Complete Genome Sequence of Escherichia coli Antibiotic-Resistant Isolate AR Bank #0349

The emergence of plasmid-mediated polymyxin resistance encoded by mcr-1 has heightened public health concerns due to the potential for rapid horizontal transfer. Here, we report the complete genome sequence of Escherichia coli AR Bank #0349, which exhibits resistance to colistin encoded by a plasmid-borne mcr-1 gene.

ABSTRACT

The emergence of plasmid-mediated polymyxin resistance encoded by mcr-1 has heightened public health concerns due to the potential for rapid horizontal transfer. Here, we report the complete genome sequence of Escherichia coli AR Bank #0349, which exhibits resistance to colistin encoded by a plasmid-borne mcr-1 gene.

ANNOUNCEMENT

Colistin (polymyxin E) is a cationic polypeptide antibiotic that disrupts the bacterial outer cell membrane of Gram-negative bacteria, causing bacterial cell death. Due to the rise in antimicrobial resistance, it is currently used as a last line of defense in the treatment of severe bacterial infections by multidrug-resistant (MDR) or extensively drug-resistant (XDR) bacteria, such as extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant Enterobacteriaceae (CRE) infections (1, 2). Due to the lack of alternative antibiotics for treatment, colistin resistance is a serious public health threat.

Colistin resistance was generally regarded to be chromosomally mediated and nontransmissible (3). However, a plasmid-mediated polymyxin resistance gene named mcr-1 was first reported in November 2015 (4). The investigators additionally demonstrated that the plasmid harboring the mcr-1 gene was stable and readily transferable. mcr-1 encodes a phosphoethanolamine (PEA) transferase that adds PEA to the lipid A of the lipopolysaccharide (LPS), masking the negatively charged phosphate groups leading to colistin resistance (5). This plasmid-mediated polymyxin resistance has heightened public health concern due to the potential for rapid horizontal transfer.

Here, we report the generation of whole-genome sequence data for Escherichia coli AR Bank #0349, obtained from the Food and Drug Administration and Centers for Disease Control and Prevention Antibiotic Resistance Isolate Bank (FDA-CDC AR Bank, CDC, Atlanta, GA, USA). The isolate was cultivated with aeration at 37°C in Luria-Bertani (LB) broth (10 g tryptone, 5 g yeast extract, and 10 g NaCl/liter) or on LB agar plates. For DNA extraction, a single colony was inoculated into LB broth and cultivated overnight with aeration at 37°C. The High Pure template preparation kit (Roche Applied Science, Indianapolis, IN) was used to extract total genomic DNA from the sample, which was subsequently used to prepare both PacBio and Illumina (San Diego, CA) libraries.

Both PacBio and Illumina MiSeq platforms were used to generate whole-genome sequence data. The PacBio library was prepared utilizing the PacBio 10-kb insert library preparation protocol (6). The library was sequenced with a PacBio RS II platform using two single-molecule real-time (SMRT) cells resulting in 183,307 total reads and an average read length of 9,274 nucleotides. A MiSeq-indexed library was created using the Nextera XT DNA sample preparation and index kits (Illumina). The library was sequenced with a MiSeq v2 500-cycle reagent kit generating 2 × 250-bp paired-end reads on the MiSeq platform (Illumina), resulting in a total of 2,414,464 reads. The reads were subsequently assessed for quality using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).

The hybrid assembler Unicycler v.0.4.4 was used to generate the complete circular chromosome assembly from the Illumina and PacBio data (7). This assembly identified the presence of a large, incompletely assembled ≥200-kb plasmid. The assembly of this large plasmid was completed from the PacBio data using Canu v.1.8, and four rounds of polishing using Pilon v.1.23 were used for error correction using the Illumina data (8, 9). Default parameters were used for all software. The final completed assembly is composed of a 4,995,442-bp circularized chromosome with a G+C content of 50.5% and the 278,067-bp plasmid pAR349 with a G+C content of 49.2%. For the chromosome, the average PacBio coverage is 279×, and the average Illumina coverage is 102×; for the plasmid, the average PacBio coverage is 247×, and the average Illumina coverage is 96×. Automated genome annotation was completed using the NCBI Prokaryotic Genome Annotation Pipeline (10). This annotation identified the presence of the mcr-1 gene on the large plasmid pAR349.

Data availability.

The whole-genome sequence for Escherichia coli AR Bank #0349 was deposited in DDBJ/ENA/GenBank with the accession numbers CP041996 for the chromosome sequence and CP041997 for the plasmid sequence. The raw reads were deposited in the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA554502 and SRA sample number SRS5353902.