Draft Genome Sequence of the Multidrug-Resistant Escherichia coli Strain LR09, Isolated from a Wastewater Treatment Plant.

We report the draft genome sequence of Escherichia coli O1:H6 strain LR09, which was isolated from a wastewater treatment plant and displays high resistance to five fluoroquinolone antimicrobials. The assembled data determine that the strain clusters with E. coli phylogroup F and harbors a plasmid conferring resistance to a broad spectrum of antibiotics.

GENOME ANNOUNCEMENT

Wastewater treatment plants provide an opportunity for the dissemination of mobile antimicrobial resistance determinants as bacteria previously exposed to antibiotics mix with naive strains (1). However, these multidrug-resistant bacteria have been otherwise poorly characterized. Here we report the draft genome sequence of Escherichia coli LR09, isolated in August 2006 from the Little Rock Wastewater Utility treatment plant in Little Rock, AR (2). This strain was isolated using media containing 200 ng µl-1 norfloxacin and was shown to be highly resistant to all fluoroquinolones tested (2), as well as to gentamicin, streptomycin, and ampicillin (all above 200 ng µl-1).

Genomic DNA was extracted from an overnight culture using the DNeasy blood and tissue kit (Qiagen). Sequencing libraries were prepared with both the TruSeq DNA sample prep kit (Illumina) and the Nextera mate pair sample prep kit (Illumina) and sequenced on the Illumina MiSeq platform, generating 12,384,122 paired-end 300-bp reads and 9,355,464 mate pair 250-bp reads, respectively. The draft genome sequence was assembled de novo with CLC Genomics Workbench version 6.5.1 (CLC bio), producing 124 contigs resulting in a total genome size of 5,338,463 bp and an N50 of 347,262 bp. Plasmid DNA was isolated using the Large-Construct kit (Qiagen) and sequenced to determine which contigs comprised plasmid DNA; however, closure of the plasmid sequences was not performed. The draft genome sequence was annotated using RAST (3) and predicted to contain 5,183 coding sequences and 88 tRNAs.

Phylogenetic analysis utilizing backbone single nucleotide polymorphisms (SNPs) from whole-genome sequences revealed that LR09 clusters with E. coli phylogroup F (4). Molecular serotyping using the wzx, wzy, and fliC loci indicated that LR09 is O1:H6. BLAST results were negative for molecular markers of known E. coli pathotypes, including toxins and toxin genes (stx1, stx, ehxA, hlyA, LT, and ST) and genes for adhesins (eae, bfpA, aggR, and pap). The genome contains two small plasmids, a 1,460-bp plasmid with 99% sequence identity to E. coli strain 1/21 plasmid pKST21 (GenBank accession no. JF436966.1) and a 7-kb plasmid encoding a colicin and a DNA restriction and modification system. In addition, LR09 possesses a 92-kb plasmid homologous to p12579_1 from E. coli O55:H7 strain RM12579 (GenBank accession no. CP003110.1). This plasmid is a lysogenic bacteriophage that remains circularized and extrachromosomal (5). A fourth plasmid harbored in LR09 is a large (>163 kb) IncF virulence plasmid exhibiting homology to pRSB225 (GenBank accession no. JX127248.1) carried by an uncultured bacterium isolated from a wastewater treatment plant in Germany (6). Along with genes encoding putative virulence factors, this LR09 plasmid carries antibiotic resistance genes identified by ResFinder (7). These include genes for resistance to aminoglycosides [strAB, aadA5, and aac(3)-IId], beta-lactam (blaTEM-1B), macrolide [mph(A)], chloramphenicol (catA1), sulfonamide (sul1), tetracycline [tet(B)], and trimethoprim (dfrA17). In addition, the genome contains an antibiotic resistance cassette conferring resistance to beta-lactam (blaOXA-1), chloramphenicol (catB3), and aminoglycosides and fluoroquinolones [aac(6′)-Ib-cr], the latter gene of which was previously shown to be responsible for the acetylation of ciprofloxacin and norfloxacin in this isolate (2).

Nucleotide sequence accession number.

The draft genome sequence of E. coli LR09 was deposited at DDBJ/EMBL/GenBank under accession no. JDVF00000000.