Cultivation of oral fusobacteria in Hitchens-Pike medium.
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Coaggregation of Fusobacterium nucleatum, Selenomonas flueggei, Selenomonas infelix, Selenomonas noxia, and Selenomonas sputigena with strains from 11 genera of oral bacteria.
Twenty-eight strains of Fusobacterium nucleatum and 41 Selenomonas strains, including S. sputigena (24 strains), S. flueggei (10 strains), S. infelix (5 strains), and S. noxia (2 strains), were tested for their ability to coaggregate with each other and with 49 other strains of oral bacteria representing Actinobacillus, Actinomyces, Bacteroides, Capnocytophaga, Gemella, Peptostreptococcus, Porphyromonas, Propionibacterium, Rothia, Streptococcus, and Veillonella species. Selenomonads coaggregated with fusobacteria and with Actinomyces naeslundii PK984 but not with any of the other bacteria, including other selenomonads. In contrast, fusobacteria coaggregated with members of all genera, although not with all strains of each species tested. Each fusobacterium strain appeared to have its own set of partners and coaggregation properties, unlike their partners, whose coaggregation properties in earlier surveys delineated distinct coaggregation groups. Coaggregations of fusobacteria with the 63 gram-negative strains were usually inhibited by EDTA, whereas those with the 27 gram-positive strains were usually not inhibited. Likewise, lactose-inhibitable coaggregations were common among some strains of fusobacteria and some strains from each of the genera containing gram-negative partners but were rarely observed with gram-positive partners. Heating the fusobacteria at 85 degrees C for 30 min completely prevented coaggregation with most partners, suggesting the involvement of a protein on the fusobacteria. Heat treatment of many of the gram-negative partners not only enhanced their coaggregation with the fusobacteria but also changed lactose-sensitive coaggregations to lactose-insensitive coaggregations. Although fusobacteria coaggregated with a broader variety of oral partner strains than any other group of oral bacteria tested to date, each fusobacterium exhibited coaggregation with only a certain set of partner strains, and none of the fusobacteria adhered to other strains of fusobacteria, indicating that recognition of partner cell surfaces is selective. The strains of F. nucleatum are heterogeneous and cannot be clustered into distinct coaggregation groups. Collectively, these results indicate that coaggregation between fusobacteria and many gram-negative partners is significantly different from their coaggregation with gram-positive partners. The contrasting variety of partners for fusobacteria and selenomonads supports the concept of coaggregation partner specificity that has been observed with every genus of oral bacteria so far examined.
Actinomyces↗
Intergeneric coaggregation of oral Treponema spp. with Fusobacterium spp. and intrageneric coaggregation among Fusobacterium spp.
A total of 22 strains of Treponema spp. including members of all four named human oral species were tested for coaggregation with 7 strains of oral fusobacteria, 2 strains of nonoral fusobacteria, and 45 strains of other oral bacteria, which included actinobacilli, actinomyces, capnocytophagae, eubacteria, porphyromonads, prevotellae, selenomonads, streptococci, and veillonellae. None of the treponemes coaggregated with any of the latter 45 oral strains or with the two nonoral fusobacteria. All treponemes, eight Treponema denticola strains, eight T. socranskii strains, four oral pectinolytic treponemes, one T. pectinovorum strain, and one T. vincentii strain coaggregated with at least one strain of the fusobacteria tested as partners. The partners consisted of one strain of Fusobacterium periodonticum, five F. nucleatum strains including all four subspecies of F. nucleatum, and a strain of F. simiae obtained from the dental plaque of a monkey. In the more than 100 coaggregations observed, the fusobacterial partner was heat inactivated (85 degrees C for 30 min), while the treponemes were unaffected by the heat treatment. Furthermore, the fusobacteria were usually inactivated by proteinase K treatment, and the treponemes were not affected. Only the T. denticola coaggregations were inhibited by lactose and D-galactosamine. None were inhibited by any of 23 other different sugars or L-arginine. Intragenic coaggregations were seen among the subspecies of F. nucleatum and with F. periodonticum, and none were inhibited by any of the sugars tested or by L-arginine. No intrageneric coaggregations were observed among the treponemes. These data indicate that the human oral treponemes show a specificity for oral fusobacteria as coaggregation partners. Such cell-to cell contact may facilitate efficient metabolic communication and enhance the proliferation of each cell in the progressively more severe stages of periodontal disease.
