Plasmid genomic epidemiology of blaKPC carbapenemase-producing Enterobacterales in Canada, 2010–2021

ABSTRACT Carbapenems are considered last-resort antibiotics for the treatment of infections caused by multidrug-resistant Enterobacterales, but carbapenem resistance due to acquisition of carbapenemase genes is a growing threat that has been reported worldwide. Klebsiella pneumoniae carbapenemase (blaKPC) is the most common type of carbapenemase in Canada and elsewhere; it can hydrolyze penicillins, cephalosporins, aztreonam, and carbapenems and is frequently found on mobile plasmids in the Tn4401 transposon. This means that alongside clonal expansion, blaKPC can disseminate through plasmid- and transposon-mediated horizontal gene transfer. We applied whole genome sequencing to characterize the molecular epidemiology of 829 blaKPC carbapenemase-producing isolates collected by the Canadian Nosocomial Infection Surveillance Program from 2010 to 2021. Using a combination of short-read and long-read sequencing, we obtained 202 complete and circular blaKPC-encoding plasmids. Using MOB-suite, 10 major plasmid clusters were identified from this data set which represented 87% (175/202) of the Canadian blaKPC-encoding plasmids. We further estimated the genomic location of incomplete blaKPC-encoding contigs and predicted a plasmid cluster for 95% (603/635) of these. We identified different patterns of carbapenemase mobilization across Canada related to different plasmid clusters, including clonal transmission of IncF-type plasmids (108/829, 13%) in K. pneumoniae clonal complex 258 and novel repE(pEh60-7) plasmids (44/829, 5%) in Enterobacter hormaechei ST316, and horizontal transmission of IncL/M (142/829, 17%) and IncN-type plasmids (149/829, 18%) across multiple genera. Our findings highlight the diversity of blaKPC genomic loci and indicate that multiple, distinct plasmid clusters have contributed to blaKPC spread and persistence in Canada.

