The Tijuana River Estuary (TRE) (N32°33′, W117°07′) is a 10-km² National Estuarine Research Reserve, National Wildlife Refuge, and Wetland of International Importance (Ramsar site #1452) on the United States-Mexico border in Imperial Beach, California, United States (Zedler et al., 1992). The 4400-km² watershed includes urban areas, agricultural land, and open chaparral and sage scrub. The South Bay International Waste Water Treatment Plant, which treats 25 million gallons of raw sewage per day from Tijuana, Mexico, is situated immediately upstream of the estuary, where it releases untreated municipal waste water into the river and wetlands during periods of heavy rain (Conway et al., 1985). Surface sediments (50 g from top 5 mm) were collected with sterile spatulas after rainfall and transported to the laboratory in sterile centrifuge tubes on ice.

AM, ampicillin; AN, amikacin; ATM, aztreonam; C, chloramphenicol; CAZ, ceftazidime; CF, cefalothin; CIP, ciprofloxacin; CL, colistin; CTX, cefotaxime; CXM, cefuroxime; D, doxycycline; ETP, ertapenem; FEP, cefepime; FOX, cefoxitin; GAT, gatifloxacin; IPM, imipenem; K, kanamycin; LOM, lomefloxacin; LVX, levofloxacin; MEM, meropenem; MXF, moxifloxacin; NA, nalidixic acid; NOR, norfloxacin; OFX, ofloxacin; PIP, piperacillin; RIF, rifampicin; S, streptomycin; SAM, ampicillin/sulbactam; SPX, sparfloxacin; SXT, sulfamethoxazole/trimethoprim; TE, tetracycline; TIC, ticarcillin; TIM, ticarcillin/clavulanic acid; TZP, piperacillin/tazobactam; ZEO, Zeocin.

In the laboratory, 50 g surface sediments were homogenized by hand. A 1-g sub-sample was added to Luria-Bertani (LB) broth amended with the antibiotics NA (20 μg/mL) and TE (5 μg/mL) followed by incubation at 37°C with shaking (150 rpm) for 7 days. A pure culture, called strain AtetA, was obtained after successive streak plates on eosin methylene blue (EMB) agar with the same antibiotics, on which it formed nucleated colonies with a metallic green sheen. Plasmids were extracted from a representative colony by alkaline lysis (Kramer and Coen, 2001) and used to transform competent Escherichia coli JM109 (Promega) by heat-shock according to the manufacturer’s instructions. Transformants were selected on LB agar amended with TE (10 μg/mL) and screened for the presence of plasmids by alkaline lysis and gel electrophoresis. Strain AtetA was identified by PCR amplification and 2X sequencing of the nearly complete 16S rRNA gene as previously described (Lane et al., 1985).

In order to capture plasmids by the exogenous method, bacteria were gently removed from 5 g wetland sediments by incubation in 25 mL sterile Na₄P₂O₇ (0.1%) for 1 h with stirring. The suspension was allowed to settle for 1 h after which 10 mL of the liquid phase was decanted. Cells were collected from the liquid phase by centrifugation at 10,000 × g for 10 min. and the pellet was re-suspended in 3 mL sterile saline (0.85% NaCl). Mating was initiated by mixing 0.5 mL donor bacteria suspension with 0.5 mL recipient bacteria [either Escherichia coli HY842 (RIF^R, S^R, ZEO^R) (Brown et al., 2013) or Pseudomonas putida KT2440 (C^R) (Nelson et al., 2002) (16-h culture in LB broth)] followed by immobilization onto a 0.22-μm nitrocellulose membrane filter. Control cultures were prepared identically with either donor or recipient bacteria alone. Filters were incubated overnight at 30°C on LB agar plates (no antibiotics). Biomass was removed with a sterile cotton swab and re-suspended in 1 mL sterile saline. Fifty μL of suspended bacteria, either donor, recipient, or the mated combination, was spread onto LB agar plates with (1) no antibiotics, (2) recipient-selective antibiotics alone (RIF, S, ZEO each at 100 μg/mL; C at 50 μg/mL), (3) plasmid-selective antibiotic alone (see below), or (4) recipient-selective antibiotics and plasmid-selective antibiotic combined, and incubated at 37°C for up to 4 days. Cycloheximide (100 μg/mL) was added to suppress fungal growth. Plasmid-selective antibiotics included AMP (100 μg/mL), or AMP (100 μg/mL) plus TE (10 μg/mL). Putative transconjugants were first confirmed to be the recipient strain by randomly amplified polymorphic DNA (RAPD) PCR using primers 208 and 272 (Mahenthiralingam et al., 1996) and then screened for the presence of plasmids by alkaline lysis and gel electrophoresis (Kramer and Coen, 2001).

