Quantitation of NTHi inside infected EpiAirway tissues The EpiAirway tissues at the ALI (#AIR-100-ABF, MatTek, Ashland, MA USA) were infected
apically with the suspensions of either the 86-028NP parent strain, or the ΔvapBC-1, ΔvapXD, and ΔvapBC-1 ΔvapXD mutants individually at ~107 CFU per insert (n = 6). The A-1210477 mw inoculation suspensions were quantified by dilution and plating for viable colony counting. The inserts were washed and the basal MM renewed daily. On day 1, 2, 4, 6 and 8 after infection, each insert was harvested as previously described [32]. Briefly, each insert was washed with D-PBS, then 300 μl of MM with gentamicin (100 μg/ml) was added apically to Captisol each insert, with 1 ml of MM with gentamicin (100 μg/ml) added basally. After 1 h of incubation at 37°C with 5% CO2, the inserts were washed 3X with D-PBS without calcium and magnesium, and 250 μl of 1% saponin in D-PBS without calcium and magnesium was added apically
to each insert and incubated at 37°C for 10 min. Subsequently, the tissues were harvested, disaggregated and diluted to 1 ml in D-PBS. The suspensions were then diluted serially and plated onto chocolate agar plates for bacterial CFU counts. NTHi survival in the chinchilla otitis media model Healthy female adult (400–600 g) chinchillas were purchased from a commercial supplier and handled in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the
National Institutes of Health. The protocol was approved by the Mercer University Institutional Animal Care and Use Committee (Assurance Number: AZD4547 order A3725-01). All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize suffering. Animals were allowed to acclimate to the vivarium for 1 week prior to challenge, and none had any visible signs of middle ear infection as detected by otoscopy. The 86-028NP parent strain and the ΔvapBC-1, ΔvapXD, and ΔvapBC-1 ΔvapXD mutants were recovered from frozen stocks and cultured for 18 h on chocolate agar at 37°C with 5% CO2. The bacteria were harvested, suspended in D-PBS containing 0.1% gelatin (D-PBSG), loaded into tuberculin syringes, and maintained on ice for the challenges. Chinchillas were anesthetized by isoflurane inhalation and each Liothyronine Sodium middle ear was injected transbullarly with 100 μl (~ 1000 CFU) of bacteria (n = 4 to 5 animals with 8 to 10 middle ears per challenge strain) or D-PBSG alone (control). Actual challenge doses were confirmed by plating followed by colony counting. On day 4 post-challenge, the animals were euthanized by cardiac exsanguination and their superior bullae were opened. Middle ear fluid was recovered, and each middle ear was washed with 1.0 ml of D-PBSG. An aliquot of each middle ear wash was diluted serially and plated on chocolate agar for CFU counts.
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