Gatifloxacin was obtained from LKT Laboratories, Inc. (St. Paul, MN). For the cell proliferation assay, gatifloxacin was solubilized in Dulbecco’s modified essential medium (Gibco, Grand Island, NY), which was then used as part of the culture medium described below. To evaluate minimum inhibitory concentration (MIC), gatifloxacin was solubilized in the broth as described below for the liquid culture of bacteria.

For plate culture, P. gulae ATCC 51700 was obtained from the American Type Culture Collection (Manassas, VA) and grown on plates containing tryptic soy agar (40 g·L⁻¹; Becton, Dickinson and Company, Franklin Lakes, NJ) supplemented with 10% defibrinated horse blood (Japan Bio serum Inc., Tokyo, Japan), hemin (5 g·L⁻¹; Sigma-Aldrich Corp., St. Louis, MO), and menadione (0.5 g·L⁻¹; Sigma-Aldrich Corp.) [14]. Preculture was performed in an anaerobic chamber (N₂: 80%, H₂: 10%, CO₂: 10%) at 37˚C. For liquid culture, the bacteria were cultured in trypticase soy broth (30 g·L⁻¹; Becton, Dickinson and Company) supplemented with 5 g·L⁻¹ of hemin and 0.5 g·L⁻¹ of menadione and incubated in an anaerobic chamber at 37˚C. The precultured colony was inoculated into a liquid broth and incubated for 2 - 4 days under the same conditions as those described for the plate culture.

Broths containing gatifloxacin were used to determine MIC. Eleven serial concentrations of gatifloxacin were predetermined, ranging from 0 M to 1 μM for P. gulae cultures. The strain was inoculated into the broth and then incubated for 2 - 3 days in an anaerobic chamber at 37˚C. MIC was defined as the lowest concentration of gatifloxacin that would inhibit visible growth of bacteria after incubation. To confirm the reliability of the data, the experiments were performed four times.

P. gulae strain was anaerobically grown at 37˚C until it reached the early stationary phase in the broth described above. The harvested cells were inoculated in the freshly prepared aseptic broth containing sufficient concentrations of gatifloxacin at 37˚C and examined for bacterial cell viability after 2 - 3 days. Bacterial cell viability was determined by adenosine triphosphate bioluminescence (ATP) assay using the BacTiter-Glo Microbial Cell Viability Assay kit (Promega, Madison, USA). A volume of BacTiter-Glo reagent equal to the volume of each bacterial suspension was added and briefly mixed. The luminescence of the solution was then recorded using the Gene Light Model GL-210A luminometer (Microtec Co., Ltd., Funabashi, Japan). The obtained value was expressed as a ratio to that at the start of incubation. The results were expressed as the mean ± standard deviation of the three experiments.

Rat BMMS cells (Sigma-Aldrich, Tokyo, Japan) were used in this study. The culture medium consisted of a modification of Minimum Essential Medium (MEM α) (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS French origin; Biowest, Nuaillé, France) and antibiotics. Cells subcultured at 37˚C in a humidified atmosphere with 5% CO₂ were suspended in MEM α at a concentration of 5.0 × 10⁴ cells per well and then incubated for 24 h at 37˚C in a humidified atmosphere with 5% CO₂. The medium was replaced with a medium containing gatifloxacin at concentrations adjusted by stepwise dilution. The culture medium was refreshed every 2 days. After the completion of culture, cell viability was determined using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) for 3 days. Briefly, a 10-μL aliquot of CCK-8 solution was added to each well. The cells were then incubated for 90 min. Absorbance was measured at 450 nm using a microplate reader (iMark Microplate Reader, Bio-Rad, CA, USA). The experiments were performed in triplicate. The data were expressed as survival rates.

Statistical analyses were performed using SPSS Statistics version 25 (SPSS Inc., Chicago, USA). The data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test, and probability (p) values < 0.05 were considered statistically significant.

As shown in Table 1, gatifloxacin showed a significant inhibitory effect on the growth of the canine periodontopathic bacteria strain tested. The MIC for P. gulae was 50 nM. These findings suggest that gatifloxacin exhibits selective inhibitory action against the growth of canine periodontopathic bacteria. The determination of MIC value of this drug is based on the turbidity of the bacterial fluid, although it is rather ambiguous to distinguish between viable or dead cells. In this case, statistical discussions are not appropriate. When our findings were compared to those of a recent study on the inhibitory effects of the drug against human periodontopathic bacteria P. gingivalis [14], which is genetically related to P. gulae, it was revealed that the MIC obtained in both studies were similar.

To further investigate the potential of gatifloxacin to inhibit the growth of periodontopathic bacteria, its bactericidal activity was assessed. As shown in Figure 2, gatifloxacin showed bactericidal activity in a concentration-dependent manner against the bacteria tested. The apparent growth from 0.25 nM to 5 nM was due to the reaction of the fluoroquinolone with the reagent, and the turbidity, shown in Table 1, was the same as that of the control (0 nM). Within this concentration range, the fluoroquinolone drug was considered to have little bactericidal effect. On using ANOVA and Tukey’s multiple comparison test, a highly significant difference was observed between cultures containing high drug concentrations and the control culture without the drug in terms of inhibitory action. The number of viable bacterial cells decreased significantly as the bacteria were exposed to gatifloxacin concentrations of >100 nM (p < 0.01). This data supports the possibility that exposure to gatifloxacin suppresses bacterial growth during incubation, indicating that the drug may demonstrate an inhibitory effect on the growth of canine periodontopathogen P. gulae. This drug is reported to exert its effect by inhibiting bacterial DNA gyrase and topoisomerase IV [19 , 20]. Against the bacteria tested in this paper, this drug is considered to have an adverse effect on a growth by a similar mechanism.

For a chronic canine periodontitis, a current professional dental treatment requires general anesthesia and surgical scaling [21 , 22]. The results in this paper support that the local administration of gatifloxacin is effective for a nonsurgical therapy.

As shown in Figure 3(a), gatifloxacin had no morphological effect on the proliferation of mammalian BMMS cells after a 3-day culture. Almost all seeded BMMS cells survived after a 1-day culture (Figure 3(b)), and 84.8% of the cells survived after a 3-day culture at 100 nM (Figure 3(c)), which is a concentration higher than the MIC of the bacteria tested. The fact that gatifloxacin is safe for mammalian cells after a 1-day culture suggests that it can be applied to other animals besides dogs. In studies of both Pneumococcal and Haemophilus strains, it is considered to be a drug that can clinically prevent the emergence of drug resistant bacteria (unpublished data from research of Kyorin Pharmaceutical Co. Ltd.) [19]. It is presumed that this reason is due to dual inhibition of both DNA gyrase and topoisomerase IV. The result of cell viability assay of gatifloxacin in rat cells was also strongly supported from the toxicity assay using normal dermal fibroblast cells derived from human adult connective tissue [14].