Sampling, Antimicrobial Resistance Testing, and Genomic Typing of E. coli in Gulls (Larus spp.) on the Kenai Peninsula, Alaska, 2016

Gull fecal samples were collected by inserting a sterile swab into recently deposited fecal material and fish skin and vent samples were collected with sterile swabs by swabbing the lateral skin surface and inside the vent walls of freshly harvested sockeye salmon. 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.

Selective screen: 10 µl incubated turbid BHI-broth was inoculated onto CHROMagar C3GR 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 resulting from selective screening approaches 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), trimethoprim-sulfamethoxazole (25 µg), amoxicillin/clavulanic acid (30/1 μg), cefotaxime (5 μg), ceftazidime (10 μg), cefepime (30 μg), and cefoxitin (30 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).

Non-selective screen: Ten µl from all turbid BHI-broth samples were inoculated on Uriselect agar plates (Bio-Rad Laboratories, France) to identify E. coli isolates. 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 resulting from selective screening approaches 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.