Table 1.

Summary of plasmid typing and subtyping schemes.

Plasmid typing schemes		Comments
Replicon typing	Inc grouping	Plasmids with similar replication machinery are often unable to stably co-exist within the same host cell (Snyder et al., 2013); this phenomenon was traditionally used to classify plasmids into incompatibility (Inc) groups. Inc grouping has been applied to plasmids from Enterobacteriaceae (Hedges and Datta, 1973), Pseudomonas aeruginosa, and Staphylococcus aureus (Taylor et al., 2004).
	Replicon probe hybridization	Couturier et al. (1988) cloned replicons representing Enterobacteriaceae Inc groups; plasmids were classified according to Southern blot hybridizations using the replicons as probes. Probe hybridization lacks specificity when closely related replicons are present, and is no longer widely used except for its application subsequent to PCR-based replicon typing (PBRT); here, amplicons derived from PCR can be used as probes to type plasmids isolated on a gel (EFSA, 2011).
	PCR-based replicon typing (PBRT)	PBRT for plasmids of the well-studied Enterobacteriaceae family currently detects 28 replicons (based on various genetic loci including rep genes and replication regulatory sequences). These PBRT types roughly correspond to traditional Inc groups, so Inc nomenclature is still used. A commercial 28-replicon PBRT kit is available (Diatheva, 2016). More recently, PBRT has been devised for Acinetobacter baumannii plasmids (Bertini et al., 2010); multiplex PCRs targeting 27 replicons are used to classify plasmids into 19 ‘GR’ types. A PBRT scheme has also been applied to plasmids of gram-positive taxa, focusing on enterococcal (Jensen et al., 2010) and staphylococcal (Lozano et al., 2012) plasmids. A closely related scheme focuses on plasmids of Enterococcus faecium (Rosvoll et al., 2010, 2012).
	Replicon subtyping	Allelic profiles are assessed at 2–6 core loci (depending on the specific scheme). Plasmids are assigned a pMLST subtype nesting within the broader replicon type. pMLST schemes are available for six common replicon types of Enterobacteriaceae plasmids (IncF, HI1, HI2, I1, N, A/C). PCR-based and in silico methods are available.
	In silico replicon typing/subtyping	Replicon and pMLST allele databases can be downloaded for local use, but user-friendly web-tools (PlasmidFinder/pMLST for replicon typing/subtyping) can run the analysis pipeline, including read assembly. The PlasmidFinder replicon database currently contains 121 reference replicons for Enterobacteriaceae plasmids; a dataset of replicons for gram-positive plasmids based on the scheme devised by Jensen et al. (2010) and Lozano et al. (2012) is also available (Carattoli et al., 2014; Center for Genomic Epidemiology, 2016). Instead of relying on read assembly and BLAST, unassembled reads can be mapped to the PlasmidFinder database or pMLST database using SRST2 (Inouye et al., 2014).
MOB typing	PCR-based MOB typing	PCR-based ‘degenerate primer MOB typing’ (DPMT) is used to type γ-Proteobacterial plasmids; 19 degenerate primer pairs target relaxase sequences to partition plasmids into five of the main MOB types identified by in silico MOB typing (Rose et al., 1998; Alvarado et al., 2012). PCR-based MOB typing has also been demonstrated for enterococcal plasmids (Goicoechea et al., 2008; Freitas et al., 2016).
	In silico MOB typing	Six N-terminal relaxase sequences are used as PSI-BLAST probes to detect relaxase sequences of transmissible plasmids, and partition plasmids into six possible MOB types (Garcillán-Barcia et al., 2009).
Other locus-targeting schemes		Other locus-based methods for plasmid typing and subtyping exist, but tend to be applicable to a more restricted set of plasmids (Guglielmini et al., 2011; Freitas et al., 2013; Compain et al., 2014; Dealtry et al., 2014a,b; Bousquet et al., 2015).
Plasmid ‘fingerprinting’ (RFLP typing)		Restriction fragment length polymorphism (RFLP) is sometimes used to subtype plasmids, especially when pMLST is unavailable. However, band patterns can be difficult to interpret, and do not provide a reliable phylogenetic marker (Laguerre et al., 1992). Shearer et al. (2011) used RFLP to assign a subset of conserved staphylococcal plasmids to three major RFLP types.