Fusobacterium watanabei sp. nov. As additional species within the genus Fusobacerium, isolated from human clinical specimens.

Eight spindle-shaped bacteria were isolated from clinical samples in Japan and investigated for their taxonomic position. Phylogenetic trees (based on 16S rRNA, rpoB, zinc protease, and gyrB gene sequence comparisons) showed distinct clustering of eight strains with the type strain of Fusobacterium nucleatum and its closely related species. In silico whole genome comparison analysis based on average nucleotide index based on BLAST (ANIb) and digital DNA-DNA hybridization (dDDH) data between our clinical isolates (PAGU 1795, PAGU 1796T, and PAGU 1797) and the type strain of the closely related species showed values of less than 92.4% and 49.5%, respectively. On the basis of its phylogenetic and genomic distinctiveness together with differential phenotypic properties and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) characteristic signal patterns, we propose Fusobacterium watanabei sp. nov., with the type strain PAGU 1796T (= GTC 21791T = CCUG 74246T).

Molecular studies of the structural ecology of natural occlusal caries.

Microbiological studies of occlusal dental biofilms have hitherto been hampered by inaccessibility to the sampling site and demolition of the original biofilm architecture. This study shows for the first time the spatial distribution of bacterial taxa in vivo at various stages of occlusal caries, applying a molecular methodology involving preparation of embedded hard dental tissue slices for fluorescence in situ hybridization (FISH) and confocal microscopy. Eleven freshly extracted teeth were classified according to their occlusal caries status. The teeth were fixed, embedded, sectioned and decalcified before FISH was performed using oligonucleotide probes for selected abundant species/genera associated with occlusal caries including Streptococcus, Actinomyces, Veillonella, Fusobacterium, Lactobacillus and Bifidobacterium. The sites showed distinct differences in the bacterial composition between different ecological niches in occlusal caries. Biofilm observed along the entrance of fissures showed an inner layer of microorganisms organized in palisades often identified as Actinomyces, covered by a more loosely structured bacterial layer consisting of diverse genera, similar to supragingival biofilm. Biofilm within the fissure proper seemed less metabolically active, as judged by low fluorescence signal intensity and presence of material of non-bacterial origin. Bacterial invasion (often Lactobacillus and Bifidobacterium spp.) into the dentinal tubules was seen only at advanced stages of caries with manifest cavity formation. It is concluded that the molecular methodology represents a valuable supplement to previous methods for the study of microbial ecology in caries by allowing analysis of the structural composition of the undisturbed biofilm in caries lesions in vivo.

Fusobacterium periodonticum, a new species from the human oral cavity.

Isolates of Fusobacterium that differ from type strains of various fusobacterial species with respect to DNA sequence, cellular fatty acid composition, and biochemical activity, were obtained from periodontitis lesions in a patient with insulin-dependent diabetes mellitus. These isolates have the following distinguishing characteristics: 28% guanine + cytosine content; 40% or less DNA homology with type strains of representative fusobacterial species; cell size, 0.5 - 1 X 4 -100 microns; absence of motility; ability to ferment glucose, fructose, and galactose, but not 25 other carbohydrates; ability to produce indole; ability to hydrolyze hippurate but not esculin; sensitivity to bile; ability to produce little or no gas; ability to utilize threonine but not lactate. We propose that the organisms be classified as a distinct species of Fusobacterium to be named Fusobacterium periodonticum. The type strain of this new species has been deposited with the American Type Culture Collection under the designation ATCC 33693.

Periodontal therapy in humans. I. Microbiological and clinical effects of a single course of periodontal scaling and root planing, and of adjunctive tetracycline therapy.

The present results showed that maarked and long-lasting changes in the subgingival microflora associated with periodontal disease could be achieved by a single course of periodontal treatment. Immediately following therapy, the total number of subgingival organisms decreased 10- to 100-fold and the proportions of cultivable Gram negative organisms and anaerobic organisms generally decreased 3- to 4-fold or more. After treatment, most periodontal pockets were populated by a scant microflora predominated by facultative Actinomyces and Streptococcus species. The kinetics of the subgingival bacterial recolonization revealed that the total cell counts and the proportions of spirochetes and Capnocytophaga species did not reach their pretreatment levels even after 6 months. Other Gram negative anaerobic species returned to pretreatment proportions after 3 to 6 months. Several Gram positive species exhibited higher posttreatment than pretreatment proportions throughout the 6 months study. The microbiological shifts paralleled significant changes in the clinical status of the periodontal tissues. Following therapy, the periodontal pocket depths decreased generally 1 to 4 mm, the gingival inflammatory index, the gingival fluid flow, and the suppurative index were generally lower, and nine of 33 test pockets examined showed apposition of alveolar bone. The microbiological and clinical changes described were exhibited by two patients treated with periodontal scaling and root planing alone and by two patients treated with the adjunctive use of systemic tetracycline therapy. In two other patients, mechanical periodontal therapy only slightly reduced the total number of subgingival organisms and the proportions of spirochetes and other Gram negative anaerobic rods. A shift in the subgingival microbial composition was achieved in these two patients after tetracycline therapy. The following model for treatment of periodontal disease is proposed: (1) Conventional therapy including thorough periodontal scaling and root planing; (2) Monitoring the subgingival flora and the clinical course; and (3) Use of antimicrobial therapy in refractory cases. Further studies are needed to develop means for rapid identification of refractory patients, and to determine the optimal antimicrobial agent, the optimal route of administration, and the optimal dosage regime.

