126 Chapter 7 Results E. coli collection To study genetic homoplasy events in suspected AmpC-producing E. coli, FOX-resistant ESBL phenotype negative E. coli isolates (n=172) were selected, as previously described by Coolen et al. (see Table S1) (Coolen et al. 2019). The entire collection was subjected to WGS, followed by de novo assembly of the sequence reads to obtain contigs. MLST and phylogroup variants To access the genetic diversity of our E. coli collection, we identified both MLST and phylogroup variants of each of the 172 E. coli isolates. A total of 75 different STs were identified, of which ST131 (8.1%, n=14), ST38 (7.0%, n=12) and ST73 (7.0%, n=12) were the most prevalent. The STs of 13 isolates are unknown. Phylogroup stratification revealed that the isolates belonged to eight different phylogroups (Table 1). Phylogroup B1, B2 and D were the most prevalent. One isolate belonged to E . coli clade IV (strain no. ampC_0128). ampC promoter and attenuator variants We examined the whole E. coli genome. However, we firstly focused on mutations in the ampC promoter and attenuator region. Previously described mutations in the ampC promoter region that according to described literature lead to ‘hyperproduction’ of AmpC were detected in 61 (35.5%) of the isolates (see Tables S1 and S2) (Caroff N, Espaze E, Gautreau D, Richet H 2000; Tracz et al. 2007; Siu et al. 2003). These isolates were, therefore, labelled as putative hyperproducers. Analysis of the promotor area (−42 to −8) resulted in 20 different variants and the wild-type (see Table 1). In the attenuator region (+17 to +37), 18 different variants were identified (see Table 1). One isolate (ampC_0128) showed an unusual promoter variant, a four nucleotide deletion (−45_−42delATCC). Moreover, an insertion (21_22insG) of unknown function was detected in the attenuator of this isolate (ampC_0128) as well (see Tables S1 and S2). pampC variants As we aimed to associate chromosomal mutations with CTX resistance, differentiation of pampC-harbouring isolates from non-pampC-harbouring isolates was required. Genomic analysis showed that 90 (52.3%) of the isolates harboured a pampC gene of which blaCMY-2 was the most prevalent (n=78). One isolate harboured two different pampC genes (blaCMY-4 and blaDHA-1) (ampC_0119). One isolate contained
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