Vancomycin

H. pylori Sydney Strain (SS1) (Lee et al., 1997 (link)) was grown on Columbia agar (Difco, Detroit, MI) supplemented with 7% horse blood with the antibiotics trimethoprim (20 μg/ml), vancomycin (6 μg/ml), cefsulodin (16 μg/ml) and amphotericin B (2.5 μg/ml) (antibiotics from Sigma-Aldrich, St. Louis, MO). The H. pylori tipα mutant (Δtipα) was grown on Columbia agar plates supplemented with 7% horse blood and 50 μg/ml kanamycin (Corning, Corning, New York). Strains were grown at 37°C in a humidified in incubator with 10% CO₂. H. pylori SS1 growth in liquid culture was performed by harvesting bacteria from Columbia blood agar plates in 1 ml of Brucella Broth (Difco, Detroit, MI) and transferred to 10 ml Brucella broth supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) and 6 μg/ml vancomycin (Sigma) or 50 μg/ml kanamycin (Corning) in T-25 flasks (Corning, New York). Liquid cultures were maintained at 37°C with 10% CO₂.

All animal experiments conducted in this study were performed in compliance with the Swiss Animal Protection Ordinance and approved by the Veterinary Office of the Canton of Zurich, Switzerland. C57BL/6 8- to 10-week-old male mice were treated with neomycin (1 g L⁻¹), vancomycin (0.5 g L⁻¹), ampicillin (1 g L⁻¹) plus aspartame (0.2%, w/v, Sigma) in drinking water for 2 days. During this period, mice were gavaged daily with 150 µL of antibiotic solution containing neomycin (1 g L⁻¹), vancomycin (0.5 g L⁻¹), ampicillin (1 g L⁻¹) and metronidazole (1 g L⁻¹) plus aspartame (0.2%, w/v, Sigma). After 1 day of recovery, mice were gavaged on two consecutive days with 250 µL (5·10⁸ CFU ml⁻¹) of recombinant E. coli EHV2 suspension in PBS. Mice were then treated with 3.5% (w/v) of DSS (molecular weight 36–50 kDa, MP Biomedicals) in drinking water for 5 days, followed by a supply of normal water until euthanasia by CO₂ inhalation on day 12 or when body weight reached the threshold of 85% of the starting weight. All supplementations and DSS treatments were performed in three independent experiments.

Bacterial strains and culture conditions used in this study are summarized in Table 1. For in vitro samples, cells were collected from overnight cultures via centrifugation at 2,000g for 5 min, washed twice in PBS, and fixed in PBS containing 4% formaldehyde and 1% NP-40 for 10 min. After fixation, cells were washed in PBS and then resuspended in PBST (PBS supplemented with 0.3% Triton X-100) for 30 min at room temperature. As optical density (OD) is unreliable in determining bacterial number densities across species, we quantified relative cell number densities through spreading 10 μL of DAPI-stained suspension between a coverslip and a glass slide and counting cells in 5 images (field of view = 440 μm × 330 μm) using epifluorescence microscopy. The resuspended bacteria were sequentially dehydrated in 50:50% methanol:PBST and then pure methanol to remove lipids. Dehydrated cells can be kept at −20°C for several months.
For vancomycin treatment, E. coli imp4213 cells were grown to early stationary phase (OD₆₀₀ = 0.8) and incubated in media containing vancomycin (Sigma-Aldrich, St. Louis, MO) at 37°C before fixation. We focused on cells during the early stationary phase because we noticed large cell-to-cell variations in expansion between dividing cells during exponential growth.

Genus and species identification were performed by gram staining, catalase test, growth in the presence of 6.5% NaCl, bile-esculin test (Merck), and fermentation of arabinose, arginine, and sorbitol (Sigma Co) (16 (link)). The phenotypic identification was confirmed by PCR using species-specific primers (Table 1), and the genomic DNA of Enterococcus was extracted by boiling. PCR amplification involved initial denaturation at 95 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 30 sec, annealing at 54 °C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 7 min (17 (link)). Vancomycin-resistant isolates were recognized by growth in bile-esculin agar containing 6 μgr/ml Vancomycin (Sigma Aldrich, Germany).

Brain–heart infusion (BHI) agar plates (Difco, Becton Dickinson and Company) supplemented with 16 g/L pancreatic digest of casein (Becton Dickinson) and varying concentrations of vancomycin (Sigma Chemical Company, St. Louis, MO, USA) were used: 3 mg/L (BHI-3) and 4 mg/L (BHI-4).
These BHI–casein–vancomycin agar plates were inoculated with 4 separate 10 µL drops of 0.5 McFarland standard suspension per isolate. After drying for 5 min, plates were incubated at 35 °C at air. Bacterial growth was visually inspected at 24 and 48 h, and the number of the visible colonies in each drop was counted. A strain was considered hVISA if at least one drop showed two or more colonies after incubation; a droplet with a count of 20 or more CFU was considered as confluent growth [19 (link)].

A total of 460 vegetable and salad samples were collected from supermarkets and groceries of various parts of Iran (Table 1). The samples were processed within an hour of collection. Samples were homogenized and 25 mL of each sample was added to 225 mL of Columbia blood agar (Oxoid, UK) supplemented with 5% horse serum (Sigma, St. Louis, MO, USA) and colistinme than esulfonate (30 mg/L), cycloheximide (100 mg/L), nalidixic acid (30 mg/L), trimethoprim (30 mg/L), and vancomycin (10 mg/L) (Sigma, St. Louis, MO, USA) and incubated for seven days at 37°C with constant shaking under microaerophilic conditions. Next, 0.1 mL of the enrichment selective broth was plated onto Columbia blood agar (Oxoid, UK) supplemented with 5% of defibrinated horse blood and 30 mg/L colistinmethanesulfonate, 100 mg/L cycloheximide, 30 mg/L nalidixic acid, 30 mg/L trimethoprim, and 10 mg/L vancomycin (Sigma, St. Louis, MO, USA) (8 (link)) and incubated for seven days at 37°C under microaerophilic conditions. Suspected colonies were identified as H. pylori based on the method described by Dunn et al. (9 (link)). For comparison, a reference strain of H. pylori (ATCC 43504) was employed. The isolates were confirmed using the PCR assay.