136 Chapter 7 Discussion We present a genome-wide analysis in which homoplastic mutations are associated with antibiotic resistance in E. coli . By comparing WGS data of 172 E. coli isolates to a reference chromosome, we were able to reconstruct the evolution of the genomes and therewith map recurrent events, allowing us to detect homoplasy associated with CTX resistance. Our foremost finding is the significant association of the −42C>T mutation, in the ampC promoter, with CTX resistance that evolved independently at least 17 times in five distinct phylogroups. The −42C>T mutation has been confirmed in former studies to result in AmpC hyperproduction in E. coli (Caroff N, Espaze E, Gautreau D, Richet H 2000; Tracz et al. 2007; Nelson and Elisha 1999). Nelson et al. demonstrated an 8 to 18 times increase in activity of AmpC when cloning the promoter upstream a lac operon (Nelson and Elisha 1999). Conversely, Caroff et al. found a decrease in expression of AmpC when cloning the promoter with a −42T>C mutation in a pKK232-8 reporter plasmid with a chloramphenicol acetyltransferase gene (Caroff N, Espaze E, Gautreau D, Richet H 2000). Tracz et al. confirmed that the −42C>T mutation has the strongest effect on the ampC promoter, resulting in a high expression of the ampC gene as detected by qRT-PCR (Tracz et al. 2007). Despite the fact that the −42C>T mutation has such a strong effect on AmpC production, the effect of the mutation on CTX MICs had not been confirmed. Moreover, the contribution of convergent evolution on this position relative to the role of the expansion of a clone with a beneficial mutation at this position has not been determined. That being the case, this study provides evidence that this −42 C>T mutation is not a result of a recombination event and most likely evolved many times independently. In the current study, we see a strong correlation between the −42C>T mutation and CTX resistance, even though there were exceptions, as not all isolates with this mutation were considered resistant according to EUCAST guidelines. As described by Coolen et al., the MIC for CTX in putative AmpC hyperproducers was generally higher than in the putative low-level AmpC producers, though the range in CTX MICs overlapped (Coolen et al. 2019). Yet, the lowest MIC measured in the isolates with the −42C>T mutation was 0.75 mg l−1, which is at the higher end of the EUCAST epidemiological cut-off values (ECOFF) distribution. The variation in phenotypical testing could be an explanation, although an interplay of AmpC hyperproduction and other strain-specific factors as previously described by Tracz et al. may also be considered (Tracz et al. 2007). In order to avoid biasing towards a single method, in our case the homoplasy-based association method, we performed the analysis using Pyseer on the same data set.
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