Complete Sequence and Molecular Epidemiology of IncK Epidemic Plasmid Encoding blaCTX-M-14

This plasmid is disseminated worldwide in Escherichia coli isolated from humans and animals.

the host, confer metabolic benefits, or enable the bacteria to colonize new environments (1).
Genes that confer resistance to antimicrobial drugs used in human or veterinary medicine are often mobilized on plasmids. One class of resistance genes encode extended-spectrum βlactamases (ESBLs), which confer resistance against many β-lactam antimicrobial drugs, leading to treatment failures (2). Within the past decade, cefotaximase-modifying (CTX-M) β-lactamases (CTX-M) have become the most prevalent ESBLs in bacteria isolated throughout the world in hospital and community settings (3). More than 85 variants have been identified (www.lahey.org/Studies), mainly in isolates of Escherichia coli that cause community-acquired urinary tract infections (4). Although clonal expansion events appear to have contributed to the spread of particular CTX-M variants, such as bla CTX-M-15 within E. coli strain O25:H4-ST131:05 (5,6), plasmids with the ability to spread efficiently, or epidemic plasmids, also are believed to be responsible for disseminating CTX-M ESBLs (7). The ability and frequency with which antimicrobial resistance genes disseminate between bacteria in humans, the environment, and animals is still debated, and the role of plasmids in this movement between ecosystems, including the food chain, is also still contested, despite mounting evidence that it occurs (8,9).
CTX-M-14 is the second most frequently identified CTX-M enzyme worldwide (10), detected in bacteria isolated from humans, animals, and the environment. CTX-M-14-producing strains show a high level of clonal diversity (11,12); therefore, dissemination has been attributed to conjugative plasmids rather than to clonal expansion of a bacterial host strain (13). In Europe, an association has been suggested between bla CTX-M-14 and plasmids of the incompatibility group IncK, or the spread of 1 particular IncK plasmid (11,13,14). In the United Kingdom in 2006, Liebana et al. described an ESBL-producing isolate from calves with diarrhea that carried bla CTX-M-14 on an IncK plasmid, denoted pCT (15,16). The plasmid spread to unrelated E. coli isolates within an index cattle farm and persisted within the environment. In this study, we report the full sequence and analysis of pCT and demonstrate the spread of pCT-like plasmids in animal and human E. coli isolates from the United Kingdom, Europe, Australia, and Asia.

Bacterial Strains
E. coli C159/11 was isolated from calves on a dairy farm in the United Kingdom in 2004 (15,16). Investigation and manipulation of the C159/11 plasmid (pCT) was conducted in an E. coli DH5α transformant created for the current study. We also investigated 15 CTX-M-14-producing E. coli isolates collected during 2006-2009 from cattle feces on farms in different geographic locations in the United Kingdom and 15 E. coli clinical isolates from England (P. Hawley, unpub. data), Germany (17), Spain (11,18), the People's Republic of China (19), and Australia (20) ( Table 1).

Plasmid Extraction and Manipulation
Plasmid pCT was extracted from E. coli DH5α transconjugants by using an alkaline sodium dodecyl sulfate Maxi preparation (21) and cesium chloride density gradient centrifugation (22). Conjugation was by solid mating on a filter (Whatman, Maidstone, UK), by using rifampin-resistant E. coli (DH5α) as a recipient and selection of transconjugants on Luria-Bertani agar containing 50 µg/mL rifampin and 8 µg/mL cefotaxime.
Susceptibility of E. coli DH5α to the antimicrobial drugs tested was also determined.

Complete Mucleotide Sequencing of pCT
The plasmid DNA sequence was determined by using a 454/Roche GS FLX analyzer (Roche, Basel, Switzerland). The de novo assembly generated 93631 bases in 7 contigs by using the 454/Roche Newbler assembly program with an N50 of 52,495 bp. The sequence represents improved high-quality draft sequence (23) with no discernable misassembles having undergone multiple rounds of computational gap closure. Annotation was completed by using Artemis

