Chocolate 2 agar

Intracellular growth of NVF1- and KU-1-passaged variants were assessed as reported previously [15 (link)]. J774.1 cells (RCB0434; RIKEN Bioresource Center, Ibaraki, Japan) were propagated in RPMI1640 medium (Wako Pure Chemicals; Osaka, Japan) containing 10% (v/v) heat inactivated fetal bovine serum (FBS) at 37 °C in 5% CO₂. The cells were grown on 24 well plates (1–2 × 10⁵ cells per well) and were infected with the bacteria at a multiplicity of infection (moi) of 100. This point was designated as time 0. After incubation for 1 h, the cells were washed and fresh 1 mL of 5% FBS-RPMI1640 with gentamicin (10 μg/mL) was added into the wells. The CFU of intracellular bacteria in the J774.1 cells were measured at 2- and 24-h post inoculation (hpi) of bacteria. The cells were washed twice with PBS and were treated with 100 μL of 1% (w/v) saponin in distilled water for 5 min to lyse the cells [28 (link)]. To measure viable bacteria, the lysed cells were serially diluted with saline and 20 μL aliquots of each dilution were spotted onto Chocolate II agar (BD). Bacterial enumeration was performed with triplicated samples at least twice independent experiment.

The SCHU P0 strain of F. tularensis subsp. tularensis (SCHU P0) was kindly provided by Dr. H. Fujita (Ohara Research Laboratory, Ohara General Hospital, Fukushima, Japan). Its precise passage history is unknown. On arrival, bacteria were cultured on chocolate II agar (Becton Dickinson, Sparks, MD, USA) at 37°C for 3 days, resuspended in saline containing 10% glycerol, and stored at −80°C until use. All work with live bacterial cultures was performed in a biosafety level 3 facility, in accordance with the regulations stipulated by the National Institute of Infectious Diseases (NIID), Japan.

Figure 1A illustrates the scheme for SCHU P0, P5, and P9 isolation. SCHU P0 was cultured to confluence for 3 days on chocolate II agar (Becton Dickinson and Co., Cockeysville, MD) from its glycerol stock and resuspended in saline. Three C57BL/6J mice were inoculated intraperitoneally with 5×10⁶ CFU ml⁻¹ of SCHU suspension and mice were sacrificed 4 days after infection. Three spleens obtained from the infected mice were homogenized. One hundred microlitter of 10% spleen homogenate was plated on chocolate II agar, and cultured for 3 days. After cultivation, the bacteria grown confluently on the plate were suspended with 1 ml of chemically defined medium (CDM). Subsequent passages were performed similarly. When mice demonstrated severe clinical signs or >20% weight loss, they were humanely sacrificed by isoflurane inhalation. If the mice did not show apparent clinical signs, they were sacrificed 4 days after infection. The inoculation dose and CFU in spleen at 4 days post-inoculation in each passage are shown in figure 1A. Spleen homogenates prepared at the 5th and 9th passages were cultured on chocolate II agar. Single colonies were then isolated and designated as SCHU P5 and P9, respectively. Bacterial stocks were prepared by cultivating respective strains in CDM at 37°C for 24 h and stored in CDM containing 10% glycerol at −80°C until use.

The FA1090 (ATCC 700825) strain of N. gonorrhoeae was obtained from the American Type Culture Collection (ATCC) (Manassas, VA). GC agar base, Chocolate II agar, dried bovine hemoglobin, brucella broth and IsoVitaleX were obtained from Becton, Dickinson, and Company (Cockeysville, MD). Heart infusion agar was obtained from Hardy Diagnostics (Santa Maria, CA). Yeast extract and dextrose were obtained from Fisher Bioreagents (Fair Lawn, NJ). Auranofin, hematin, pyridoxal, and nicotinamide adenine dinucleotide (NAD) were obtained from Chem-Impex International (Wood Dale, IL). Protease peptone and VCNT supplement were purchased from Oxoid (Lenexa, KS). Phosphate-buffered saline (PBS) was obtained from Corning (Manassas, VA). Ceftriaxone, azithromycin and saponin were obtained from TCI America (Portland, OR) Tween 80 was obtained from Acros Organics (Fair Lawn, NJ). Estradiol pellets (5-mg, 21-day controlled-release) were purchased from Innovative Research of America (Sarasota, FL). Dacron swabs were purchased from the Medical Packaging Corporation (Camarillo, CA).

Neisseria gonorrhoeae clinical isolates (Table S1) used in this study were obtained from the CDC. S. aureus, MRSA, E. faecalis, E. faecium, and L. monocytogenes strains were obtained from the American Type Culture Collection (ATCC). E. coli BW25113 and JW25113 were obtained from the Coli Genetic Stock Center (CGSC), Yale University, USA. Media and reagents were purchased from commercial vendors: Brucella broth, chocolate II agar, cation-adjusted Mueller Hinton broth, tryptic soy broth (TSB), and tryptic soy agar (TSA) (Becton, Dickinson and Company, Cockeysville, MD, USA); yeast extract and dextrose (Fisher Bioreagents, Fairlawn, NJ, USA), proteose-peptone, nicotinamide adenine dinucleotide (NAD), agarose and tetracycline (Sigma-Aldrich, St. Louis, MO, USA); hematin, Tween 80, pyridoxal, linezolid and gentamicin sulfate (Chem-Impex International, Wood Dale, IL, USA); Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and phosphate-buffered saline (PBS) (Corning, Manassas, VA, USA); and azithromycin (TCI America, Portland, OR, USA). Human colorectal adenocarcinoma epithelial cells (Caco-2) (ATCC HTB-37) was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Compounds were synthesized from commercial sources in our laboratory.