Fusobacterium canifelinum sp. nov., from the oral cavity of cats and dogs

Fourteen strains of Gram-negative, anaerobic, fluoroquinolone-resistant, non-sporulating rods were isolated from various infections in cats and dogs, as well as from wounds in humans after cat- or dog-bites. These strains were characterized by sequencing of the 16S-23S rDNA internal transcribed spacer (ITS) regions, 16S rDNA, DNA-DNA hybridization, phylogenetic analysis, and phenotypic tests. The results indicate that the novel strains belong to a distinct species, closely related to Fusobacterium nucleatum. The species Fusobacterium canifelinum sp. nov. is proposed, with strain ATCC BAA 689T as the type strain.     

Serum antibodies of periodontitis patients compared to the lipopolysaccharides of Porphyromonas gingivalis and Fusobacterium nucleatum

Serum antibody titers against the lipopolysaccharides (LPSs) of Porphyromonas gingivalis and Fusobacterium nucleatum were compared between 9 periodontitis patients and 24 healthy persons. The IgG titers against the LPSs of P. gingivalis ATCC 33277(T) and W50 were clearly higher in the patients than in the healthy persons. However, IgM titers against the LPSs of P. gingivalis strains were relatively low, and no significant difference was observed between the patients and healthy persons. On the other hand, IgG and IgM titers against the LPS of Fusobacterium nucleatum JCM 8532(T) in some patients were significantly higher than those in the healthy persons, although the difference in IgG titers was not large compared to that of the LPS of P. gingivalis. These results suggest that the antibody measurement of patients' sera against the LPS of periodontal bacteria can be applied for the diagnosis of periodontitis.     

成人牙周炎龈下套氧菌分离鉴定及药敏试验结果分析

分离并鉴定了329例成人牙周炎龈下优势厌氧菌群,并对不同病程中的菌群变迁、厌氧菌的药物敏感性进行了分析.成人牙周炎龈下标本中厌氧菌阳性检出率为97.9%,其中以牙龈紫质单胞菌检出率最高(38.5%),具核梭杆菌次之(18.9%).随着牙周病变程度的加重,牙龈紫质单胞菌、具核梭杆菌、产黑色素普氏菌、星群厌氧链球菌、厌氧消化链球菌的检出率增高(P＜0.05),小韦荣球菌的检出率下降(P＜0.01),表明前5种厌氧菌在AP发病过程中有重要作用,小韦荣球菌与之无关.替硝唑、甲硝唑和克林霉素对438株革兰氏阴性厌氧菌的MIC90分别为1～8,2～8和4～16 mg/L,对278株革兰氏阳性厌氧菌的MIC90分别为16～32,16～64和4～16 mg/L,表明替硝唑和甲硝唑体外抗革兰氏阴性厌氧菌效果优于克林霉素,抗革兰氏阳性厌氧菌作用不如克林霉素.     

Phylogenetic analysis of Porphyromonas species isolated from the oral cavity of Australian marsupials

Porphyromonas species are frequently isolated from the oral cavity and are associated with periodontal disease in both animals and humans. Black, pigmented Porphyromonas spp. isolated from the gingival margins of selected wild and captive Australian marsupials with varying degrees of periodontal disease (brushtail possums, koalas and macropods) were compared phylogenetically to Porphyromonas strains from non-marsupials (bear, wolf, coyote, cats and dogs) and Porphyromonas gingivalis strains from humans using 16S rRNA gene sequence analysis. The results of the phylogenetic analysis identified three distinct groups of strains. A monophyletic P. gingivalis group (Group 1) contained only strains isolated from humans and a Porphyromonas gulae group (Group 2) was divided into three distinct subclades, each containing both marsupial and non-marsupial strains. Group 3, which contained only marsupial strains, including all six strains isolated from captive koalas, was genetically distinct from P. gulae and may constitute a new Porphyromonas species.     

Genotypic and phenotypic characterization of fusobacteria from Chinese and European patients with inflammatory periodontal diseases

Phylogenetic and antigenic studies were performed on 48 human oral Fusobacterium strains from Chinese patients with either necrotizing ulcerative gingivitis (NUG) or gingivitis and on 23 Fusobacterium nucleatum or Fusobacterium periodonticum strains from European periodontitis patients. Alignment of partial 16S rRNA gene sequences resulted in a phylogenetic tree that corresponded well with the current classification of oral fusobacteria into F. periodonticum and several subspecies of F. nucleatum, in spite of much minor genetic variability. F. periodonticum, F. nucleatum subsp. animalis and a previously undescribed phylogenetic cluster (C4), that may represent an additional F. nucleatum subspecies, constituted discrete clusters distinct from the remainder of F. nucleatum with high bootstrap values. Chinese and European strains differed markedly with regard to their respective classification patterns, suggesting a predominance of F. peridonticum and F. nucleatum susp. animalis over F. nucleatum subsp. nucleatum and F. nucleatum subsp. fusiforme/vincentii in samples from China. Antigenic typing enabled the association of many previously described serovars with distinct phylogenetic clusters and when applied directly to uncultured clinical samples confirmed the differential distribution of oral Fusobacterium taxa in Chinese and European samples. Bacteria from cluster C4 and F. nucleatum subsp. animalis were significantly more prevalent and accounted for higher cell numbers in NUG than in gingivitis samples, suggesting a possible association of these rarely observed taxa with NUG in Chinese patients.     