Endopeptidase K↗
Fusobacterial infections in children.
A total of 243 strains of Fusobacteria species was recovered from 226 of 1399 (16%) specimens obtained from 213 children. The strains included 65 (27%) Fusobacterium sp., 144 (59%) Fusobacterium nucleatum, 25 (10%) Fusobacterium necrophorum, five (2%) Fusobacterium varium, three (1%) Fusobacterium mortiferum, and one (0.4%) Fusobacterium gonidiaformans. Most Fusobacteria species were recovered from patients with abscesses (100), aspiration pneumonia (24), paronychia (15), bites (14), chronic sinusitis (ten), chronic otitis media (nine), and osteomyelitis (eight). Predisposing conditions were noted in 32 (15%) of the cases. These included immunodeficiency in nine (4%), steroid therapy in eight (4%), previous surgery in six (3%), diabetes in six (3%) and malignant neoplasms in five (2%). Fusobacteria sp. was the only isolate in 16 (8%) instances while mixed infections were encountered in 197 (92%) patients. The organisms most commonly isolated with Fusobacteria sp. were anaerobic cocci (155), pigmented Prevotella sp. and Porphyromonas sp. (95), Bacteroides fragilis group (80), Escherichia coli (43) and Bacteroides sp. (39). Most strains of B. fragilis group and E. coli were recovered from intra-abdominal infections and skin and soft tissue infections proximal to the rectal area. Most pigmented Prevotella sp. and Porphyromonas sp. were recovered from oropharyngeal and pulmonary sites and from sites around the head and neck. Antimicrobial therapy was administered to all patients; surgical drainage was performed in 85 (40%). All patients, except two who died, recovered. These findings illustrate the prevalence of Fusobacteria sp. associated with infections in children.
Adolescent↗
Helicobacter pylori adheres selectively to Fusobacterium spp.
Helicobacter pylori strains ATCC 43504 and ATCC 43629 were tested for their ability to coaggregate with 79 strains of bacteria representing 16 genera. All except two of the strains were of human origin, and most of the strains were isolated from the oral cavity. The helicobacters failed to coaggregate with all strains except the fusobacteria. Several coaggregations were partially or completely inhibited by lactose. Strong coaggregation was seen with each of four subspecies of Fusobacterium nucleatum and with Fusobacterium periodonticum ATCC 33693, all of human dental plaque origin. In contrast, the helicobacters failed to coaggregate with non-plaque isolates, Fusobacterium mortiferum ATCC 25557 and Fusobacterium ulcerans ATCC 49185. Heat treatment of the fusobacteria inactivated their ability to coaggregate, whereas heating of the Helicobacter partners had no effect, suggesting the presence of an adhesin on the fusobacteria and a corresponding receptor on the helicobacters. The potential ability of H. pylori to colonize the oral cavity by adhering selectively to the ubiquitous fusobacteria gives credence to the possibility that dental plaque may serve as a reservoir for this pathogen outside of the stomach.
Bacterial Adhesion↗
Corncob formation between Fusobacterium nucleatum and Streptococcus sanguis.
Corncob formation in dental plaque was believed to be limited to strains of Bacterionema matruchotii and Streptococcus sanguis. We observed recently that strains of Fusobacterium nucleatum also interacted with S. sanguis to form corncobs. Since the fusobacteria are among the first anaerobic filaments to colonize subgingival plaque, these interactions could serve as a connecting link between the transformation of supra- to subgingival plaque. To further characterize these interactions, quantitative in vitro studies of the kinetics of corncob formation of the fusobacteria were undertaken. These studies indicated that fewer streptococci were needed to saturate F. nucleatum strain 364 compared to strain 10953. Corncob formation with both strains was enhanced with increasing pH up to pH 8, at which point autoaggregation of the streptococci occurred. Variation in ionic strength and divalent cations had little effect on the interaction, and EDTA suppressed aggregate formation only slightly. Detergents at concentrations above 0.05% also inhibited corncob formation. Electron micrographs suggested that attachment of the cocci to the fusiforms was mediated through localized tufts of fimbriae, as they are in the Bacterionema system. However, although both trypsin and heat treatment of the streptococci inhibited corncob formation with fusobacteria, the effects were not as complete as those seen in Bacterionema species. Unlike the Bacterionema model, trypsin and heat treatment of the fusobacteria resulted in inhibition of corncob formation. These results suggest that several different receptors may be involved in corncob formation.
