Antimicrobial Resistance Testing and qPCR Detection of Antimicrobial Resistance Genes in Surface Water and Gull (Larus spp.) Feces on the Kenai Peninsula, Alaska, 2021

Gull fecal samples were collected by inserting a sterile swab into recently deposited fecal material and surface water samples were collected with sterile 500 mL bottles. Swabs were subsequently placed into vials with bacterial transport media, and all samples were kept cool on ice packs, for approximately 4–48 hours until frozen at -80 Celsius, or for water samples, filtered.

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 and K. pneumoniae. All plates were incubated in aerobic conditions for 18–24 hours at 36 degrees Celsius. One randomly selected pure E. coli or K. pneumoniae colony, confirmed by MALDI-TOF, was chosen from each plate displaying evidence for E. coli or K. pneumoniae growth and tested for antibiotic susceptibility. Antimicrobial susceptibility testing was performed equivalently on all 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 or K. pneumoniae isolates. All plates were incubated in aerobic conditions for 18–24 hours at 36 degrees Celsius. One randomly selected pure E. coli or K. pneumoniae colony, confirmed by MALDI-TOF, was chosen from each plate displaying evidence for E. coli or K. pneumoniae 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 and K. pneumoniae 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 300 base pair read lengths.

DNA was extracted from all isolates using the PowerFecal Pro DNA Kit (Qiagen, Venlo, Netherlands). Extracted DNA were run for qPCR using the Microbial DNA qPCR Array according to manufacturer instructions (Qiagen, Verlo, Netherlands). qPCR was performed on a CFX96 thermal cycler for 45 amplification cycles (Bio-Rad, Hercules, CA USA).