gene sequences from fish and selected animals were aligned against human fecal Bacteroides isolates to compare and characterize the variable regions within the 16S rRNA gene sequence. Conserved sequences between 4 variable regions were deleted and the truncated sequences were combined to develop a hyper-variable genomic segment (HVGS). The cladogram created from truncated sequences show a clear separation of Bacteroides from human feces and those from animal sources. The proposed strategy was field tested by collecting water samples from central Arizona source waters and three different recreational ponds. PCR using HF134 and HF183 primer sets was performed and sequences from positive reactions were aligned against human Bacteroides sequences to identify the source of contamination. Based on PCR results, the source of fecal contamination was presumptively identified as either human or from another source. For samples testing positive using the HF183 primer set (8/13), fecal contamination was presumed to be from human sources, but to confirm the results, PCR products were sequenced and aligned against the four variable regions and then incorporated within the truncated cladogram. As expected, the sequences from water samples with human fecal contamination grouped in a separate clade. A variability matrix, developed after exclusion of conserved sequences among the four regions, was utilized to establish discrete groupings for sequences within the truncated cladogram, generally differentiating Bacteroides isolates from varying host animals, but most importantly, separating Bacteroides from human feces from Bacteroides from other animals. The proposed strategy offers a new tool box method for MST and a step-wise methodology essential for identifying human sources of fecal pollution.

Original languageEnglish (US)
Pages (from-to)897-905
Number of pages9
JournalScience of the Total Environment
Volume572
DOIs
StatePublished - Dec 1 2016

Fingerprint

genetic marker
Animals
Contamination
Water
animal
feces
water
Pollution
Genes
pollution
gene
Ponds
Fish
contamination
Assays
genomics
pond
assay
matrix
methodology

Keywords

  • 16S RNA
  • Bacteroides
  • Cladogram
  • Sequence truncation
  • Source tracking

ASJC Scopus subject areas

  • Environmental Engineering
  • Environmental Chemistry
  • Waste Management and Disposal
  • Pollution

Cite this

A tool box strategy using Bacteroides genetic markers to differentiate human from non-human sources of fecal contamination in natural water. / Kabiri, Leila; Alum, Absar; Rock, Channah; McLain, Jean E.; Abbaszadegan, Morteza.

In: Science of the Total Environment, Vol. 572, 01.12.2016, p. 897-905.

Research output: Contribution to journalArticle

@article{1c6cf1b59a344c029bc315976982a06b,
title = "A tool box strategy using Bacteroides genetic markers to differentiate human from non-human sources of fecal contamination in natural water",
abstract = "Bacteroides genetic markers have been widely used to identify fecal pollution of water originating from human and animal sources. Many of the assays currently used for detecting human-specific Bacteroides produce false positive results. The focus of this study was to develop a microbial source tracking (MST) tool box strategy for differentiating Bacteroides from human and animal sources. Bacteroides 16S rRNA gene sequences from fish and selected animals were aligned against human fecal Bacteroides isolates to compare and characterize the variable regions within the 16S rRNA gene sequence. Conserved sequences between 4 variable regions were deleted and the truncated sequences were combined to develop a hyper-variable genomic segment (HVGS). The cladogram created from truncated sequences show a clear separation of Bacteroides from human feces and those from animal sources. The proposed strategy was field tested by collecting water samples from central Arizona source waters and three different recreational ponds. PCR using HF134 and HF183 primer sets was performed and sequences from positive reactions were aligned against human Bacteroides sequences to identify the source of contamination. Based on PCR results, the source of fecal contamination was presumptively identified as either human or from another source. For samples testing positive using the HF183 primer set (8/13), fecal contamination was presumed to be from human sources, but to confirm the results, PCR products were sequenced and aligned against the four variable regions and then incorporated within the truncated cladogram. As expected, the sequences from water samples with human fecal contamination grouped in a separate clade. A variability matrix, developed after exclusion of conserved sequences among the four regions, was utilized to establish discrete groupings for sequences within the truncated cladogram, generally differentiating Bacteroides isolates from varying host animals, but most importantly, separating Bacteroides from human feces from Bacteroides from other animals. The proposed strategy offers a new tool box method for MST and a step-wise methodology essential for identifying human sources of fecal pollution.",
keywords = "16S RNA, Bacteroides, Cladogram, Sequence truncation, Source tracking",
author = "Leila Kabiri and Absar Alum and Channah Rock and McLain, {Jean E.} and Morteza Abbaszadegan",
year = "2016",
month = "12",
day = "1",
doi = "10.1016/j.scitotenv.2016.07.188",
language = "English (US)",
volume = "572",
pages = "897--905",
journal = "Science of the Total Environment",
issn = "0048-9697",
publisher = "Elsevier",

}

TY - JOUR

T1 - A tool box strategy using Bacteroides genetic markers to differentiate human from non-human sources of fecal contamination in natural water

AU - Kabiri, Leila

AU - Alum, Absar

AU - Rock, Channah

AU - McLain, Jean E.

