128 Chapter 7 Reference chromosome To be able to reconstruct an accurate phylogeny, we selected E. coli isolate ampC_0069, one of the strains of the study, to use as self-constructed reference chromosome for SNP calling. The reference chromosome was constructed through a hybrid assembly of n=4423109 2×150 bp Illumina NextSeq 500 paired-end reads together with n=218 475 PacBio Sequel SMRT subreads (median 5640 bp). This resulted in a high-quality full circular chromosome of E. coli isolate ampC_0069, with a size of 5056572 bp. This isolate belongs to ST648 and contains a plasmid-encoded blaCMY-42. The full circular chromosome was uploaded to GenBank under accession number CP046396 and was used for further analysis. Genome annotation with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) identified 4720 coding sequences. SNP calling The mapping of the reads of all isolates to the reference chromosome E. coli ampC_0069 (accession no. CP046396) resulted in a coreSNP alignment containing 314200 variable core SNP positions. For further details per isolate see Table S3. To validate our SNP calling method, we compared the ampC_0069 Illumina NextSeq 500 paired-end reads to the reference chromosome of ampC_0069, resulting in 0 SNPs detected, supporting that the SNP calling data and method produce no false positives. Phylogenetic tree based on coreSNP The coreSNP alignment was used for further analysis. Fig. 2 illustrates the approximately maximum-likelihood phylogenetic tree of all 172 isolates based on the coreSNP alignment. The tree has a robust topology as indicated by the SH-like (Shimodaira–Hasegawa like) branch support, only three positions have a value ≤60% (M. N. Price, Dehal, and Arkin 2010). When focusing on the ampC promoter mutations, they were most prevalent in phylogroups B1, B2 and C, although they were present in all phylogroups except phylogroup E that lacked mutations in either the promoter or attenuator region. Interestingly, two positions previously highlighted by Tracz et al., −42 and −32, are only mutated in the absence of a pampC gene, even in isolates with a similar MLST ST (ST12, ST88 and ST131). The −42C>T mutation, which results in an alternate displaced promoter box and, therefore, leads to increased resistance (Tracz et al. 2007), is present in 24 isolates in five distinct phylogroups and in 17 separate phylogenetic branches, indicating that this mutation is homoplastic. Additionally, the −32T>A mutation in the promoter, previously also associated with resistance [6], is present in 20 isolates in three distinct phylogroups and in 14 separate phylogenetic branches. To quantify the level of homoplasy, we calculated the consistency index.
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