The transmissibility of each plasmid was tested with the filter-mating protocol described above using E. coli HY842 (RIF^R, S^R, ZEO^R) or E. coli JM109 (NA^R) as recipients.

Escherichia coli JM109 with and without plasmids was repeatedly (n = 5) subjected to antimicrobial susceptibility testing (AST) against a broad range of antibiotics by the disk diffusion method according to CLSI protocols and standards (Clinical and Laboratory Standards Institute, 2015, 2017) with disks obtained from BD or Oxoid. Minimum inhibitory concentrations (MICs) were estimated with Etests (bioMérieux). E. coli ATCC 25922 was tested for quality control.

Plasmid copy numbers were determined in E. coli JM109 according to the quantitative polymerase chain reaction (qPCR) method of Loftie-Eaton et al. (2014). Total cellular DNA was extracted from 5, 50, and 500 μL of early stationary phase cells with the GenElute Bacterial Genomic DNA Kit (Sigma–Aldrich) according to the manufacturer’s instructions. Chromosomal and plasmid DNA (0.04 ng/μL) was quantified in triplicate from each extraction by qPCR with primers (750 nM each) targeting either the atpB gene of E. coli (Yano et al., 2012), which exists on the chromosome in single copy, or one of the single-copy antibiotic resistance genes encoded on each plasmid: tetA on pLNU-11 and pTRE-1611 (Guarddon et al., 2011), bla_OXA-1 on pTRE-131 (Knapp et al., 2010), and bla_CTX-M-55 pTRE-2011 (Ellem et al., 2011). Amplification efficiency [E = 10^(-1/m)] for all primer pairs was greater than 1.96 based on 10-fold serial dilutions of total DNA (R² > 0.99). Gel electrophoresis and melting curve analysis of the qPCR products confirmed amplification specificity. Oligonucleotide PCR primers are described in Supplementary Table S1.

Total DNA was extracted using the GenElute Bacterial Genomic DNA Kit (Sigma), following the manufacturer’s instructions. Full genome sequencing was performed by the IBEST Genomic Resources Core using a MiSeq sequencer (Illumina, San Diego, CA, United States) and 250 bp Paired-End Sample Preparation Kits (Illumina, San Diego, CA, United States). Prior to analysis, duplicate read pairs were removed using a custom Python script. Sequencing adapters and low-quality bases were removed using the software package Seqyclean¹. All remaining reads were mapped to the chromosome of either E. coli O157:H7 (BA000007.2) or P. putida KT2442 (NC_002947.3) using Bowtie2. The unused reads (i.e., non-chromosomal reads) were then assembled de novo with Newbler. Automated annotation was performed by the IGS Analysis Engine (University of Maryland, School of Medicine, Institute for Genome Sciences) and then curated manually. Any open reading frames shorter than 100 amino acids in length with no alignments to previously annotated sequences were not included in the final annotations. Plasmid maps were constructed using Geneious v. 8.1.3 (Biomatters Ltd.).

