Antimicrobial activities of Garcinia kola on oral Fusobacterium nucleatum and biofilm

The extracts from the root, bark and seed of Garcinia kola are currently used in traditional medicine in Nigeria. The aim of this study was to evaluate the inhibitory activity of crude extracts of G. kola on Fusobacterium nucleatum isolated from the oral cavity. Methanol and aqueous extracts were prepared from the seed and the minimal inhibitory concentration was evaluated by the agar dilution method, using a Wilkins-Chalgren agar supplemented with horse blood (5%), hemin (5 mu g/ml) and menadione (1 mu g/ml). Antimicrobial activity of plant extracts on microbial biofilms was determined in microtiter plates. The seed of G. kola demonstrated significant inhibitory action on F. nucleatum isolates at a concentration of 1.25 and 12.5 mg/ml for amoxicillin resistant strain. It was able to inhibit the microbial biofilm formed by the association of F. nucleatum with Porphyromonas gingivalis ATCC 33277, Aggregatibacter actinomycetemcomitans ATCC 33384 and Prevotella intermedia ATCC 2564 at a concentration of 25 mg/ml. The in-vitro inhibitory effect of G. kola on F. nucleatum population suggests a potential role for its use in oral hygiene.

Effect of Fusobacterium nucleatum on the T and B cell responses to Porphyromonas gingivalis in a mouse model

T cell cytokine profiles and specific serum antibody levels in five groups of BALB/c mice immunized with saline alone, viable Fusobacterium nucleatum ATCC 25586, viable Porphyromonas gingivalis ATCC 33277, F. nucleatum followed by P. gingivalis and P. gingivalis followed by F nucleatum were determined. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-10 by dual colour flow cytometry and the levels of serum anti-F. nucleatum and anti-P. gingivalis antibodies determined by an ELISA. Both Th1 and Th2 responses were demonstrated by all groups, and while there were slightly lower percentages of cytokine positive T cells in mice injected with F. nucleatum alone compared with the other groups immunized with bacteria., F nucleatum had no effect on the T cell production of cytokines induced by P gingivalis in the two groups immunized with both organisms. However, the percentages of cytokine positive CD8 cells were generally significantly higher than those of the CD4 cells. Mice immunized with F nucleatum alone had high levels of serum anti-E nucleatum antibodies with very low levels of P. gingivalis antibodies, whereas mice injected with P gingivalis alone produced anti-P. gingivalis antibodies predominantly. Although the levels of anti-E nucleatum antibodies in mice injected with E nucleatum followed by P. gingivalis were the same as in mice immunized with F nucleatum alone, antibody levels to P. gingivalis were very low. In contrast, mice injected with P. gingivalis followed by F nucleatum produced equal levels of both anti-P. gingivalis and anti-F nucleatum antibodies, although at lower levels than the other three groups immunized with bacteria, respectively. Anti-Actinobacillus actitiomycetemcomitans, Bacteroides forsythus and Prevotella intermedia serum antibody levels were also determined and found to be negligible. In conclusion, F nucleatum immunization does not affect the splenic T cell cytokine response to P. gingivalis. However, F nucleatum immunization prior to that of P. gingivalis almost completely inhibited the production of anti-P gingivalis antibodies while P. gingivalis injection before F. nucleatum demonstrated a partial inhibitory effect by P. gingivalis on antibody production to F. nucleatum. The significance of these results with respect to human periodontal disease is difficult to determine. However, they may explain in part differing responses to P. gingivalis in different individuals who may or may not have had prior exposure to F. nucleatum. Finally, the results suggested that P. gingivalis and F. nucleatum do not induce the production of cross-reactive antibodies to other oral microorganisms.

Plasmid profile in oral Fusobacterium nucleatum from humans and Cebus apella monkeys

Fusobacterium nucleatum is a strict anaerobe and is indigenous of the human oral cavity. This organism is commonly recovered from different monomicrobial and mixed infections in humans and animals. In this study, the plasmid profile, the plasmid stability and the penicillin-resistance association in oral F. nucleatum isolated from periodontal patients, healthy subjects and Cebus apella monkeys were evaluated. Forty-five F. nucleatum strains from patients, 38 from healthy subjects and seven from C. apella were identified and analyzed. Plasmid extraction was performed in all the isolated strains. These elements were found in 26.7% strains from patients and one strain from C. apella. Strains from healthy subjects did not show any plasmid. Most of strains showed two plasmid bands ranging from 4 to 16 Kb, but digestions with endonucleases showed that they belonged to a single plasmid. The plasmid profile was similar and stable in human and monkey strains. Also, plasmids were classified into three groups according to size. Two strains were positive to beta-lactamase production and no plasmid DNA-hybridization with a beta-lactamase gene probe was observed, suggesting a chromosomal resistance.

