147 In silico estimates of plasmid copy number and increased resistance in CMY-2-producing E. coli Introduction The increase in antimicrobial resistance has become a major public health threat (Paterson 2006; J. D. Pitout and Laupland 2008). Enterobacterales cause a considerable number of nosocomial and community-acquired bacterial infections, and resistance to the commonly used beta-lactam antibiotics may complicate optimal antibiotic treatment (Harris 2015; Rodríguez-Baño et al. 2018). The plasmid-encoded resistance genes play a central role in this phenomenon, as the horizontal spread of mobile genetic elements may cause transmission of resistance genes (Philippon, Arlet, and Jacoby 2002). The emergence of extended-spectrum beta-lactamase producing Enterobacterales (ESBLE) has been the subject of numerous studies during the last decades. Increasingly other plasmid-encoded beta-lactamases, such as plasmid-mediated AmpC (pAmpC) betalactamases, are being reported (Park et al. 2013; Mata et al. 2010; Miró et al. 2013). The most prevalent pampC genes found in E. coli in Western Europe are blaCMY-2 -like genes (P Bogaerts et al. 2010; Stéphane Corvec et al. 2007). Production of this beta-lactamase is associated with resistance to third-generation cephalosporins and most beta-lactam/beta-lactamase inhibitor combinations, although Minimal Inhibitory Concentrations (MICs) tend to differ between isolates (Kurpiel and Hanson 2012; Edquist et al. 2013; Skalweit et al. 2013). It is known that increased plasmid copy numbers influence antibiotic susceptibility in several bacterial species. San Millan et al. showed an increase in the copy number of a ROB-1 beta-lactamase gene in Haemophilus influenzae under antibiotic pressure, which resulted in higher ampicillin resistance levels (Millan et al. 2015). Subsequently, Santos-Lopez et al. revealed an inverse mechanism in which lower plasmid copy numbers resulted in lower MICs in H. influenzae (SantosLopez et al. 2017). Elevated copy plasmid numbers have been associated with increased resistance in carbapenemase-producing Enterobacterales (Shen et al. 2020; Becker et al. 2016). This increase in MIC can occur with other resistance mechanisms, e.g., a decrease in membrane porins or changes in the promoter sequence of the beta-lactamase. Nonetheless, Shen et al. confirmed copy number increase as an independent cause of increased resistance as well (Shen et al. 2020). Kurpiel et al. have shown that E. coli strains with an increased copy number of an IncI1 plasmid harbouring a blaCMY2 gene were associated with increased resistance to piperacillin-tazobactam (Kurpiel and Hanson 2012). It is unknown if copy numbers of blaCMY-2 encoded plasmids may influence MICs of third-generation cephalosporins such as cefotaxime or ceftazidime. Becker et al. referred to variations in copy number of small plasmids being related to differences in sequencing depth in whole genome sequencing (WGS) data (Becker et al. 8
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