PCR Amplification and Sequencing
Boiled bacterial cell lysates provided template DNA, 1 µL of which was added to PCR ReddyMix Master mixture (Abgene, Epson, UK). Typically, PCR conditions were 30 cycles of 95°C for 30 sec, 51°C for 30 sec, and 72°C for 30 sec. PCR was used to detect bla CTX-M-14 and insertion sequences ISEcp1 and IS903 as described (24)(25)(26). All primers used are shown in Table   2. To determine whether the pCT bla CTX-M-14 shares a common insertion site with bla CTX-M-14 on other plasmids, PCRs were designed to amplify the sequence from bla CTX-M-14 into both pCT flanking genes.
Using the pCT sequence, we designed primer pairs to amplify novel regions of pCT for rapid identification of potential pCT-like plasmids in CTX-M-14-producing bacteria ( Table 2).
PCRs to amplify DNA encoding the putative sigma factor, pilN gene, and shufflon recombinase were developed into a multiplex PCR. An additional primer pair was designed to a unique region of pCT and compared with other known sequences for amplification across coding sequences (CDSs) pCT008-pCT009 for further discrimination of pCT-like plasmids. Sequencing of the relaxase gene has been reported to further categorize plasmids within the IncI complex (11,28).
A modified primer pair was designed for this region (nikB) by using sequence data from pCT and other related sequenced plasmids. Resulting amplicons were sequenced by using BigDye Terminator version 3.1 cycle sequencing (Applied Biosystems, Foster City, CA, USA) at the functional genomics laboratory of the University of Birmingham (Birmingham, UK) sequences were aligned by using MEGA 4.0 (29) for phylogenetic analysis (30). Primers amplifying group 9 bla CTX-M genes were used as a positive control in each instance. The complete DNA sequence of plasmid pCT was assigned GenBank accession no. F868832.

Features of pCT
The bla CTX-M-14 -carrying plasmid isolated from E. coli C159/11 was demonstrated to be conjugative by successful transfer to E. coli DH5α by using filter mating and previously determined to be of the incompatibility group IncK (16). Analysis of transconjugants showed resistance to β-lactam antimicrobial drugs as the only transferrable resistance phenotype. Wholeplasmid sequencing showed that pCT was 93,629 bp ( Figure 1) with an average G+C content of 52.67%. Annotation of the plasmid showed 115 potential protein CDSs, 89 of which were homologous to proteins of known function (Technical Appendix, www.cdc.gov/EID/17/4/pdfs/10-1009-Techapp.pdf). No genes known to play a role in determining virulence were identified, and sequencing confirmed bla CTX-M-14 as the only antimicrobial drug resistance gene on pCT. The pCT bla CTX-M-14 gene is found between insertion sequences ISEcp1 and IS903 as described (31).
Most of the identified putative coding open reading frames are typical for an IncK plasmid backbone (32). Two conjugal transfer systems were identified. The first is the more commonly described tra operon, which encodes the primary pilus for conjugation transfer. The pCT tra locus is analogous to the tra operon of R64/ColIb-P9 and is found in a similar conformation, although minor differences existed between the 3 plasmids throughout. The second is the pil locus encoding a thin pilus, which is believed to increase conjugation rates in liquid media. The tip of this thin pilus is variable, and the exact nature of the expressed epitope is determined by the orientation and order of pilV shufflon components (pCT_094-pCT_098), which can be inverted by the action of the recombinase protein (pCT_093) encoded downstream.
Analysis of the pCT plasmid assembly showed that this region was present in multiple forms (data not shown), which is consistent with site-specific recombination mediated by a shufflon recombinase. The pCT shufflon potentially differs from that of the other closely related plasmids (pO113, pO26_vir and pSERB1) because each of these apparently has an inactive shufflon, which can be attributed to the absence of the recombinase or an insertion element within this CDS. The antirestriction and segregation genes on pCT are typical of this type of plasmid.

Comparison of pCT with Other Plasmids
When we compared complete sequences deposited in GenBank of plasmids carrying bla CTX-M genes with pCT, we found that outside the bla CTX-M gene those plasmids with different replication mechanisms, such as those within the IncFII group or of the IncN group (pKP96) (33), have almost no DNA homology to pCT. The only other bla CTX-M carrying IncI complex plasmid to be sequenced and deposited in GenBank thus far is a bla CTX-M-3 carrying IncI plasmid pEK204 (EU935740) (32). pEK204 shares sequence conservation over ≈60% of the pCT genome, including most of the core IncI complex-related genes for replication or transfer.
Further similarities were found in the minimal carriage of resistance genes (Figure 2, panel D).
The pCT genome was also compared with other IncI group sequenced plasmids to identify regions considered core or backbone and to determine novel encoded genes. IncI complex reference plasmids R64 (AP005147) and ColIb-P9 (AB021078) shared 99% identity with 64% of the pCT sequence primarily within genes involved in replication and conjugation ( Figure 2, panel C). Other plasmids compared with pCT included R387, the Sanger IncK reference plasmid, which shared a high percentage identity to pCT across IncK-specific core genes ( Figure 2, panel B).
We investigated novel genes by identifying regions of the pCT sequence absent from those plasmid sequences with most homology to pCT: pO26_vir (FJ38659), pO113 (AY258503), and the partially sequenced pSERB1 (AY686591), none of which carry bla CTX-M genes. Both pO26_vir (168,100 bp) and pO113 (165,548 bp) are large plasmids that carry several virulence genes and share 99% homology with 85% and 83% of the pCT genome, respectively (online Technical Appendix Table; Figure 2, panel A). pSERB1 also has high DNA sequence identity (91% of the pSERB1 deposited genome sequence has 99% identity to two thirds of the pCT sequence), however, because this plasmid is deposited in GenBank only as a partial sequence, the total identity cannot be assessed.