caused by multidrug-resistant Gram-negative bacteria.Following the use of carbapenems in clinical practice, the emergence of carbapenem-resistant pathogens poses a great threat to human health (1).Carbapenem-resistant Enterobacterales have been reported worldwide as a consequence largely of the acquisition of carbapenemase genes (2).
Of the different classes of carbapenemases, Klebsiella pneumoniae carbapenemase (bla KPC ) is the most commonly identified in many countries including the United States and Canada [reviewed in references (3)(4)(5)(6)].bla KPC was the dominant type of carbapene mase isolated from 2010 to 2014 by the Canadian Nosocomial Infection Surveillance Program (CNISP), where they comprised 50%-83% of all carbapenemases analyzed per year (6).From 2017 to 2021, the rates of carbapenemase-producing Enterobacterales infections in Canada increased by 166% (from n = 20 in 2017 to n = 55 in 2021), and bla KPC remains the most common carbapenemase type detected in Canada (7).
Advances in genome sequencing have enabled detailed characterization of complete bla KPC -encoding plasmids in recent years (13,15).bla KPC is associated with a variety of plasmid types, including the narrow-host-range IncF-type plasmids which were crucial for the success of K. pneumoniae clonal complex 258 (3,12,15).bla KPC has also been found on broad-host-range plasmids with IncN, IncR, IncX, ColRNA, IncA/C, or IncP replicons around the world (3,13,15,(17)(18)(19)(20).Many of these plasmids can have multiple replicons, undergo large rearrangements, and encode other genetic features that ensure their persistence (20).
Here, we applied whole genome sequencing to characterize the molecular epidemiol ogy of bla KPC carbapenemase-producing isolates collected by the Canadian Nosocomial Infection Surveillance Program from 2010 to 2021.Using combined short-read and long-read sequencing of selected representatives to generate complete bla KPC -encoding plasmids, we investigated the diversity of carbapenemase-encoding plasmids among these isolates across Canada and compared them to the global context of bla KPC .
To investigate where these plasmids fit within a global data set, we clustered our 202 complete plasmids alongside 34,513 plasmids present in PLSDB (25) using the MOBcluster tool from MOB-suite (24,26).Altogether, the plasmids clustered into 10,996 distinct primary clusters, and our bla KPC plasmids grouped into a subset of 28 primary clusters which all contained representative plasmids from PLSDB.
From the above data, we observed 10 primary clusters which represented 87% (175/202) of the Canadian bla KPC plasmids from this study.The primary clusters generally reflected incompatibility groups and the major trends of each are summarized in Table 1.Within the global plasmid data set, the presence of bla KPC varied between our top primary clusters (Fig. 4); some primary clusters tended to be frequently associated with bla KPC (AA014, AA013, AA029, and AA109), whereas others appeared to encode bla KPC more sporadically (AA085 and AA017).Some clusters had a tight distribution of plasmid sizes (AA013 and AA042) whereas others had a broader size distribution (AA085 and AA002).The percentage of core/soft core genes (defined as genes found in >95% plasmids) varied from 3% (AA042) to 28% (AA014) across the primary clusters, indicating some tend to have more diverse or dynamic gene content than others.We also evaluated the core genome size of the Canadian plasmids within each of these primary clusters as we suspected that the Canadian plasmids would be more closely related to each other than to global plasmids.As expected, the percentage of core genes within the Canadian plasmids was higher than the percentage predicted in the global data set with one exception (AA042) (Fig. 4).Several primary clusters had an average of five or more antimicrobial resistance genes (AA002, AA017, and AA085).Certain primary clusters had broad host ranges (AA002, AA007, and AA124), whereas others were only found in a single genus (AA085 and AA109).The dominant type of transposon was Tn4401b except for the IncF clusters (AA085 and AA029) where Tn4401a was dominant.
Not surprisingly, all primary clusters with the exception of AA042 contain features that support their stability and persistence in the host cell.Of the genes found in >95% of plasmids in each cluster, between one and five genes (representing 2%-33% of genes per cluster) are involved in stability/transfer/defense which includes genes such as partition/stability genes (e.g., parA, parM, and stbB), anti-restriction genes (e.g., ardA, ardB, and ardR), and SOS-inhibition genes (e.g., psiA and psiB).Different primary clusters had different proportions of stability/transfer/defense gene content; some harbored a single anti-restriction protein (AA124), whereas some encoded two anti-restriction proteins, a plasmid stability protein, an endonuclease, and a stability protein (AA029).Values indicate the most common genotype in the cluster and may not apply to all plasmids in the cluster. b Values obtained from MOB-suite (24,26).Mobility is assigned based on the presence of relaxase (mobilizable) and/or MPF proteins (conjugative) or absence of both (non-mobilizable).
c ARGs = antimicrobial resistance genes.
d Core genes represent the number of genes present in >95% of plasmids in the cluster, divided by the total number of non-redundant genes in the cluster.

