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Immunoelectron microscopic localization of lipopolysaccharides in the cell wall of Bacteroides oralis and Fusobacterium nucleatum.

Lipopolysaccharides (LPS) have been extracted and purified from two anaerobic gram-negative bacteria: Bacteroides oralis and tfusobacterium nucleatum. Chemical analysis of the preparations showed a great proportion of neutral sugars, mainly glucose, in LPS of B. oralis. In rabbits, LPS of B. oralis induced both immunoglobulin M and G antibodies in contrast to LPS of F. nucleatum, to which only immunoglobulin M antibodies were produced. An immunohistochemical method with horseradish peroxidase-labeled antibodies was used to localize LPS antigens at the ultrastructural level. An electron-dense reaction product, representing an immune complex consisting of bacterial surface antigens and specific rabbit immunoglobulin labeled with peroxidase, was surrounding the cell wall, whereas appropriate controls were negative. The findings of the present study show that LPS of Bacteroides are probably bound to a complex, including glucans, in the outer membrane of the cell wall. LPS of Fusobacterium resemble LPS of other gram-negative bacteria.

Antigens, Bacterial

Capnocytophaga: new genus of gram-negative gliding bacteria. III. Physiological characterization.

Sixty-eight strains of capnophilic fusiform Gram-negative rods from the human oral cavity were subjected to extensive physiologic characterization, tested for susceptibility to various antibiotics, and the mol-percent guanine plus cytosine of each isolate determined. The characteristics of the isolates were compared with 10 fresh and 2 stock isolates of Fusobacterium nucleatum. The isolates clearly differed from the Fusobacterium species on the basis of mol-percent guanine plus cytosine, end products, growth in a capnophilic environment and fermentation of carbohydrates. All of the gliding isolates required CO2 and formed acetate and succinate, but not H2S, indole or acetylmethylcarbinol. All fermented glucose, sucrose, maltose and mannose. The organisms may be differentiated on the basis of fermentation of additional carbohydrates, hydrolysis of polymers and reduction nitrate. Three species are proposed: Capnocytophaga ochracea, Capnocytophaga sputigena and Capnocytophaga gingivalis. Ten isolates did not fit into the proposed species.

Adult

Survival of anaerobic bacteria in common laboratory diluents.

The survival of six species of anaerobic bacteria was studied in simple or commercially available diluents. Bacteroides fragilis and Fusobacterium nucleatum showed excellent survival in all diluents including distilled water. Fusobacterium mortiferum survived well in all diluents except water and water supplemented with 0.1% gelatain. Clostridium perfringens survived best in phosphate-buffered saline with gelatin. Peptococcus asaccharolyticus required gelatin added to the basic diluent, and Streptococcus intermedius showed excellent survival only in minimal essential medium with gelatin. These diluents could provide effective and economical alternatives to more complex and costly diluents often used in work with anaerobic bacteria.

Anaerobiosis

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.

Actinomyces

The predominant cultivable microflora of advanced periodontitis.

A study on the predominant cultivable microorganisms harbored in the base of deep periodontal pockets of eight patients aged 34-48 years was carried out using the roll tube culture technique. From a total of 475 isolates, 425 (89.5%) were obligate anaerobes, 356(74.9%) were Gram-negative, and 441(92.8%) were categorized as rods. Bacteroides melaninogenicus and Fusobacterium nucleatum constituted the majority of the isolates in seven samples, but their mutual proportions differed considerably between the samples. One sample was dominated by Actinomyces species. Some periodontopathic potentials of the predominating Gram-negative rods are summarized.

Actinomyces

Lethality for mice and chick embryos, pyrogenicity in rabbits and ability to gelate lysate from amoebocytes of Limulus polyphemus by lipopolysaccharides from Bacteroides, Fusobacterium and Veillonella.

Phenol-water extracted lipopolysaccharides (LPS) from Veillonella, Fusobacterium nucleatum, Bacteroides fragilis and Bacteroides melaninogenicus were lethal for mice and 11-days-old chick embryos, pyrogenic in rabbits, and gelated Limulus amoebocyte lysate. Mouse lethality was considerably enhanced by actinomycin-D. In all test systems the endotoxin activity of Veillonella and Fusocbacterium LPS was comparable to that of LPS from Salmonella enteritidis, which was included as a reference endotoxin. The endotoxicity of the Bacteroides LPS was very low. While nanograms of the Veillonella and Fusobacterium LPS killed the chick embryos and gelated the Limulus lysates, microgram amounts of the Bacteroides LPS were needed to give positive reaction in the same test systems. As much as 74 microgram of the most active B. fragilis LPS were required to give a typical biphasic fever response in rabbits. A significant correlation was found between all test results (r = 0.90-0.98, p less than 0.001).

Animals

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.

Actinomyces

Hepatic necrobacillosis. Report of a case resembling metastatic tumor.

