108 Chapter 6 a maximum difference of two SNPs. Furthermore, only one gene was variably present between the different plasmids, and no rearrangements were observed apart from a small, highly variable region. This area is the formerly described highly variable shufflon region at the C-terminal end of the PilV protein (Brouwer et al. 2015; 2019). A high degree of similarity between IncI1–pST12 plasmids was previously reported (Pietsch et al. 2018; Roer et al. 2019; Castellanos et al. 2019; Shirakawa et al. 2020). However, all of the studies either contained only plasmids extracted from one E. coli sequence type (ST131) or the included plasmids were primarily of poultry origin (Roer et al. 2019; Castellanos et al. 2019). All of the studies used either gene presence/ absence-based or SNP-based analysis, but not both, possibly missing subtle differences between various plasmids. Shirakawa et al used a combination of short-read sequence data of different blaCMY-2-containing plasmids from Japanese poultry and human origin, together with plasmid sequence data retrieved from the National Center for Biotechnology Information nucleotide database (https://www.ncbi.nlm.nih.gov/) to perform an extensive plasmid comparative analysis(Shirakawa et al. 2020). Their clustering analysis showed a high similarity among the IncI1–pST12 plasmids as well; however, this study did not provide further detail on the SNP differences of possible rearrangements within the plasmid sequences. Moreover, these studies predominately used in silico reference-based plasmid reconstructions of short-read sequence data rather than performing a hybrid assembly of both short- and long-read sequence data. A recent study by Valcek et al. on IncI1–pST3 and IncI1–pST7 plasmids showed that using combined long-read and short-read sequencing data improves the accuracy of a full plasmid analysis, e.g., of rearrangements (Valcek et al. 2019). The current study is the first study describing plasmid differences using both gene presence/absence-based and SNP-based analysis. Moreover, rearrangements between the different plasmids could be detected such as those shown in the hypervariable region, which were missed in previous studies based on only short-read sequences. Several studies have described outbreaks with blaCMY-2-harbouring Enterobacteriaceae (Matsumura et al. 2015; Wendorf et al. 2015; I. F. Huang et al. 2005; Kameyama et al. 2015). Since the blaCMY-2 is predominantly located on plasmids, horizontal transfer of the plasmid in an outbreak can go undetected if only typing of the bacterial chromosome is performed. Distinguishing epidemiologically related and unrelated plasmids is essential to confirm plasmid transmission in an outbreak. Therefore, statements on the horizontal transfer of these plasmids based on genetic identity should be made with caution. However, given the conservation of the IncI1–pST12 plasmids, they could instead be used as a tool to monitor the speed and breadth of spread of these plasmids through populations, either different in place of origin or bacterial host.
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