Canadian bla KPC -encoding plasmids in the top primary clusters
Given the high proportion of conserved genes among Canadian plasmids within each primary cluster (Fig. 4), we focused on the features of these Canadian plasmids separately from the other PLSDB plasmids present in each primary cluster.The primary clusters containing the most bla KPC -encoding Canadian plasmids were IncL/M replicons (AA002) and IncN replicons (AA007, AA014, and AA017) (Table 1) and are examined in more detail below.
The Canadian plasmids in primary cluster AA002 (IncL/M) were classified into three secondary clusters: AL008 (49/62, 79%), AL013 (8/62, 13%), and AL001 (1/62, 1.6%) (Fig. 5).The first secondary cluster (AL008) had >99% nucleotide identity.They were 72.3 kb isolated from K. pneumoniae species complex, K. aerogenes, K. oxytoca, C. freundii complex, E. cloacae complex, E. coli, and Raoultella planticola between 2016 and 2020 from three sites in the same province.These plasmids harbor bla KPC-3 on Tn4401b-3 and do not encode any other resistance genes.The next secondary cluster (AL013) was also shared among multiple genera with >99% nucleotide identity.They were 109.8 kb isolated from E. coli, K. pneumoniae species complex, and E. cloacae complex from one Pangenome size was calculated for (A) Canadian and PLSDB plasmids, and (B) Canadian plasmids only.Gene categories represent genes found in 99%-100% of plasmids (core), 95%-99% of plasmids (soft core), 15%-95% of plasmids (shell), and 0%-15% of plasmids (cloud).(C) Plasmid length in base pairs and prevalence of bla KPC among plasmids in each primary cluster.site between 2011 and 2021.In addition to bla KPC-3 on Tn4401b-3, this plasmid also encodes ant(2″)-Ia, bla SHV-30 , dfrA7, qacE, qnrB2, tetA, and two copies of sul1.Structurally, the secondary clusters have similar backbones of transfer and replication genes (Fig. 5).AL008 and AL013 have Tn4401b-3 inserted on the same strand, but AL013 contains many additional integration/excision mobile elements, antimicrobial resistance genes, and hypothetical coding sequences.The AL001 plasmid is structurally similar to AL008 plasmids, but has a different locus of insertion of Tn4401b-3 on the opposite strand and has a pemKI toxin-antitoxin system.The prevalence of these plasmids among different genera and the presence of conjugation genes (MOBP relaxase and MPF_I mating pair formation protein) suggests that regional horizontal transmission has contributed to these secondary clusters' persistence.
Three primary clusters (AA007, AA014, and AA017) were identified as having IncN-type replicons with similar features (Table 1).Each cluster corresponds to a different type of replicon: IncN (AA007), IncN2 (AA017), and IncN3 (AA014).All plasmids in primary cluster AA007 were grouped in a single secondary cluster (31/31, 100%), as did all plasmids in primary cluster AA017 (7/7, 100%); in contrast, plasmids in primary cluster AA014 were split into two secondary clusters: AL059 (11/13, 84%) and AL060 (2/13, 15%).The first secondary cluster (AL059) was isolated from E. coli, K. pneumoniae, and E. cloacae complex from five sites in one province between 2014 and 2020.These plasmids encode bla KPC-3 on Tn4401b-2 and no other resistance genes with the exception of a single plasmid with a multi-resistance gene island.The other secondary cluster (AL060) was found in C. freundii complex and K. pneumoniae complex at two sites in one province.This group has no resistance genes aside from bla KPC-2 on Tn4401a-2.Primary cluster AA017 plasmids were isolated from E. coli, K. pneumoniae complex, E. cloacae complex, and Citrobacter farmeri from two sites in one province between 2016 and 2020.Aside from bla KPC-2 on Tn4401b-1, other resistance genes found in this cluster include bla TEM-1B , dfrA25, mph(A), qacE, qnrB2, and sul1.Plasmids in the largest IncN primary cluster (AA007) were isolated from E. coli, K. pneumoniae complex, Klebsiella oxytoca, Klebsiella michiganensis, Raoultella ornithinolytica, E. cloacae complex, C. freundii complex, Citrobacter amalonaticus, Citrobacter koseri, and Serratia marcescens.These plasmids were isolated from 2012 to 2020 from nine sites in four provinces.This group contains bla KPC-3 on Tn4401b with many of them encoding different combinations of aac(6′)-Ib, aph(3″)-Ib, aadA1, bla OXA-9 , bla TEM-1A , dfrA14, qacE, qnrS1, sul1, sul2, and tetD.The prevalence of IncN plasmids in multiple genera from multiple sites in the same province (primary clusters AA017 and AA014) or across multiple provinces (AA007) along with encoded conjugation genes (MOBF relaxase and MPF_T mating pair formation protein) suggests there has been widespread horizontal transmission of these primary clusters in Canada.