AU - Abbaszadegan, Morteza

PY - 2016/12/1

Y1 - 2016/12/1

N2 - Bacteroides genetic markers have been widely used to identify fecal pollution of water originating from human and animal sources. Many of the assays currently used for detecting human-specific Bacteroides produce false positive results. The focus of this study was to develop a microbial source tracking (MST) tool box strategy for differentiating Bacteroides from human and animal sources. Bacteroides 16S rRNA gene sequences from fish and selected animals were aligned against human fecal Bacteroides isolates to compare and characterize the variable regions within the 16S rRNA gene sequence. Conserved sequences between 4 variable regions were deleted and the truncated sequences were combined to develop a hyper-variable genomic segment (HVGS). The cladogram created from truncated sequences show a clear separation of Bacteroides from human feces and those from animal sources. The proposed strategy was field tested by collecting water samples from central Arizona source waters and three different recreational ponds. PCR using HF134 and HF183 primer sets was performed and sequences from positive reactions were aligned against human Bacteroides sequences to identify the source of contamination. Based on PCR results, the source of fecal contamination was presumptively identified as either human or from another source. For samples testing positive using the HF183 primer set (8/13), fecal contamination was presumed to be from human sources, but to confirm the results, PCR products were sequenced and aligned against the four variable regions and then incorporated within the truncated cladogram. As expected, the sequences from water samples with human fecal contamination grouped in a separate clade. A variability matrix, developed after exclusion of conserved sequences among the four regions, was utilized to establish discrete groupings for sequences within the truncated cladogram, generally differentiating Bacteroides isolates from varying host animals, but most importantly, separating Bacteroides from human feces from Bacteroides from other animals. The proposed strategy offers a new tool box method for MST and a step-wise methodology essential for identifying human sources of fecal pollution.

AB - Bacteroides genetic markers have been widely used to identify fecal pollution of water originating from human and animal sources. Many of the assays currently used for detecting human-specific Bacteroides produce false positive results. The focus of this study was to develop a microbial source tracking (MST) tool box strategy for differentiating Bacteroides from human and animal sources. Bacteroides 16S rRNA gene sequences from fish and selected animals were aligned against human fecal Bacteroides isolates to compare and characterize the variable regions within the 16S rRNA gene sequence. Conserved sequences between 4 variable regions were deleted and the truncated sequences were combined to develop a hyper-variable genomic segment (HVGS). The cladogram created from truncated sequences show a clear separation of Bacteroides from human feces and those from animal sources. The proposed strategy was field tested by collecting water samples from central Arizona source waters and three different recreational ponds. PCR using HF134 and HF183 primer sets was performed and sequences from positive reactions were aligned against human Bacteroides sequences to identify the source of contamination. Based on PCR results, the source of fecal contamination was presumptively identified as either human or from another source. For samples testing positive using the HF183 primer set (8/13), fecal contamination was presumed to be from human sources, but to confirm the results, PCR products were sequenced and aligned against the four variable regions and then incorporated within the truncated cladogram. As expected, the sequences from water samples with human fecal contamination grouped in a separate clade. A variability matrix, developed after exclusion of conserved sequences among the four regions, was utilized to establish discrete groupings for sequences within the truncated cladogram, generally differentiating Bacteroides isolates from varying host animals, but most importantly, separating Bacteroides from human feces from Bacteroides from other animals. The proposed strategy offers a new tool box method for MST and a step-wise methodology essential for identifying human sources of fecal pollution.

KW - 16S RNA

KW - Bacteroides

KW - Cladogram

KW - Sequence truncation

KW - Source tracking

UR - http://www.scopus.com/inward/record.url?scp=84994017979&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84994017979&partnerID=8YFLogxK

U2 - 10.1016/j.scitotenv.2016.07.188

DO - 10.1016/j.scitotenv.2016.07.188

M3 - Article

VL - 572

SP - 897

EP - 905

JO - Science of the Total Environment

JF - Science of the Total Environment

SN - 0048-9697

ER -