The genome sequence of plasmid pTRE-2011 was completed using the Pacific Biosciences Single Molecule Real Time (SMRT) sequencing platform at Washington State University’s Molecular Biology and Genomics Core. The plasmid was sequenced on a PacBio RS instrument (Pacific Biosciences, Menlo Park, CA, United States) using SMRT sequencing technology.

Plasmid sequences were compared for genomic similarity to fully sequenced plasmids published in GenBank (Benson et al., 2014). Contiguous regions of plasmid backbone genes (those involved in plasmid replication, conjugation, mating pair formation, partitioning, or stability) were used to identify other similar reference plasmids in the GenBank database using NCBI BLAST based on total BLAST alignment scores. Comparative genomic analysis of plasmid organization was performed using Mauve (Darling et al., 2010).

Identification of putative plasmid incompatibility groups, along with insertion sequences and integrons, was performed using a variety of web-based resources. Plasmid incompatibility groups were identified using PlasmidFinder 1.3 and specific plasmid multi-locus sequence type (pMLST) was identified with pMLST-1.4 (Carattoli et al., 2014). Insertion sequences were identified using ISFinder (Siguier et al., 2006), and INTEGRALL was used to identify integrons and gene cassettes (Moura et al., 2009).

Phylogenetic analysis of the amino acid sequence of the AmpC-like β-lactamase found on plasmid pLNU-11 was performed using MEGA v. 7 (Kumar et al., 2016) using MUSCLE alignments (Edgar, 2004). The best-fit evolutionary model was determined based on the minimum Bayesian Information Criterion (BIC) and Akaike Information Criterion (AIC), which were computed for all 56 amino acid evolution models in MEGA using an initial neighbor joining tree. The best fit evolutionary model was then used to identify a maximum likelihood phylogenetic tree. Reliability of the best fit tree was assessed by identifying branches conserved in other best fit maximum likelihood phylogenetic trees fit to 500 bootstrap replicates.

The sequence type of the native host organism of plasmid pTRE-2011 was determined using its complete genomic sequence (determined with the PacBio platform) and the MLST tool (Larsen et al., 2012) hosted at the Center for Genomic Epidemiology².

The plasmid nucleotide sequences reported in this study have been deposited in the GenBank database³ under the accession numbers KX863568-KX86357. The nearly complete 16S rRNA gene sequence of Citrobacter freundii strain AtetA has been deposited in the GenBank database under the accession number KF245926.1. The ampC-like gene found on plasmid pLNU-11 has been deposited in the GenBank database under the accession number KY018700 and has been assigned the allele designation bla_WDC-1 by NCBI. The wild type host of plasmid pLNU-11, C. freundii strain AtetA, also carries an ampC-like β-lactamase gene on its chromosome whose nucleotide sequence has been deposited in the GenBank database under the accession number KX863576.

The first plasmid, pLNU-11, was found in strain AtetA, which was selectively enriched from TRE wet-season sediments in LB broth supplemented with the antibiotics NA (20 μg/mL) and TE (5 μg/mL). A pure colony type was purified through successive passes on EMB agar, where it formed metallic green colonies typical of Gram-negative, lactose-fermenting bacteria. The 16S rRNA gene of strain AtetA shared >99% sequence similarity (1432/1435 bp) with C. freundii strain JCM 24061 (AB548826), warranting designation as a C. freundii strain. A plasmid preparation of C. freundii AtetA showed four distinct bands upon agarose gel electrophoresis. Additionally, a tetracycline-resistant E. coli JM109 transformant of this plasmid preparation possessed a plasmid with the same electrophoretic mobility as the largest of the four bands in the C. freundii plasmid preparation. The pLNU-11 plasmid copy number was estimated to be approximately 15 copies per cell in E. coli JM109 (Table 1).

Mating experiments between either C. freundii AtetA or E. coli JM109 (pLNU-11) as donor and E. coli HY842 as recipient were unsuccessful, suggesting that plasmid pLNU-11 is not self-transmissible (i.e., not conjugative). This observation was further validated by examination of the plasmid DNA sequence, which lacks any conjugative transfer genes (Figure 1).