Adhesiveness↗
Betalactamase-producing microorganisms in recurrent tonsillitis.
In 73% of 167 patients with recurrent tonsillitis, colonization with betalactamase-producing microorganisms was found. Betastreptococci group A were recovered in 6% of the patients and group C and G streptococci in 23%. Other microorganisms found were Staphylococcus aureus in 42%, Haemophilus species in 52%, bacteroides species in 80% and fusobacteria in 40%. Ninety-eight per cent of Staph. aureus, 60% of bacteroides species and 10% of fusobacteria were betalactamase-producing. Phenoxymethylpenicillin (1 g twice a day for ten days) diminished 50% of group A, C and G streptococci. No other microorganisms were affected by this antibiotic. Clindamycin (0.15 g four times a day for ten days) eradicated Staph. aureus, group A, C and G streptococci, bacteroides and fusobacteria. Haemophilus species were not affected by clindamycin. Tinidazole (1 g once a day for 10 days) diminished bacteroides and fusobacteria. Aerobic microorganisms, Staph. aureus, streptococci and Haemophilus, were not affected by tinidazole.
Adolescent↗
Impact on peritonsillar infections and microflora of phenoxymethylpenicillin alone versus phenoxymethylpenicillin in combination with metronidazole.
In a double-blind study, 20 patients with peritonsillar abscesses were treated with 2 g phenoxymethylpenicillin b.i.d. for ten days together with needle aspiration, incision and daily drainage, and 20 patients were treated with 2 g phenoxymethylpenicillin b.i.d. and 0.8 g metronidazole b.i.d. for ten days together with needle aspiration, incision and daily drainage. Group A beta-hemolytic streptococci were isolated from pus in 20 of the patients with peritonsillar abscesses, in five of these together with indigenous oropharyngeal aerobic and anaerobic microorganisms. Pure anaerobic bacteria were found in nine abscesses, together with indigenous aerobic microorganisms in eight, and together with group A, C and G streptococci in five. In one patient heavily colonized with beta-lactamase-producing Staphylococcus aureus, Haemophilus parainfluenzae and Bacteroides, group A beta-streptococci failed to be eradicated. In the penicillin group, nine of 18 patients harboured beta-lactamase producing Bacteroides strains in the tonsils on the day of admission. On the third and tenth days of treatment all patients harboured beta-lactamase producing Bacteroides strains in the tonsils, while in the penicillin + metronidazole group, only one out of 17 patients still harboured beta-lactamase producing Bacteroides strains. None of the patients harboured beta-lactamase producing fusobacteria on the day of admission. In the penicillin group, however, beta-lactamase producing fusobacteria were recovered from three patients on the tenth day of treatment. No beta-lactamase producing fusobacteria were recovered from the penicillin + metronidazole group.(ABSTRACT TRUNCATED AT 250 WORDS)
Abscess↗
Dietary guar gum and pectin stimulate intestinal microbial polyamine synthesis in rats.
