121 Genome-wide analysis in E. coli unravels homoplasy associated with cefotaxime resistance the promoter and attenuator region were described (Tracz et al. 2007). Tracz et al. used a two-step quantitative reverse-transcriptase PCR (qRT-PCR) to determine the effect of promoter/attenuator variants on ampC expression. Various mutations were related to different delta–delta cycle threshold values in the qRT-PCR and corresponding variations in FOX resistance. An interesting observation that emerged from this study was that the −32T>A and the −42C>T mutation were the major alterations that strengthened the ampC promoter. Both result in a consensus −35 box. Although it is known that AmpC hyperproduction leads to FOX resistance, as studied by Tracz et al., the effects of various mutations on resistance to a 3GC antibiotic such as CTX have not been explored. This is relevant because CTX is commonly used in the treatment of patients with severe E. coli infections, often in combination with selective digestive tract decontamination in intensive care units (de Smet et al. 2009; Aardema et al. 2020). While previous research mainly focused on the chromosomal AmpC resistance mechanism and the impact of AmpC hyperproduction, there is a lack in knowledge and understanding of the evolutionary origin of these promoter/attenuator variants. Notably, it is unexplored how the two most prominent promoter mutations, −32T>A and −42C>T, are distributed over the E. coli phylogeny and therewith how often they occur. More precisely, literature shows selective pressure can lead to convergent evolution that results in the reoccurrence of a mutation in multiple isolates independently and in separate lineages (Wake, Wake, and Specht 2011). This phenomenon is named homoplasy (Crispell, Balaz, and Gordon 2019). A consistency index can be calculated to quantify homoplasy by dividing the minimum number of changes on the phylogeny by the number of different nucleotides observed at that site minus one, effectively quantifying how often the same mutation occurred in a phylogenetic tree (Kluge and Farris 1969). One can use the consistency index to recognize genomic locations subjected to homoplasy, and relate the SNP positions that are inconsistent with the phylogeny to antibiotic resistance, as has been done, for example, in multiple studies on Mycobacteria spp. (Farhat et al. 2013; Mortimer, Weber, and Pepperell 2018; Ruesen et al. 2018; Miotto et al. 2014). In the present study, we hypothesize that some of the mutations in the ampC promoter/ attenuator region are homoplastic and are associated with CTX resistance. To test our hypothesis, we performed genome-wide homoplasy analysis and combined it with a genome-wide analysis of polymorphisms associated with CTX resistance by constructing an E. coli reference chromosome and combining it with whole-genome sequencing (WGS) data of 172 both FOX resistant and ESBL-negative E. coli isolates from human and animal origin previously collected by our research group (Coolen et al. 2019). 7
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