Detection of pCT-like Plasmids in Animal and Human Isolates
Fifteen CTX-M-14-producing E. coli isolates collected from different cattle farms around the United Kingdom were examined for pCT-like plasmids with a series of PCRs that amplified characteristic regions of pCT. Veterinary isolates I779 and I780 were obtained 2 years apart and from different geographic areas in the United Kingdom. These isolates carried plasmids encoding all 6 pCT regions that were investigated (bla CTX-M-14 , nikB, putative sigma factor, rci, pilN, and pCT008-009) and when compared with pCT had identical flanking regions adjacent to bla CTX-M-14 . Therefore, these plasmids were deemed pCT-like. Fifteen CTX-M-14-producing E. coli human clinical isolates from England, Germany (17), Spain (11,18), Australia (20), and China (19) also were examined for pCT (Table 1) sequences with >98% DNA identity to the pCT nikB sequence and clustered when these sequences were used to construct a phylogenetic tree (Figure 3). nikB sequences from non-pCT-like plasmids clustered further from pCT within the phylogenetic tree ( Figure 3). This analysis further supports the hypothesis that pCT has disseminated broadly between bacteria in animal and human ecosystems.

Discussion
We report the complete sequence of a bla CTX-M-14 -carrying IncK plasmid, pCT, from an E. coli isolate from a cattle farm in the United Kingdom (16). Within the 115 putative CDs, there is an absence of any genes known to play a role in determining virulence of the host and the absence of any other antimicrobial drug resistance genes except for bla CTX-M-14 . Therefore, the persistence and spread of pCT cannot be attributed to coselection associated with pressure from non-β-lactam antimicrobial drugs. This finding suggests that pCT persistence and dissemination have been driven by either constant β-lactam exposure or that pCT can remain stable within a population in the absence of any antimicrobial drug selective pressure. There has been much speculation about the role of the type IV pilus and its shufflon found in plasmids and possible role(s) in adhesion of the host bacterium to surfaces and eukaryotic epithelial cells in vitro and in biofilm formation (34). The thin pilus might also aid conjugation in a liquid environment by anchoring donor and recipient cells (35). This system may have played a role in the persistence of E. coli C159/11, which originally was found to remain within slurry and on the floor of cow sheds during the longitudinal farm study from which it was identified (16). These attributes of the type IV pilus might contribute to the persistence of pCT within bacteria isolated from the UK farm and in animals and humans throughout the world. Comparison of the genome of pCT with other bla CTX-M -encoding plasmids showed no conserved regions outside the β-lactamase gene. Therefore, no single feature of the plasmid backbone appears responsible for the spread of bla CTX-M genes, and the acquisition of these genes is unlikely to have been a single event. Homology was highest between pCT and 4 plasmids (pO26_vir, pO113, pSERB1, and TP113). pO26_vir was identified in a Shiga toxin-producing E. coli strain 026:HII and encodes several virulence genes not found on pCT, including genes for the production of a hydrolase, catalase, and a hemolysin transport protein. pO113 was isolated from another hemolysin-producing EHEC O113:H21 E. coli sample from a patient in Australia (36). The finding that pCT is most closely related to 2 plasmids that carry an array of virulence genes is of concern because of the potential for recombination between these plasmids, creating       (19). A neighbor-joining tree was constructed by using complete deletion modeling and computed by using the maximum composite likelihood method (30). The phylogenetic tree was linearized assuming equal evolutionary rates in all lineages. Circles, nikB sequences from plasmids isolated from veterinary isolates from the United Kingdom; triangles, nikB sequences of plasmids from Escherichia coli isolated from humans; squares, nikB sequences of plasmids obtained from GenBank or the Sanger Institute; shaded shapes, plasmids identified as pCT-like by using PCR in this study; asterisks, plasmids encoding bla CTX-M-14 . pCT, IncK plasmid. Scale bar indicates nucleotide substitutions per site.