Mueller hinton broth (mhb)

MIC was determined using the broth microdilution method described by Hancock [30 ] with some modifications. Briefly, bacterial colonies grown from the MHA were prepared in MHB (Oxoid) as a 0.5 McFarland suspension (which is equal to 1 × 10⁸ CFU/mL), and was further 200-fold diluted by the MHB (5 × 10⁵ CFU/mL). The bacterial suspension (50 µL) was mixed with 2-fold serial dilutions of peptide (5.5 µL) achieving final concentrations of 256 to 1 µM using a round-bottom polypropylene (PP) 96-well plate (Greiner Bio-One). The plate was incubated at 37 °C for 18 h including incubating peptide free-bacteria as positive growth control, and MHB as negative growth control. After the incubation, the lowest concentration with no visible growth was defined as a MIC value. The assay was performed in a duplicate manner.

The inoculum was prepared as described in disk diffusion assay and was then diluted 10-fold to reach a final concentration of 5×10⁶ CFU/ml. The antimicrobial activity against the following MRSA strains was examined: Reference strain of MRSA (ATCC 43300), clinical isolates of MRSA strains (37 and 1). The MIC of all EOs was determined by broth microdilution method using 96-well microtiter plates according to the standard protocols of (CLSI, 2014) with some modification. Briefly, the inoculum was prepared as described above. A2-fold serial dilution of each EO stock (50 µl) in MHB (Oxoid, UK) was prepared in 96-well microplates except the last two columns, which served as negative controls (bacterial inoculum and MHB without EO). Fifty µL of prepared bacterial suspensions (1×10⁶ CFU/ml) were added to each well to reach a final concentration of approximately 5×10⁵ CFU/ml. After 24h of incubation at 37°C, MIC was determined as the lowest concentration of the EO inhibiting visible bacterial growth. The MBC was determined by subculturing 100 µl onto MHA (Oxoid, UK) from wells showing no turbidity next to the MIC well. The MHA was incubated at 37°C for 24h and the lowest concentration without apparent microbial growth was considered as the MBC. Values are the averages of three independent experiments.

UriSelect 4™ agar (Bio-Rad, Watford, UK) plates were used for enumeration and isolation of E. coli from LIC. Vancomycin (16 mg/ml) was added to inhibit the concomitant growth of Enterococcus on this media. An aliquot of 200 mg (±20 mg) of LIC was suspended in 1 ml of phosphate-buffered saline (PBS; Oxoid, Hampshire, UK) and vortexed for 5 min to homogenize. A 10-fold dilution series (from undiluted to 10⁻⁶) was prepared, and 100 μl from each dilution spread onto agar for overnight incubation at 37°C. Cloacal swabs were moistened with PBS and swabbed directly onto UriSelect 4™ agar for isolation of E. coli. Following incubation, E. coli were identified as pink colonies according to manufacturer's instruction. Bacterial counts in original LIC were back calculated based on the total number of colonies countable on UriSelect 4™ agar.
For each sample originating from a specific chicken intestine, eight presumptive E. coli colonies were selected at random for each LIC sample and sub-cultured to confirm purity. Species identification was later confirmed by PCR, using the protocol described by Le Devendec et al. (18 (link)) Isolates were stored in Mueller Hinton Broth (MHB; Oxoid, Hampshire, UK) with 25% glycerol (Fisher Scientific, Loughborough, UK) at −80°C for further analysis.

The cultivation of C. jejuni for transcriptomic analysis was carried out as previously described [50 (link)]. Mueller Hinton Agar (MHA; Oxoid, Basingstoke, UK) was inoculated with each isolate to form a bacterial lawn, which was incubated at 37 °C for 48 h microaerobically. The resulting lawn was resuspended in 1ml of Mueller-Hinton Broth (MHB, Oxoid, Basingstoke, UK), and the optical density was adjusted to 0.3 OD₅₉₅. A total of 85 µL from this suspension was transferred to a biphasic culture flask containing 8 mL of MHA and 8 mL of MHB, which was supplemented with or without 100 µM of each hormone. All samples were incubated at 37 °C microaerobically, until they reached 0.1 OD₅₉₅ (16–20 h) [50 (link)].

