U.S. drinking water regulations require that water samples be examined for fecal contamination using U.S. EPA-approved methods. Cultivation of membrane filtered water samples on MI agar has been used as a reliable method for detecting such contamination because of its ability to simultaneously detect and quantify both total coliforms and Escherichia coli specifically. MI agar is a selective and differential medium, containing cefsulodin to inhibit Gram-positive bacteria and noncoliform Gram-negative bacteria and indoxyl-β-D-glucuronide and 4-methylumbelliferyl-β-D–galactopyranoside for the presumptive identification of E. coli and other coliform bacteria, respectively (1). Although most coliform bacteria other than E. coli do not express β-glucuronidase activity, some strains of Klebsiella have been reported to express this enzyme and appear indistinguishable from E. coli on MI agar (2, 3). It is important to identify false-positive bacteria on MI agar to improve water quality monitoring techniques and risk assessments for the fecal contamination of water. A suspect blue colony of E. coli was isolated from a river water sample in 2012 on MI agar and was identified as R. planticola by rpoB gene sequencing (4). To understand the metabolic function of R. planticola strain CHB, whole genome sequencing was performed. The isolate was cultured in 10 mL Luria-Bertani broth overnight at 37°C. The overnight culture of 1 to 5×10⁸ CFU/mL was pelleted and resuspended in 350 µL PBS. The genomic DNA was extracted by mixing with an equal amount of 2×UNEX nucleic acid extraction buffer (Microbiologics, MN) as per manufacturer’s instructions followed by a final purification step using a polyvinylpolypyrrolidone (PVPP) spin column (Spin-IV-HRC columns, Zymo Research Corporation, Orange, CA).

The whole genome was sequenced using the Illumina MiSeq for paired-end 300×300 library preparation (MR DNA, Shallowater, TX). The genome sequence was assembled using DNAStar SeqMan NGen resulting in 18 contigs and a total length of 5.8 Mb. The genome had coverage of 422× (N₅₀ of 561 Kb) and a G+C content of 55.4%. Annotation was performed using the RAST (Rapid Annotation using Subsytems Technology) server (5). The RAST server listed closest neighbors based on functional comparison of genome sequences, including E. coli AA86 (score 544), E. coli 96.0107 (score 284), Klebsiella sp. 1_1_55 (score 275), Klebsiella pneumoniae 342 (score 274), Klebsiella oxytoca 10 to 5,246 (score 250), and Klebsiella variicola At-22 (score 265). RAST predicted 5,462 coding sequences and 101 RNAs representing 572 subsystems. Several genes were identified that were associated with resistance to heavy metals (arsenic, cobalt, zinc, chromium, cadmium, mercury, and copper), as well as resistance to antibiotics [fluoroquinolones, fosfomycin, β-lactamase, multiple antibiotic resistance (mar) locus, the mdtABCD multidrug resistance cluster, and multidrug resistance efflux pumps]. Identification of antimicrobial resistance genes and carbohydrate metabolism of this bacterium will facilitate formulation of novel chromogenic/fluorogenic agar media to support exclusive growth and specific identification of E. coli.