The 36-kb pLNU-11 plasmid genome (KX863568) comprises a putative IncP-6 replicon, with 36 open reading frames, only six of which have been associated with essential backbone function, namely the parA/B/C operon, repA, kfrA, and mobA (Figure 1). Members of this incompatibility group are all mobilizable plasmids ranging in size from 13 to 57 kb. They are capable of replicating in a broad host-range extending beyond E. coli and P. aeruginosa. Relatively few IncP-6 plasmids have been fully sequenced, including Rms149 (AJ877225), pCOL-1 (KC609323), and p10265-KPC (KU578314) from clinical strains of P. aeruginosa (Haines et al., 2005; Naas et al., 2013; Dai et al., 2016); pRIO-5 (JF785550) from a clinical strain of Serratia marcescens (Bonnet et al., 2000; Bonnin et al., 2012); pRSB105 (DQ839391), a plasmid captured from a German WWTP (Schlüter et al., 2007); and pHH2-227 (JN581942), an uncultured IncW/IncP-6 hybrid plasmid that was exogenously captured from an arable soil (Heuer et al., 2009; Król et al., 2013).

The backbone regions of pLNU-11 showed high sequence similarity to multiple known plasmids of both clinical and environmental origin. The greatest similarities (>97% identity) were observed with the S. marcescens bla_GES-5 plasmids pG5A4Y201 (KJ541069) and pG5A4Y426 (KJ541068), and the E. coli bla_GES-5 plasmids pG5A4Y217 (KJ541071) and pG5A4Y413 (KJ541070) from nosocomial infections in Canadian hospitals (Boyd et al., 2015); Rms149, first identified in a clinical strain of P. aeruginosa in Germany (Haines et al., 2005); pRSB105; and the Aeromonas hydrophila plasmid pKPC2 (KR014106) from China (unpublished GenBank citation). Like pLNU-11, each of these similar plasmids lacks genes known to be involved in plasmid transfer, such as the tra or trb operons, implying that they are all mobilizable plasmids that require the presence of a conjugative plasmid to be transferred. Mauve alignments showed the backbone genes to be highly conserved, interrupted by accessory regions not necessarily shared by one another, and all were predicted to be mobilizable rather than self-transmissible, typical of IncP-6 plasmids (Supplementary Figure S1).

The plasmid pLNU-11 genome shows remnants of multiple insertion events, with dozens of intact or partial mobile elements dispersed throughout, similar to the genomes of other IncP-6 plasmids. For example, it possesses numerous partial and complete transposases from transposon families Tn3, IS5, IS4, and IS66. Additionally, a partial ISCR transposase is located between the bla_WDC-1 and czcD genes, upstream of the 3′ conserved sequence of a class 1 intregron. The integron, however, is missing the 5′ conserved sequence, which would normally possess the integrase and the attI cassette integration site. It appears that, after integrating several cassettes, pLNU-11 lost a portion of its genome beginning within the ISCR transposase and continuing upstream to include the integrase and attI site.

Plasmid pLNU-11 contains a unique accessory region encoding predicted resistance against the tetracyclines (tetA/R), sulfonamides (folP, dfrA10), quaternary ammonium compounds (qacEΔ1), β-lactams (ampC-type β-lactamase), and aminoglycosides (GCN-5-like acetyl transferase). Additionally, it encodes a collection of genes predicted to be involved in heavy metal resistance [an nrbE-like major facilitator superfamily (MFS) protein, czcD, and arsR].

