Effect of azithromycin on a red complex polymicrobial biofilm

Abstract

Azithromycin has recently gained popularity for the treatment of periodontal disease, despite sparse literature supporting efficiency in treating periodontal bacterial biofilms. The aim of this study was to evaluate the effect of azithromycin on biofilms comprised of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia in comparison to an amoxicillin and metronidazole combination. P. gingivalis W50, T. denticola ATCC35405, and T. forsythia ATCC43037 grown under anaerobic conditions at 37°C were aliquoted into 96-well flat-bottom plates in different combinations with addition of azithromycin or amoxicillin + metronidazole at various concentrations. For the biofilm assay, the plates were incubated at 37°C anaerobically for 48 h, after which the biofilms were stained with crystal violet and measured for absorbance at AU₆₂₀. In this model, polymicrobial biofilms of P. gingivalis + T. denticola, P. gingivalis + T. forsythia, and T. denticola + T. forsythia were cultured. Combination of all three bacteria enhanced biofilm biomass. Azithromycin demonstrated a minimal biofilm inhibitory concentration (MBIC) of 10.6 mg/L, while the amoxicillin + metronidazole combination was more effective in inhibiting biofilm formation with a MBIC of 1.63 mg/L. Polymicrobial biofilm formation was demonstrated by combination of all three red complex bacteria. Azithromycin was ineffective in preventing biofilm formation within a clinically achievable concentration, whereas the combination of amoxicillin and metronidazole was more effective for this purpose.

Keywords: Red complex bacteria; antibiotic therapy; azithromycin; biofilm; periodontal disease.

Figure 1.

Formation of mono- and polymicrobial biofilms in a 96-well plate model after 48 h of incubation at 37°C under anaerobic condition. Native bacterial growth with addition of uncultured growth medium and no antibiotic served as controls. Adherent biofilms were stained with 0.1% crystal violet and the optical density at AU₆₂₀ was measured. Data represent the mean AU₆₂₀ value of a minimum of three biological replicates.

Figure 2.

Effect of azithromycin up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Azithromycin at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h under anaerobic conditions. Data points represent the mean AU₆₂₀ value of a minimum of three biological replicates. Note the categorical scale.

Figure 3.

Effect of amoxicillin + metronidazole up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Amoxicillin + metronidazole in a 1:1 ratio at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h at 37°C anaerobically. Data points represent the mean AU₆₂₀ value of a minimum of three biological replicates. Note the categorical scale.

Figure 4.

Effects of azithromycin and amoxicillin + metronidazole (1:1 ratio) up to 5.0 mg/L on formation polymicrobial biofilms after 48 h of anaerobic incubation at 37°C in a 96-well plate model. Azithromycin and amoxicillin + metronidazole (1:1 ratio) at concentrations 0–100 mg/L were incubated with bacterial cultures. Data points represent the mean AU₆₂₀ value of a minimum of three biological replicates and the standard deviation. *p < 0.05, Student’s paired t-test.