Oxantel disrupts polymicrobial biofilm development of periodontal pathogens

Abstract

Bacterial pathogens commonly associated with chronic periodontitis are the spirochete Treponema denticola and the Gram-negative, proteolytic species Porphyromonas gingivalis and Tannerella forsythia. These species rely on complex anaerobic respiration of amino acids, and the anthelmintic drug oxantel has been shown to inhibit fumarate reductase (Frd) activity in some pathogenic bacteria and inhibit P. gingivalis homotypic biofilm formation. Here, we demonstrate that oxantel inhibited P. gingivalis Frd activity with a 50% inhibitory concentration (IC50) of 2.2 μM and planktonic growth of T. forsythia with a MIC of 295 μM, but it had no effect on the growth of T. denticola. Oxantel treatment caused the downregulation of six P. gingivalis gene products and the upregulation of 22 gene products. All of these genes are part of a regulon controlled by heme availability. There was no large-scale change in the expression of genes encoding metabolic enzymes, indicating that P. gingivalis may be unable to overcome Frd inhibition. Oxantel disrupted the development of polymicrobial biofilms composed of P. gingivalis, T. forsythia, and T. denticola in a concentration-dependent manner. In these biofilms, all three species were inhibited to a similar degree, demonstrating the synergistic nature of biofilm formation by these species and the dependence of T. denticola on the other two species. In a murine alveolar bone loss model of periodontitis oxantel addition to the drinking water of P. gingivalis-infected mice reduced bone loss to the same level as the uninfected control.

FIG 1

Correlation of fold changes for the genes upregulated by oxantel treatment and heme limitation. Both heme limitation and oxantel treatment were achieved by using P. gingivalis cells growing in anaerobic, planktonic continuous culture at identical growth rates. The symbols represent the genes shown as upregulated in Table 4.

FIG 2

Effect of oxantel on polymicrobial biofilm development. Polymicrobial biofilms composed of P. gingivalis (gray bars), T. denticola (white bars), and T. forsythia (black bars) were cultured in OBGM in 12-well flat-well plates for 48 h in the presence or absence of oxantel. The polymicrobial biofilm was removed from the substratum after being washed and resuspended in OBGM prior to bacterial cell numbers being determined by real-time PCR. The data points represent the mean and standard deviation of five replicates. A one-way ANOVA with Dunnett's test showed that the numbers of bacterial cells for each species were significantly different (P < 0.05) between the three oxantel concentrations.

FIG 3

Effect of oxantel on a polymicrobial biofilm composed of P. gingivalis, T. forsythia, and T. denticola cultured in OBGM in a static assay for 48 h. Total (dark gray bars), live (light gray bars), and dead (white bars) cells were determined by flow cytometric analysis of LIVE/DEAD-stained cells. A one-way ANOVA with Dunnett's test showed that the numbers of total and live bacterial cells were significantly different (P < 0.05) between the three oxantel concentrations. There was no significant difference in dead bacterial cell numbers for the three oxantel concentrations.

FIG 4

Effect of oxantel and amoxicillin treatment on alveolar bone loss in mice orally infected with P. gingivalis. Three groups of mice were orally infected with P. gingivalis W50 prior to addition of amoxicillin or oxantel to the drinking water, while the remaining three groups were sham inoculated. Mice were killed 29 days after the last oral inoculation and 46 days after the start of amoxicillin or oxantel treatment. The maxillae were removed and defleshed, and alveolar bone loss was determined in the right-hand maxillae by computer-assisted image analysis (43). The data are presented as means plus standard deviations (n = 12) and were analyzed by one-way ANOVA with Dunnett's test. Values that were significantly different (P < 0.05) from the value for the group inoculated with the P. gingivalis W50 wild-type strain are indicated by an asterisk.