132 Chapter 7 CTX-resistance measurements CTX MIC measurements from Coolen et al. in relation to the genotype of the E. coli isolates are shown in Table S1 (Coolen et al. 2019). Eighty-four of ninety (93.3 %) pampC-harbouring E. coli were CTX resistant (MIC >2 mg l−1) based on EUCAST clinical breakpoints. Twenty-two of sixty-one (36.1%) isolates categorized as putative hyperproducers based on Tracz et al. were CTX resistant, primarily isolates with the −42 (n=15, 62.5% of isolates with −42 mutation) or −32 mutation (n=2, 10.0% of isolates with −32 mutation). The pampC genes never occurred simultaneously with the −42 or −32 mutations in any of these isolates. As depicted in Fig. 2, the nonpampC strains with a phenotype of CTX >2 mg l−1 were present in all phylogroups, although CTX-resistant isolates with the −42 or −32 mutation were predominantly present in phylogroups B1, B2 and C. Genotype to phenotype To be able to link E. coli chromosomal mutations to CTX resistance, we excluded all E. coli isolates with a plasmid containing an ampC beta-lactamase gene. The association of SNPs with the CTX-resistance phenotype (MIC >2 mg l−1) was tested in the remaining 82 isolates using Fisher’s exact test. After FDR correction to 0.05, 45998 significant positions were found (see Fig. 4, ring b). Mutation C>T on position −42 of the ampC promoter was found to be significantly associated with CTX resistance (FDR=0.034). However, position −32 A>T was not significantly associated with CTX resistance (FDR=1). Homoplasy-based association analysis Combining the outcome of the homoplasy analysis with the significant CTX-resistanceassociated positions results in genomic positions associated with CTX resistance that have evolved multiple times independently. After selecting the lowest scoring consistency index positions, ≤0.05882353, 24 relevant genomic positions were identified that had both a low consistency index and a significant association with CTX resistance. Most notably, 1 of these 24 positions is position −42. Only 2 mutations of those 24 that were located in genes were non-synonymous: a (conservative) missense mutation in the type II secretion system protein L (gspL) gene leading to a Ser330Thr alteration, and a mutation in the hydroxyethylthiazole kinase (thiM) gene resulting in a Thr122Ala alteration according to the annotation of E. coli strain ampC_0069 (accession no. CP046396). In addition to the non-synonymous mutation found on the gspL gene, eight synonymous mutations are
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