Canadian bla KPC -encoding plasmids in the other primary clusters
Plasmids in primary cluster AA042 are small in size (10-20 kb) and grouped into four secondary clusters: AL143 (2/21, 10%), AL144 (17/21, 81%), AL149 (1/21, 5%), and AL150 (1/21, 5%).The largest secondary cluster (AL144) had multiple replicon types identified by MOB-suite, with the most common being ColRNAI_rep_cluster_1987 and rep_clus ter_2335.These plasmids were isolated from 14 sites across three provinces from C. freundii complex, E. coli, K. pneumoniae complex, and S. marcescens between 2011 and 2021.These plasmids did not contain other resistance genes and most (14/17, 82%) were classified as "mobilizable" by MOB-suite, which indicates they encoded a relaxase or recognizable oriT sequence.The core genes in this group include all components of the Tn4401b transposon including bla KPC-3 which results in integration/excision genes being common in this secondary cluster.The small size of mobilizable plasmids in this primary cluster and their prevalence at various sites indicate that bla KPC spread may be influenced by these smaller vectors.
We detected a novel replicon type in primary cluster AA109, designated as rep_clus ter_2268 by MOB-typer, which now has a novel replicon designation of repE(pEh60-7) in PlasmidFinder (personal communication).We obtained complete plasmid sequences for seven isolates which grouped into a single secondary cluster.These plasmids were only found in the Enterobacter cloacae species complex, specifically Enterobacter hormaechei subsp.hoffmannii ST316, at a single site in one province from 2011 to 2020, indicating a very stable plasmid or reservoir of this strain.Occurrences of this plasmid are intermittent from 2011 to 2016 and appear to become more prevalent from 2016 to 2018 with fewer detected in recent years.SNVPhyl (28) was run to analyze SNV differences in the ST316 bla KPC -harboring isolates from this site over the 8-year time span (n = 51), and a maximum of 62 SNVs was identified between isolates using 88.51% of the genome.The novel repE(pEh60-7) plasmids encode bla KPC-3 in Tn4401b-3 but did not encode any other resistance genes.While they were classified as non-mobilizable by MOB-suite, they do encode a mating pair formation protein and other genes annotated as conjugation machinery which may not be represented in current databases (Fig. 6).Further work will need to be done to determine if these plasmids are conjugative.These plasmids also encode for stability/transfer/defense proteins including a SOS-inhibition protein, stability partition proteins, the hok-sok toxin-antitoxin system, and an anti-restriction protein.There were two PLSDB plasmids that grouped in the same primary AA109 cluster but in different secondary clusters, and their gene content is quite diverse compared to the Canadian plasmids (70% identity to NZ_AP022432.1 and 43% identity to NZ_CP080472.1)(Fig. 6).Given these plasmids are only found in one specific sequence type, clonal transmission is likely driving this primary cluster's persistence.
Primary cluster AA124 plasmids were classified as IncX5 replicons grouped into two secondary clusters: AL404 (1/10, 10%) and AL405 (9/10, 10%).The largest secondary cluster (AL405) encoded bla KPC-3 on Tn4401b-3.These plasmids were isolated from K. pneumoniae complex, Serratia liquefaciens, E. cloacae complex, and Klebsiella ascorbata from four sites in two provinces between 2015 and 2021.Eight of these nine plasmids are >99% identical and do not contain any resistance genes aside from bla KPC-3 on Tn4401b-3.These plasmids are predicted to be conjugative based on the presence of the MOBP relaxase and MPF_T mating pair formation protein.The wide range of genera suggests that these IncX5 plasmids have disseminated horizontally across the country.

Epidemiology of bla KPC -encoding plasmid clusters across Canada
Long-read data are essential for resolving plasmid structures (32), but this can be costly for large surveillance data sets when performed alongside short-read sequencing, which then makes it difficult to analyze plasmid populations at a broad scale.Consequently, we explored MOB-recon (24) as a tool to predict plasmid cluster presence in isolates with incomplete bla KPC -encoding contigs (635/829, 76.6%) using a database containing a subset (~20%) of our isolates that were long-read sequenced.We successfully assigned the majority of bla KPC -encoding contigs to an existing plasmid primary cluster that we defined above, demonstrating that MOB-recon is a feasible approach to predict plasmid cluster membership without long-read sequencing all isolates.
Given the majority of bla KPC -encoding contigs were predicted to be of plasmid origin, we examined the temporal and geographic patterns of all bla KPC -encoding plasmids in Canada from 2010 to 2021 (Fig. 7).Geographically, the incidence of bla KPC -encoding isolates is much higher in the Central region than the West (Central: n = 777/829, 93.7%; West: n = 52/829, 6.3%).The three primary clusters that have persisted over the longest time frame are IncN (AA007), AA002 (IncL/M), and AA0029/AA085 (IncF).Occurrences of AA002 (IncL/M) plasmids were low until 2016, but appear to be found more frequently for the remainder of the study period.Some of the earliest detected plasmids were AA029/AA085 (IncF) plasmids, but these were observed less frequently from 2020 onward, perhaps as AA002 (IncL/M) plasmids increased in prevalence in the Central region.The AA007 (IncN) plasmids appear to be established in the population.Since 2018, there has been an increase in replicons in the "Other" category or those plasmids that do not fall into the top 10 primary clusters, particularly in the West region.This may indicate that other plasmids are becoming dominant in the West region, and further investigation into smaller Western-specific clusters (such as plasmids with IncP6 replicons) is required.
We examined the epidemiology of the three secondary clusters in primary cluster AA002 (IncL/M) in more detail, as this is the only primary cluster wherein we had a substantial number of plasmids in multiple secondary clusters (Fig. 5D).From an epidemiological perspective, AL013 plasmids have been present for a longer time frame at low abundances with an increase in prevalence in 2021, whereas AL008 plasmids have appeared consistently since 2016.It appears that secondary cluster AL001 may be spreading to the Western region, as this replicon was detected initially in the West in 2010 but not again until 2021 (Fig. 5D).The independent samples collected over long time periods indicate that these plasmids seem to be stably circulating in these regions.