The effects of two highly fermentable dietary fibers (guar gum and pectin) on the type and concentrations of cecal polyamines as affected by the intestinal microflora were studied in groups of germ-free (n = 10/group) and conventional rats (n = 6/group). Both germ-free and conventional rats were randomly assigned to one of three treatments as follows: 1) fiber-free control diet, 2) control diet + 10% guar gum and 3) control diet + 10% pectin. In germ-free rats, guar gum and pectin had no effect on cecal polyamine concentrations. Putrescine was confirmed to be the major endogenous polyamine within the gut lumen. In cecal contents of conventional rats, both guar gum and pectin led to the appearance of cadaverine and to elevated putrescine concentrations in comparison with the fiber-free control diet (1.35 +/- 0.15 and 2.27 +/- 0.32, respectively, vs. 0.20 +/- 0.03 micromol/g dry weight, P < 0.05). The cecal cadaverine concentration was higher in pectin- than in guar-fed rats (8.20 +/- 0.89 vs. 1.92 +/- 0.27 micromol/g dry weight, P < 0.05). Counts of total bacteria, bacteroides, fusobacteria and enterobacteria were higher (P < 0.05) in rats fed guar gum and pectin. Bifidobacteria were found exclusively in guar-fed rats. In vitro studies on selected species representing the numerically dominant population groups of the human gut flora (bacteroides, fusobacteria, anaerobic cocci and bifidobacteria) were examined for their ability to synthesize intracellular polyamines. These experiments demonstrated the ability of bacteroides, fusobacteria and anaerobic cocci to synthesize high amounts of putrescine and spermidine. Calculations based on these results suggest that the intestinal microflora are a major source of polyamines in the contents of the large intestine.
Animals↗
Characterization of the novel Fusobacterium nucleatum plasmid pKH9 and evidence of an addiction system.
Fusobacterium nucleatum is an important oral anaerobic pathogen involved in periodontal and systemic infections. Studies of the molecular mechanisms involved in fusobacterial virulence and adhesion have been limited by lack of systems for efficient genetic manipulation. Plasmids were isolated from eight strains of F. nucleatum. The smallest plasmid, pKH9 (4,975 bp), was characterized and used to create new vectors for fusobacterial genetic manipulation. DNA sequence analysis of pKH9 revealed an open reading frame (ORF) encoding a putative autonomous rolling circle replication protein (Rep), an ORF predicted to encode a protein homologous to members of the FtsK/SpoIIIE cell division-DNA segregation protein family, and an operon encoding a putative toxin-antitoxin plasmid addiction system (txf-axf). Deletion analysis localized the pKH9 replication region in a 0.96-kbp fragment. The pKH9 rep gene is not present in this fragment, suggesting that pKH9 can replicate in fusobacteria independently of the Rep protein. A pKH9-based, compact Escherichia coli-F. nucleatum shuttle plasmid was constructed and found to be compatible with a previously described pFN1-based fusobacterial shuttle plasmid. Deletion of the pKH9 putative addiction system (txf-axf) reduced plasmid stability in fusobacteria, indicating its addiction properties and suggesting it to be the first plasmid addiction system described for fusobacteria. pKH9, its genetic elements, and its shuttle plasmid derivatives can serve as useful tools for investigating fusobacterial properties important in biofilm ecology and pathogenesis.
Amino Acid Motifs↗
Accuracy and reproducibility of the 4-hour ATB 32A method for anaerobe identification.
The ATB 32A system (API System SA, La Balme les Grottes, Montalieu-Vercieu, France) was evaluated for use in the identification of 214 anaerobes. Organisms included 73 isolates of the Bacteroides fragilis group, 24 Bacteroides spp., 10 fusobacteria, 43 clostridia, 28 cocci, and 36 gram-positive, nonsporeforming rods. With the concomitant use of Gram stain, pigmentation, catalase testing, and aerobic growth, the ATB 32A system correctly identified 97% of the B. fragilis group isolates, 88% of Bacteroides spp., 50% of fusobacteria, 74% of clostridia, 100% of cocci, and 86% of the gram-positive, nonsporeforming rods. Overall, 188 strains (88%) were correctly identified, with 18 (8%) requiring extra tests, other than the four mentioned above, for correct identification. Eight strains were misidentified, including one Bacteroides sp., three fusobacteria, one Clostridium sp., and three gram-positive, nonsporeforming rods. Reproducibility was very good, with 12 of 14 strains (86%) tested in triplicate yielding identical correct results on each of three occasions and 2 strains (14%) yielding identical correct results on two occasions. There was a low-probability identification (including the correct species) on the third testing. The ATB 32A system represents a worthwhile advance in systems used for the identification of clinically significant anaerobic bacteria.
Bacteria, Anaerobic↗
Cellular fatty acids in Fusobacterium species as a tool for identification.
