Intracellular replication of fusobacteria requires new actin filament formation of epithelial cells

We examined survival and replication of fusobacteria inside epithelial cells. Subconfluent cultures of HaCaT keratinocytes were infected with five bacterial strains representing three Fusobacterium species: F. nucleatum, F. necrophorum, and F. mortiferum. Adhesion and invasion of the bacteria were assayed before and after antibiotic treatment that killed the adhered and extracellular bacteria. The number of live fusobacteria was examined by bacterial culturing after sonication of the epithelial cells. The role of host cell cytoskeleton functions was examined by treating the epithelial cells with cell function inhibitors. Number of viable epithelial cells was measured with the CellTiter96 kit. The tested Fusobacterium species adhered to and invaded the epithelial cells, and multiplied intracellularly for several hours. Thereafter, the intracellular number of bacteria rapidly declined. Concomitantly, viable fusobacteria were detected in the culture medium. Treatment of the infected epithelial cells with an actin formation inhibitor markedly reduced the number of living intracellular fusobacteria. Newly formed actin filaments were seen by confocal microscopy in the epithelial cells associated with the invaded bacteria. Fusobacteria infection did not reduce the number of viable epithelial cells in culture. Thus, fusobacteria are able to adhere to and invade epithelial cells, and survive under aerobic conditions. This property may enable them to survive in mucosa and participate in various disease processes of oral and pharyngeal tissues.     

Gingival crevice microbiota from Chinese patients with gingivitis or necrotizing ulcerative gingivitis

The objective of this study was to quantitatively compare the bacterial population structure in plaque from the gingival margin of two groups of 21 Chinese patients with gingivitis or necrotizing ulcerative gingivitis (NUG). Subjects were recruited in four dental clinics in Eastern China. Samples were quantitatively assessed by immunofluorescence and fluorescent in situ hybridization for taxa known to be associated with periodontal diseases. The analyses showed that the fusiform taxa (Fusobacterium nucleatum/Fusobacterium periodonticum, Leptotrichia buccalis, Tannerella forsythensis, and Capnocytophaga sp.), Campylobacter rectus, Prevotella intermedia, Prevotella nigrescens, Selenomonas sputigena, and treponemes were present in both groups with high prevalence. Porphyromonas gingivalis and Actinomyces gerencseriae were much more prevalent in the NUG group. Quantitatively, most taxa, including P. gingivalis, F. nucleatum and the treponemes, accounted, on average, for < 3% of the total bacterial cell number. Only P. intermedia/P. nigrescens, P. gingivalis, S. sputigena, A. gerencseriae, and the sum of all monitored suspected periodontal pathogens were significantly increased in the NUG group. The present study demonstrates for both groups a highly diverse plaque composition and suggests that, etiologically, the overall concentration and the concerted effects of the entire group of opportunistic pathogens thriving in NUG-associated plaque are of prime importance.     

Subgingival microbiome dynamic alteration associated with necrotizing periodontal disease: A case report

Rationale:Necrotizing periodontal diseases (NPDs) are a group of infectious diseases varying in severity, and microorganisms are responsible for these diseases. Currently, the oral microbiota in early disease has been poorly investigated; thus, the causative pathogen and dynamic alteration of the microbiome in NPDs remain unclear.Patient concerns:We report a case of a 33-year-old female patient with severe gingival pain and localized necrotizing ulcerative gingival lesions. Conventional therapy was performed, but the necrotizing lesion continued to develop.Diagnoses:X-ray examination showed marginal alveolar bone loss in the involved teeth. Histological examination of a biopsy from the gingival lesion showed chronic inflammatory cell infiltration in the tissue, and no cancer cells were observed. Subgingival swabs were taken from the ulcerative gingiva and the gingiva that was not yet affected, and the composition of the microbiota was analyzed by targeted pyrosequencing of the V3-V4 hypervariable regions of the small subunit ribosomal RNA. We found that Neisseria spp., Corynebacterium spp., and Prevotella spp. were clearly enriched in the lesion site. However, Fusobacteria was more abundant in the not-yet-affected gingiva, and Leptotrichia spp. were the most abundant phylotype.Interventions:After clinical assessment, a tooth with poor prognosis was extracted, and minocycline hydrochloride was locally administered in the involved tooth pocket every day. Additionally, the patient received 100 mg of hydrochloric acid doxycycline twice per day.Outcomes:Remarkable improvement was obtained after 3 days, and the lesion completely healed after 1 week. The follow-up examination 1 year later showed a complete recovery with no recurrent episodes of pain.Lessons:Changes in the subgingival microbiome can occurr before clinical symptoms appears, and Fusobacteria may be involved in the imbalance of the subgingival flora in the early stage of NPDs. Moreover, Neisseria is a potential bacterial candidate that deserves further study.     