DISCUSSION
We examined the prevalence and distribution of bla KPC -producing Enterobacterales and their plasmids in Canada from 2010 to 2021.Our results suggest that a combina tion of regional clonal transmission (AA109, AA029, and AA085), regional horizontal transmission (AA017, AA124, AA002, and AA014), widespread horizontal transmission (AA007), and other means of mobilization (AA013 and AA042) has contributed to bla KPC prevalence in Canada.
Here, we report on a novel bla KPC plasmid, repE(pEh60-7), isolated exclusively from E. hormaechei subsp.hoffmannii ST316, which has been stably circulating in this population for over 8 years.There have been limited reports on the E. cloacae complex ST316 lineage elsewhere; one study identified nine E. hormaechei ST316 isolates encoding bla KPC-2 in the United Kingdom between 2014 and 2016 as part of a larger reference study, however, the plasmid content was not reported (33).Otherwise, eight isolates [one each from references (34)(35)(36), and five from reference (37)] of E. hormaechei ST316 have been recently been reported to carry bla NDM-1 in Asia.Although conjugation genes were present, the narrow host range of the plasmid suggests that bla KPC persistence is likely due to the ST316 clone and not driven by the plasmid.
We report on some plasmid clusters that are found across a variety of species, supporting evidence of horizontal transfer of bla KPC .One such primary cluster was AA002, which contained the IncL/M plasmids isolated from Citrobacter spp., Escherichia spp., Enterobacter spp., Klebsiella spp., and Raoultella spp.isolates.The secondary cluster AL008 plasmids had a single resistance gene, but secondary cluster AL013 plasmids encoded fluoroquinolone resistance genes, additional β-lactamase genes, and other antimicrobial resistance genes which may favor their persistence in hospital environ ments.In addition, all IncL/M plasmids encoded a ParB-like partitioning/stability protein, an anti-restriction protein, a restriction endonuclease (Mrr), single-stranded DNA-binding protein (Ssb), and the RelB antitoxin although no RelE toxin was detected in any plasmid sequence.These features likely contribute to their long-term persistence and stability in Canada.However, bla KPC -encoding IncL/M replicons appear to be rare as they are notably absent from other recent bla KPC surveillance studies (13,15,38,39).One study reported that bla KPC-2 being associated with IncL/M replicons a multi-clonal K. pneumo niae outbreak in a region in Argentina, but the relatedness between plasmids was not investigated (40).IncL/M plasmids are typically associated with bla NDM , bla IMP , and bla OXA -type carbapenemases (3,12,13,(41)(42)(43), although a select few cases have been reported in diverse hosts (19,(44)(45)(46)(47).The pNE1280 IncL/M plasmid, which was one of the first IncL/M plasmids described carrying bla KPC-2 (45), clustered in the AL001 secondary cluster, which was not well represented in the Canadian data set.We demonstrated ongoing bla KPC -encoding IncL/M plasmid transfer across multiple genera which merits further investigation of IncL/M-type replicons as potential vectors for long-term bla KPC persistence.
In contrast to the IncL/M plasmids, the IncN plasmid family had the broadest phylogenetic and geographical distribution across Canada, which is unsurprising given their broad host ranges and their long association with bla KPC genes (3,48,49).bla KPCencoding IncN plasmids have been linked to multi-center outbreaks in Columbia (50) and Germany (51), both facilitated by intra-and inter-species horizontal plasmid transfer.Other bla KPC -encoding IncN plasmids implicated in inter-patient transfer grouped in the same secondary clusters as the Canadian plasmids here [plasmid 12 (49) and pYDC107_70 (52) in AA007/AL033; pKPC-SMH (53) in AA014/AL059], indicating high sequence similarity among Canadian plasmids to those found across the globe.Similar to IncL/M plasmids, all IncN plasmids encoded partitioning/stability proteins (StbA, StbC, and StdB), at least two anti-restriction proteins (ArdA, ArdB, and ArdR), an endonuclease (EcoRII and PemK), and other antimicrobial resistance genes.Given these features are also common in IncL/M plasmids, further investigation is required to explain why the IncN plasmids have a broader geographic and host range distribution in Canada than the IncL/M plasmids.
The IncF-type replicons observed here [IncFIB(pQil)/IncFII(K) and IncFIB(K)/IncFII(K)] are common in bla KPC -producing K. pneumoniae species complex isolates detected in other nationwide surveillance programs (12,13,15,17,18,38,39,54). IncF-type replicons have been implicated in clinical outbreaks of bla KPC -producing K. pneumoniae in the Netherlands (55) and the United States (56) as well as cross-species plasmid transmission in the United Kingdom (57).The clonal lineage ST258/512 accounts for 39.6% (106/268) of bla KPC -producing K. pneumoniae isolates in our collection (Fig. 1) which were isolated across 3 provinces and 12 sites.This frequency is lower than frequencies observed in Colombia [47% (18)], in the United States [74% CRACKLE-2 (5)], and across Europe [73% EuSCAPE (13), 76% Europe/Israel (39)], but is higher than frequencies reported in the United Kingdom [20% (15)] and Argentina [0% (40)].Our findings support that IncF-type plasmids in K. pneumoniae ST512 and ST258 are important in Canada for bla KPC persistence in the clonal group ST258/512, although there is more diversity in the sequence type of bla KPC -encoding K. pneumoniae isolates in Canada compared to other parts of the world.Interestingly, IncF-type plasmids were less common in recent years (2020 and 2021), indicating other plasmid types such as IncL/M and IncN-type replicons may be replacing them in the Canadian landscape.
