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(12) United States Patent (10) Patent No.: US 9,725,770 B2 Andersen Et Al USO0972577OB2 (12) United States Patent (10) Patent No.: US 9,725,770 B2 Andersen et al. (45) Date of Patent: Aug. 8, 2017 (54) METHODS AND COMPOSITIONS FOR FOREIGN PATENT DOCUMENTS IDENTIFICATION OF SOURCE OF MICROBAL CONTAMINATION IN A WO WO 99.22023 * 5/1999 WO WO 2007/018563 * 2/2007 SAMPLE WO WO 2007/039.319 * 4/2007 (71) Applicant: The Regents of the University of California, Oakland, CA (US) OTHER PUBLICATIONS (72) Inventors: Gary L. Andersen, Berkeley, CA (US); Bavykin et al., Applied and Environmental Microbiology 67 (2): Eric A. Dubinsky, Berkeley, CA (US) 922 (2001).* Bernhard et al., Applied and Environmental Microbiology 66 (4): (73) Assignee: The Regents of the University of 1587 (Apr. 2000).* Bernhard et al., Applied and Environmental Microbiology 66 California, Oakland, CA (US) (10): 4571 (Oct. 2000).* (*) Notice: Subject to any disclaimer, the term of this Brodie et al., Applied and Environmental Microbiology 72 (9) : 6288 (2006).* patent is extended or adjusted under 35 Brodie et al., PNAS 104 (1): 299 (2007).* U.S.C. 154(b) by 795 days. Cho et al., Applied and Environmental Microbiology 67 (8): 3677 (2001).* (21) Appl. No.: 13/787,500 DeSantis et al., FEMS MicrobiologyLab 245 :271 (2005).* Guschin et al., Applied and Environmental Microbiology 63 (6): (22) Filed: Mar. 6, 2013 2397 (1997).* (65) Prior Publication Data Kildare et al., Water Research 41:3701 (2007).* Palmer et al., Nucleic Acids Research 34 (1): e5 (2006).* US 2014/02001 49 A1 Jul. 17, 2014 Peplies et al., Applied and Environmental Microbiology 69 (3) : 1397 (2003).* Related U.S. Application Data Peplies et al., Applied and Environmental Microbiology 72 (7) : 4829 (2006).* (60) Provisional application No. 61/607.340, filed on Mar. Rompre et al., J. of Microbiological Methods 49: 31 (2002).* 6, 2012. Sagaram et al., Applied and Environmental Microbiology 75 (6): 1566 (2009).* (51) Int. Cl. Small et al., Applied and Environmental Microbiology 67 (10): CI2O I/68 (2006.01) 4708 (2001).* C7H 2L/00 (2006.01) Wilson et al., Applied and Environmental Microbiology 68 (5) : (52) U.S. Cl. 2535 (2002).* CPC .................................... CI2O 1/689 (2013.01) Yi-Bo et al., Progress in Biochemistry and Biophysics 36 (8): 1025 (58) Field of Classification Search (2009).0.* CPC ...... C12Q 1/68; C12O 1/6813; C12O 1/6816; Dubinsky et al., Water Research 105: 56 (2016).* CO7H 21AOO See application file for complete search history. * cited by examiner (56) References Cited Primary Examiner — Ethan C Whisenant U.S. PATENT DOCUMENTS (74) Attorney, Agent, or Firm — Knobbe, Martens, Olson & Bear, LLP 2004/0033547 A1* 2, 2004 Field .................... C12O 1/6876 435/732 2007/O122831 A1* 5/2007 Bachoon .............. C12O 1/6806 435/6.15 (57) ABSTRACT 2009/0203032 A1* 8, 2009 Shanks .................. C12O 1/689 435/6.15 Herein are described 1058 different bacterial taxa that were 2009, 0291858 A1* 11/2009 Andersen ................ GO6F 19,20 unique to either human, grazing mammal, or bird fecal 506,10 wastes. These identified taxa can serve as specific identifier 2011/0143334 A1* 6, 2011 Roscoe .................... C12O 1/04 taxa for these sources in environmental waters. Two field 435/5 2012/01652.15 A1 6, 2012 Andersen ............. C12O 1/6837 tests in marine waters demonstrate the capacity of phyloge 506.9 netic microarray analysis to track multiple sources with one 2012fO190025 A1 7, 2012 Blackwood ............ C12O 1/689 teSt. 435/6.11 2012/0264.637 A1* 10/2012 Wiener-Kronish ... C12O 1/6883 506.9 12 Claims, 7 Drawing Sheets U.S. Patent Aug. 8, 2017 Sheet 1 of 7 US 9,725,770 B2 goose peiica fast gui WWWWWWWWWW igai gui igui WWWWWWWWWWWWWW pigeot nepigeon igui gai: peiican peiica gait gui Sewage s&88g8 is88:a:: $83.838 $88838 Sewage 8883:38 Sex: 88 is&a is&as it in 8888 888& U.S. Patent Aug. 8, 2017 Sheet 3 of 7 US 9,725,770 B2 U.S. Patent Aug. 8, 2017 Sheet 4 of 7 US 9,725,770 B2 s s s S. s e s exeieties caecifix U.S. Patent Aug. 8, 2017 Sheet S of 7 US 9,725,770 B2 Sn00000?º?u?NAO";e snopopol??uz,q??H• 30anos36eMºs» U.S. Patent Aug. 8, 2017 Sheet 6 of 7 US 9,725,770 B2 Fig. 5 Human :30 A & 20 k 38 383 Bird 83 : 8 83 too 83 63 20 38 Enterocarts wPNitri U.S. Patent Aug. 8, 2017 Sheet 7 Of 7 US 9,725,770 B2 Fig. 6 itra 20 : 3. 3 Grazer 8: 6 : 48 & 8 8 3. 20 8 & 8 3 to 1111 33 { 8:8 Enterocaccias (APNf60 in US 9,725,770 B2 1. 