concisus isolated from the oral cavity of a healthy human (LMG778

concisus isolated from the oral cavity of a healthy human (LMG7788; = CCUG 13144; = ATCC 33237). Isolates were collected from people residing in the Chinook Health Region of Southwestern Alberta, Canada. learn more These isolates were originally collected as part of a larger study [35]. Scientific and ethics approval for stool collection was obtained from the Regional Ethics Committee of the former CHR and from the University of Lethbridge Human Subject Research Committee. Campylobacter jejuni 81-167 [36] was used as a positive pathogen control for all pathogenicity assays. In addition, the non-pathogenic Escherichia coli HB101 was used as a negative pathogen control for measuring

epithelial IL-8 expression in response to the presence of bacteria. Isolates were stored at -80°C in Columbia broth (Difco, Detroit, MI) containing 40% glycerol. With the excepiton of E. coli which was grown in an aerobic enviornment, inocula of C. concisus for cell culture assays were prepared by growing isolates for 14-16 h in Columbia broth (37°C, 100 rpm) in a microaerobic atmosphere (consisting of 5% O2, 10% CO2, 30% H2 and balance nitrogen). 16S rRNA gene sequence Genomic DNA was extracted using a DNAeasy Tissue kit (Qiagen Inc., Doramapimod manufacturer Mississauga, ON) according to the manufacture’s

instructions. The 16S rRNA gene was PCR amplified using the primers UNI27F and UNI1492R [37] (Table 5) and the resultant product was used as template for sequencing. A BigDye Terminator kit (Applied Biosystems, Foster City, CA) along with universal primers (Table KPT330 5) were used for sequencing the near full-length 16s rRNA gene according to the manufacturer’s instructions. Sequence Phospholipase D1 reactions were separated with an ABI 3130 automated DNA sequencer (Applied Biosystems). Sequences were analyzed using Sequencher software (Gene Codes, Ann Arbor, MI) and compared directly with the NCBI

non-redundant nucleotide database using BLASTN. Table 5 Primers and adaptors used in this study. Targeta Primer/Adaptor Sequence (5′ to 3′) Size (bp) Reference — Bgl II adaptor1 CGGACTAGAGTACACTGTC — [38] — Bgl II adaptor2 GATCGACAGTGTACTCTAGTC — [38] — Csp6 I adaptor1 AATTCCAAGAGCTCTCCAGTAC — [38] — Csp6 I adaptor2 TAGTACTGGAGAGCTCTTGG — [38] — BLG2F-0 6-fam-GAGTACACTGTCGATCT — [38] — CSP61-A GAGCTCTCCAGTACTACA — [38] Universal 16S rRNA gene UNI27F AGAGTTTGATCCTGGCTCAG — [37]   UNI338F ACTCCTACGGGAGGCAG — [37]   UNI1100R AGGGTTGCGCTCGTTG — [37]   UNI1492R TACGG(C/T)TACCTTGTTACGACT — [37] C. concisus 23S rRNA gene MUC1 (forward) ATGAGTAGCGATAATTGGG — [11]   CON1 (reverse) CAGTATCGGCAATTCGCT 306 [11]   CON2 (reverse) GACAGTATCAAGGATTTACG 308 [11] C. concisus cpn gene (primary primers) Ccon-cpn_66f TATCGAAGTGAAACGTGGCA 357 [35]   Ccon_cpn_423r GCTCAAGCACTGGCAATAAG — [35] C. concisus cpn gene (nested primers) Ccon_cpn_72f AGTGAAACGTGGCATGGATA 270 [35]   Ccon_cpn_342r GCATCTTTTCAGGGTTTGTG — [35] C.

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