Iso sensitest broth

The hemolytic activity was identified by observing the zone around the growth on Columbia blood agar containing 5% sheep blood (Oxoid Ltd., United Kingdom) after being anaerobically incubated at 37°C for 48 h. The bile salt deconjugation activity was determined by observing the precipitation in or around the growth on MRS containing 0.5% taurodeoxycholic acid, or 0.5% glycodeoxycholic acid after 72 h of incubation at 37°C under anaerobic conditions (Dashkevicz and Feighner, 1989 (link)). The resistance phenotype of L. pentosus 9D3 was investigated as recommended by the European Food Safety Authority (EFSA FEEDAP, 2018 (link)). The strain’s susceptibility to seven antimicrobials, i.e., ampicillin, gentamicin, kanamycin, erythromycin, clindamycin, tetracycline, and chloramphenicol was determined. The minimum inhibitory concentration (MIC) for each antimicrobial was evaluated using the microdilution method following the international standard (International Organization for Standardization (ISO) and International Dairy Federation (IDF), 2010 ). The MICs were determined in LSM broth [90% ISO-sensitest broth (Oxoid Ltd., United Kingdom): 10% MRS], after incubation of the strain at 37°C under anaerobic conditions (10% H₂, 10% CO₂, 80% N₂) for 48 h.

Minimal inhibitory concentration of the emulsions was determined by the modified 96 well plate Resazurin assay^{23 (link)}. Briefly, stock solutions of the PA emulsions were serially diluted into Iso-sensitest broth (Oxoid Ltd., Hants, UK) containing Resazurin (Biotium, Hayward, CA) (0.001 g/ml) and inoculated with ca. 5 Log CFU/ml of Salmonella. Serial dilutions were performed to obtain PA concentrations ranging from 500 mM to 2.73 mM of each emulsion. The 96 well plates were incubated for 24 h at 37 °C and the wells containing the serial dilutions of the emulsions were observed for a change in color from blue to pink indicating bacterial growth. The lowest dilution at which no bacterial growth was determined (blue well) was considered the minimum inhibitory concentration. The MICs of the individual surfactants at concentrations of 1%, 0.1% and 0.01% (w/v) and PA without surfactant that was suspended in water by vortexing for 1 min were determined as comparative controls using the modified 96 well plate Resazurin assay. Each test was performed three times and an average of the three MIC values were used.

Overnight broth cultures of each isolate were prepared in 10 mL of Iso-Sensitest broth (ISB; Oxoid). Time–kill assays were set up in 10 mL ISB with starting inocula of 10⁶ cfu/mL as per a previously published protocol.²⁰ (link) A separate experiment to investigate inoculum effect with AB14 was conducted using starting inocula ranging between 10⁷ and 10⁸ cfu/mL.
Concentrations of colistin used ranged from 0.25 to 8192 mg/L (colistin concentrations 1024–8192 mg/L were prepared directly in broth without the use of antibiotic stock solution) and fusidic acid concentrations from 1 to 8192 mg/L. Concentrations chosen ranged from suboptimal to maximal (or 8192 mg/L) bactericidal activity for each single- or dual-agent condition. Bacterial colony counts were determined by plating out 100 μL aliquots (with serial dilutions in sterile PBS where appropriate) onto unsupplemented Iso-Sensitest Agar (ISA), with manual counts performed following incubation under aerobic conditions at 37°C for 18–24 h.

Strains used in this study are listed in Table 7. Bacterial strains were grown overnight at 37°C in Lennox broth (Sigma-Aldrich, UK) or Iso-Sensitest broth (Oxoid, UK). All chemicals and antibiotics were supplied by Sigma-Aldrich, UK. Antimicrobial disks were supplied by Oxoid, UK.

MICs were determined using Etest strips (bioMérieux, Marcy-l’Étoile, France), or Liofilchem MIC test strips (liofilchem srl, Roseto degli Abruzzi, Italy). Cultures were prepared from single colonies in Müller-Hinton broth and overnight incubation. The cultures were then diluted in 0.9% saline 1:20 for a standard inoculum density of 10⁸ cfu/ml (equivalent to 0.5 McFarland), or 1:1000 for a lower inoculum density of approximately 10⁶ cfu/ml. Heteroresistance is only detectable at the higher inoculum density. The prepared cell suspensions were streaked onto Müller-Hinton agar using a sterile cotton swab. Etest strips were applied, and the plates were incubated for 20 h, after which the MIC was read according to manufacturer instructions. For protamine sulfate, MICs were determined in glass vials using broth microdilution in ISO-Sensitest broth (Oxoid, Hants, UK; Ref. CM0473) supplemented with 0.2% glucose, as previously described [17 (link)].

Animal experiments were approved by the University of Nottingham animal welfare and ethical review body, under UK Home Office Licence 40/3399. Foxp3-green fluorescent protein (GFP) C57/BL6 mice (JAX strain B6.Cg-Foxp3^tm2(EGFP)Tch/J)28 (link) were infected by oral gavage on three alternate days with doses of 1 × 10⁹H pylori strain B128 7.13 in 100 µL Isosensitest broth (Oxoid).11 (link) After 3 weeks mice were humanely killed, their spleens were removed and rubbed through a sterile disposable 100 μm cell strainer. Cells were enriched using an EasySep Mouse CD4 T cell enrichment kit (StemCell Technologies, Vancouver, Canada), and sorted into GFP⁺ and GFP⁻ populations using a MoFlo XDP flow cytometer (Beckman Coulter). Infection was confirmed using quantitative culture of stomach tissue homogenate, as described previously.29 (link) The median colonisation density for the infected mice was 1.31×10⁵ colony-forming units/g (range 0.625–3.88×10⁵) and colonies were confirmed as H pylori by urease tests and Gram staining.

The minimum inhibitory concentration (MIC) of a series of heavy metals was determined in L. garvieae IPLA 31405 and its plasmid-free derivatives by inoculating the strains into LSM (90% IsoSensitest broth and 10% MRS broth; both from Oxoid; Thermo Scientific) [26 (link)](Klare et al., 2005). Two-fold increasing concentrations (from 0.03 to 2048 μg/ml) of the following metal salts were assayed: cadmium (CdSO₄·8H₂O), cobalt (CoCl₂·6H₂O), copper (CuSO₄·5H₂O), iron (FeC₆H₅O₇·5H₂O and FeSO₄·7H₂O), lead (Pb(NO₃)₂), magnesium (MgSO4·7H₂O), manganese (MnSO4·H₂O), mercury (HgCl₂) and zinc (ZnSO4·7H₂O). A bacterial suspension corresponding to McFarland standard 1 in sodium chloride (0.9%) was prepared and diluted 1:1000. This was then used to inoculate metal-containing LSM broth to obtain a final cell concentration of ~10⁵ cfu/ml. Readings were recorded after 24 h of incubation at 30°C; the MICs were taken as the lowest concentration at which growth was completely inhibited.