A 47-year-old man had multiple pyogenic liver abscesses from which the anaerobic bacteria Fusobacterium nucleatum and Peptostreptococcus were isolated. The clinical course, diagnostic evaluation, and appearance of the lesions at surgery suggested metastatic tumor. Histologically, the lesions consisted of necrotic liver tissue surrounded by zones of acute and chronic inflammation and fibrous connective tissue closely resembling the lesions of hepatic necrobacillosis attributable to F necrophorum. Treatment with high doses of penicillin was curative.

Bacterial Infections

Survival of anaerobic and aerobic bacteria on cotton swabs in three transport systems.

The capacity of aerobic and anaerobic bacteria to survive on cotton swabs placed into a dry gassed-out CO2-filled tube (DGT), dry sterile aerobic tube (DAT), and a tube containing a modified Stuarts' transport medium (MST), was assessed. Pseudomonas aeruginosa increased in numbers by 2 and 3 logs when stored in MST and DAT, respectively. The viability of P. aeruginosa, although retarded when compared to MSA and DAT, was not adversely affected by the CO2 environment in the DGT. The MST maintained relatively constant numbers of Streptococcus pyogenes during the 48-h storage period. The DAT and the DGT were unable to maintain the viability of S. pyogenes. Staphylococcus aureus, when stored in a DGT, DAT, or MST, was maintained in relatively constant numbers throughout the entire storage period. Of the four anaerobic bacteria evaluated (Bacteroides fragilis ssp. thetaiotaomicron, Bacteroides melaninogenicus ssp. asaccharolyticus, Fusobacterium nucleatum, and Peptostreptococcus anaerobius), only B. fragilis ssp. thetaiotamicron survived the 48-h storage period in the DGT. Under these test conditions the DGT did not adequately maintain the viability of the majority of anaerobic bacteria tested (when held on cotton swabs). However, the MST did maintain the viability of all species tested for at least the first 2 h of storage.

Aerobiosis

Ultrastructural localisation of lipopolysaccharide-binding sites with peroxidase-conjugated lipopolysaccharides.

The localisation of lipopolysaccharide-binding sites on erythrocytes with peroxidase-coupled LPS is described. LPS was isolated from Fusobacterium nucleatum (Fus MC-8) by phenol-water extraction. The LPS was coupled to horseradish peroxidase by the two-step method of Avrameas and Ternynck (1971). The biological and serological activities of the conjugated LPS were compared with those of the native material. Peroxidase could be coupled to LPS without significant loss of endotoxic or serological activity. The LPS-peroxidase conjugate could be demonstrated on erythrocytes by light and electron microscopy.

Animals

Microbiota of gingivitis in man.

A study on the predominant cultivable microorganisms inhabiting gingival crevices affected with a chronic gingivitis was carried out using the roll tube culture technique. Samples were obtained from nine individuals 25--42 years of age. Gram-positive rods make up 29.1% of the isolates and included mainly Actinomyces naeslundii, Actinomyces israelii, and Actinomyces viscosus. Streptococcus mitis and Streptococcus sanguis together made up 26.8% of the cultivable organisms. Peptostreptococcus averaged 3.0% of the organisms recovered. Gram-negative anaerobic rods constituted 25.0% of the total isolates with Fusobacterium nucleatum, Bacteroides melaninogenicus ss. intermedius. Bacteriodes ochraceus, other Bacteroides species, Selenomonas sputigena, and Campylobacter sputorum as the most predominant isolates. Haemophilus parainfluenzae averaged about 14% and Veillonella species 4.3% of the cultivable microflora. The data presented indicate that the subgingival microflora of a chronic gingivitis differs from those of healthy periodontium and advanced adult and juvenile periodontis. This might suggest that different infectious processes may be operative in various clinical entities of periodontal disease.

Actinomyces

Compact liquid nitrogen storage system yielding high recoveries of gram-negative anaerobes.

A simple and compact system suitable for the preservation of fragile gram negative anaerobes and other bacteria in liquid N2 has been developed. Polypropylene straws used as specimen containers can be used easily within glove bags of anaerobic chambers, and their small size greatly increases the number of cultures which can be stored. Ancillary equipment and methods developed are described. The overall system was tested, using Streptococcus mutans, Fusobacterium nucleatum, and Selenomonas sputigena. Various basal suspending fluids and cryoprotective supplements were studied. With fast rates of freezing and thawing, survival recoveries of the test microorganisms ranged from 80 to 100 percent of the input colony-forming units in a complex medium broth base without cryoprotective agent addition, and they consistently were 100 percent when 0.4 mM polyvinylpyrrolidine was used. Overall, cryoprotection by polyvinyl pyrrolidine was superior to that from glycerol or dimethyl sulfoxide, the latter yielding recoveries similar to or less than those obtained with no cryoprotectant additive. All microorganisms were recoverable after storage for 1 year.

Dimethyl Sulfoxide

Anaerobic pulmonary infections.

