Sampling, Antimicrobial Resistance Testing, and Genomic Typing of Carbapenemase Producing E. coli in Gulls (Larus spp.) in Alaska, 2016

Gull fecal samples were collected by inserting a sterile swab into recently deposited fecal material. Swabs were subsequently placed into vials with bacterial transport media, and kept cool on ice packs, for approximately 4–48 hours until frozen at -80 degrees Celsius.

Samples were inoculated, using a sterile cotton swab, into a vial containing 2 ml brain heart infusion broth (BHI-broth; Becton, Dickinson, USA), supplemented with vancomycin (16 mg/L, ICN Biomedicals Inc., USA) and incubated for 18-24 hours at 36 degrees Celsius for enrichment. 10 µl incubated turbid BHI-broth was inoculated onto CHROMagar Supercarba plates (CHROMagar, France) to screen for E. coli. All plates were incubated in aerobic conditions for 18–24 hours at 36 degrees Celsius. One randomly selected pure E. coli colony, confirmed by MALDI-TOF, was chosen from each plate displaying evidence for E. coli growth and tested for antibiotic susceptibility. Antimicrobial susceptibility testing was performed equivalently on all E. coli isolates in accordance with the European Committee on Antibiotic Susceptibility (EUCAST) disc diffusion method (Matuschek et al. 2014) using the following set of antibiotic discs (obtained through Oxoid Ltd, Basingstoke, Hampshire, England): ampicillin (10 µg), cefadroxil (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), gentamicin (10 µg), mecillinam (10 µg), meropenem (10 µg), nalidixic acid (30 µg), nitrofurantoin (100 µg), piperacillin-tazobactam (36 µg), tetracycline (30 µg), trimethoprim (5 µg), and trimethoprim-sulfamethoxazole (25 µg). Inhibition zone diameters were interpreted according to EUCAST breakpoints (Matuschek et al. 2014). There are currently no defined clinical breakpoints for chloramphenicol, nalidixic acid, and tetracycline for E. coli isolates, therefore the inhibition zone diameters were interpreted by breakpoints defined by the Normalized Resistance Interpretation method (Kronval et al. 2003).

DNA was extracted from all antimicrobial resistant E. coli isolates using the MagnaPure compact nucleic acid isolation kit (Roche, Mannheim, Germany). Multiplexed DNA libraries were prepared for whole genome sequencing using the Nextera DNA Flex library preparation kit (Illumina, San Diego, USA) according to manufacturer’s instructions. Paired-end sequencing was performed using the MiSeq or HiSeq 4000 platform (Illumina, San Diego, USA) using either 150, 250, or 300 base pair read lengths. Isolates representing distinct antibiotic resistant profiles were also sequenced with the PacBio Sequel platform (Pacific Biosciences, Menlo Park, USA).