EM Imaging of Oral Bacteria: Insights into EDSL and Biofilm

Studies on the ultrastructure of intact bacteria and detailed structures of polymicrobial biofilms rely on electron microscopy. Objectives: (1) To investigate the relationship between the previously reported electron dense surface layer (EDSL) and colonial pigmentation, both of which were associated with active gingipains on Porphyromonas gingivalis cell surface. (2) To provide spatial and structural insights into the biofilm formed by the periodontal red complex species including P. gingivalis, Treponema denticola and Tannerella forsythia. Methods: (1) P. gingivalis wild type and isogenic mutant strains with various colonial pigmentation were recovered from blood agar and analysed by cryo-EM, including ATCC 33277 (wild type), and pg1626, lptO and porQ mutants in the ATCC 33277 background strain. (2) Flow cell biofilms that were established using the red complex, or P. gingivalis and T. denticola alone, were analysed using different in situ SEM techniques. Results: (1) The degree of colonial pigmentation was associated with the presence/thickness of EDSL observed in the various strains. However, the cryo-EM results excluded the association of the P. gingivalis EDSL with the reported thick haem dimer deposit of up to the μm range. (2) The individual red complex strains can be distinguished by conventional SEM that removed the glycocalyx to reveal the backbone structures of the biofilms. Using this technique, numerous non-cell novel structures became visible. Conclusions: Cryo-EM analysis excluded the black pigment derived from the haem dimer deposit on the P. gingivalis cell surface being electron dense. In situ conventional SEM confirmed the abundance of P. gingivalis vesicles and the synergistic growth of P. gingivalis and T. denticola in the red complex biofilm and revealed the role of T. denticola in providing the basal and bridging structure of the biofilm colonies. The detailed species-specific structural features provided a better understanding of the biofilm maturation pathway and species-specific survival strategies.