2 METHODS AND COMPOSITIONS FOR is one of many potential sources of FIB, and monitoring IDENTIFICATION OF SOURCE OF results are often confounded by inputs from a variety of MICROBAL CONTAMINATION IN A wildlife and non-fecal sources (Field, K. et al., Water SAMPLE Research 2007, 41, (16), 3517-3538; Boehm, A. B., Entero cocci concentrations in diverse coastal environments exhibit CROSS-REFERENCE TO RELATED extreme variability. Environmental Science & Technology APPLICATIONS 2007, 41, (24), 8227-8232; Boehm, A. B., Covariation and Photoinactivation of Traditional and Novel Indicator Organ This application is a non-provisional application of and isms and Human Viruses at a Sewage-Impacted Marine claims priority to U.S. Provisional Patent Application No. 10 Beach. Environmental Science & Technology 2009, 43, (21), 61/607.340, filed on Mar. 6, 2012, hereby incorporated by 8046-8052; Yamahara, K. M.; Layton, B. A.; Santoro, A. E.; reference in its entirety. This application is also related to Boehm, A. B., Beach sands along the California coast are U.S. patent application Ser. No. 13,379,839, filed on Dec. diffuse sources of fecal bacteria to coastal waters. Environ 20, 2011, which is also incorporated by reference in its mental Science & Technology 2007, 41, (13), 4515-4521). entirety. 15 FIB are common in most warm-blooded animals, and many studies demonstrate that FIB occur in several environmental STATEMENT OF GOVERNMENTAL SUPPORT Sources aside from feces, including soils and sediments, algal wrack and beach sands. Ibid. Thus water bodies often This invention was made with government Support under contain measurable amounts of FIB even where anthropo Contract No. DE-AC02-05CH11231 awarded by the U.S. genic inputs are absent, and the presence of FIB provides an Department of Energy, and under Grant No. ES013515 insufficient indication of health risk without additional awarded by the National Institute of Health. Further funding Source tracking data. and support provided by the California State Water Shortcomings of the current FIB monitoring approach Resources Control Board Clean Beaches Initiative (07-576 combined with widespread development and implementa 550-0), and the County of Marin under Work for Others 25 tion of Total Maximum Daily Load (TMDL) requirements Agreement LB08004214, the US Environmental Protection for microbiological pollution are fueling interest in micro Agency and the City of Dana Point. The government has bial source tracking (MST) methods (Santo Domingo, J. W.; certain rights in the invention. Bambic, D. G.; Edge, T. A.; Wuertz, S., Quo Vadis source tracking? Towards a strategic framework for environmental REFERENCE TO TABLE APPENDIX 30 monitoring of fecal pollution. Water Research 2007, 41, (16), 3539-52. USEPA, Microbial Source Tracking Guide Table 1 is a list of the unique bird feces taxa identified. Document. In Washington, D.C., 2005; p 131). Many Table 2 is a list of the unique grazer feces taxa identified. approaches to Source tracking are under development, most Table 3 is a list of the unique sewage and human feces of which rely on single phenotypic or genotypic biomarkers taxa identified. 35 to measure sources (Field, K., et al., Water Research 2007, The Tables 1, 2, and 3 are hereby incorporated by refer 41, (16), 3517-3538; Santo Domingo, J. W. et al., Quo Vadis ence in their entirety. Source tracking? Towards a strategic framework for envi ronmental monitoring of fecal pollution. Water Research BACKGROUND OF THE INVENTION 2007, 41, (16), 3539-52). A limitation of single targets is that 40 no single assay is known to be 100% specific for any one Field of the Invention type of waste (Field, K., et al., Water Research 2007, 41, The present invention relates to identification of unique (16), 3517-3538; Santo Domingo, J. W. et al., Quo Vadis identifying sequences and taxa which can identify specific Source tracking? Towards a strategic framework for envi organism sources of contamination in samples, especially ronmental monitoring of fecal pollution. Water Research environmental samples, and methods and compositions that 45 2007, 41, (16), 3539-52), and MST based on single targets find use thereof. is entirely dependent on the fate of one biomarker once it Related Art enters receiving waters (Bae, S.; Wuertz, S., Rapid decay of Beach closures and public health advisories have a major host-specific fecal Bacteroidales cells in seawater as mea economic impact on coastal communities whose economies sured by quantitative PCR with propidium monoazide. are based largely on tourism from beach recreation. Most 50 Water Research 2009, 43, (19), 4850-4859: Balleste, E.; closings and advisories are triggered by water samples that Blanch, A. R. Persistence of Bacteroides Species Popula exceed microbial water quality standards for fecal indicator tions in a River as Measured by Molecular and Culture bacteria (FIB), usually culturable coliforms, E. coli or Techniques. Applied and Environmental Microbiology 2010, enterococci that are considered a proxy for human health 76, (22), 7608–7616; Walters, S. P.; Field, K. G., Survival risk in recreational waters. Because the direct measurement 55 and persistence of human and ruminant-specific fecal Bacte of all human pathogens is often impractical and unreliable roidales in freshwater microcosms.
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