A novel AmpC-like β-lactamase was discovered on pLNU-11 upstream of uvrD, dfrA10, qacEΔ1, folP, and GCN-5 (Figure 1). The novel allele shares less than 62% amino acid sequence identity with other characterized enzymes, namely those from the PDC class of β-lactamase found in P. aeruginosa. The new AmpC-like β-lactamase on pLNU-11 has been designated WDC-1 (wetland-derived class C β-lactamase) by the NCBI. It shares high amino acid sequence identity (>99% coverage and >93% identity) to three uncharacterized AmpC-like proteins in GenBank (WP_061904073.1, WP_021699607.1, and WP_025297965.1m). In fact, there are over 100 entries of this protein in GenBank, however, all are located in chromosomal sequences from different species of Pseudomonas. Together, these four proteins form a distinct sub-class of β-lactamases (Figure 2). The bla_WDC-1 gene on pLNU-11 appears to be the first allele of this protein found on a plasmid. Disk diffusion and Etest results indicate that WDC-1 is primarily an oxacillinase and cephalothinase (Table 2 and Supplementary Table S2). Note that, like many Citrobacter spp., C. freundii AtetA carries a chromosomal bla_AmpC allele distinct from bla_WDC-1 found on pLNU-11 (“C. freundii strain AtetA AmpC” in Figure 2).

The original host of pLNU-11, C. freundii AtetA, was resistant (as defined by CLSI breakpoints) to β-lactams (AM, CF), tetracyclines (D, TE), (fluoro)quinolones (LOM, MXF, NA), an aminoglycoside (S), and sulfonamide (SXT) (Table 2), and showed intermediate resistance to three other (fluoro)quinolones (CIP, GAT, OFX) and a β-lactam (FOX) (intermediate phenotypes not shown in Table 2). The transformant E. coli JM109 (pLNU-11) showed resistance to CF, D, and TE, though decreased susceptibility relative to E. coli JM109 without plasmid was also observed to AM, SAM, and SXT (Table 2). The resistance to (fluoro)quinolones or S observed in C. freundii AtetA could not be explained by pLNU-11, suggesting that resistance to these antibiotics may have been chromosomally encoded or located on one of the smaller plasmids. Plasmid-mediated resistance to NA cannot be tested in E. coli JM109 since it is naturally resistant to NA. Although pLNU-11 encodes a GCN-5-like acetyltransferase, no S resistance was observed in the JM109 transformant, suggesting that the gene might not be functional.

Plasmid pTRE-1611 was captured from wetland bacteria into P. putida KT2440 as recipient under selection for TE resistance. As P. putida KT2440 is broadly resistant to antibiotics, we attempted to transfer pTRE-1611 to E. coli HY842 by mating, but were unsuccessful, suggesting that it is not self-transmissible. The plasmid was transferred by heat-shock transformation to E. coli JM109 where resistance profiles could be more readily studied. Additional mating experiments between E. coli JM109 (pTRE-1611) and E. coli HY842 were also unsuccessful. Although, pTRE-1611 was originally captured by conjugation, this behavior was not demonstrated again, suggesting that it was likely mobilized into P. putida KT2440 by a self-transmissible plasmid that was either not co-transferred or was subsequently lost from the recipient. Plasmid pTRE-1611 was maintained in E. coli JM109 at approximately 3 copies per cell (Table 1).

Plasmid pTRE-1611 conferred resistance to E. coli JM109 against three β-lactam drugs (AM, PIP, TIC), a tetracycline (TE), and an aminoglycoside (S) (Table 2). Decreased susceptibilities to SAM and SXT were also noted, though resistant breakpoints were not surpassed.

The 46-kb pTRE-1611 genome (KX863570) has a putative IncR replicon and 39 open reading frames. Although IncR plasmid replicons were first described in Salmonella strains (García-Fernández et al., 2009), they have since been more commonly found in Klebsiella pneumoniae (e.g., Compain et al., 2014), and are often associated with the epidemic metallo-β-lactamase NDM-1 (e.g., Kocsis et al., 2016). IncR replicons are characteristically non-conjugative. Plasmid pTRE-1611 does not carry bla_NDM-1.

