Iso sensitest

Susceptibility to ampicillin, chloramphenicol, clindamycin, erythromycin, gentamycin, kanamycin, metronidazole, nitrofurantoin, norfloxacin, streptomycin, tetracycline, trimethoprim/sulfamethoxazole, and vancomycin was assessed according to the European Union Commission recommendations for probiotic safety (16 , 17 ).
The minimum inhibitory concentrations (MICs) were determined by the Etest^® method (BioMérieux, Marcy l'Etoile, France). The assay was performed by suspending individual colonies picked up from fresh cultures on LSM agar (90% Iso-Sensitest (Oxoid Ltd., Basingstoke, UK) and 10% MRS (Oxoid)) plates incubated for 24 h at 37°C in an anaerobic glove chamber (Concept 400, Biotrace, Bridgend, UK) to McFarland standard 1 in sterile 0.85% NaCl solution. The suspension was swabbed in three directions on LSM agar and allowed to dry before applying the Etest strips (18 (link), 19 (link)). Plates were incubated at 37°C for 48 h under anaerobic conditions before final results were recorded. MICs were read directly from the test strip according to the instructions of the manufacturer. Strains showing MICs less than EFSA's breakpoints were considered sensitive; otherwise, they were considered resistant (16 , 17 ).

After 1, 3, or 6 weeks, mice were euthanized. Stomachs were carefully halved longitudinally along the greater and lesser curvatures and rinsed in sterile phosphate-buffered saline. Half of each stomach was manually disrupted on ice in 750 µL of Iso-Sensitest (Oxoid, Basingstoke, UK) broth/15% glycerol, serially diluted, and plated on Galaxo Selective Supplement A plates (blood agar base 2 (Oxoid) plates supplemented with 10% defibrinated horse blood, 10 µg/mL vancomycin, 20 µg/mL bacitracin, 4 µg/mL amphotericin B, 2.5 IU/mL polymyxin B sulfate and 1.07 µg/mL nalidixic acid). The other half of the stomach was formalin fixed, paraffin embedded and sectioned, then stained with hematoxylin-eosin or periodic acid–Schiff/Alcian blue. The hematoxylin-eosin–stained sections were scored for severity of inflammation and spasmolytic polypeptide-expressing metaplasia (SPEM) by the same qualified histopathologist (P. V. K.) in a blinded manner. Sections were assigned scores of 0 (absent), 1 (mild), 2 (moderate), or 3 (severe). Because inflammation and SPEM were patchy in the mouse gastric mucosa after 1–6 weeks of infection, the length of each was measured and normalized to the total length of corpus examined, to quantify the extent of inflammation and SPEM for each mouse.