Complete Genome Sequences of IncI1 Plasmids Carrying Extended-Spectrum β-Lactamase Genes

Extended spectrum beta-lactamases (ESBLs) confer resistance to clinically relevant antibiotics. Often, the resistance genes are carried by conjugative plasmids which are responsible for dissemination. Five IncI1 plasmids carrying ESBLs from commensal and clinical Escherichia coli isolates were completely sequenced and annotated along with a non-ESBL carrying IncI1 plasmid.

treatment of bacterial infections in human and veterinary medicine. Resistance genes are often carried by mobile genetic elements such as plasmids through which resistance is spread within an environment (1). Extended spectrum beta-lactamases (ESBLs) are multidrug-resistant proteins which can hydrolyze a variety of clinically relevant antibiotics such as penicillins and cephalosporins. Frequently, ESBLs are carried by plasmids of the incompatibility group I1 which are present in Escherichia coli from diverse environments such as feces from animals in bio-industry and wildlife, human clinical samples, and food stuffs intended for human consumption (2)(3)(4)(5)(6). In general, IncI1 plasmids measure 90 to 120 kb and carry various antibiotic-resistant genes and plasmid addiction systems (7,8).
Next-generation whole-genome sequencing of the plasmids was performed by shotgun XL pyrosequencing (454-Roche XL sequencer). De novo assembly of the reads was performed using Newbler v2.5.3. Scaffolds were built by custom scripts using synteny of contigs determined by BLAT v34 (10) mapping on reference sequence R64 (7). These scaffolds (artificial chromosomes), which also included any non-homologous contigs with reference R64, were compared using customized Nucmer (MUMmer v3.07 [11]) scripts allowing sequence comparison and curation in Artemis and ACT (12,13). PCR and subsequent Sanger sequenc-ing (ABI-Prism GA3130) were performed to close any gaps between the scaffolded contigs. Final annotation of the plasmids was performed with the NCBI Prokaryotic annotation pipeline (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/) and was manually curated.
The plasmids sequenced here range in size between 89,503 and 110,137 bp, the GϩC content of ranges between 49.5% and 51.4% and between 101 and 129 coding sequences (CDSs) were predicted per plasmid. In addition to beta-lactamases, which are present on all plasmids except additional plasmid pE17.16, various resistance genes are present. Resistance to aminoglycosides, sulfonamide, and trimethoprim is carried by pESBL-283, pESBL-305, and pESBL-315, whereas pESBL-12 carries resistance to aminoglycosides and pE17.16 carriess resistance to aminoglycosides and sulfonamide.
These nucleotide sequences confirm that there is high conservation in the backbone of IncI1 plasmids.
Nucleotide sequence accession numbers. Genome sequences have been submitted to GenBank under the accession numbers listed in Table 1.