Intracellular Azithromycin Accumulation Suppresses Porphyromonas Gingivalis in Infected Gingival Fibroblasts

Objectives: Porphyromonas gingivalis (Pg) can invade gingival fibroblasts and epithelium, allowing it to evade the host immune system and re-colonize pockets after debridement. Azithromycin (AZM) is active against Pg and is actively accumulated inside human cells. We previously demonstrated that AZM accumulation helps eliminate Aggregatibacter actinomycetemcomitans from infected gingival epithelial monolayers. In this study, we used a similar approach to determine whether AZM is as effective as amoxicillin (AMX) plus metronidazole (MET) at eliminating infections by Pg strain W83 from cultured gingival fibroblasts.
Methods: Cultured gingival fibroblast monolayers were infected with Pg W83 for 90 minutes at a 100:1 multiplicity of infection. Extracellular bacteria were killed by treatment with teicoplanin (100µg/ml). Infected cells were then incubated for 4 hours with therapeutic concentrations of AMX (4µg/ml), AMX (4µg/ml) + MET (10µg/ml) or AZM (8µg/ml). Under these conditions, the intracellular concentration of AZM was 87µg/ml. Previous studies indicate that AMX and MTZ do not concentrate inside cells. Infected cells treated in the absence of any antibiotic served as the negative control while moxifloxacin (3.2µg/ml) served as the positive control. Viable intracellular Pg were released by cell lysis and plated on reduced blood agar plate for enumeration. Antimicrobial activity against planktonic Pg was also evaluated.
Results: After treatment with AZM, survival of Pg W83 inside fibroblasts was 37.8 ± 5.22% compared to the negative control, while survival was essentially unchanged after treatment with AMX or AMX+MET. AZM killed significantly more Pg than the other regimens (p<0.01). With planktonic Pg, survival after four hours of treatment with AZM, AMX or AMX+MET was 55.9 ± 10.5%, 84.9 ± 1.87% and 84.2 ± 2.42%, respectively.
Conclusions: Intracellular accumulation helps AZM eliminate Pg W83 from infected gingival fibroblasts. In contrast, AMX or MET appear to be less effective against Pg-infected fibroblasts than against planktonic bacteria.