Small cryptic plasmids are often overlooked (58) and underrepresented in plasmidmediated antimicrobial resistance studies (59).ColRNA-type replicons smaller than 22 kb were the third largest bla KPC -encoding primary clusters in our data set.ColRNA-type plasmids were predicted to be found in >90% of carbapenemase-producing Klebsiella pneumoniae isolates across Europe (13,60) and around the world (61), although these authors did not investigate if any carbapenemase genes were found on these plas mids.Similarly, ColRNAI replicons were present in >50% of bla KPC -producing isolates in Colombia (18).Our MOB-recon results agree with these data and predict that 69% (568/829) of Canadian isolates contain ColRNAI-type or rep_cluster_2335 plasmids, the majority (524/568, 92%) of which do not encode bla KPC (data not shown).These plasmids can be important vectors for transferring antimicrobial resistance genes between isolates, including the transfer of bla KPC -type carbapenemases (19,31,62).Experimen tally, ColRNAI plasmids encoding bla KPC and Tn4401 could mobilize between strains and participate in Tn4401 transposition events within a patient (63).About half the plasmids we sequenced in this primary cluster do not encode a relaxase but they could be mobilized by the relaxase of a co-resident plasmid if the oriT sequence has enough similarity as shown in a previous study (64).Given their high prevalence and ability to mobilize resistance elements, these small plasmids are likely contributing to bla KPC dissemination and merit further investigation as potential vectors for bla KPC and other antimicrobial resistance spread across the globe.
Plasmid clustering and analyses require a methodology that is tolerant to a small amount of changes given the plasticity of plasmid sequences.Clustering plasmids with MOB-cluster provided a way to analyze subsets of similar plasmids, and MOB-recon allowed us to predict the prevalence of these plasmid clusters in isolates with incomplete assemblies.Canadian plasmids are grouped in two or three secondary clusters within each primary cluster (Mash distance <0.025), with typically a single secondary cluster containing the majority of Canadian plasmids in that primary cluster, indicating many of them have near duplicate sequences (26,65) and this provided a starting point for investigating clusters of interest in more detail.
Analyzing broad-scale plasmid populations is desirable for surveillance programs, and ideally, every isolate would be sequenced to obtain complete plasmid sequences.Short-read sequencing alone results in many plasmids remaining fragmented (32) but performing long-read sequencing in addition to short-read on large collections of isolates can be cost and time-prohibitive, so we explored using MOB-recon to predict putative plasmids in incomplete assemblies after long-read sequencing a subset of isolates.There is no standardized method for estimating plasmid presence/containment in fragmented assemblies.It is important to recognize that any reference-based method for plasmid reconstruction involving mapping to reference sequences to infer presence assumes that plasmid structures are relatively conserved, and this approach can produce misleading results if plasmid plasticity is high (66,67).Similar studies have used short-read mappers such as BWA/Bowtie2/SMALT (13,43,68,69) or k-mer-based tools (60,70) to determine plasmid prevalence in incomplete assemblies, typically using a select few plasmids as references.We use a homology-based clustering approach with MOB-recon (24) to reconstruct plasmids using a relatively large set of complete Canadian plasmids (1,856 plasmids).Previous work has shown that MOB-recon performed well compared to other tools (58,71,72), and that Enterobacterales plasmid replicons and mobility genes are well represented in the MOB-suite database (58).Using this approach, we were able to predict plasmids for the majority of incomplete bla KPC -encoding contigs (605/635, 95%).For several primary clusters that were only present in specific sequence types [IncF-type, AA029/AA085 and repE(pEh60-7), AA109], those bla KPC -encoding contigs that were predicted to be part of plasmids in those primary clusters were from isolates with matching sequence types, which further supports this methodology for plasmid analysis in large-scale Enterobacterales surveillance data and eliminates the requirement to perform long-read sequencing on every single isolate.
In summary, there are multiple distinct clusters of bla KPC -encoding plasmids that circulated in Canada from 2010 to 2021.Certain plasmid clusters spread by horizontal transmission and were found in multiple genera in multiple provinces whereas others persisted through clonal dissemination of the host organism.Our findings highlight the need to integrate targeted long-read sequencing into carbapenemase-producing organism surveillance to generate complete plasmid assemblies and demonstrate that plasmid clustering can facilitate analyses of a large number of plasmids.The characteriza tion of bla KPC plasmids is not only important for outbreak control but also for epide miological surveillance of antimicrobial resistance, as plasmids encoding bla KPC genes typically carry other antimicrobial resistance genes that can be exchanged between species.

