Plasmid 116 (2021) 102578 Available online 6 May 2021 0147-619X/© 2021 Published by Elsevier Inc. Interference of ISEcp1-bla CTX-M-1 with the shuffon rearrangement in IncI1 plasmids Milan S. Stosic a, *, 1 , Marianne Sunde a , Solveig Sølverød Mo a , Amar Anandrao Telke a , Knut Rudi b a Section for Food Safety and Animal Health Research, Department of Animal Health, Welfare and Food Safety, Norwegian Veterinary Institute, Ullevålsveien 68, Oslo 0454, Norway b Norwegian University of Life Sciences, Chemistry, Biotechnology and Food science department (IKBM), Campus Ås, Universitetstunet 3, Ås 1433, Norway A R T I C L E INFO Keywords: Shuffon Shuffon rearrangement IncI1 plasmids ISEcp1-bla CTX-M-1 -orf477 ABSTRACT IncI1 plasmids are known disseminators of the extended-spectrum cephalosporin resistance (ESC) gene bla CTX-M- 1 , among species of the Enterobacteriaceae family. In several IncI1 plasmids, this gene was found incorporated into the transposition unit, ISEcp1-bla CTX-M-1 -orf477, interrupting a shuffon region, a hallmark of IncI1 conjugative plasmids. The shuffon diversifes pilV gene that encodes the adhesine-type protein found on the tip of the conjugative pilus. To further elucidate the shuffon rearrangement, we examined to what extent the shuffon rearrangement was affected by the transposition-unit insertion. As expected, the interrupted shuffons generated a lower number of shuffon variants and exhibited an altered segment-deletion pattern compared to the non- interrupted shuffon. Interestingly, segment-loss frequency of the interrupted shuffons was distinctive in different plasmid hosts. Finally, the analysis of the 3 ′ end of the pilV gene revealed that shuffon rearrangement favoured segment A to complete pilV partial open reading frame regardless of the shuffon. Thereby, it could be assumed that the A-segment has greater importance during conjugation, however, this remained a hypothesis. Further exploration of shuffon rearrangement and its importance in the plasmid-recipient selection during conjugation would be benefcial as the knowledge could be applied in developing a strategy to limit IncI1 mediated antimicrobial resistance dissemination. 1. Introduction The discovery that IncI1 conjugative plasmids contribute to the dissemination of extended-spectrum cephalosporin (ESC) resistance genes increased scientifc interest for this plasmid group (Carattoli, 2011). The host-range of IncI1 plasmids encompasses only the members of the Enterobacteriaceae family (Carattoli et al., 2018). However, several IncI1-encoded addiction systems enable their long-term persistence in bacterial communities even without antimicrobial selective pressure (Hagbø et al., 2019). A previous study concluded that dissemination and persistence of IncI1 plasmids carrying bla CTX-M-1 were responsible for the high occurrence of ESC-resistant Escherichia coli clones in pigs. Inter- estingly, the same study suggested that a four-year period would be necessary to signifcantly reduce/eliminate the ESC-resistant E. coli after the elimination of antimicrobial selection pressure (Abraham et al., 2018). Moreover, IncI1 plasmids associated with bla CTX-M-1 are also globally distributed in broiler production (Carattoli, 2011; Carattoli et al., 2018; Touzain et al., 2018; Niero et al., 2018). A recent study confrmed that these plasmids also circulate among E. coli originating from Norwegian broiler production (Mo et al., 2020). More specifcally, the detected bla CTX-M-1 /IncI1 plasmids belonged to sequence type (ST) 7, ST3, and ST42. As ST3 and 42 are closely related, they were grouped in the same clonal complex (CC), CC-3. Further characterization of the CC-3 plas- mids confrmed previous fndings; the ESC-resistance gene, bla CTX-M-1 , was a component of the transposition unit, ISEcp1-bla CTX-M 1 -orf477, located within a shuffon region (Carattoli et al., 2018; Mo et al., 2020). The shuffon was the frst multiple inversion system discovered on plasmid R64, a reference plasmid of the IncI1 incompatibility group (Brouwer et al., 2015; Komano, 1999). Several similar systems were * Corresponding author at: Section for Food Safety and Animal Health Research, Department of Animal Health, Welfare and Food Safety, Norwegian Veterinary Institute, Ullevålsveien 68, Oslo 0454, Norway. E-mail address: milansto@oslomet.no (M.S. Stosic). 1 OsloMet - Oslo Metropolitan University, Faculty of Health Sciences, Pilestredet campus, Pilestredet 32 – 54, 0130 Oslo, Norway. Contents lists available at ScienceDirect Plasmid journal homepage: www.elsevier.com/locate/yplas https://doi.org/10.1016/j.plasmid.2021.102578 Received 29 January 2021; Received in revised form 24 April 2021; Accepted 4 May 2021