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Applied and Environmental Microbiology, September 2006, p. 5915-5926, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.02453-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Integrated Analysis of Established and Novel Microbial and Chemical Methods for Microbial Source Tracking

Anicet R. Blanch,^1* Lluís Belanche-Muñoz,² Xavier Bonjoch,¹ James Ebdon,³ Christophe Gantzer,⁴ Francisco Lucena,¹ Jakob Ottoson,⁵ Christos Kourtis,⁶ Aina Iversen,⁷ Inger Kühn,⁷ Laura Mocé,¹ Maite Muniesa,¹ Janine Schwartzbrod,⁴ Sylvain Skraber,⁴ Georgios T. Papageorgiou,⁶ Huw Taylor,³ Jessica Wallis,³ and Joan Jofre¹

Department of Microbiology, University of Barcelona, Avda. Diagonal 645, Barcelona, Spain,¹ Departament de Llenguatges i Sistemes Informàtics, Universitat Politècnica de Catalunya, Jordi Girona 1-3, Barcelona, Spain,² EPHRU, School of the Environment, University of Brighton, Brighton, United Kingdom,³ Laboratoire de Chimie Physique et Microbiologie pour l'Environnement (LCPME), UMR 7564 CNRS/UHP-Nancy I, Faculté de Pharmacie, 5 rue Albert Lebrun, 54000 Nancy, France,⁴ Water and Environmental Microbiology, SMI, Swedish Institute for Infectious Disease Control, SE 171 82 Solna, Sweden,⁵ State General Laboratory, Microbiological Section, Kimonos 44, 1451 Nicosia, Cyprus,⁶ Microbiology and Tumor Biology Center, Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden⁷

Received 17 October 2005/ Accepted 28 June 2006

Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.

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* Corresponding author. Mailing address: Department of Microbiology, University of Barcelona, Avda. Diagonal 645, Barcelona, Spain. Phone: 34 934029012. Fax: 34 934039047. E-mail: ablanch{at}ub.edu.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, September 2006, p. 5915-5926, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.02453-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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