Síndrome de Lemierre: relato de caso / Lemierre syndrome: case report

A síndrome de Lemierre é uma doença rara, mais comum em jovens, causada frequentemente pelo Fusobacterium necrophorum. Inicia-se com faringite e propaga-se até a veia jugular interna, promovendo uma fonte de bacteremia contínua e êmbolos sépticos pulmonares. Manifestações clínicas incluem febre, alterações respiratórias e massa cervical. O diagnóstico é realizado por tomografia computadorizada e duplex scan, além de hemocultura ou cultura direta. O tratamento é realizado com antibióticos beta-lactâmicos resistentes a beta-lactamases, sendo a cirurgia raramente necessária. Paciente do sexo feminino, 34 anos, com quadro de orofaringite, evoluiu em 48 horas com queda do estado geral, febre, aumento de volume e dor em região cervical esquerda...

Lemierre syndrome is a rare disease. It often affects young adults and is most frequently caused by Fusobacterium necrophorum. The initial event is pharyngitis, which extends to the internal jugular vein, serving as source of continuous bacteremia and septic pulmonary emboli. Clinical manifestations include fever, respiratory distress, and swollen cervical lymph nodes. Diagnosis is established based on blood culture or direct blood culture and confirmed by computed tomography and/or duplex scan. Treatment consists of administration of beta-lactamase resistant beta-lactam antibiotics...

[Proportion and frequency of 3 kinds of anaerobic bacteria in relation to the gingival index]. / Proportion et fréquence de trois genres bactériens anaérobies en fonction de l'indice gingival.

The proportion and frequency of three groups of particular bacteria (Bacteroides, Fusobacterium and spirochetes) were studied in relation to the gingival index on the sites from which they were sampled. Seventy eight samples collected from 40 individuals were studied together with measurements of the gingival index and the pocket or sulcus depth. The samples were dispersed by ultrasonic treatment, diluted and cultured on a non-selective medium. The identification of the species was done by biochemical tests and by analysis of the metabolic products with gas chromatography. As a whole, our results support the hypothesis of an important change in the microbial flora from a clinical healthy status (with predominant Gram positive facultative anaerobic bacteria) to disease (with predominant Gram negative strict anaerobic bacteria). Furthermore the results suggest that Bacteroides gingvalis as well as Fusobacterium nucleatum and spirochetes through their high frequency and important proportion at the affected sites played an etiological role in the pathogenesis of gingivitis and periodontis Man.

Qualitative changes in dental plaque formation related to removable partial dentures.

A study was performed on the microbial composition of dental plaque developing on fifteen abutment teeth in nine partially edentulous subjects. Bacteria were counted in samples of plaque which had formed on vestibular surfaces, and on proximal surfaces facing edentulous spaces, during a period when dentures were not worn, and then for a period when dentures were being worn. It was found that the presence of removable partial dentures favoured a proliferation of spiral organisms.

Pleomorphism of fusobacteria isolated from the cockroach hindgut.

Fusobacteria are commonly isolated from the hindgut of the cockroach Eublaberus posticus . Eleven strains isolated from E. posticus by us were keyed to four species, Fusobacterium necrophorum, F. varium , F. gonidiaformans , and F. prausnitzii , using current taxonomic criteria. With the exception of F. gonidiaformis , all species showed rods with swollen centers and large bodies. The pleomorphism of F. varium was examined by phase microscopy and scanning and transmission electron microscopy. The pleomorphic process begins with a gradual swelling at the center of the rod until a large round body is formed. Some of these round bodies then fragment, giving rise to rod-shaped cells. When 10% yeast extract was added to growth media, pleomorphism was not induced. A dialyzable factor was found to account for this observation. Fermentation of [1-14C]glutamic acid gives rise to butyrate labeled in the carboxyl carbon, indicating that butyrate is formed by the hydroxyglutarate pathway which may be characteristic for the genus Fusobacterium.

Surface ultrastructure of some oral bacteria.