Surveillance period and PCR confirmation of bla KPC carbapenemase gene
CNISP is a sentinel surveillance system which collects epidemiological and linked microbiology data from 90 Canadian acute-care hospitals across 10 provinces and 2 territories.Enterobacterales isolates non-susceptible to a carbapenem (as determined by the submitting site) isolated from patients from 2010 to 2021 were voluntarily submitted to the National Microbiology Laboratory (Winnipeg, Canada) by Canadian hospitals and provincial public health laboratories for carbapenemase gene detection.Multiplex PCR to confirm the carbapenemase gene bla KPC was conducted as previously described (54).A total of 829 isolates encoding bla KPC were collected from 2010 to 2021 from 34 hospitals (Table S1), with one hospital submitting 38% (317/829) of all isolates.Where applicable, the Central region refers to the provinces of Ontario and Québec, and the West region refers to the provinces of British Columbia, Alberta, Saskatchewan, and Manitoba.See the Supplemental Materials for additional information about the surveillance program and isolate eligibility criteria.

Whole genome sequencing
All 829 isolates were sequenced with Illumina MiSeq platforms, and 155 of these were additionally sequenced using Oxford Nanopore Technologies (ONT).Isolates for ONT long-read sequencing represented about 20% of all bla KPC cases stratified across all provinces in Canada.For sites with known outbreaks, we took representative isolates to prevent oversampling of clonal cases.The average Illumina depth of coverage was 123×, and the average ONT depth of coverage was 75×.Genomes were trimmed and filtered before assembly with Unicycler v0.5.0 (73).A total of 111 chromosomes and 1,856 plasmids were completed and circularized in our data set.Details are in the Supplemen tary Materials, and details on bla KPC -encoding plasmids can be found in Table S2.

Plasmid clustering and containment analysis
MOB-suite primary cluster designations are a useful way to broadly cluster plasmids for epidemiological studies, and so plasmids assigned to different primary MOB-clus ters are sufficiently unrelated to not be considered as part of an epidemiologically relevant transmission event (26,65).However, plasmids that share the same primary cluster designation can be examined in more detail through higher resolution subtyp ing such as secondary cluster designations.If two plasmids are assigned to the same secondary cluster, they have near duplicate sequences and are sufficiently related to be strong candidates for outbreak investigations (26,65).In addition to secondary cluster designation, epidemiological data are required to best assess direct plasmid transmis sion.
For plasmid clustering analysis, the PLSDB v2021_06_23v2 (25) database was downloaded and clustered alongside the 202 circular bla KPC -encoding plasmids in this study using MOB-cluster from the MOB-suite v3.1.4package (24,26).For plasmid containment analysis, all 1,856 circular plasmids completed in this study (including the 202 bla KPC -encoding plasmids) were clustered using MOB-cluster to create a custom Canadian plasmid database.All 829 isolates were screened for plasmids with MOB-recon using this custom database, and the output was filtered to focus on the reconstructed plasmids containing bla KPC .Further details on plasmid clustering and containment are provided in the Supplementary Materials.

