Computational protocol: Identification and Characterization of Cefotaxime Resistant Bacteria in Beef Cattle

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Protocol publication

[…] The 23 cefotaxime resistant bacteria were analyzed for the presence of nine different ESBL genes () and taxonomic identification was conducted at the species level. Genomic DNA was extracted with a Qiagen DNA mini kit and used as a template for multiplex polymerase chain reaction (PCR) to amplify nine ESBL genes [] and 16S rRNA gene [] using the primer sets shown in (). The PCR conditions for all reactions were: 95°C for 5 minutes for initial denaturation, 30 cycles of 95°C for 30 seconds, 55°C for 35 seconds, 72°C for 90 seconds, and a final extension at 72°C for 7 minutes. All products were resolved on 1% agarose gel stained with ethidium bromide and visualized with a UV gel doc system (Bio-Rad, USA). The PCR products from the most frequently observed genes bla-TEM, bla-CTX-M, and the 16SrRNA were eluted using QIAEX II Gel Extraction Kit (Qiagen Inc, Germany) and sequenced by the Interdisciplinary Center for Biotechnology Research (ICBR) at University of Florida. The online NCBI nucleotide BLAST program was used to compare the homology of the sequences from the isolates with ESBL genes and 16S rRNA sequences of other organisms []. The sequences of the ESBL genes and 16S rRNA from the isolates were aligned and a maxium likelihood tree was constructed using the Jukes and Cantor model in MEGA version 6.0 software with 1000 bootstrap replications with a bootstrap value of 0.95 (95%) []. Tree annotations were performed using FigTree (version 1.4.2.). […]

Pipeline specifications

Software tools MEGA, FigTree
Application Phylogenetics
Organisms Bos taurus, Bacteria, Homo sapiens
Diseases Bacterial Infections
Chemicals Cefotaxime, Cephalosporins