Genetic diversity of oral Fusobacterium nucleatum isolated from patients with different clinical conditions

The genetic diversity of 23 oral Fusobacterium nucleatum isolated from 15 periodontal patients, eight from seven healthy subjects, nine from nine AIDS patients and two from two Cebus apella monkeys were analyzed. EcoRI restricted the bacterial DNA and 28 ribotypes grouped from A to J groups were obtained. Isolates formed 24 ribotypes which were contained into A, B, C, D, E and F groups, and three reference strains and two clinical isolates of A. actinomycetemcomitans, and E. coli CDC formed four different ribotypes into the G, H, I and J groups. Moreover, from nine F. nucleatum from AIDS patients, six were ribotyped as group C and three as group D. By using ribotyping we distinguished F. nucleatum recovered from different sources. It is possible that isolates from AIDS patients may contain some phenotypic or genotypic factor did not observed in this study.

Fusobacterium necrophorum causing infective endocarditis and liver and splenic abscesses

A 25-year-old male without prior co-morbidities was admitted to hospital with Fusobacterium necrophorum bacteremia, where he was found to have liver and splenic abscesses. Further evaluation with echocardiography revealed a bicuspid aortic valve with severe insufficiency and a 1.68 x 0.86 cm vegetation. The patient required abscess drainage, intravenous antimicrobial therapy and aortic valve replacement. Complete resolution of the infection was achieved after valve replacement and a prolonged course of intravenous antimicrobial therapy. A brief analysis of the patient's clinical course and review of the literature is presented.

Rendimento dos meios seletivos para bactérias do gênero Fusobacterium

Meios seletivos para Fusobacterium foram, comparados no aspecto de sua eficiência no isolamento dos microrganismos fusiformes e quanto à inibição da flora concomitante. Material não calcificado do sulco gengival foi homogeneizado e diluído em tampão de fosfato (0,067 M - pH 7,2); 0,1 ml foi semeado em agar sangue, no meio de Omata & Disraely, meio de Omata & Disraely sem sôro, meio de McMarthy & Snyder, agar sangue com vancomicina, meio de Onisi, meio de Onisi com sôro de cavalo e meio de Onisi com pH 7,0. Em todos os casos a incubação foi realizada em anaerobiose, em jarra tipo Brewer, com 95% nitrogênio e 5% gás carbônico, durante 4 dias. O meio de McCarthy & Snyder mostrou-se tão eficiente quanto o agar sangue em relação ao isolamento de Fusobacterium, ainda inibindo a flora concomitante, o meio de Omata & Disraely, com cristal violeta e estreptomicina, foi de certo modo inibidor também para Fusobacterium. O meio de Onisi, sugerido, sem sôro, possibilitou o isolamento de pequeno número de fusobactérias, provàvelmente as amostras não exigentes de proteínas naturais.

Ultrastructural changes of Fusobacterium nucleatum as a defense mechanism against human neutrophil peptide-1 - A Transmission Electron Microscopy (TEM) study

Aims: The aim of this work was to assess the ultrastructural changes, cellular proliferation, and the biofilm formation ability of F. nucleatum as defense mechanisms against the effect of HNP-1. Materials and methods: The type strain of F. nucleatum (ssp. nucleatum ATCC 25586) and two clinical strains (ssp. polymorphum AHN 9910 and ssp. nucleatum AHN 9508) were cultured and incubated with four different test concentrations of recombinant HNP-1 (1, 5, 10 and 20 µg/ml) and one control group (0 µg/ml). Bacterial pellets from each concentration were processed for TEM imaging. Planktonic growth was assessed and colony forming units (CFU) were measured to determine the cellular proliferation. Scrambled HNP-1 was used for confirmation. Results: TEM analyses revealed a decrease in the outer membrane surface corrugations and roughness of the strain AHN 9508 with increasing HNP-1 concentrations. In higher concentrations of HNP-1, the strain AHN 9910 showed thicker outer membranes with a number of associated rough vesicles attached to the outer surface. For ATCC 25586, the treated bacterial cells contained higher numbers of intracellular granules with increasing the peptide concentration. Planktonic growth of the two clinical strains were significantly enhanced (P<0.001) with gradually increased concentrations of HNP-1. None of the planktonic growth results of the 3 strains incubated with the scrambled HNP-1 was statistically significant. HNP-1 decreased the biofilm formation of the two clinical strains, AHN 9910 and 9508, significantly (P<0.01 and P<0.001; respectively). Conclusions: The present in vitro study demonstrates that F. nucleatum has the ability to withstand the lethal effects of HNP-1 even at concentrations simulating the diseased periodontium in vivo. The increase in planktonic growth could act as defense mechanisms of F. nucleatum against HNP-1.