Adhesion of Streptococcus sanguis, Fusobacterium nucleatum and an Actinomyces sp. to enamel and epon and their interspecies cohesion was studied with scanning and transmission electron microscopy. For adhesion studies enamel or epon was coated with salivary macromolecules and then cells of S. sanguis and in some experiments also with F. nucleatum or Actinomyces sp. Cells of S. sanguis were seen scattered over the surface of a thin "pellicle" that was heavily stained, and F. nucleatum and Actinomyces sp. adhered to S. sanguis or directly to the "pellicle". For studies of cohesion S. sanguis was brought to cohere with F. nucleatum or Actinomyces sp. and then processed for transmission electron microscopy. The morphology of the cell surface structures involved was studied in negatively stained preparations or in thin sections of material treated with ruthenium red or poststained with uranyl and lead salts, phosphotungstic acid or periodic acid-thiocarbohydrazide-osmium tetroxide. S. sanguis demonstrated a fuzzy coat of fimbriae that seemed to unfold in areas of contact with other cells, while cells of F. nucleatum had 6-10 polar pilus-like fimbriae, which appeared to be instrumental in cohesion, as did a dense coat of long, slender fimbriae that covered cells of Actinomyces sp.

Anaerobic bacteria on the mucosal epithelium of the murine large bowel.

Anaerobic bacteria can be detected at population levels of 10(11) organisms per g of cecum or colon in adult mice from four different colonies widely spaced in the United States. Most of these microorganisms are oxygen-intolerant fusiform-shaped bacteria. At least one type of these tapered, rod-shaped bacteria can be seen in layers in the epithelial mucin in frozen-section histological preparations of the large bowels of mice. In addition, such microorganisms can be seen within 0.5 mum of the epithelium in ultrathin sections of colon or cecum examined in an electron microscope. These fusiform-shaped bacteria predominate in the mucin layers. However, spiral-shaped microorganisms can be found as well near the mucosal epithelia in ultrathin sections of colon. Also, such organisms can be seen in negatively-stained preparations of washings of the colonic mucosal epithelia examined in an electron microscope. At least three types of spiral-shaped organisms, including both spiral-shaped bacteria and spirochetes, can be found in preparations from mice from three of the four colonies. Such spiral-shaped microorganisms can be detected at population levels as great as 10(9) organisms per g of cecum or colon in anaerobic cultures of the large bowels of mice from all four colonies. One anaerobic spiral bacterium was isolated in pure culture. This particular organism was found by immunofluorescence to be intermingled with the fusiform-shaped bacteria in the mucin on the mucosal epithelium in the mouse large bowel.

Effect of sonic treatment on pure cultures and aggregates of bacteria.

Pure cultures of a variety of bacteria were treated with ultrasonic energy using a sonic probe. Fractions of organisms killed at different sonic energies were calculated, and Streptococcus mutans was 600 times more resistant than Fusobacterium nucleatum, the most sensitive organism tested. The effects of sonic treatment on aggregates of bacteria were examined, and the results were interpreted as a model of the events that probably occur during the sonic dispersion of dental plaque.

Unusual Sphaerophorus species from the large intestine of man.

An obligately anaerobic, gram-negative microorganism identified as a Sphaerophorus species was recovered from the fecal material of two cancer (chronic myelogenous leukemia and idiopathic thrombocythemia) patients receiving cobalt radiation therapy. The organism, isolated on sheep blood-agar, exhibited extreme pleomorphism (rods, filaments, and spheroids) and was a major component of the anaerobic fecal microflora. In one patient the numbers of Sphaerophorus species (designated as isolate 6-13-68), Bacteroides species, and Clostridium perfringens declined after irradiation; however, they were stable in this same patient after a second therapeutic dose of radiation. The numbers of anaerobes in the other patient remained fairly consistent after radiation. The biochemical and morphological characteristics and carbohydrate fermentation reactions of isolate 6-13-68 most closely resembled those of Sphaerophorus ridiculosis.

Antibiotic susceptibility patterns as aids in classification and characterization of gram-negative anaerobic bacilli.

Patterns of susceptibility of gram-negative anaerobic bacilli to antibiotics have been found to be distinctive and of significant help in classification and identification. Five major groups of gram-negative anaerobic bacilli have been defined on the basis of morphological and biochemical criteria. Antibiotic sensitivity patterns conform to these groupings and provide additional taxonomic criteria. The Bacteroides fragilis group is resistant to penicillin G, whereas the other groups are generally sensitive. B. fragilis strains are relatively sensitive to erythromycin, whereas the Sphaerophorus necrophorus group is resistant. B. melaninogenicus strains, B. oralis, and Fusobacterium are all more sensitive to kanamycin and neomycin than the other two groups. Kanamycin is more active against Fusobacterium strains than neomycin, but less active against all other groups. Colistin or polymyxin B is useful for distinguishing between the resistant B. fragilis and the sensitive S. necrophorus. Antibiotic susceptibility determinations may be more readily performed in clinical laboratories than certain biochemical tests recommended for differentiation of the gram-negative anaerobic bacilli and may serve as helpful adjuncts to morphological and biochemical observations in classifying and characterizing these organisms. The use of standardized procedures for antibiotic susceptibility tests is essential if comparable results are to be obtained in different laboratories.