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(12) Patent Application Publication (10) Pub. No.: US 2016/0040215 A1 Henn Et Al US 201600.40215A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0040215 A1 Henn et al. (43) Pub. Date: Feb. 11, 2016 (54) METHODS FOR PATHOGEN DETECTION Related U.S. Application Data AND ENRICHMENT FROM MATERALS AND (60) Provisional application No. 61/781,854, filed on Mar. COMPOSITIONS 14, 2013. (71) Applicant: SERES THERAPEUTICS, INC., Publication Classification Cambridge, MA (US) (51) Int. Cl. (72) Inventors: Matthew R. Henn, Somerville, MA CI2O I/68 (2006.01) (US); John Grant Aunins, Doylestown, GOIN33/569 (2006.01) PA (US); David Arthur Berry, (52) U.S. Cl. Brookline, MA (US); David N. Cook CPC .......... CI2O 1/689 (2013.01); G0IN33/56911 Brooklyn, NY (US) (2013.01) (21) Appl. No.: 14/776,676 ( 57 ) ABSTRACT Provided are methods and compositions for characterization (22) PCT Filed: Mar. 14, 2014 of bacterial compositions for the maintenance or restoration of a healthy microbiota in the gastrointestinal tract of a mam (86). PCT No.: PCT/US1.4/29539 malian Subject, and the resulting characterized compositions. Provided are methods of characterizing bacterial composi S371 (c)(1), tions including Subjecting the compositions to various detect (2) Date: Sep. 14, 2015 ing processes. Patent Application Publication Feb. 11, 2016 Sheet 1 of 19 US 2016/0040215 A1 FIGURE 1 Patent Application Publication Feb. 11, 2016 Sheet 2 of 19 US 2016/0040215 A1 FIGURE 2 1 AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTA 51 ACACATGCAAGTCGAACGGTAACAGGAAGAAGCTTGCTCTTTGCTGACGA 1 O1 GTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATA 151 ACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGG 2O1 GGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAG 251 TAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAG 3 O1 GATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGG 351 CAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCC 4 O1 GCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGA 451 AGGGAGTAAAGT'TAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGC 5 O1 ACCGGCTAACTCGTGCCCAGGCATGCGCAGGAATACGGAGGTGCAAGCGT 551 TAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAG 6 O1 ATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGC 651 TTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGT 7 O1 AGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACT 751. CACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGT 8O1 AGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTG 851 GCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAA 9 O1 GGTTAAAACT CAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATG 951 TGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCAC 1 OO1 GGAAGTTTTCAGAGATGAGAATGTGCCTTCGGGAACCGTGAGACAGGTGC 1 O 51 TGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCA 11 O1 ACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACT CAA 1151. AGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCA 12 O1. TCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAA 1251. AGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGT 13 O1 CCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAAT 1351 CGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCG 14 O1 CCCGMCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTT 1451 CGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAAC 15 O1. AAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTTA Patent Application Publication Feb. 11, 2016 Sheet 4 of 19 US 2016/0040215 A1 FIGURE 4 x & s SSSs i 3 Patent Application Publication Feb. 11, 2016 Sheet 5 of 19 US 2016/0040215 A1 FIGURE 5 Pure Spore Strain Standard S. 25 a 35 a 1.5 s 55 6 65 7 C. bifermentaris Log CFU/mL) Patent Application Publication Feb. 11, 2016 Sheet 6 of 19 US 2016/0040215 A1 FIGURE 6 Donor A u. DOnOr B SS Swiss SS 836SEty: Patent Application Publication Feb. 11, 2016 Sheet 7 of 19 US 2016/0040215 A1 FIGURE 7 i09 Ettifoxgai patetics (SAMfer arritoi & GAMffs rufis: r=83 Patent Application Publication Feb. 11, 2016 Sheet 8 of 19 US 2016/0040215 A1 FIGURE 8 1.00E+07 OE-i-S to e : & +). S v MY $ : 85C Donor A for B Door C Donor Sample Patent Application Publication Feb. 11, 2016 Sheet 9 of 19 US 2016/0040215 A1 FIGURE 9 ySQEyrne & + iysozyme Patent Application Publication Feb. 11, 2016 Sheet 10 of 19 US 2016/0040215 A1 FIGURE 10 eas poYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY$ Ss 4.0c x |-------------------------------------------------------------------------------- E 2.8 . .&- s : se : 1.30 : rs&c.S. s 8 s 3. O.E. sooooooo-oooooooooooooooooooooooooooooooooooo--------------------s------------------- 5 . O . s: S.O. S. s spike titer log CFU frn Patent Application Publication Feb. 11, 2016 Sheet 11 of 19 US 2016/0040215 A1 FIGURE 11 Sequences Per Sample Patent Application Publication Feb. 11, 2016 Sheet 12 of 19 US 2016/0040215 A1 FIGURE 12 $33. xOS (sar S$3: , 38&S SE R O9 Ds D14tiss ga: 338 Patent Application Publication Feb. 11, 2016 Sheet 13 of 19 US 2016/0040215 A1 FIGURE 13 Bacteroides fragilis group species s s s SES s$........................................................................................................................ $8 ........... s s S S.E & s Patent Application Publication Feb. 11, 2016 Sheet 14 of 19 US 2016/0040215 A1 FIGURE 14 8 t a 8.8 : 8.8 s s s & 8.8 : x 8.8 : 8.8 s 3. 