Antimicrobial effect of human serum on oral Fusobacterium nucleatum isolates from humans and monkeys

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Plasmid profile in oral Fusobacterium nucleatum from humans and Cebus apella monkeys

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of antimicrobial subinhibitory concentrations on hemolytic activity and bacteriocin-like substances in oral: Fusobacterium nucleatum

Fusobacterium nucleatum is considered for its role in colonization of initial and late microorganisms in dental plaque and for its coaggregation with other bacterial species. It is known that action of different antimicrobial substances may interfere with either virulence factors or with host-bacteria interaction. The goal of this study was to examine the influence of subinhibitory concentrations of chlorhexidine, triclosan , penicillin G and metronidazole on hemolytic activity and bacteriocin-like substance production of oral F. nucleatum. A high resistance to penicillin G was observed and 63% of the isolates were β-lactamase positive. All the tested isolates were susceptible to metronidazole. F. nucleatum isolates grown with or without antimicrobials were alpha-hemolytics. Bacteriocin-like substance production was increased in isolates grown with penicillin G. Impaired production of hemolytic or antagonic substances can suggest a role in the regulation of oral microbiota.

Isolation and molecular identification of Fusobacterium nucleatum from Nigerian patients with oro-facial infections.

Fusobacterium nucleatum is one of the most common anaerobic bacteria present in the oral cavity and is often isolated from infections involving other body sites. To characterise F. nucleatum strains from patients attending a teaching hospital in Nigeria in order to provide information on the methods for accurate identification of anaerobes in clinical specimen. Fusobacterium nucleatum specie from 50 patients presenting with oro-facial infections were studied by culture on Fusobacterium selective agar and fastidious anaerobe agar. The isolates were characterised based on colonial morphology, microscopy, lipase production, susceptibility to kanamycin and colistin and resistance to vancomycin. Biochemical tests were performed using a commercial test kit. The identity of the isolates was confirmed based on molecular characterization performed using polymerase chain reaction (PCR) analysis. Forty-eight (96%) F. nucleatum isolates were obtained from the 50 patients by culture and all the isolates were identified by colonial appearance and microscopy based on their unique spindle shape with tapered ends. Only 26 (54.2%) of the 48 isolates were identified by commercial API 20A test kit while PCR confirmed the identity of all the isolates. Anaerobes are involved in human infections and their study is quite cumbersome due to tedious nature and high cost of the techniques involved. Cultural method is reliable in the isolation and identification of F. nucleatum species. PCR is a rapid and simple method that can complement the phenotypic identification of anaerobes and would assist in their full identification.

Occurrence and antimicrobial susceptibility of Porphyromonas spp. and Fusobacterium spp. in dogs with and without periodontitis

The occurrence of Porphyromonas gulae, Porphyromonas macacae, Fusobacterium nucleatum and Fusobacterium canifelinum in subgingival plaque from dogs with and without periodontitis as well as their antimicrobial susceptibility were evaluated. From 50 dogs with periodontitis were identified 38 P. gulae, 8 P. macacae, 26 F. nucleatum and 15 F. canifelinum, and from 50 dogs without periodontitis were identified 15 P. gulae, 12 F. nucleatum and 11 F. canifelinum. All strains were susceptible to most of the antibiotics tested, however, different resistance rates to clarithromycin, erythromycin and metronidazole among strains were observed. The role of P. gulae, P. macacae, F. nucleatum and F. canifelinum in periodontal disease of household pets needs to be defined to a better prevention and treatment of the canine periodontitis. (c) 2012 Elsevier Ltd. All rights reserved.

Genome sequence of Fusobacterium nucleatum subspecies polymorphum - a genetically tractable fusobacterium.

Fusobacterium nucleatum is a prominent member of the oral microbiota and is a common cause of human infection. F. nucleatum includes five subspecies: polymorphum, nucleatum, vincentii, fusiforme, and animalis. F. nucleatum subsp. polymorphum ATCC 10953 has been well characterized phenotypically and, in contrast to previously sequenced strains, is amenable to gene transfer. We sequenced and annotated the 2,429,698 bp genome of F. nucleatum subsp. polymorphum ATCC 10953. Plasmid pFN3 from the strain was also sequenced and analyzed. When compared to the other two available fusobacterial genomes (F. nucleatum subsp. nucleatum, and F. nucleatum subsp. vincentii) 627 open reading frames unique to F. nucleatum subsp. polymorphum ATCC 10953 were identified. A large percentage of these mapped within one of 28 regions or islands containing five or more genes. Seventeen percent of the clustered proteins that demonstrated similarity were most similar to proteins from the clostridia, with others being most similar to proteins from other gram-positive organisms such as Bacillus and Streptococcus. A ten kilobase region homologous to the Salmonella typhimurium propanediol utilization locus was identified, as was a prophage and integrated conjugal plasmid. The genome contains five composite ribozyme/transposons, similar to the CdISt IStrons described in Clostridium difficile. IStrons are not present in the other fusobacterial genomes. These findings indicate that F. nucleatum subsp. polymorphum is proficient at horizontal gene transfer and that exchange with the Firmicutes, particularly the Clostridia, is common.