Antibiofilm Effects of Azithromycin and Erythromycin on Porphyromonas gingivalis

Abstract

Antibiotic resistance of biofilm-grown bacteria contributes to chronic infections, such as marginal and periapical periodontitis, which are strongly associated with Porphyromonas gingivalis. Concurrent azithromycin (AZM) administration and mechanical debridement improve the clinical parameters of periodontal tissue in situ. We examined the in vitro efficacy of AZM against P. gingivalis biofilms. The susceptibilities of adherent P. gingivalis strains 381, HW24D1, 6/26, and W83 to AZM, erythromycin (ERY), ampicillin (AMP), ofloxacin (OFX), and gentamicin (GEN) were investigated using a static model. The optical densities of adherent P. gingivalis cells were significantly decreased by using AZM and ERY at sub-MIC levels compared with those of the controls in all the strains tested, except for the effect of ERY on strain W83. AMP and OFX inhibited P. gingivalis adherent cells at levels over their MICs, and GEN showed no inhibition in the static model. The effects of AZM and ERY against biofilm cells were investigated using a flow cell model. The ATP levels of P. gingivalis biofilms were significantly decreased by AZM at concentrations below the sub-MICs; however, ERY was not effective for inhibition of P. gingivalis biofilm cells at their sub-MICs. Furthermore, decreased density of P. gingivalis biofilms was observed three-dimensionally with sub-MIC AZM, using confocal laser scanning microscopy. These findings suggest that AZM is effective against P. gingivalis biofilms at sub-MIC levels and could have future clinical application for oral biofilm infections, such as chronic marginal and periapical periodontitis.

Inhibition of polysaccharide synthesis by the sinR orthologue PGN_0088 is indirectly associated with the penetration of Porphyromonas gingivalis biofilms by macrolide antibiotics

“…All bacterial strains used in this study are listed in Table 1. Similar to our previous studies (Yamaguchi et al, 2010;Yamamoto et al, 2011Yamamoto et al, , 2013, P. gingivalis cells were grown anaerobically (10 % CO 2 , 10 % H 2 and 80 % N 2 ) in Gifu anaerobic medium (GAM; Nissui) on Soybean-Casein-Digest agar (SCD agar; Nissui). For selection and maintenance of antibioticresistant strains, antibiotics were added to the medium at concentrations as follows: 100 mg ampicillin ml 21 ; 20 mg chloramphenicol ml 21 ; 200 mg gentamicin ml 21 ; or 0.7 mg tetracycline ml 21 .…”

“…We determined the MICs and MBICs for AZM (Tocris Bioscience) and ERY (Wako Pure Chemical Industries) as previously reported (Maezono et al, 2011). Briefly, to measure macrolide MICs for planktonic P. gingivalis, we dispensed 100 ml of culture into 96-well U-bottom microplates (Becton Dickinson).…”

“…The epsA-O and yqxM-sipWtasA operons are controlled by the repressor SinR (Chu et al, 2006;Lopez et al, 2009;Winkelman et al, 2009). We revealed previously that the number of genes that are expressed .1.5-fold was highest at the later stage of biofilm formation by P. gingivalis ATCC 33277 (Yamamoto et al, 2011). The gene encoding PGN_0088 (a sinR orthologue, which inhibits carbohydrate synthesis in P. gingivalis biofilms) was down-regulated by the greatest extent (Table S1, available in the online Supplementary Material).…”

“…Furthermore, although chlorhexidine gluconate effectively reduces the viability of P. gingivalis in biofilms (Noiri et al, 2003), EPS remaining after chlorhexidine treatment serve as a scaffold for the attachment of bacterial cells to form a new biofilm . In contrast, subMIC levels of AZM and ERY inhibit P. gingivalis biofilms formed using static models, and AZM inhibits flow-cell biofilms (Maezono et al, 2011). Maezono et al (2011) used confocal laser scanning microscopy (CLSM) to demonstrate that treatment of flow-cell biofilms with subMIC levels of AZM reduces the adherence of P. gingivalis, although dead cells were undetected.…”

“…In contrast, subMIC levels of AZM and ERY inhibit P. gingivalis biofilms formed using static models, and AZM inhibits flow-cell biofilms (Maezono et al, 2011). Maezono et al (2011) used confocal laser scanning microscopy (CLSM) to demonstrate that treatment of flow-cell biofilms with subMIC levels of AZM reduces the adherence of P. gingivalis, although dead cells were undetected. Therefore, subMIC levels of AZM may not influence cell survival but may influence the maintenance of the biofilm structure.…”