The main cause of anaerobic pulmonary infections is aspiration of saliva, upper airway secretions or gastric content. Predisposing conditions include prominent dental disease, chronic upper respiratory tract infections and reduced consciousness. Fusobacterium nucleatum, Bacteroides melaninogenicus and anaerobic Gram-positive cocci are the most frequently encountered organisms. The clinical presentations are lung abscess, lung gangrene and empyema, which all tend to take a slow and indolent course. Preferred localization are dependent lung segments, most often on the right side. For bacteriological examination in these infections, only transtracheal aspirate and aspirate from the lung or pleural space are considered adequate. In 26 cases of empyema treated in our hospital during the last 3 years, adequate specimens had been taken in 19. Fifteen had been adequately examined, and anaerobes were cultured in 6. Among 29 abscesses treated during the same period, adequate specimens had been taken in only 14, and 11 had been properly examined. Seven specimens grew anaerobes on culture. In prospective studies of transtracheal aspirate in 15 chronic bronchitics without emphysema, anaerobes were not demonstrated. In 11 patients with bronchiectasis, anaerobic bacteria were cultured in 3. Finally, no anaerobic bacteria could be demonstrated in the transtracheal aspirate from 76 patients with acute exacerbation of chronic bronchitis. Anaerobic, pulmonary infections do not represent an intriguing medical problem in our region. However, knowledge of these infections is necessary to secure adequacy in collection of specimens and in their bacteriological examination.

Bacterial Infections

[In vitro susceptibility of bacteroidaceae to cefoxitin and cephalothin (author's transl)].

The activity of the new cephamycin C derivatives cefoxitin against 102 clinical isolates of Bacteroidaceae (95 Bacteroides strains and 7 Fusobacterium-Sphaerophorus cultures) was determined by tube dilution and standardized agar diffusion tests and compared to that of cephalothin. Cefoxitin was more active than cephalothin against 56 Bacteroides fragilis strains, 19 Bacteroides thetaiota-omicron strains and two Bacteroides splanchnicus strains. The difference seemed less pronounced with five Bacteroides vulgatus and four Bacteroides variabilis cultures. Three Bacteroides melaninogenicus and six Bacteroides oralis strains displayed similar sensitivity to both cephalosporins. At concentrations of 32 microng/ml or less, cefoxitin inhibited 91% and cephalothin 33% of the Bacteroides strains. Two strains of Sphaerophorus varius were likewise much more sensitive to cefoxitin than to cephalothin (MIC values 8 micron8/ml of cefoxitin and 256 microng/ml of cephalothin). Three other Sphaerophorus strains (the species necrophorus and freundii/mortiferum) and two of Fusobacterium fusiforme (Fusobacterium nucleatum) strains were very similar in their susceptibility to both cephalosporins. Statistical analysis of the relationship between MIC and zone size diameter showed relatively high correlation coefficients for cefoxitin and cephalothin (r=-0.741 and r=-0.703 respectively).

Bacteroidaceae

Molecular epidemiology and phylogeographic architecture of oncogenic intracellular bacteria in cervical cancer patients across Northern China.

BACKGROUND: Oncogenic intracellular bacteria, including Chlamydia trachomatis, Mycoplasma genitalium, and Fusobacterium nucleatum, have emerged as significant contributors to cervical carcinogenesis. Despite growing interest in microbial oncology, the molecular epidemiological landscape and phylogeographic distribution of these pathogens in Northern China remain poorly characterized. This study aimed to determine the prevalence, co-infection patterns, genotypic diversity, and spatial phylogeographic clustering of oncogenic intracellular bacteria among cervical cancer patients across five provinces of Northern China. METHODS: A cross-sectional, multi-center study was conducted between March 2022 and November 2024 across Shaanxi, Heilongjiang, Beijing, Shandong, and Inner Mongolia. Cervical swab specimens were collected from 1247 confirmed cervical cancer patients. Pathogen detection was performed using multiplex real-time polymerase chain reaction, 16S rRNA gene amplicon sequencing, and whole-genome sequencing. Phylogeographic analyses employed maximum likelihood and Bayesian evolutionary inference frameworks. Statistical analyses included multivariate logistic regression and geographic information system-based spatial clustering. RESULTS: The overall prevalence of at least one oncogenic intracellular bacterium was 68.3% (n&#xa0;=&#xa0;852). Chlamydia trachomatis was the most prevalent pathogen detected in 41.2% of participants. Co-infection with two or more bacteria was identified in 29.7% of cases and was independently associated with advanced-stage cervical cancer (adjusted odds ratio&#xa0;=&#xa0;2.87; 95% confidence interval: 1.94 to 4.23; p&#xa0;<&#xa0;0.001). Phylogeographic analysis revealed three distinct molecular clades with evidence of bidirectional gene flow between Shaanxi and Heilongjiang. Whole-genome sequencing identified 14 novel virulence gene variants not previously characterized in Chinese clinical isolates. CONCLUSIONS: Oncogenic intracellular bacteria are highly prevalent and genotypically diverse among cervical cancer patients in Northern China. The identified phylogeographic clustering and novel virulence variants have direct implications for regional screening programs, targeted antimicrobial strategies, and the development of region-specific molecular diagnostic panels.

Cervical cancer