Identification of fusobacteria from clinical specimens currently requires analysis of metabolic end products by gas-liquid chromatography in addition to certain biochemical and enzymatic tests because of the relative biochemical inactivity of these bacteria. Even the finding of pointed, thin gram-negative cells on Gram-stained slides can no longer be relied on for identification of Fusobacterium nucleatum, since at least four other species of fusobacteria have been seen to exhibit similar morphology. We examined 46 clinical isolates and six American Type Culture Collection type strains of fusobacteria by conventional methods and by the Microbial ID Systems MIDI software package for analyzing cellular fatty acid patterns measured by capillary column gas-liquid chromatography. Distinctive patterns of major fatty acids could be used to reliably identify most clinical isolates to the species level. The MIDI system identified 89% of the isolates correctly and provides an alternative to conventional methods.
Bacterial Typing Techniques↗
Comparison of two popular methods for removal and killing of bacteria from dentures.
A study was undertaken to compare the efficacy of a soaking solution (Efferdent Extra-Strength Denture Cleanser Tablets) to mechanical cleaning with a denture paste (Advanced Formula Dentu-Creme Denture Cleaning Paste) to remove and kill plaque bacteria from removable dentures. The study was conducted in a randomized, four-way crossover fashion with 18 subjects. At each clinic visit, subjects were randomized to one of four treatment regimens: 1) no treatment; 2) brushing with denture paste; 3) soaking in Efferdent; 4) brushing followed by soaking in Efferdent. Microbiological sampling for plaque bacteria was made before and after each treatment. Aliquot samples of 10-fold serial dilutions were plated on supplemented Schaedler Agar (for total anaerobes) and on CVE agar (for fusobacteria). Analysis of covariance was performed on the log10 transformed scores at posttreatment using the pre-treatment scores as covariates. Significant treatment effects were: F(3,41) = 81.60, p less than 0.001 for anaerobes and F(3,50) = 104.38, p less than 0.001 for fusobacteria. Pairwise comparisons using Tukey hsd post hoc tests showed that for total anaerobes, treatments 1 and 2 yielded higher scores than treatments 3 and 4. For fusobacteria, treatment 1 greater than 2 greater than 3 or 4; no difference between treatments 3 and 4. The results demonstrated the superior performance of Efferdent over Dentu-Creme.
Analysis of Variance↗
[Identification of Bacteroides and Fusobacterium using antibiotic- and bile-impregnated disks].
Fifty-nine strains of gram-negative non-sporulating anerobic bacteria were identified to the genus. Comparison of the data on the biochemical identification with the phenotypes by sensitivity to antibiotics and bile showed, that the bacteroids of the fragilis group had the same phenotype. Sensitivity was determined with anaerodisks impregnated with antibiotics and bile. The other species of bacteroids and fusobacteria had several phenotypes. However, these phenotypes being non-specific for the species were characteristic of the genus: none of the bacteroid species had the fusobacteria phenotype. The presented table of the bacteroid and fusobacteria phenotypes by sensitivity to antibiotics and bile may be useful in identification of these organism.
Anti-Bacterial Agents↗
Validation of 16S rDNA sequencing in microdissected bowel biopsies from Crohn's disease patients to assess bacterial flora diversity.
The bowel flora is implicated in Crohn's disease (CD) pathogenesis but its precise role is still unclear. Several non-mutually exclusive hypotheses have been proposed: an unidentified persistent pathogen; excessive bacterial translocation; an immune system abnormality in response to normal bacteria; or a breakdown in the balance between protective and harmful bacteria. These hypotheses can be tested by identifying bacteria in specific microscopic bowel structures or lesions. The present paper describes a novel technique to assess bacterial flora diversity in bowel biopsies, by combining laser capture microdissection with broad-range 16S rDNA sequencing. Fifty-four samples comprising histologically normal and pathological mucosa, MALT, ulcers, submucosal lymphangiectasias, epithelioid granulomas, and lymph nodes were microdissected out of 30 bowel biopsies from five CD patients. Bacterial 16S rDNA was successfully amplified by PCR in all samples, and PCR products from 15 samples were selected for cloning and sequence analysis. A total of 729 bacterial DNA sequences were analysed, which could be attributed to six different phyla (Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Fusobacteria, and Planctomycetes). DNA from typical bowel bacteria (Enterobacteriaceae, Clostridiales, Bacteroidetes, Fusobacteria) was detected in all microdissected areas. It was thus convincingly demonstrated that 16S rDNA sequencing can be combined with microdissection to study the bowel flora. However, no specific persistent pathogen causal for CD was identified. The results suggest that Enterobacteriaceae may initiate or colonize ulcers in CD. Translocation of bacteria through established mucosal lesions or as a result of increased permeability may be involved in the evolution towards chronic inflammation and in the establishment of persistent lesions. Further study is needed to confirm these preliminary findings.
