Sampling and Resistance and Genomic Typing of Cephalosporin-resistant E. coli in Gulls (Larus spp.) and Bald Eagles (Haliaeetus leucocephalus) in Southcentral Alaska, 2016

Samples were collected by inserting a sterile swab into recently deposited faecal material or directly into the cloaca of live-captured gulls caught using noose carpets. Swabs were subsequently placed into a vial with chilled Luria broth (BD, Sparks, Maryland, USA), and kept cool on ice packs, for approximately 4–48 hours until frozen at -80 Celsius.

Fecal material from samples were inoculated, using a sterile cotton swab, into a vial containing 2ml 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 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), 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).

Selective screen for putative cephalosporin resistance: Ten µl from each of 76 turbid BHI-broth samples were inoculated on CHROMagar C3G R plates (CHROMagar 4, Paris, France) to selectively culture for bacteria exhibiting extended-spectrum cephalosporin resistance (ESBL and pAmp-C). To confirm ESBL- or AmpC phenotypes, E.coli isolates from the CHROMC3G-plates were spread on Muller-Hinton agar (Department of Clinical Microbiology, Uppsala University Hospital) according to EUCAST disc diffusion method for antimicrobial susceptibility testing. Five antibiotic discs, amoxicillin/clavulanic acid (30/1 μg), cefotaxime (5 μg), ceftazidime (10 μg), cefepime (30 μg), and cefoxitin (30 μg), were placed on the plate according to standard protocol at the Department of Clinical Microbiology in Uppsala University Hospital. Specific patterns of bacterial growth around the antibiotic discs were used to identify ESBL/AmpC-phenotypes (Jarlier, 1988 and Polsfuss, 2011).

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 NexteraXT library preparation kit (Illumina, San Diego, USA) according to manufacturer’s instructions. Paired-end sequencing was performed using the MiSeq platform (Illumina, San Diego, USA) using either 250 or 300 base pair read lengths.