The pathogenetic role of rod‐shaped bacteria containing intracellular granules in the vellus hairs of a patient with perioral dermatitis: A comparison with perioral corticosteroid‐induced rosacea

The cause of perioral dermatitis is still unknown. We previously reported that rod‐shaped bacteria (RB) containing intracellular granules were detected in cases of perioral dermatitis at a high incidence. The aim of this study was to study further the role of RB in perioral dermatitis. Altogether 10 patients with perioral dermatitis and eight patients with perioral corticosteroid‐induced rosacea, who were referred to our department from 2009 to 2014, were examined for the presence of RB, using the tape‐stripping toluidine blue method. RB were detected on the surfaces of the roots of vellus hairs from lesions in nine of the 10 patients with perioral dermatitis. In contrast, RB were not detected in any of the eight patients with perioral corticosteroid‐induced rosacea. No RB were found in the perioral areas of other types of facial dermatitis, including atopic dermatitis and seboerrheic dermatitis or in 16 healthy controls. We treated four of the patients with perioral dermatitis with minocycline hydrochloride and five with cefcapene pivoxil hydrochloride hydrate. Three of the patients with perioral dermatitis who were treated with minocycline hydrochloride were cured in 3 to 8 weeks, while the five patients treated with cefcapene pivoxil hydrochloride hydrate were cured in 2 to 9 weeks. These results strongly suggest that RB (possible fusobacteria) play an important role in perioral dermatitis and that this is probably a distinct clinical entity from corticosteroid‐induced rosacea. Cefcapene pivoxil hydrochloride hydrate seems to be an effective treatment for perioral dermatitis associated with RB.     

Isolation of fusobacteria from the oral cavities of malnourished Nigerian children living in agricultural and herding villages

A previous study demonstrated the presence and possible involvement of Fusobacterium necrophorum in the pathogenesis of noma lesions of children living in agricultural and herding villages in northwestern Nigeria. In order to determine if F. necrophorum was part of the oral flora of malnourished children with no noma lesions, a study of the fusobacteria present in the oral cavities of 30 children, 2-6 years of age in Sokoto State, was undertaken. Swabs taken of the oral cavity were cultured on selective fusobacteria medium using conventional anaerobic microbiological techniques. F. nucleatum was recovered from each child and F. necrophorum was isolated from the oral cavity of only one child. The presence of F. nucleatum and the lack of F. necrophorum, except in one case, suggests that the latter is not normal flora in the children at risk for noma. F. necrophorum, a putative trigger organism for noma may gain a foothold only when certain staging conditions (i.e., lowered host resistance and/or oral lesion) are present.     

Cytokine, elastase and oxygen radical release by Fusobacterium nucleatum-activated leukocytes: a possible pathogenic factor in periodontitis

Periodontitis is characterised by tissue destruction caused by reactive oxygen species (ROS) and proteolytic enzymes, which are released by the interaction between bacteria and phagocytes. We estimated the ability of Fusobacterium species to induce release of tissue destructive and proinflammatory mediators from in vitro activated peripheral leukocytes. ROS was measured with the nitroblue tetrazolium (NBT) method, elastase with a specific chromogenic substrate and cytokines, including interleukin 1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha), and interleukin 8 (IL-8) with a sandwich ELISA method. Various clinical isolates of unopsonized Fusobacterium species stimulated the neutrophils to an increased NBT- reduction. IL-1beta, TNFalpha, IL-8 and elastase were released in significantly higher levels from neutrophils stimulated by Fusobacterium species. In conclusion, unopsonized Fusobacterium species can induce increased production of oxygen radicals, cytokines and elastase from leukocytes activated in vitro.