Mueller hinton

Samples were collected from the Oslo Fjord at the shore near a small village called Hvitsten. Samples from rocks, seaweed, sand, mud and seawater were spread onto BHI, LB, TH and Mueller–Hinton agar (Becton, Dickinson and Company) and incubated at room temperature for 5 days to obtain bacterial colonies. A volume of 100 µl S. pneumoniae culture with OD₅₅₀=0.3 was added to 5 ml of melted TH soft agar [0.75 % (w/v) agar] holding 47 °C. The soft agar was then mixed by vortexing for 2 s, before it was gently spread on top of colonies formed by the marine bacteria as described above. After anaerobic incubation at 37 °C overnight, the plates were inspected for colonies surrounded by inhibition zones. For detection of inhibition zones surrounding Lysinibacillus sp. OF-1 colonies, the same protocol with TH soft agar was used for all streptococcal indicator species. BHI soft agar was used when P. brenneri, B. subtilis, S. aureus and M. smegmatis were the indicators, while LB soft agar was used for E. coli and GM17 soft agar for L. lactis and E. faecalis.

Three ATCC reference strains for ETEST FO and four ATCC reference strains for AD, as recommended by CLSI M100-Ed31 (2021) (19 ) and EUCAST v11.0 QC table (4 ), were tested as quality controls on each day of comparative testing on Mueller-Hinton (Becton, Dickinson and Company; Franklin Lakes, USA). The following ATCC strains were tested with ETEST FO: Enterococcus faecalis ATCC 29212 (range CLSI 32 to 128 μg/mL), Staphylococcus aureus ATCC 29213 (range CLSI/EUCAST 0.5 to 4 μg/mL), Escherichia coli ATCC 25922 (range CLSI/EUCAST 0.5 to 2 μg/mL). Pseudomonas aeruginosa ATCC 27853 (range CLSI/EUCAST 2 to 8 μg/mL) was solely used for AD.

Mueller-Hinton (Beckton-Dickinson, Sparks, MD, USA) and LB-plates (Merck, Darmstadt, Germany) were prepared with 13 g/l agar (Merck), and RH-plates were made by autoclaving 100 ml milliQ water with 7.5 g agar, cooling it to 55°C and mixing with 500 ml modified RPMI-medium (Sigma, R7388) prewarmed to 55°C. +U, +C, and +Mg, indicates addition of 20 mg/l uracil, 10 mg/l chloramphenicol, and 5 mM MgCl₂, respectively.

All C. jejuni strains were routinely grown at 37°C on Mueller-Hinton (MH, Becton Dickinson) agar plates supplemented with 10 μg/ml vancomycin for 1–2 passages in a HERAcell 150i incubator (ThermoFisher Scientific) in a microaerobic environment (10% CO₂, 5% O₂, 85% N₂). Agar plates were further supplemented with marker-selective antibiotics (20 μg/ml chloramphenicol, 250 μg/ml hygromycin, 50 μg/ml kanamycin) for selection of transformed clones. Bacteria were then transferred to Brucella Broth (BB, Becton Dickinson) liquid cultures in T25 flasks (Corning) by inoculation from plate to a final OD₆₀₀ of 0.005 and grown under agitation at 140 rpm and 37°C.

The bacterial strains used in this study are listed in Table 1. Unless otherwise indicated, bacteria were grown for 16 hours in Mueller-Hinton (MH) (Becton, Dickinson, Franklin Lakes, NJ) medium at 37°C on a rotary shaker at 225 rotations per minute (rpm) and then used to inoculate (1:100) fresh MH, fresh human serum (MP Biomedicals, Solon, OH) or bovine pulmonary surfactant (Infasurf, ONY, Amherst, NY) medium and processed, as described below.

Metronidazole, ciprofloxacin, clindamycin, neomycin, and ampicillin were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Vancomycin was acquired from MSD & Co., Inc. (Hangzhou, China). Luria-Bertani (LB) medium was purchased from Thermo Fisher Scientific (Shanghai, China). Mueller-Hinton (MH) and brain heart infusion (BHI) media were acquired from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). ¹⁸F-FDG and ¹⁸F were obtained from the Department of Nuclear Medicine, First Affiliated Hospital of Xiamen University. Staphylococcus aureus RN450 and Escherichia coli BW25113 were from frozen stocks of the Laboratory of Microbial Pathogens, Xiamen University. Probiotics (30 billion CFU/capsule; Island’s Miracle, USA) were purchased from Amazon. Female C57BL/6 mice and male hamsters were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), and housed at the Laboratory Animal Center of Xiamen University.