54 Chapter 4 WGS analyses Sequence reads were demultiplexed and merged to obtain fastq files for each sample. Reads were quality assessed and adapter trimmed by Trim_galore (version 0.4.1)19 followed by a custom NextSeq read cleaning script to remove reads containing six or more As and Gs introduced by the sequencing chemistry (Martin 2011). Read coverage was calculated by dividing the number of sequence bases for each sample by the length of E. coli K-12 strain C3026 (RefSeq: NZ_CP014272.1). Samples not exceeding 30× read coverage were excluded for further analyses and samples containing >120× read coverage were subsampled to 120×. Reads were de novo assembled to create contigs by SPAdes (version 3.11.1) using default settings and k-mer sizes 21, 41, 61, 81 and 101 (Bankevich et al. 2012). MLST STs were derived from the contigs using mlst (version 2.5 pubMLST, 31 October 2017) (Jolley and Maiden 2010; Seemann, T, n.d.). Plasmid-mediated ampC detection To detect pampC genes, contigs were BLASTed (version 2.2.30+) against the ResFinder database (2018-02-16) using abricate (version 0.5) (Altschul et al. 1990; Camacho et al. 2009; Seemann, T, n.d.). Genes that had a coverage of ≥90% and a sequence identity >75% were interpreted as present. To circumvent the absence of genes due to wrong assembly, pampC genes were validated using KMA (version 0.14.3) with the ResFinder database (2018–02-16), which is a method that uses raw sequences as input (Clausen, Aarestrup, and Lund 2018). Genes were marked present if KMA matched >90% coverage and >90% identity. Finally, pampC genes were considered present if both methods reported an identical gene and the strain was labelled pampC accordingly. Detection of ampC hyperproducer genotype The promoter and attenuator region of campC was extracted from all samples to obtain a similar 271 bp fragment, as described by Peter-Getzlaff et al (Peter-Getzlaff et al. 2011). The sequence of each strain was aligned against the promoter/attenuator region of the campC gene of the E. coli K-12 strain MG1655 (GenBank accession number U00096.3) using AliView (version 1.23) (Larsson 2014). Strains were labelled cAmpC hyperproducer when promoter mutations were found, as reported by Caroff et al and Tracz et al (Caroff N, Espaze E, Gautreau D, Richet H 2000; Tracz et al. 2007).
RkJQdWJsaXNoZXIy MTk4NDMw