Contents lists available at ScienceDirect Plasmid journal homepage: www.elsevier.com/locate/yplas Short Communication Identification of a novel lineage of plasmids within phylogenetically diverse subclades of IncHI2-ST1 plasmids Piklu Roy Chowdhury a,b, ⁎ , Mathieu Fourment a , Matthew Z. DeMaere a , Leigh Monahan a , John Merlino c,d , Thomas Gottlieb c,d , Aaron E. Darling a , Steven P. Djordjevic a a The Ithree Institute, University of Technology Sydney, City Campus, Ultimo, NSW 2007, Australia b NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Sydney, Australia c Department of Microbiology and Infectious Diseases, Concord Hospital, NSW Health Pathology, Hospital Road, Concord 2139, NSW, Australia d Faculty of Medicine, University of Sydney, NSW, Australia ABSTRACT IncHI2-ST1 plasmids play an important role in co-mobilizing genes conferring resistance to critically important antibiotics and heavy metals. Here we present the identification and analysis of IncHI2-ST1 plasmid pSPRC-Echo1, isolated from an Enterobacter hormaechei strain from a Sydney hospital, which predates other multi- drug resistant IncHI2-ST1 plasmids reported from Australia. Our time-resolved phylogeny analysis indicates pSPRC-Echo1 represents a new lineage of IncHI2-ST1 plasmids and show how their diversification relates to the era of antibiotics. 1. Introduction The IncHI2-ST1 subgroup of IncHI2 plasmids play a significant role in the dissemination of carbapenemase-encoding genes, including bla IMP, bla VIM, bla NDM-1, bla KPC and bla OXA-48 (Beyrouthy et al., 2018; Chavda et al., 2016; Peirano et al., 2018; Samuelsen et al., 2018), and in the evolution of Carbapenem-resistant Enterobacterales (CRE). IncHI2 plasmids are broad host range, large (> 250 kb) conjugative plasmids that mobilize metal and drug resistance genes within Gram-negative pathogens. IncHI2-ST1 plasmids are most frequently reported to play a central role in the evolution of complex resistance phenotypes within disease-causing strains of the Enterobacter cloacae complex and other genera belonging to Enterobacterales. Surveillance strategies based on the accurate identification of plasmid lineages are essential for monitoring plasmid-mediated spread of drug resistance genes in emerging pathogens like Enterobacter hor- maechei, and other Gram-negative members belonging to the ESKAPEE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumo- niae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli). A targeted plasmid double locus (pDLST) scheme, which subtypes the IncHI2 plasmids into 15 sequence types (ST), was proposed in 2010 to detect and track the lateral movement of IncHI2 plasmids (Garcia-Fernandez and Carattoli, 2010). IncHI2-ST1 plasmids have been reported from pathogens isolates from humans, companion animals, chicken and pigs and known to disseminate clinically-im- portant antimicrobial resistance genes (Abraham et al., 2016; Fang et al., 2018; Garcia-Fernandez and Carattoli, 2010). However, the limited availability of complete IncHI2-ST1 plasmid sequences has prevented detailed analysis of their phylogeny. Hybrid genome assembly algorithms that combine short and long read sequence data assist efforts in rapid assembly of large plasmids (McKinnon et al., 2018; Wyrsch et al., 2019) and improve the resolution of phylogenetic analysis. To date, only three complete multi-drug re- sistance (MDR) IncHI2-ST1 plasmid sequences from Australia (pMS7884A, pIMP4-SEM1, and pAUSMDU141–1) are publicly acces- sible in GenBank. All three plasmids were isolated in 2015 from dif- ferent genera of pathogenic Enterobacterales along the east coast of Australia (Table 1), two of which carried bla IMP-4 . In this report, we present a comprehensive genetic characterisation and phylogenenetic analysis of a 339.9 kb MDR-IncHI2-ST1 plasmid, pSPRC-Echo1, which predates the three Australian MDR-plasmids reported recently. To the best of our knowledge, this is the first report on time resolved phylo- geny analysis of this important group of plasmids. Our analysis iden- tifies a new lineage of IncHI2-ST1 plasmids, and details the ancestry of subsets of IncHI2-ST1 plasmids available in public repositories. 2. Methods Plasmid pSPRC-Echo1 (BioProject PRJNA494598, Accession number CP032842) was recovered from an extended-spectrum ß-lac- tamase positive (ESBL+), third generation cephalosporin resistant Enterobacter hormaechei strain C15117 from the Burns Unit at Concord https://doi.org/10.1016/j.plasmid.2019.03.002 Received 10 December 2018; Received in revised form 22 February 2019; Accepted 13 March 2019 ⁎ Corresponding author at: The Ithree Institute, University of Technology, Sydney City Campus CB04.07, Ultimo, NSW 2007, Australia. E-mail address: Piklu.Bhattacharya@uts.edu.au (P. Roy Chowdhury). Plasmid 102 (2019) 56–61 Available online 15 March 2019 0147-619X/ © 2019 Elsevier Inc. All rights reserved. T