Characterization of β-lactamases and multidrug resistance mechanisms in Enterobacterales from hospital effluents and wastewater treatment plant Plasmids of isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca and Citrobacter spp.
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The library for NGS sequencing was prepared using Swift 2S Turbo DNA Library Kits (Swift Biosciences, Ann Arbor, Michigan, United States). Briefly, 100 ng genomic DNA was fragmented, end prepped and adapter ligated. Magnetic beads size selection was performed to select 250-300 bp insert size fragments followed by the library amplification according to the manufacturer instructions. The quality of the library was checked on 4200 TapeSation System using D1000 Screen Tape (Agilent Technologies, Palo Alto, California, USA) and the quantity was measured on Qubit 3.0. (Thermo Scientific, Waltham, Massachusetts, USA). Illumina sequencing was performed on NovaSeq 6000 instrument (Illumina, San Diego, California, USA) with 2x151 run configuration. Quality control (QC), trimming, and filtering of 150 pb paired-end raw reads were performed in preprocessing step. The QC analysis was performed with FastQC (Andrews et al. 2010). The Phred-like quality scores (Qscores) were set to >30. Poor quality reads, adapters at the ends of reads, limited skewing at the ends of reads were eliminated by using Timmomatic (Bolger et al. 2014). Since data contained genomic DNA debris, identification of plasmid-derived contigs were performed after de novo assembly of cleaned reads. Plasmids were assembled with the following parameters via Spades: --plasmid flag was turned on, and every other parameters were used as default (Antipov et al. 2016.). For plasmid identification genes characteristically encoded in plasmids for each strains were determined based on literature and aligning them for the contigs using locally the Blast+ (Altschul et al. 1990). Prokaryotic gene finding were performed by Glimmer using Bacterial, Archaeal, and Plant Plastid Code. Glimmer uses Interpolated Markov Models (IMMs) to identify the coding regions and to distinguish them from non-coding DNA which enabled identified genes to be annotated (Delcher et al. 1999). Functional annotation and Gene Ontology (GO) analysis were carried out using OmixBox.Biobam as follows: sequences were blasted against NCBI nr (non-redundant) database (taxID: 2Bacteria) applying blastn configuration locally. To retrieve GO terms associated with the 10 Hits obtained by the Blast search GO mapping and annotation were performed. GeneBank identifiers (gi), the primary blast Hit ids, were used to retrieve UniProt IDs making use of a mapping file from PIR (Non-redundant Reference Protein Database) including PSD, UniProt, Swiss-Prot, TrEMBL, RefSeq, GenPept and PDB. Accessions were searched directly in the dbxref table of the GO database. BLAST result accessions were searched directly in the gene-product table of the GO database; GO annotations were specified according to GO terms: molecular function, cellular component and biological process (Götz et al. 2008).