Tryptone soy broth (tsb)

Escherichia coli O157:H7 strain ATCC^® 43888^TM, a human fecal isolate obtained from the American Type Culture Collection, was used throughout this study. This strain lacks the genes for Shiga toxins I and II, and was chosen as a laboratory surrogate for EHEC O157:H7 strains because of its attenuated virulence. Stationary phase cultures were obtained by inoculating test tubes containing 4 ml of Tryptone Soy Broth (TSB; Oxoid, Basingstoke, UK) with a single colony grown on a Tryptone Soy Agar (TSA) plate, and then incubated aerobically with shaking (300 rpm) for 18 h at 37°C.

We inoculated faecal calf samples and worker stool samples on MacConkey’s agar (Oxoid, Hampshire, UK), and the plates were incubated at 37 ℃ for 24 h. Lactose fermenter (pink in colour) colonies were then subcultured on eosin methylene blue agar (EMB; Oxoid) and were incubated under the same conditions. For milk samples, ten dilutions on Tryptone Soy broth (TSB; Oxoid) were incubated at 37 ℃ for 6 h, and then an inoculum of each sample was cultured on MacConkey’s agar, followed by EMB agar at 37 ℃ for 24 h each. Suspected E. coli colonies on EMB (green metallic sheen in colour) were biochemically confirmed by API-20E (bioMérieux, Marcy-l’Etoile, France). All EPEC isolates were serotyped using polyvalent and monovalent O-antisera sets (Denka Seiken Co., Tokyo, Japan) according to the manufacturer’s instructions.

A collection of 27,990 stool samples from outpatients with acute diarrhea was obtained from 11 sentinel hospitals in Shenzhen, China between 2010 and 2014. From this collection, a total of 393 S. Typhimurium clinical isolates were identified by using standard biochemical and serological tests. Isolates obtained within 2 months with the same antibiotic susceptibility and PFGE pattern were confirmed as outbreak isolates by epidemiological investigation. The outbreak isolates were excluded for this study. So based on the PFGE and epidemiological data of the 393 S. Typhimurium isolates, 207 strains with 168 different PFGE patterns and other 33 food isolates recovered from product of poultry or livestock, ready to eat food, and aquatic products (Table S1) were selected for this study, The stock cultures were maintained at −80°C in tryptone soy broth (TSB; Oxoid Ltd., Cambridge, U.K.) supplemented with 20% glycerol.

K. pneumoniae KP/01, used as a host for bacteriophage infection, was isolated from a human clinical diabetic-foot sample from a male patient in May 2016 and identified by National Institute of Diabetes using the VITEK method for identification (Cairo, Egypt). Other clinical isolates of K. pneumoniae (n = 21), Proteus mirabilis (n = 18) and E. coli (n = 15) were also isolated by National Institute of Diabetes, for bacteriophage host-range analysis, from wound infection samples and provided to the microbiology research lab at Zewail City. Isolates were kept in tryptone soy broth (TSB; Oxoid, England) containing (w/v) 20% of glycerol, at −80°C. In the following experiments, bacterial strains were grown on tryptic soy agar (TSA; Oxoid, England) overnight, and isolated colonies of bacteria were grown at 37°C, in TSB, to reach OD₆₀₀ approximately 0.3.

Streptococcus oralis (ATCC 9811; American Type Culture Collection, Manassas, VA, USA) was cultured overnight on Tryptone Soy Agar (TSA) plates at 37 °C. A few colonies were inoculated into Tryptone Soy Broth (TSB) (Oxoid Limited, Hamsphire, UK) supplemented with 10% yeast extract (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and 50 mM glucose (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) at 37 °C under constant shaking. Aggregatibacter actinomycetemcomitans (DSM 11123; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) or Porphyromonas gingivalis (DSM 20709; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were cultured for 48 h on fastidious anaerobe agar (FAA) plates (LabM, Heywood, UK), supplemented with 5% sheep blood at 37 °C under anaerobic conditions (80% N₂, 10% H₂, 10% CO₂). A few colonies of A. actinomycetemcomitans or P. gingivalis were inoculated overnight into brain heart infusion medium (BHI; Oxoid, Wesl, Germany) supplemented with 10 μg/mL vitamin K (Roth, Karlsruhe, Germany) under anaerobic conditions [36 (link)]. Treponema denticola (DSM 14222; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) was cultured in new oral spirochete (NOS) medium at 37 °C for 72 h under static anaerobic conditions [37 (link)].

The following strains were used in this study; Pseudomonas aeruginosa PAO1, methicillin-resistant Staphylococcus aureus (MRSA 1004A)⁷⁵ , polymyxin B-resistant (PMB^R) Escherichia coli PN47 (carrying the colistin resistance plasmids mcr-1 and mcr-3)^{76 (link)} and polymyxin B-sensitive (PMB^Sens) E. coli IR57. Bacterial colonies were sub-cultured on LB agar plates supplemented with/without 2 µg/ml polymyxin B (Sigma-Aldrich). Overnight bacterial cultures were grown in Tryptone Soy Broth (TSB; Oxoid) for 37 °C at 120 rpm. Biofilms were grown in cation-adjusted Mueller Hinton Broth (MHB; LabM) with/without supplementation with the antibiotic polymyxin B.

Bacterial strains MB3936 (STEC O26; stx1+ stx2+ eae+) and MB4378 (STEC O138; stx2e+) were used in this study, both were isolated from humans. Both strains carry single copies of the tested genes. Both strains were stored at −80 °C using Pro-Lab Microbank cryovials (Pro-Lab, Richmond Hill, ON, Canada) according to the manufacturer’s instructions. Strains were cultured on Tryptone Soy Agar (TSA; Oxoid Ltd., Basingstoke, Hampshire, UK) and incubated at 37 °C for 24 h. A single colony from these culture plates was transferred into Tryptone Soy Broth (TSB; Oxoid). After incubation at 37 °C for 24 h the genomic DNA (gDNA) was purified using DNeasy Blood & Tissue kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instruction for Gram-negative bacteria with an additional RNase step, and eluted in a final volume of 200 µL elution buffer. The concentration of the gDNA was measured using a Quantus^TM fluorometer (Promega, Madison, WI, USA). The following formula was used for calculation of the mass (M) of one genome:
$M=n\times 1.096\times {10}^{-21} \frac{gram}{bp}$
For E. coli strain O157:H7 EDL933, the genome length (n) was determined as 5.53 × 10⁶ bp [25 (link)].
Both gDNA preparations were diluted in nuclease-free water (Qiagen) to 10⁶ copies/μL and stored as stock template at −20 °C until use.