2.2. Antibacterial Activity

Methods for Dilution Antimicrobial Susceptibility testing developed in 2006 is the official method used in many clinical microbiology laboratories for routine antimicrobial susceptibility testing. Nowadays, many accepted and approved standards are published by the Clinical and Laboratory Standards Institute (CLSI) for bacteria and yeast testing.

Although not all fastidious bacteria can be tested accurately by this method, the standardization has been made to test certain fastidious bacterial pathogens like Streptococci, Haemophilus influenzae, Haemophilus parainfluenzae, Neisseria gonorrhoeae and Neisseria meningitidis, using specific culture media, various incubation condition [24,25]. E. faecalis, E. faecium, S. epidermidis and S. aureus were maintained in tryptic soy broth (TSB). S. mutans was maintained in brain heart infusion broth (BHI) and incubated at 37 °C for 24 h. A. viscosus was maintained in tryptic soy broth (TSB), S. sobrinus was maintained brain heart infusion (BHI), F. nucleatum was maintained in reinforced clostridial (RCM), P. gingivalis were cultured in tryptic soy broth (TSB) at 37 °C for 24–72 h in anaerobic conditions (10% H₂, 10% CO₂, and balanced N₂) and then transferred to a 96-well plate of 0.5 McFarland standard. Serial twofold dilutions of compounds were prepared in the TSB, RCM or BHI. The MIC value was defined as the lowest compound concentration required to inhibit bacterial growth visibly after the indicated time. Oxytetracycline was used as a positive control.

The minimum bactericidal concentration was obtained using a microdilution assay according to the standard with little modification. ⁵ After bacteria were cultured in the same manner as in the MIC test, bacterial cultures were inoculated onto the agar plates and incubated at 37 °C for 24–72 h, and the bacterial colonies were counted. The lowest concentration of compound that inhibited the growth of bacteria was considered to be the minimum bactericidal concentration. The experimental and control groups were assigned to the wells in triplicate.

Broth inocula with 0.5 McFarland standard were spread onto the sterile plate by cotton swabs. P. gingivalis was cultured in tryptic soy broth supplemented with 10% defibrinated horse blood and brain heart infusion (BHI) at 37 °C for 24 h in anaerobic conditions (10% H₂, 10% CO₂, and balanced N₂). Then, 8-nm sterile paper discs were impregnated with KHQ 711–718 100 μg/mL was added, and the sizes of the inhibition zones were measured after 24 h [26,27].

The growth curve was obtained using the previously described 96-well microplate. P. gingivalis was cultured in tryptic soy broth (TSB) at 37 °C for 24–72 h in anaerobic conditions (10% H₂, 10% CO₂, and balanced N₂). KHQ 711–718 MIC and ½ MIC concentrations were added, and then the cells were incubated at 37 °C. Growth was evaluated by measuring OD₆₀₀ using a microplate reader at time zero and after 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66 and 72 h of incubation [28].