Effects of sub-minimal inhibitory concentrations of antibiotics on the morphology and surface hydrophobicity of periodontopathic anaerobes.

It has been reported that sub-minimal inhibitory concentrations (sub-MICs) of antibiotics are capable of altering bacterial surface properties and phenotype. In this study, the effects of sub-MICs of certain antibiotics on surface hydrophobicity, cell morphology, and protein profile were ascertained using Fusobacterium nucleatum, Porphyromonas gingivalis and Treponema denticola strains, which are pathogenic bacterial species in periodontal diseases. The MICs of antibiotics were determined by culturing bacteria in media supplemented with serially diluted antibiotic solutions, and sub-MIC of antibiotics was used. The effect of sub-MIC of antibiotics on cell morphology was determined by scanning electron microscopy. Microscopic observation of F. nucleatum and P. gingivalis grown at a sub-MIC of amoxicillin revealed cell enlargement. T. denticola grown at a sub-MIC of doxycycline also showed cell elongation. The relative surface hydrophobicity determined by measuring the ability of the bacteria to absorb n-hexadecane revealed an increase in surface hydrophobicity of F. nucleatum grown at sub-MIC of penicillin and amoxicillin, but a decrease with metronidazole; whereas increased hydrophobicity was observed in T. denticola grown at sub-MIC of doxycycline, metronidazole and tetracycline. The surface hydrophobicity of P. gingivalis increased only when grown in sub-MIC of metronidazole. The protein expression profile of the treated bacteria differed from their respective controls. These results confirmed that sub-MIC concentrations of antibiotics can affect the phenotype, surface properties and morphology of periodontal pathogenic anaerobic bacteria.

Comparative analysis of motility and other properties of Treponema denticola strains.

The periodontitis-associated pathogen Treponema denticola is a spirochetal bacterium that swims by rotating its cell body like a corkscrew using periplasmic flagella. We compared physiologic and pathogenic properties, including motility, in four strains of T. denticola. Phase-contrast microscopy showed differential motility between the strains; ATCC 35404 showed the highest motility, followed by ATCC 33521, and the remaining two strains (ATCC 35405 and ATCC 33520) showed the lowest motility. Transmission electron microscopy showed that the low motility strains exhibited extracellular flagellar protrusions resulting from elongated flagella. Treponemal flagellar filaments are composed of three flagellins of FlaB1, FlaB2 and FlaB3. FlaB1 expression was comparable between the strains, whereas FlaB2 expression was lowest in ATCC 35404. FlaB3 expression varied among strains, with ATCC 35405, ATCC 33520, ATCC 33521, and ATCC 35404 showing the highest to lowest expression levels, respectively. Additionally, the low motility strains showed faster electrophoretic mobility of FlaB3, suggesting that posttranslational modifications of these proteins may have varied, because the amino acid sequences of FlaB3 were identical between the strains. These results suggest that inappropriate expression of FlaB2 and FlaB3 caused the unusual elongation of flagella that resulted in decreased motility. Furthermore, the low motility strains grew to higher bacterial density, and showed greater chymotrypsin-like protease activity, and more bacterial cells associated with gingival epithelial cells in comparison with the high motility strains. There may be a relationship between motility and these properties, but the genetic factors underlying this association remain unclear.

Porphyromonas gingivalis and Treponema denticola synergistic polymicrobial biofilm development.

Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. The aims of this study were to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia, as well as the role of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms, and a strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. In addition there was a morphological change of T. denticola in polymicrobial biofilms when compared with homotypic biofilms, suggesting reduced motility in homotypic biofilms. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola.

Native cellular architecture of Treponema denticola revealed by cryo-electron tomography.

Using cryo-electron tomography, we are developing a refined description of native cellular structures in the pathogenic spirochete Treponema denticola. Tightly organized bundles of periplasmic flagella were readily observed in intact plunge-frozen cells. The periplasmic space was measured in both wild-type and aflagellate strains, and found to widen by less than the diameter of flagella when the latter are present. This suggests that a structural change occurs in the peptidoglycan layer to accommodate the presence of the flagella. In dividing cells, the flagellar filaments were found to bridge the cytoplasmic cylinder constriction site. Cytoplasmic filaments, adjacent to the inner membrane, run parallel to the tightly organized flagellar filaments. The cytoplasmic filaments may be anchored by a narrow plate-like structure. The tapering of the cell ends was conserved between cells, with a patella-shaped structure observed in the periplasm at the tip of each cytoplasmic cylinder. Several incompletely characterized structures have been observed in the periplasm between dividing cells, including a cable-like structure linking two cytoplasmic cylinders and complex foil-shaped structures.