Adolescent↗
Comparative antibacterial activity of the new cephalosporin cefcanel against anaerobic bacteria.
The activity of cefcanel against anaerobic cocci, Clostridium perfringens, Bacteroides fragilis, Bacteroides spp. and fusobacteria was determined by the agar dilution method and compared with the activity of cefaclor, cephalexin, cefadroxil, phenoxymethylpenicillin and ampicillin. Cefcanel showed good activity against Clostridium perfringens, Bacteroides spp. and fusobacteria (MIC90 = 1-4 mg/l). Against anaerobic cocci its MIC90 value was 16 mg/l, and against Bacteroides fragilis, 32 mg/l. Cefcanel has an antibacterial activity that warrants investigation in clinical trials.
Ampicillin↗
Selective suppression of the anaerobic oropharyngeal microflora with local metronidazole.
Anaerobic microorganisms of the normal oropharyngeal flora have been shown to be the main pathogens in orofacial infections of odontogenic origin. Reduction of the density of anaerobes in the oral cavity as a prophylactic measure before oral surgery may be a rational way to reduce the frequency of post-operative infections. This report describes a local antibiotic regimen that can reduce anaerobic pathogens in the oral cavity. Over a period of 7 days, 10 healthy individuals applied locally 1.5-2 g of an antibiotic preparation consisting of 0.5% metronidazole in 99.5% Orabase paste three times daily. The paste had antibacterial activity against obligate anaerobic micro-organisms such as bacteroides, fusobacteria and leptotrichia, known pathogens in orofacial infections. Fusobacteria and leptotrichia were eliminated in all subjects. Bacteroides species were eliminated in five subjects and significantly decreased in three subjects. The aerobic microflora was not affected. No new colonisation of the oropharynx was observed during the treatment period. The pre-treatment numbers of different micro-organisms were re-established within 2-9 days after the withdrawal of metronidazole treatment.
Adult↗
Effects of Streptococcus crista and human saliva on the viability of Fusobacterium nucleatum ATCC 10953.
Associations with facultative species might help planktonic oral anaerobes survive when they traverse saliva. This study investigated whether co-aggregation with Streptococcus crista ATCC 51110 enhanced the viability of Fusobacterium nucleatum ATCC 10953. Two tubes each of co-aggregates and Fus. nucleatum ATCC 10953 alone were prepared. One of each set was resuspended in buffer, and the other in clarified saliva from each of 20 donors. After 1 h, cells were stained for viability. The median percentage of viable Fus. nucleatum ATCC 10953 in buffer with Strep. crista (86%) was significantly higher (p=0.04) than in buffer alone (77%). A similar trend was observed for saliva with Strep. crista (81%) and saliva alone (74%), although that difference was not significant (p=0.41). The median percentage of Fus. nucleatum co-aggregated in buffer (44%) was significantly reduced after incubation in saliva (16%) (p<0.0002). No such change was seen when saliva was replaced with purified salivary proline-rich glycoprotein, which can bind both species. For co-aggregate suspensions, there was no difference in the viability of fusobacteria that were or were not in direct contact with Strep. crista. In both cases, viability was significantly reduced in saliva relative to buffer. Strep. crista may enhance the viability of planktonic Fus. nucleatum ATCC 10953, but it is not yet clear whether that requires co-aggregation. Transmission of fusobacteria through saliva could depend on the interplay between protective factors, such as the presence of streptococci, and antimicrobial factors, which kill cells or disassociate co-aggregates.
Anti-Bacterial Agents↗