8. E,& E. & s: & 8: & 38 S Patent Application Publication Feb. 11, 2016 Sheet 15 of 19 US 2016/0040215 A1 FIGURE 15 & SE& 8335:8:nsists 85-883 : & S.S.S, SH s3. 8 : S8, St. 8 tiss So Sir & 3 ses. 3 is sis 838 as 13 Fis 8s: & Six cirg is train s :: Seg & 38. & S. Sess :S 3S Sag is Patent Application Publication Feb. 11, 2016 Sheet 16 of 19 US 2016/0040215 A1 FIGURE 16 Y S & Saftgie w w w w w & SFSpse 3 was s : Sarge 3 Sarpie 4 8 Pre heat 5 ming Pre heat is ming Pre heat sec 60c MacConkey (O2) BBA (O2) Bile-escuin (-02) Featre Patent Application Publication Feb. 11, 2016 Sheet 17 of 19 US 2016/0040215 A1 FIGURE 17 s: S. 1. E -: S. S &ssw8SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS &SSSSSSSSSSSS& SS: ,SSS. - ^SS: Risassics is: w8Six Sis: 8 & Es: 3. R} is 683 2538 336 338 3000 fississioritis Patent Application Publication Feb. 11, 2016 Sheet 18 of 19 US 2016/0040215 A1 FIGURE 18 &&&&&&&&&&&&&&&&&&? Donations Colored by Donor) &&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& E ECF ESES ES EE-i 3-3 Donations (Colored by Donor) Patent Application Publication Feb. 11, 2016 Sheet 19 of 19 US 2016/0040215 A1 FIGURE 19 US 2016/0040215 A1 Feb. 11, 2016 METHODS FOR PATHOGEN DETECTION limited number of species; they also tend to be limited in the AND ENRICHMENT FROM MATERALS AND number of species provided in a given probiotic product. As COMPOSITIONS Such, they do not adequately replace or encourage replace ment of the missing natural microflora of the GI tract in many RELATED APPLICATIONS situations. For example, despite routine inoculation with Bifi dobacterium, Lactobacillus, Lactococcus, and Streptococcus 0001. This application is related to U.S. Provisional Appli species, significant changes in the bacterial species composi cation No. 61/781,854, filed Mar. 14, 2013, which is incor tion of monozygotic twin pairs were not observed (McNulty porated by reference in its entirety. et al. (2011) Sci. Transl. Med. 3(106):106. 0006 Thus practitioners have a need for a method of popu REFERENCE TO ASEQUENCE LISTING lating a subject's gastrointestinal tract, and other niches, with 0002 This application includes a Sequence Listing sub a diverse and useful selection of microbiota in order to altera mitted electronically as a text file named 26335PCT SE dysbiosis. In response to the need for durable, efficient, and QUENCELISTING.TXT, created on Mar. 14, 2014 with a effective compositions and methods for treatment of diseases, size of 4,196,119 bytes. The sequence listing is incorporated restoring or enhancing microbiota functions by providing a by reference. multi-component bacterial composition with a diverse and/or complex microbial composition is a solution. Assessing mul BACKGROUND tivalent compositions to verify their safety, identity, viability, 0003 Mammals are colonized by microbes in the gas potency and purity for the treatment of mammalian Subjects is trointestinal (GI) tract, on the skin, and in other epithelial and required to assure the compositions are of the appropriate tissue niches Such as the oral cavity, eye Surface and vagina. In quality and consistency to meet global regulatory standards. particular, the gastrointestinal tract harbors an abundant and A particular challenge for multi-component compositions is diverse microbial community. It is a complex system, provid the detection of microbial contaminants at low levels in the ing an environment or niche for a community of many differ composition (e.g. see Temmerman etal 2003 Identification of ent species or organisms, including diverse strains of bacteria. antibiotic susceptibility of bacterial isolates from probiotic Hundreds of different species may form a commensal com products. Int J. of Food Microbiology 81:1-10 and Temmer munity in the GI tract in a healthy person, and this comple man etal 2003 Development and Validation of a nested-PCR ment of organisms evolves from the time of birth to ultimately denaturing gradient gel electrophoresis method for taxo form a functionally mature microbial population by about 3 nomic characterization of bifidobacterial communities). Due years of age. Interactions between constituents of these popu to the complex nature of the microbial compositions there is lations, between them and Surrounding environmental com a lack of techniques to appropriately characterize a beneficial ponents, and between microbes and the host, e.g. the host microbial composition for therapeutic and other health uses. immune system, shape the community structure, with avail ability of and competition for resources affecting the distri SUMMARY
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