Buffered peptone water (bpw)

Gram-negative bacteria were isolated using standard cultivation techniques for each species (Murray et al., 2007 ). One swab from each bird or environmental source was first placed in 10 ml of buffered peptone water (Difco Laboratories, Detroit, MI, USA) and was incubated at 37 °C for 24 h. Following incubation, a loop of turbid buffered peptone water was streaked for isolation on MacConkey agar (Difco Laboratories, Detroit, MI, USA) and incubated at 37 °C for up to 48 h. A well-isolated colony on MacConkey agar was then re-streaked for isolation on eosin methylene blue agar (i.e., a single isolate was collected per sample) BD Diagnostic System, Franklin Lakes, NJ, USA) and incubated for 24–48 h at 37 °C. Presumptive Escherichia coli colonies on eosin methylene blue agar were confirmed as Escherichia coli if negative for citrate metabolism on Simmons citrate agar (Thermo Fisher Scientific, Waltham, MA, USA) and positive for indole production in tryptophan broth (Thermo Fisher Scientific, Waltham, MA, USA). Confirmed isolates were stored on nutrient agar (Thermo Fisher Scientific, Waltham, MA, USA) for additional testing.

Isolation of Salmonella was performed according to the United States Department of Agriculture (USDA) standard methods for rinsing whole-bird samples [29 ]. Each sample was aseptically placed in a sterile plastic bag containing 400 mL of buffered peptone water (BPW, Difco, Detroit, MI, USA). The whole duck was then rinsed by shaking for 2 min. Next, 30 mL rinsed fluid of each sample was vortex-mixed in 30 mL of BPW for 15 s, followed by incubation at 37 °C for 18–24 h. Then, 0.1 mL portions of BPW enrichment broth were transferred to 10 mL of Rappaport–Vassiliadis broth (RV; BD, USA), and 0.5 mL portions subjected to pre-enrichment were transferred into 10 mL of tetrathionate broths (TT; BD, Franklin Lakes, NJ, USA) and continued to be incubated for 24 h at 41.5 °C. The selective cultures were streaked on xylose–lysine–desoxycholate agar (XLD; BD, Franklin Lakes, NJ, USA) and bismuth sulfite agar (BSA; BD, Franklin Lakes, NJ, USA) plates, followed by incubation at 37 °C for 24 h. Typical colonies were selected for biochemical tests, and polyvalent antisera for O and H antigens (BD, Franklin Lakes, NJ, USA) in order to identify Salmonella isolates. Salmonella ATTC 14028, Salmonella ATCC 13076, and Escherichia coli ATCC 8389 were used as the quality control standards for the isolation procedure. All isolated Salmonella strains were stored at −80 °C for further analyses [30 ].

Samples (2.5 g) were homogenized in 22.5 mL of buffered peptone water (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The homogenates were plated on CHROMagar™ ECC agar (CHROMagar, Paris, France) without and with 100 μg mL^-1 ampicillin (Sigma-Aldrich, St. Louis, MO, USA) to determine the abundance (CFU g^-1) of Escherichia coli and coliforms (excluding E. coli), as well as β-lactam-resistant E. coli and coliforms (excluding E. coli), respectively [17 (link)]. After incubation at 37 °C for 20 h, E. coli and coliform colonies were counted according to their colony colors (E. coli, blue colony; other coliforms, red colony) and morphologies. Up to three E. coli or coliform colonies were isolated from each agar plate. Means and standard errors were calculated for each type of sample. Additionally, to isolate other Escherichia species from samples wherein E. coli colonies did not grow, precultures with 2.5 and 1 g of samples were inoculated in 22.5 mL of Luria-Bertani broth (Invitrogen, Carlsbad, CA, USA) and Colilert-18 (IDEXX, Westbrook, ME, USA), respectively, and incubated at 37 °C for 20 h. Then, the preculture was streaked onto CHROMagar ECC agar without and with 100 μg mL^-1 ampicillin and incubated at 37 °C for 20 h. Up to three E. coli or coliform colonies were isolated from each agar plate.

A random selection of 221 samples (56 chicken, 54 ground turkey, 55 ground beef, and 56 pork chops) was processed for isolation of E. coli per the 2018 NARMS Retail Meat Surveillance protocol [79 ]. Briefly, 25 g of each sample was placed in Whirl-Paks containing 250 mL buffered peptone water (Becton Dickinson, Franklin Lakes, NJ, USA) and hand massaged for 3 min. A total of 50 mL of rinsate was then added to 50 mL double-strength MacConkey broth (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated at 35 °C for 24 h. Following overnight enrichment, a loopful (10 µL) was streaked to a MacConkey plate and incubated at 35 °C for 24 h. One suspect E. coli colony based on typical colony morphology was streaked to purity on blood agar plates and incubated overnight at 35 °C. Isolates were confirmed as E. coli using biochemical tests (indole positive and oxidase negative), and banked in Brucella broth with 15% glycerol, frozen, and shipped on dry-ice to the FDA’s Center for Veterinary Medicine (CVM) for antimicrobial susceptibility testing and whole-genome sequencing.

Samples were processed per the NARMS Retail Meat Surveillance protocol. Briefly, 25 g of each sample in 250 ml buffered peptone water (Becton Dickinson, Franklin Lakes, NJ, United States) was hand massaged for 3 min or placed on a mechanical shaker at 200 rpm for 15 min. Fifty milliliters of rinsate was added to 50 ml of double-strength lactose broth (Becton Dickinson, Franklin Lakes, NJ, United States) and incubated at 35°C for 24 h. After overnight enrichment, 0.1 ml of lactose broth was transferred to 9.9 ml Rappaport-Vassiliadis R10 (RVR10) broth (Becton Dickinson, Franklin Lakes, NJ, United States) and incubated at 42°C for 16–20 h. The RVR10 enrichments were then streaked onto XLT-4 (Remel, Lenexa, KS, United States) and Hektoen Enteric (Becton Dickinson, Franklin Lakes, NJ, United States) agars and incubated at 35°C for 18–24 h. Up to two suspect Salmonella colonies based on colony morphology from each selective agar were then streaked to purity on blood agar plates. Isolates were shipped on dry ice to the FDA’s Center for Veterinary Medicine (CVM) for antimicrobial susceptibility testing and WGS.

In the laboratory, 10g of each fecal sample was weighted in a 710mL Whirl Pak^® bag (Whirl-Pak, Madison, Wisconsin) and 90mL of buffered peptone water (Becton Dickinson, New Jersey, United States) was added. The mixture was placed in a commercial stomacher for 2 minutes at 230 rpm. Thereafter, the mixture was incubated at 42°C overnight prior to isolation of E. coli and Salmonella.