Microscopy Analyses Reveal the Parasitism of Entamoeba gingivalis in Periodontitis: An Observational Study

Abstract

Background: Whereas the periodontal microbiota is well described, its non-bacterial component needs a better understanding. Metagenomic analyses show a strong increase of the protozoan Entamoeba gingivalis in inflamed periodontal pockets. Its presence is associated with periodontal deterioration and pockets typically 3mm or more in depth. The aim of this study is to observe the amoeba within the biofilm during active disease. Materials and Methods: Here, we present results of a phase contrast microscopy-based observation of amoebae in periodontal patients’ subgingival plaque from infected sulcus. Plaque samples from deepest part of sulcus are picked up and spread out between blade and coverslip in patient saliva medium. We relate significant behavior of the parasite during active periodontal disease. Results: From low power observation, parasite is quite frequent. Once targeted, high power observation confirms amoeban anatomy and presence. We observed a high degree of amoeba locomotion and movements toward specific environments within the subgingival plaque. This was accompanied by the formation of “channels” within the biofilm. We also present evidence of adhesion to human cells as well as characteristically parasitic behavior. Specifically, we observed the intrusion of amoeba pseudopods into leukocytes coupled with a decrease in leukocyte intracellular granular activity. We documented both single trogocytic processes and trogocytosis through multiple pseudopods. In addition to leukocytes, we also observed trogocytosis of red blood cells. Parasitic behavior was also evident from the observation of amoebae digesting the nuclei of multiple vacuolar white blood cells, simultaneously. Following trogocytosis, polynuclear neutrophils had the appearance of ghost cells. Finally, we show evidence for amoeba nesting and reproduction within periodontal pockets. Conclusion: Phase contrast microscopy of periodontal biofilms strongly suggests that E. gingivalis escapes the first lines of innate defenses and promotes a pathological state. Trogocytosis and exonucleophagy processes targeting neutrophils could consequently disrupt neutrophil extracellular traps activity and normal apoptotic function, a vital component of wound healing. This study points to Entamoeba gingivalis as a microbe involved in the inflammatory process during periodontitis and as a driver of the disease rather than a harmless commensal species.