2 thiophenecarboxylic acid hydrazide

Mice were euthanized at 3, 6, 9, 12 or 20 weeks by CO₂ asphyxiation, after H37Ra challenge. Then, the lungs of the individual mice were homogenized and serially diluted in PBS. The number of viable bacteria was determined on 7H10 agar supplemented with 10% OADC, 10 μg/mL amphotericin B (Sigma) and 2 μg/mL 2-thiophenecarboxylic acid hydrazide (Sigma). The bacterial numbers were counted after 2–3 weeks of incubation at 37 ℃. The protective efficacies are expressed as the log10 reduction in the bacterial count in immunized mice compared with the bacterial counts in each group.

At 5 and 10 weeks after the HN878 challenge, five mice per group were euthanized with CO₂, and the lungs and spleens were homogenized. The number of viable bacteria was determined by plating serial dilutions of the organ (left lung or half spleen) homogenates onto Middlebrook 7H11 agar (Difco Laboratories, Detroit, MI) supplemented with 10% OADC (Difco Laboratories), amphotericin B (Sigma-Aldrich, St. Louis, MO) and 2 μg/ml 2-thiophenecarboxylic acid hydrazide (Sigma-Aldrich). Colonies were counted after 2–3 weeks of incubation at 37°C. For the histopathological analysis, the superior lobes of the right lung were stained with hematoxylin and eosin and assessed for the severity of inflammation. The level of inflammation in the lungs was evaluated using the ImageJ (National Institutes of Health, Bethesda, ML) software program, as described previously [32 (link)]. In addition, the inflammatory responses were assessed based on lesion size and constitution of immune cells.

Disease phenotype and severity were evaluated through histopathology and by measuring bacterial growth in the lung and spleen. To determine how well they had been protected, the organs were removed 30 d after infection. For lung histopathology, the right superior lobes were fixed overnight in 10% formalin and embedded in paraffin. Lungs were sectioned at a thickness of 4–5 μm and stained with H&E. For bacterial growth analysis, the lung and spleen were homogenized, and serially diluted samples were plated onto Middlebrook 7H10 agar plates (Becton Dickinson, Franklin Lakes, NJ) supplemented with 10% OADC (Difco Laboratories, Detroit, VA), 2 μg/ml 2-thiophenecarboxylic acid hydrazide (Sigma-Aldrich, St. Louis, MO) and amphotericin B (Sigma-Aldrich). After 4 weeks of incubation at 37 °C, bacterial colonies were counted. Data are presented as log₁₀ colony-forming units (CFU) per organ.

Sixteen weeks after the HN878 challenge, six to seven mice per group were euthanized by CO₂ asphyxiation, and the lungs and spleens were homogenized. The number of viable bacteria was determined by plating serial dilutions of the organ (left lung or half spleen) homogenates onto Middlebrook 7H11 agar (Difco Laboratories, Detroit, MI) supplemented with 10% OADC (Difco Laboratories), amphotericin B (Sigma-Aldrich, St. Louis, MO) and 2 μg/ml 2-thiophenecarboxylic acid hydrazide (Sigma-Aldrich). Colonies were counted after 4 weeks of incubation at 37°C. For the histopathological analysis, the superior lobes of the right lung were stained with H&E and assessed for the severity of inflammation. The level of inflammation in the lungs was evaluated using the ImageJ software (National Institutes of Health, Bethesda, MD), as described previously [56 (link)]. In addition, the inflammatory responses were assessed based on lesion size and constitution of immune cells. The data on CFUs and assessment of lung inflammation are reported as the median log₁₀ CFU ± interquartile range (IQR).

Mice were infected under strict barrier conditions in a BSL-3 facility at the Avison Biomedical Research Center, Yonsei College of Medicine. Briefly, mice were challenged with the pre-calibrated Mtb H37Rv (ATCC 27294) via aerosol using an airborne infection apparatus (Glas-Col, USA), and ~200 viable bacteria were delivered. Mice at 4 weeks post-infection were used. For the bacterial growth analysis, the lungs were homogenized, and serially diluted samples were plated onto Middlebrook 7H11 agar plates (Becton Dickinson, USA) supplemented with 10% OADC (oleic acid albumin dextrose catalase; Difco Laboratories, USA), 2 μg/mL 2-thiophenecarboxylic acid hydrazide (Sigma-Aldrich, USA), and amphotericin B (Sigma-Aldrich). The bacterial colonies were counted after 3–4 weeks of incubation period at 37°C. The animal ethics committee of Yonsei University College of Medicine approved all of the experimental protocols used (2016-0178 and 2018-0218).