Horse blood agar plates

H. pylori bacterial strains 60190 (ATCC 49503, CagA+/VacA+), 26695 (ATCC 700392, CagA+/VacA−), and Tx30a (ATCC 51932, CagA−) were obtained from the American Type Culture Collection (Manassas, VA, USA) and then treated. Bacteria were cultured on 5% horse blood agar plates (Oxoid Ltd, Basingstoke, UK) in humidified incubators with 5% CO₂ at 37 °C. The H. pylori strains (both CagA+ and CagA−) were inoculated into Brucella broth containing 5% FBS under microaerophilic conditions (5% O2, 10% CO2 and 85% N2) at 37 °C. For H. pylori infection, SNU1, AGS, MGC-803, and MKN1 cells were seeded into six-well plates with antibiotic-free cell culture medium and cultured to a confluency of 80–90%. The H. pylori bacterium was harvested, resuspended with phosphate-buffered saline (PBS) and added to the gastric cancer cells at a multiplicity of infection ratio of 100:1 as previously described^{35 (link)}. The H. pylori-infected gastric cancer cells were incubated for either 6, 12, 24, 36, or 72 h, and then isolated.

H pylori bacterial strains 60190 ((ATCC 49503, CagA+) and Tx30a (ATCC 51932, CagA−) were got from American Type Culture Collection. Bacteria were routinely cultured on 5% horse blood agar plates (Oxoid Ltd, Basingstoke, UK) in humidified incubators, which provided an atmosphere of 5% CO₂ at 37°C. GC cells were trypsinized, resuspended in normal growth medium and seeded into 6 well plates. Three duplicate wells were prepared for each experimental condition. When the cells reached 70% confluence they were serum-starved for 24 h prior to the addition of HP at a multiplicity of infection of 100. Cells were co-cultured with the bacteria for 24 h before either RNA or DNA extraction or protein measurements were performed.

The study included all pneumococcal isolates identified at Landspítali in 2011–2017, except isolates from nasopharynx and throat. Repeat isolates of the same phenotype (same serotype and antibiogram) from the same patient within 30 days were excluded as they were considered to represent the same infection. The pneumococci were cultured and identified from routine patient specimens using conventional methods, i.e. plated on two 5% horse blood agar plates (Oxoid, Hampshare, UK), one incubated in a 5% CO₂ and the other one anaerobically. Identification was done using optochin test (and bile solubility if unclear). All isolates from 2016–2017 were also identified with MALDI-Tof.
The patients were divided into five age groups: 0–1, 2–6, 7–17, 18–64 and ≥65 years old. The isolates were grouped according to the sampling site as follows: middle ear (ME: swabs and pus from middle ear, or otorrhoea), lower respiratory tract (LRT: sputum, bronchiolar lavage, pleural fluid), sterile body fluids (SBF: blood, cerebrospinal fluid and joint fluid) and other sampling sites (OSS: mostly specimens from conjunctiva and sinuses).