Sterile cotton swab

A total of 608 samples were collected from May 2015 till June 2016. They were 300 food samples, 238 surfaces and utensils samples, and 40 hand samples. Food samples were collected aseptically using sterile spoons and placed into Stomacher bags (Grade, UK). The targeted surfaces and an area ranging from 20 to 100 cm² (according to the dimension of the surface to be sampled) were swabbed by sterile cotton swabs (Oxoid, UK), premoistened into a 2 mL sterile Brain heart Infusion solution (Oxoid, UK) then transported to the laboratory in ice boxes (4°C) [14 (link)]. The biological samples were collected from bare-hand food handlers using swabs as recommended by Evancho et al. [15 (link)]. All samples were obtained during the food preparation process.

All bacterial isolates (53 SS-O157, 6 control O157 and 20 bovine E. coli) with intermediate-resistant and resistant AST phenotype were selected for MIC testing against respective antibiotics using the MIC strips (Etest®, BioMe'rieux Inc., Durham NC). Hence, isolates were tested for MIC against 11 antibiotics, in triplicate for each antibiotic (results represented as average MIC μg/mL), based on the CLSI guidelines [34 ] (). Briefly, four purified bacterial colonies were inoculated in trypticase soy broth (TSB; Becton Dickinson/Thermo Fisher Scientific, Rockford, IL) to reach a McFarland turbidity of ∼0.5 (concentration, ∼10⁵ CFU/mL). The inoculum was spread-plated onto the Mueller-Hinton Agar (MHA) (Becton Dickinson/Thermo Fisher Scientific) plates using sterile cotton swabs (Thermo Fisher Scientific). Within 15 min of inoculation, the MIC Strips (Etest®) were placed onto MHA plates. The plates were incubated at 37°C for 16–18 h and the MIC (μg/mL), corresponding to the elliptical zone of inhibition around the strip (), was directly read from the strips. E. coli (ATCC®25922™) was used as a control to validate assay conditions and the quality of antibiotics [34 ].

Microbial DNA was extracted from 231 breast milk samples using a modified protocol from the DNeasy Powerfood Microbial kit (Qiagen, UK) (formerly PowerFood™ Microbial DNA Isolation kit, MoBio, Carlsbad, CA) as previously outlined^{22 (link)}. Briefly, milk samples were subjected to initial centrifugation of 4000g × 30 min at 4 °C, the fat layer removed with a sterile cotton swab (Thermo Fisher Scientific, Inc.), and the supernatant was discarded. Cell pellets were washed twice with phosphate buffered saline (Sigma Aldrich) and treated with 90 µL of 50 mg/mL lysozyme (Sigma Aldrich) and 50 µL of 5 KU/mL mutanolysin (Sigma Aldrich) followed by incubation at 55 °C × 15 min. Samples were subsequently treated with 28 µL of 20 mg/mL proteinase k (Qiagen, UK) and incubated further at 55 °C for 15 min followed by using the DNeasy Powerfood Microbial kit protocol. Negative controls using sterile molecular water (Sigma Aldrich) were extracted as above.

For Methods 1–4, 2.5 ml of milk was used for extractions and 0.5 ml used for Methods 5 and 6. Microbial DNA was extracted from Methods 1–5 milk samples using a modified protocol from the PowerFood^TM Microbial DNA Isolation kit (MoBio, Carlsbad, CA). Briefly, samples were subject to an initial centrifugation 4000 g x 30 min at 4°C, the fat layer was removed with a sterile cotton swab (Thermo Fisher Scientific, Inc.) and supernatant discarded. Cell pellets were washed with phosphate‐buffered saline (Sigma Aldrich) and centrifuged at 13,000 g × 1 min at room temperature. A second wash step was performed using the same process. Samples were treated with 90 µl of 50 mg/ml lysozyme (Sigma Aldrich) and 50 µl of 5 KU/ml mutanolysin (Sigma Aldrich) followed by incubation at 55°C × 15 min. Samples were subsequently treated with 28 µl of 20 mg/ml proteinase k (Qiagen, UK) and incubated further at 55°C × 15 min followed by the Mobio PowerFood^TM Microbial DNA Isolation kit protocol. Method 6 used the Milk Preservation and Isolation Kit (Norgen BioTek) as per the manufacturer's instructions with the addition of the optional 2‐h lysozyme step. Two negative controls using sterile molecular water (Sigma Aldrich) were extracted as above, one with each kit.