155 In silico estimates of plasmid copy number and increased resistance in CMY-2-producing E. coli Discussion The present study aimed to evaluate whether an increased estimated copy number of blaCMY-2 carrying plasmids is associated with reduced susceptibility for CTX, CAZ, and TZP in ESBL-negative E. coli isolates. Based on the assumption that differences in sequencing depth between chromosomal and plasmid-encoded DNA sequence data correlate with plasmid copy number, an estimated plasmid copy number (ePCN) was calculated. A significantly higher ePCN of the blaCMY-2 gene was found in isolates with high MIC values for CTX, CAZ, and TZP compared to isolates with low MIC values. The results in the present study are in accordance with the study of Kurpiel et al. (Kurpiel and Hanson 2012), although using another method, describing an association between an increased plasmid copy number of the blaCMY-2 gene and higher MICs for piperacillin-tazobactam using real-time reverse transcriptase PCR and Southern hybridization. Molecular analysis showed alterations in the sequence of the loop region of the Inc antisense RNA between low- and high-resistant strains, which might be associated with plasmid copy number regulation in IncI1 plasmids. The present study detected fewer acquired resistance genes other than blaCMY-2 in the high-MIC group for CAZ and TZP compared to the isolates in the low-MIC group. It is known that the presence of plasmids can have a negative effect on the fitness of bacteria (San Millan and MacLean 2017; Hall et al. 2017). The occurrence of multiple plasmids simultaneously seems to have a higher fitness cost than the presence of a single plasmid (Gama, Zilhão, and Dionisio 2018). The presence of specific antibiotic genes on the plasmid may also influence the bacterium’s fitness as well (Rajer and Sandegren 2022). The results of the current study indicate that in the presence of one high copy number resistance gene, the presence of multiple resistance genes may pose a disadvantage for the bacterium. The interactions between multiple plasmids and the interactions with the micro-organism containing them are complex, and further studies are needed to unravel the complexity of these mechanisms. In the present study, WGS data are used to estimate the plasmid copy number and relate this to the phenotypical susceptibility of E. coli isolates. Multiple methods have been developed to estimate plasmid copy numbers, from luciferase-based densitometry to quantitative PCR techniques and, more recently, droplet digital PCR (Providenti et al. 2006; Coronado et al. 1994; C. Lee et al. 2006; Plotka, Wozniak, and Kaczorowski 2017). These methods have proven to be accurate in determining plasmid copy numbers. However, they can be labour intensive and time-consuming. New improvements in WGS, automation, and bioinformatics pipelines make it easier to upscale the number 8
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