The simple backbone of plasmid pTRE-1611 consists of the repB, parA/B, umuC/D, and stbA genes and is absent of any tra or trb genes necessary for conjugation. Additionally, it encodes the vapB/C type II toxin-antitoxin system, along with phosphoglucomutase, pecM, and an isochorismatase-like hydrolase, each of which has been implicated in resistance or virulence (Ugalde et al., 2000; Maruyama and Hamano, 2009; Burska and Fletcher, 2014). Antibiotic resistance genes include sulII, tetA/R, strA/B, and bla_TEM-1. Each of the resistances and decreased susceptibilities observed in E. coli JM109 (Table 2) could be accounted for with these four resistance genes or pairs of genes.

Like plasmid pLNU-11, pTRE-1611 is littered with several diverse transposons from the families Tn3, IS3, IS110, IS91, IS5, IS6, and IS1, some partial and others complete. A small portion of a class 1 integrase is present downstream of sulII along with what appears to be an ISCR2 element (Poirel et al., 2009) located upstream of sulII (Figure 1). It is not uncommon for ISCR2 elements to be associated with a 3′ conserved sequence of an integron, including sulII, but lacking the 5′ conserved sequence (Toleman et al., 2006).

The small backbone regions of pTRE-1611 only shared similarity (>85% coverage, >98% identity) with the K. pneumoniae plasmids pKP1034 (KP893385), pK245 (DQ449578), pKPN5 (CP000650), and pWSZBR (CP015991), all of which lack tra/trb operons and thus would be predicted to be incapable of self-transmission. Of these plasmids, only pK245 could be tracked to a source, a clinic in Tainan, Taiwan (Chen et al., 2006). The others were only recently submitted to GenBank (2015 or later) and had not been published as of May 2017. Mauve alignment of pTRE-1611 with the above four plasmids highlights the similar backbones with distinct accessory regions that include tetA/R, pecM, and bla_TEM-1 (Supplementary Figure S2).

The third plasmid, pTRE-131, was also captured from wetland bacteria, but now into E. coli HY842 as recipient with selection for AM resistance. pTRE-131 was readily transferred back and forth between E. coli HY842 and E. coli JM109 by conjugation, establishing it as a self-transmissible plasmid. Remarkably, the 48-kb plasmid was apparently maintained at approximately 240 copies per E. coli JM109 cell (Table 1). This unusually high copy number was confirmed repeatedly, and in line with a much stronger plasmid band on agarose gels compared to the other plasmids. Whereas the high copy number may be imposing a high cost to its bacterial host, it very likely also affords the plasmid greater opportunity to persist within a population absent of a selective pressure.

Escherichia coli JM109 (pTRE-131) displayed resistance to only AM and TIC (Table 2), but susceptibility was decreased to many other classes of antibiotics including fluoroquinolones, chloramphenicol, aminoglycosides, and sulfonamides, qualifying it for multi-drug resistance status.

The pTRE-131 plasmid genome (KX863569) has a putative IncN3 broad-host-range replicon (Götz et al., 1996) with 55 open reading frames. The backbone comprises repA, stbA/C, parA, ardB/K, ssb, traA/B/C/D/E/F/G/I/K/L/O, fip, trbC, eex, korA/B, trwB, typical of IncN replicons (Figure 1). The backbone regions of pTRE-131 share high similarity (>93% identity, >95% coverage) with multiple plasmids in GenBank including the K. pneumoniae plasmids p0801-IMP (KT345947), pJIE137 (EF219134), pKPC-SMH (KT148595) and pTR3 (JQ349086), the Enterobacter hormaechei plasmids p34998-53 (KT148595) and p34983-59.134kb (CP010378), and the E. coli plasmids pACT2-NDM-1 (KP826703), pEC448_OXA163 (CP010382), pNDM-ECS01 (KJ413946) and p271A (JF785549). All other alignments to this backbone produced less than 65% coverage during alignment.