FIG 1
FIG1 Summary of genera (inner ring), species (middle ring), and multi-locus sequence types (MLST; outer ring) of bla KPCencoding isolates included in the study (829 total isolates).MLST profiles found in three or fewer isolates were grouped into "other".Not all labels are displayed.

FIG 2
FIG 2 Proportion of isolates encoding antimicrobial resistance genes identified by StarAMR, then categorized by drug class is presented across the top four genera.Values represent proportion of isolates encoding genes belonging to a certain antimicrobial class."N" indicates the number of isolates in that genus.The "Other" genera include Serratia spp., Raoultella spp., Pseudescherichia spp., Pantoea spp., Morganella spp., Kluyvera spp., and Hafnia spp.

FIG 3
FIG 3 Characteristics of 202 bla KPC -encoding plasmids sequenced in this study.Groups on the x-axis correspond to primary cluster IDs generated among the plasmid database PLSDB and Canadian bla KPC -encoding plasmids."Other*" indicates SNVs found in fewer than three plasmids or Tn4401 absence."Other**" indicates either truncated, alternative isoforms or absence of Tn4401."Other***" indicates Hafnia, Kluyvera, Morganella, Raoultella, or Serratia genera."Other/ unknown****" indicates replicons (n = 26) found in fewer than five plasmids or replicons that could not be typed.

FIG 4
FIG 4 Pangenome size and bla KPC prevalence among top 10 MOB-suite primary clusters identified among PLSDB and Canadian bla KPC -encoding plasmids.

FIG 5
FIG 5 Diversity of secondary clusters within primary cluster AA002 (IncL/M).Plasmid structures of (A) secondary cluster AL008, (B) secondary cluster AL013, and (C) secondary cluster AL001 which grouped within the AA002 (IncL/M) primary cluster.Coding sequences are colored by function, and not all coding sequences are labeled.(D) Date of positive culture and region of plasmids from primary cluster AA002 (IncL/M).Date of positive culture was grouped into year-month bins.The Central region represents the Canadian provinces of Ontario and Québec, and the West region represents British Columbia, Alberta, Saskatchewan, and Manitoba."N" indicates the number of isolates that contained assembled or predicted plasmids in the respective secondary clusters.

FIG 6
FIG 6 Comparison of p11A1314040_B_KPC, representative of plasmids with novel replicon repE(pEh60-7) and reference NZ_CP088228.1 (pEh60-7), NZ_CP080472.1 (PLSDB), and NZ_AP022432.1 (PLSDB) from cluster AA109.Reference sequences were aligned using blastn+ and an e-value cut-off of 0.1.NZ_CP080472.1 and NZ_AP022432.1 were placed in the same primary cluster by MOB-cluster as the reference p11A1314040_B_KPC but in different secondary clusters.Genes are colored by functional classes determined by mobileOG-db, and genes without annotations are hypothetical proteins.Not all gene labels are displayed.

FIG 7
FIG 7 Epidemiology of Canadian bla KPC -encoding plasmid primary clusters from 2011 to 2021.Date of positive culture was grouped into year-month bins.The Central region represents the Canadian provinces of Ontario and Québec, and the West region represents British Columbia, Alberta, Saskatchewan, and Manitoba."Other" indicates plasmids which did not group in the top 10 primary clusters.

TABLE 1
Summary features of top 10 Canadian bla KPC -

encoding plasmid clusters among PLSDB Primary cluster ID No. Canadian plasmids Median size (kb) Predicted mobility a,b Replicon type a,b Relaxase type a,b MPF type a,b No. genera Core genes (%) d bla KPC allele a bla KPC frequency Tn4401 variant a Mean no. ARGs per plasmid c Plasmid taxonomic unit (PTU)
a