.pone.0081330.gPost-Fecal Transplant Microbiota CharacterizationTable 1. RCDI patient study population.Case [#] 1 2 three 4 5 6a* 6b* eight 9 ten 11 12 13Sex F F F F F FAge 65 65 61 56 76RCDI duration [months] 18 six five 12 72 8Donor Husband Husband Buddy Friend Buddy Son Brother Daughter Husband Husband Wife Husband Husband WifeTime to resolution of symptoms [days] Stick to up [months] two 3 two three 2 three 4 3 two three 3 2 3 2 17 17 17 16 16 12 12 26 21 22 19 7Inciting antibiotic Beta-lactam1 + lincosamide2 multiple Lincosamide2 Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones3 Unknown Lincosamide2 + fluoroquinolone4 Clindamycin Unknown Lincosamide2 Unknown UnknownF F F M F F M72 63 61 68 41 795 6 11 6 12 12 four.*#6a had a relapse of RCDI a single month soon after productive FMT and received a second FMT 3 months immediately after the first (#6b). Within the NCBI brief read archive, samples referred to as #6b are designated as #7 samples.Formula of 2-Methyl-5-nitropyridin-3-amine 1 Penicillin; two clindamycin; 3 ciprofloxacin; four levofloxacin. doi:ten.1371/journal.pone.0081330.tbead beating in tubes with Lysing Matrix B (0.1 mm silica spheres, MP Biomedicals, Solon, OH, USA), at 6 m s21 for 40 s at area temperature within a FastPrep-24 (MP Biomedicals). The resulting crude lysate was processed using the ZR Fecal DNA mini-prep kit (Zymo, Irvine, CA, USA) according to the manufacturer’s recommendation.2′-O-MOE-U manufacturer The samples have been eluted with 100 ml of ultra pure water into separate tubes.PMID:33723702 DNA concentrations in the samples have been measured employing the Quant-iT PicoGreen dsDNA assay kit (Molecular Probes, Invitrogen, Carlsbad, CA, USA).Sequence processing and analysis16S rRNA sequence reads had been processed with QIIME [40] and CloVR [41], working with the automated CloVR-16S pipeline as described inside the corresponding standard operating process [42]. Briefly, making use of the QIIME split_libraries.py tool sequences have been binned determined by sample-specific barcodes, trimmed by removal of barcode and primer sequences and filtered for good quality, employing the default parameters, except for “–barcode-type “variable_length”. Chimeric sequences were removed with UCHIME [43] making use of MicrobiomeUtilities (http://microbiomeutil.sourceforge.net/) and the rRNA16S.gold.fasta reference database. Reads had been clustered into operational taxonomic units (OTUs) applying a similarity threshold of 95 . On average, OTUs have been classified applying the RDP Naive Bayesian Classifier [44] using a score filtering threshold of 0.5. Rarefaction curves were calculated based on OTU counts employing the rarefaction.single routine of the Mothur package [45]. Hierarchical clustering, boxplots, and statistical calculations (Wilcoxon rank sum tests, Jensen-Shannon divergence etc.) have been performed in R. Differentially abundant OTUs have been determined with Metastats [46]. Phylogenetic trees were created with FastTree2 [47] making use of trimmed alignments generated with NAST. Dot plots to evaluate phylogenetic distances and Jensen-Shannon divergence among sample pairs and changes in relative abundance of particular taxonomic families more than time had been generated with Prism5 (version 6 for Mac, GraphPad Computer software, San Diego CA, USA).Amplification and sequencingIn short, hypervariable regions V1 3 of the bacterial 16S rRNA gene have been amplified with primers 27F and 534R as described previously [39]. DNA amplification of 16S rRNA genes was performed applying AccuPrime Taq DNA polymerase Higher Fidelity (Invitrogen) and 50 ng of template DNA inside a total reaction volume of 25 ml, following the AccuPrime solution protocol. Reactions were run i.