The pTRE-131 genome contains several partial insertion sequences, a complete Tn3-like transposon, and a complex ISCR1 element embedded within a class 1 integron (Figure 1). The integron begins in the 5′ conserved region with an integrase, followed by five resistance genes: aac(6′)-Ib, bla_OXA-1, catB, qacEΔ1, and sulI. The last two genes in the list, qacEΔ1 and sulI, typically form the 3′ conserved sequence of the integron. However, in the case of pTRE-131, the first sulI gene is followed by an ISCR1 element and four more accessory genes: sapA, qnrB, qacEΔ1, and sulI, forming the 3′ conserved region and ending the integron. The sapA-like gene has been implicated as a virulence factor involved in resistance to host antimicrobial peptides (Parra-Lopez et al., 1993), while the others confer resistance to aminoglycosides (aac(6′)-Ib), β-lactams (bla_OXA-1), chloramphenicol (catB), disinfectants (qacEΔ1), sulfonamides (sulI), and (fluoro)quinolones (qnrB). The resistance phenotype of E. coli JM109 (pTRE-131) (Table 2) indicates that each of the drug resistance genes in the genome (Table 1 and Figure 1) is functional.

Mauve alignments with the top five best aligning backbones show that only pJIE137 carries the complex ISCR1 element present in pTRE-131 (Supplementary Figure S3), however, the cat, bla_OXA-1 and aac(6’)-Ib genes are not present in pJIE137. Instead, pJIE137 carries a dfrA12, followed by an unknown protein and aadA2 located in the same position. Of the database entries annotated with source information, this backbone appears to be globally dispersed with examples from Australia (Partridge et al., 2012), Thailand (Netikul et al., 2014), eastern Canada (Tijet et al., 2016), and Singapore (Chen et al., 2012). This report now adds the southwestern United States to its expanding distribution map.

During a bi-parental mating experiment with wetland bacteria as plasmid donor, a homogeneous colony type appeared on a control plate containing RIF, S, ZEO, and CTX that was inoculated with only environmental bacteria. The pure culture, named strain TRE-T11, contained two high-molecular-weight plasmids. The first, pTRE-2011, was easily transferred to E. coli HY842 or E. coli JM109 by mating with CTX, where it was maintained at an average of fewer than one copy per cell (Table 1). Strain TRE-T11 produced metallic green colonies on EMB agar, and biochemical evidence using the API 20E rapid identification kit (bioMérieux) indicated that it is a strain of E. coli (data not shown). The second plasmid was sequenced with the PacBio platform and found to carry no antibiotic resistance genes or other accessories. It was therefore not studied further.

The genome of strain TRE-T11 was sequenced using PacBio and identified as the broadly distributed E. coli ST744 by multi-locus sequence typing. ST744 distribution includes Europe (Guenther et al., 2012; Brolund et al., 2014; Wagner et al., 2014; Hasman et al., 2015; Loncaric et al., 2016), South(east) Asia (Hasan et al., 2012; Stoesser et al., 2012; Cai et al., 2014; Ho et al., 2016), Australia (Sidjabat et al., 2014; Abraham et al., 2015), and North Africa (Belmahdi et al., 2016). To our knowledge, this is the first report of ST744 in the Americas.

Escherichia coli TRE-T11 demonstrated resistance to no fewer than 23 of the antibiotics tested, including aminoglycosides (AN, K, and S), a monobactam (ATM), β-lactams (CAZ, CF, CTX, CXM, FOX, PIP, and TIC), fluoroquinolones (CIP, GAT, LOM, LVX, MXF, NOR, OFX, and SPX), a polymyxin (CL), tetracyclines (D and TE), and a sulfonamide (SXT) (Table 2). Sixteen of these resistance traits transferred to E. coli JM109 after conjugation (Table 2), including some of the β-lactams (CF, CTX, PIP, and TIC), the monobactam (ATM), most of the fluoroquinolones (CIP, GAT, LOM, LVX, MXF, NOR, and OFX), the two tetracyclines (D and TE), one of the aminoglycosides (K), and the sulfonamide (SXT). E. coli JM109 (pTRE-2011) was also resistant to ampicillin with and without the β-lactamase inhibitor sulbactam (AM and SAM) as well as chloramphenicol (C), neither of which was detected in the original E. coli TRE-T11 host (Table 2). Furthermore, a statistically significant decrease in zone of inhibition was observed in E. coli JM109 (pTRE-2011) for the antibiotics CL, FEP, and TIM, although CLSI clinical resistance breakpoints were not achieved (Table 2). Much, but not all, of the resistance phenotype of E. coli TRE-T11 could be transferred with plasmid pTRE-2011.

