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286and Proteobacteria (3%) (Figure 2).Laboratory procedureDuring cholecystectomy, bile samples were obtained under sterile conditions from the fundus of the gallbladder using an 18G needle and 20 ml syringe. A portion of each was immediately frozen at -80℃ for metagenomic analysis, and the remaining bile samples were sent to the bacteriologic labora-tory for culture. Samples were inoculated onto 5% sheep blood trypticase soy agar (Nissui Pharma-ceutical, Japan), Drigalski agar (Nissui Pharmaceu-tical) and Anaero Columbia agar RS (Becton Dick-inson and Company, Japan), and incubated in an atmosphere containing 10% CO2 at 35℃ for 5 days. The isolates were identified using the MALDI biotyper (Bruker Co., Ltd., Japan) over a score of 1.700. The minimum inhibitory concentrations (MICs) of antimicrobial agents were determined using the microdilution method, according to the guidelines of the Clinical & Laboratory Standards Institute (CLSI, M100-S25). The antimicrobial agents tested included amikacin (AMK), amino-benzylpenicillin (ABPC), arbekacin (ABK), aztre-onam (AZT), cefazolin (CEZ), ceftazidime (CAZ), ciprofloxacin (CIP), colistin (CST), erythromycin (EM), gentamicin (GM), imipenem (IPM), levo-floxacin (LVFX), meropenem (MEPM), oxacillin (OXA), penicillin (PCG), piperacillin (PIP), teico-planin (TEIC) and vancomycin (VCM). Genomic DNA of bacterial isolates was extracted using DNeasy Blood & Tissue kits (QIAGEN, Tokyo, Japan). Genomic libraries were prepared using Nextera XT DNA kits (Illumina, San Diego, CA). Paired-end sequencing was performed using MiSeq Reagent Kit v3 (600-cycles). Quality trimming, filtering and assembly of the obtained sequence reads were performed using CLC Genomics Work-bench v11 (QIAGEN, Hilden, Germany). The assembled genome sequence data were searched for genes associated with drug resistance, using the ABRicate program (https://github.com/tsee-mann/abricate) and data from the National Center for Biotechnology Information (NCBI), the Compre-hensive Antibiotic Resistance Database (CARD), and the ResFinder databases.DNA was extracted from bile samples using DNeasy PowerSoil Kits (QIAGEN). Each library was prepared in accordance with “Illumina 16S Metagenomic Sequencing Library Preparation Guide” with a primer set (27Fmod: 5ʼ- AGR GTT TGA TCM TGG CTC AG -3ʼ and 338R: 5ʼ- TGC CTC CCG TAG GAG T -3ʼ) targeting the V1–V2 region of the 16S rRNA gene. The 251 bp paired end sequencing of the amplicon was performed on a MiSeq. The obtained paired end sequences were merged using PEAR (http://sco.h-its.org/exelixis/web/software/pear/). Subsequently, 30,000 reads per sample were randomly sampled using seqtk (https://github.com/lh3/seqtk) for taxonomic assign-ment. These sampled sequences were clustered into operational taxonomic units (OTU) defined as 97% similarity using UCLUST version 1.2.22q. Representative sequences for each OTU were clas-sified taxonomically using RDP Classifier version 2.2 with the Greengenes database (gg_13_8).The isolate harbored 10 genes associated with drug resistance, including AAC(6’)-Ie-APH(2”)- Ia, ant(9)-Ia, blaI, blaR1, blaZ, ermA, mecA, mecI, mecR1, and tetM.Because E. coli is the organism most frequently isolated from bile samples of patients with chole-cystitis, it has been regarded as a cause of this condition. Other microorganisms isolated from the bile samples of patients with cholecystitis include Enterobacter, Enterococcus, Klebsiella, Strepto︲coccus, and Pseudomonas spp.4). These pathogens are thought to enter the gallbladder from the duodenum in a retrograde manner, although there is other possibility that they enter the gall bladder via the portal vein through the hepatic sinusoids and space of Disse5). Gallbladder stones play an important role in the pathological conditions observed in patients with cholecystitis.Pathologically, XGC is characterized by thick-ening of the gallbladder wall, mimicking advanced gallbladder carcinoma1). Although these patholog-ical changes are thought to be due to intense acute or chronic inflammation, the pathogenesis of XGC remains unclear. XGC is often associated with gall-stones. Gallstones cause ulceration of the gallbladder mucosa, rupture of Rokintansky-Aschoff sinuses, and eventually xanthogranulomatous changes6).Next-generation DNA sequencing has enabled analysis of the microbiota in the biliary tracts of patients with various diseases, including bacterial Discussion