Gantrez^® S-97 (GAN) (acid form of methylvinylether and maleic anhydride copolymer) (Mw = 1.2 × 106 Da), was provided by Ashland (Tadworth, Surrey, UK). Hyabest^®(S) LF-P (sodium hyaluronate 99.9% purity, MW 250–400 kDa range) was obtained from Kewpie Corporation Fine Chemical Division (Tokyo, Japan). Methylene blue (MB) was purchased from Sigma–Aldrich (Steinheim, Germany). Poly(vinyl chloride) (PVC) sheets (unplasticised) with a thickness of 0.2 mm were obtained from Goodfellow Ltd (Cambridge, UK). Phosphate-buffered saline (PBS), tryptone soya broth (TSB), quarter-strength Ringer’s solution (QSRS) and Mueller-Hinton broth (MHB) were obtained from Oxoid Ltd (Hampshire, UK). Proteus mirabilis ATCC 35508 and Staphylococcus aureus ATCC 6538 (LGC Standards, Middlesex, UK) were maintained on cryopreservative beads (Protect Bacterial Preservation System, Technical Service Consultants Ltd., UK) in 10% glycerol at −80 °C and cultivated in MHB at 37 °C when required for the microbiological assessments.

Phosphate-buffered saline (PBS) and VCM were purchased from Sigma-Aldrich (St. Louis, MO, USA) and RFB from Pharmacy Biotech AB (Uppsala, Sweden). The pure phospholipids, dimyristoyl phosphatidyl choline (DMPC), dimyristoyl phosphatidyl glycerol (DMPG), dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoyl phosphatidyl ethanolamine covalently linked to poly(ethylene glycol)2000 (DSPE-PEG) were purchased from Lipoid (Ludwigshafen, Germany). Rhodamine covalently linked to phosphatidylethanolamine (Rho-PE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Thiazolyl blue tetrazolium bromide (MTT), crystal violet (CV), and glycerol were obtained from Panreac AppliChem, ITW Reagents (Darmstadt, Germany). Culture media Mueller–Hinton Agar (MHA) and Mueller–Hinton Broth (MHB) were obtained from Oxoid, Ltd. (Basingstoke, UK) and Tryptic Soy Broth (TSB) from Biokar (Pantin, France). The fluorescent stain SYTO 9 was obtained from Molecular Probes (Eugene, OR, USA). D(+)-glucose monohydrate was acquired from Merck KGaA (Darmstadt, Germany). Dimethyl sulfoxide (DMSO) and ethanol absolute anhydrous were obtained from Carlo Erba Reagents S.A.S. (Val de Reuil, France). All other reagents were of analytical grade.

Bacterial isolates used in this study are listed in Table 2. Strain 38 was obtained from Mark Wilcox, University of New South Wales Australia and WBG525 was obtained from Warren Grubb, Curtin University, Australia. Isolates 12-334-07086 and 14-268-06583 were obtained from the Capital District Health Authority, Halifax, NS, Canada. The canine otitis isolates were obtained from Luisa De Martino, University of Naples, Italy. The bovine mastitis isolates were obtained from the Canadian Mastitis Culture Collection, Montreal, Canada. All isolates were routinely cultured from frozen stocks (-80°C) and maintained on Trypticase Soy Agar (TSA, Sigma-Aldrich) or Blood Agar (BA, Becton Dickinson). Liquid cultures were grown in Mueller-Hinton Broth MHB (Oxoid) or Roswell Park Memorial Institute Medium (RPMI 1640, Sigma-Aldrich) supplemented with 2% (w/v) glucose, buffered with 0.165M 3-(N-morpholino)-propanesulfonic acid. Partially deferrated MHB and RPMI, extracted with FEC1 to remove excess Fe (MHB-FEC1 and RPMI-FEC1) were prepared using FEC1 as previously described (Holbein and Mira de Orduña, 2010 (link)). Cultures were grown at 35–37°C with shaking. All experiments were executed following approved biosafety and biosecurity standards (Public Health Agency of Canada) in a level 2 biosafety laboratory.