The 144-kb pTRE-2011 plasmid genome (KX863571) (Figure 1) consists of 160 open reading frames including a putative IncFIB/FII multi-replicon backbone (ST F18:A-:B1 according to the pMLST method of Carattoli et al., 2014) containing sopA/B, ssbF, 24 tra genes, nine trb genes, finO, multiple rep genes, and umuD. The backbone is highly conserved with other known plasmids. The largest contiguous backbone region shares >99% identity (100% coverage) with the globally distributed E. coli plasmids pMR0516mcr (KX276657), p3PCN033 (CP006635), and pCERC3 (KR827684), identified in the United States, China, and Australia, respectively (Liu et al., 2015; McGann et al., 2016; Moran et al., 2016).

Plasmid pTRE-2011 conferred resistance to an extensive list of antibiotics in E. coli JM109 (Table 2). Identifiable resistance genes (Table 1) are predicted to confer resistance to β-lactams, tetracyclines, aminoglycosides, sulfonamides, and amphenicols. Transferred resistance to fluoroquinolones and decreased sensitivity to colistin, however, could not be readily explained by the plasmid genome.

The bla_CTX-M-55 gene of plasmid pTRE-2011 is associated with an ISEcp-1 insertion sequence, common among the bla_CTX-M genes (Karim et al., 2001; Rossolini et al., 2007). ISEcp1 provides a highly active Enterobacteriaceae promoter within its 3′ sequence (Karim et al., 2001), leading to overexpression of downstream genes (Wang et al., 2013). Facile transposition of bla_CTX-M genes by ISEcp1 between bacterial chromosomes and plasmids, including across genus boundaries (Lartigue et al., 2006; Mahrouki et al., 2012), may account for their epidemic global spread in recent decades.

Several toxin-antitoxin systems were identified including the hok/sok antitoxin (hok toxin was not observed), pemI/K, vapB/C, and cmi/a. These may account for the plasmid’s persistence despite such low copy numbers. Virulence genes were also discovered in the pTRE-2011 genome sequence including an iron acquisition system consisting of the sitA/B/D genes, fecC/D, enolase, shiF, lucA/C/D, and srr; the protease gene ompT; the hemolysin gene hlyF; and the putative virulence gene mig-14.

Mauve alignments with pTRE-2011 and the most similar plasmids (based on backbone similarity) in the GenBank database demonstrated that the largest contiguous region of backbone genes of pTRE-2011 were present in at least one of the other known reference plasmids. The large contiguous backbone of pTRE-2011 showed 100% identity to that of pMR0516mcr (Supplementary Figure S4), a 226-kb IncF plasmid from an E. coli urinary tract pathogen (McGann et al., 2016). The DNA sequence of pMR0516mcr appears to have two separate backbone regions, only one of which aligns well with plasmid pTRE-2011 described here, suggesting that pMR0516mcr is the result of a pTRE-2011-like plasmid combining with another distinct replicon to create a large, multi-replicon genome. Although the pTRE-2011 genome does not include an mcr-1-like colistin resistance gene, as seen in pMR0516mcr, the original host of pTRE-2011, E. coli TRE-T11, was indeed resistant to colistin and the transconjugant E. coli JM109 (pTRE-2011) showed decreased susceptibility to colistin (Table 2), suggesting that there may be an alternative mechanism such as a generalized efflux pump that has not been recognized.