122 Chapter 7 Methods Isolate selection, DNA isolation, library preparation and DNA sequencing One hundred and seventy-two both FOX resistant (MIC >8 mg l−1) and ESBL-negative E. coli isolates previously used by our study group were selected in the present study (Table S1, available with the online version of this article) (Coolen et al. 2019). To summarize the methods, DNA isolation was performed as previously described (Coolen et al. 2019), library preparations were performed using an Illumina Nextera XT library preparation kit, and DNA sequencing was performed using an Illumina NextSeq 500 to generate 2×150 bp paired-end reads or 2×300 bp reads on an Illumina MiSeq. De novo assembly was also performed identically to the method as described previously [16] using SPAdes version 3.11.1 (Bankevich et al. 2012). Phylogroup and multilocus sequence typing (MLST) Phylogroup stratification was performed using ClermonTyping version 1.4.0 (Beghain et al. 2018). MLST sequence types (STs) were derived from the contigs using Mlst version 2.5 PubMLST (31 October 2017) (Jolley and Maiden 2010; Seemann, T, n.d.). Obtaining the ampC promoter/attenuator region To detect the promoter/attenuator region, a custom blast database was created using the 271 bp fragment as described by Peter-Getzlaff et al. using E. coli K-12 strain ER3413 (accession no. CP009789.1) (Altschul et al. 1990; Peter-Getzlaff et al. 2011). ABRicate version 0.8.9 was used to locate matching regions per sample and these were extracted and converted into multi-fasta format using a custom Python script (Seemann, T, n.d.). Strains were labelled AmpC putative hyperproducer when promoter mutations were found, as previously identified by Caroff et al., Siu et al. and Tracz et al. (Caroff N, Espaze E, Gautreau D, Richet H 2000; Tracz et al. 2007; Siu et al. 2003). pampC detection Detection of pampC genes was performed by using ABRicate version 0.5 and ResFinder database (16/02/2018) as described by Coolen et al. (Coolen et al. 2019). PacBio single-molecule real-time (SMRT) sequencing of an E. coli isolate To enable an accurate SNP analysis, a reference chromosome of an E. coli isolate from our collection (ampC_0069) was constructed using PacBio SMRT sequencing. For sequencing, genomic DNA (gDNA) was extracted using a bacterial gDNA isolation kit (Norgen Biotek). A single E. coli isolate was subjected to DNA shearing using Covaris
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