63 Development of an algorithm to discriminate between plasmid- and chromosomal-mediated AmpC pAmpC might be useful, although discriminatory performance was more prominent when using disc diffusion as compared with MIC testing by Etest strips (Edquist et al. 2013). In their study, a multiplex PCR was performed to detect pampC genes, but there was no verification for cAmpC hyperproducers in the strain collection used. Our WGS results reliably show that phenotypic hyperproduction of cAmpC beta-lactamase can be caused by mutations in the ampC promoter region (Edquist et al. 2013). No conclusions can be drawn about mutations resulting in elevated AmpC production, in addition to previously mentioned mutations. However, there is evidence that alterations of the AmpC beta-lactamase34,35 or changes in membrane permeability may lead to differences in cephalosporin resistance (Martínez-Martínez 2008; Nordmann, Poirel, and Nordmann 2007; Mammeri, Galleni, and Nordmann 2009). Further analysis on the incorrectly classified campC isolates is needed to exclude causes of cephalosporin resistance, other than the known promoter mutations. ACT-1, DHA-1, DHA-2 and CMY-13 are inducible while other pAmpC betalactamases (such as CMY-2) are constitutively expressed (Jacoby 2009; Philippon, Arlet, and Jacoby 2002; Reisbig, Hossain, and Hanson 2003). Reisbig et al previously reported that absence of the ampD gene in combination with the inducible ACT-1 pampC gene increased MICs of 3GCs (Reisbig, Hossain, and Hanson 2003). If we assume that the inducible blaDHA group might have a similar mechanism, we would expect higher ceftazidime and cefotaxime MICs in the absence of ampD; our strains with blaDHA showed lower MICs of 3GCs compared with the non-inducible blaCMY2 genes, so we can infer that ampD might be present; however, further analysis on the influence of ampD on blaDHA expression is needed. Additionally, Reisbig et al described the contribution of plasmid copy number of ACT-1 and MIR-1 pampC genes to 3GC resistance (Reisbig, Hossain, and Hanson 2003). They concluded that plasmid copy number probably impacts MIC values for pampCpositive strains; however, this was not substantiated (Hanson 2003). We were able to accurately detect pampC-positive strains even without measuring plasmid copy numbers. A strength of the present study is that ST information on E. coli in our datasets was provided, which made it possible to exclude clonal origin, in contrast to other studies (Polsfuss et al. 2011; Edquist et al. 2013; Aarestrup et al. 2010). Though ST131, ST73 and ST38 predominated, a wide variety of STs was represented in our collection (Table S2). This is in line with other studies that report higher prevalence of ST131 and ST73 in human samples and ST38 in animal samples (Pietsch et al. 2018; Doumith et al. 2012; Miajlovic et al. 2016). 4
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