不同浓度臭氧水对牙周致病菌的体外抑菌作用

摘要：目的 探讨不同浓度臭氧水对4种牙周致病菌的体外抑菌效果。方法 采用定量悬液法,分别用1.02 ppm、2.03 ppm和3.88 ppm三种浓度臭氧水,0 ppm无臭氧水溶液（阴性对照组）及3%双氧水（阳性对照组）,对常见的4种牙周致病菌牙龈卟啉单胞菌（Porphyromonas gingivalis）、具核梭杆菌（Fusobacterium nucleatum）、中间普氏菌（Prevotella intermedia）和黏性放线菌（Actinomyces viscosus）分别作用30 s、60 s后,观察不同作用时间下3种不同浓度臭氧水的抑菌效果。结果 对P. gingivalis、F. nucleatum、P. intermedia和A. viscosus作用30 s和60 s,与阴性对照组比较,1.02 ppm、2.03 ppm和3.88 ppm三种浓度臭氧水均具有明显的抑菌效果,3.88 ppm臭氧水与3%双氧水对4种细菌作用60 s的抑菌效果相同。 结论 实验所用的3种不同浓度臭氧水对牙周致病菌P. gingivalis、F. nucleatum、P. intermedia和A. viscosus均有抑菌作用,浓度越高,抑菌效果越好,臭氧水应用于牙龈牙周疾病临床防治有一定的价值和可行性。     

Identification and localization of extraradicular biofilm-forming bacteria associated with refractory endodontic pathogens

Bacterial biofilms have been found to develop on root surfaces outside the apical foramen and be associated with refractory periapical periodontitis. However, it is unknown which bacterial species form extraradicular biofilms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biofilms. Twenty extraradicular biofilms, used to identify bacteria using a PCR-based 16S rRNA gene assay, and seven root-tips, used to observe immunohistochemical localization of three selected bacterial species, were taken from 27 patients with refractory periapical periodontitis. Bacterial DNA was detected from 14 of the 20 samples, and 113 bacterial species were isolated. Fusobacterium nucleatum (14 of 14), Porphyromonas gingivalis (12 of 14), and Tannellera forsythensis (8 of 14) were frequently detected. Unidentified and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected. In the biofilms, P. gingivalis was immunohistochemically detected in all parts of the extraradicular biofilms. Positive reactions to anti-F. nucleatum and anti-T. forsythensis sera were found at specific portions of the biofilm. These findings suggested that P. gingivalis, T. forsythensis, and F. nucleatum were associated with extraradicular biofilm formation and refractory periapical periodontitis.     

Synergy in biofilm formation between Fusobacterium nucleatum and Prevotella species

The formation of biofilm by anaerobic, Gram-negative bacteria in the subgingival crevice plays an important role in the development of chronic periodontitis. The aim of this study was to characterize the role of coaggregation between Fusobacterium nucleatum and Prevotella species in biofilm formation. Coaggregation between F. nucleatum and Prevotella species was determined by visual assay. Effect of co-culture of the species on biofilm formation was assessed by crystal violet staining. Effect of soluble factor on biofilm formation was also examined using culture supernatant and two-compartment co-culture separated by a porous membrane. Production of autoinducer-2 (AI-2) by the organisms was evaluated using Vibrio harveyi BB170. Cells of all F. nucleatum strains coaggregated with Prevotella intermedia or Prevotella nigrescens with a score of 1-4. Addition of ethylenediamine tetraacetic acid or l-lysine inhibited coaggregation. Coaggregation disappeared after heating of P. intermedia or P. nigrescens cells, or Proteinase K treatment of P. nigrescens cells. Co-culture of F. nucleatum ATCC 25586 with P. intermedia or P. nigrescens strains increased biofilm formation compared with single culture (p < 0.01); co-culture with culture supernatant of these strains, however, did not enhance biofilm formation by F. nucleatum. Production of AI-2 in Prevotella species was not related to enhancement of biofilm formation by F. nucleatum. These findings indicate that physical contact by coaggregation of F. nucleatum strains with P. intermedia or P. nigrescens plays a key role in the formation of biofilm by these strains.     

A selective medium for Fusobacterium spp.

J.S. BRAZIER, D.M. CITRON AND E.J.C. GOLDSTEIN, 1991. A new selective medium (JVN) for the isolation of Fusobacterium spp. from clinical material is described. The medium incorporates josamycin, vancomycin and norfloxacin (at 3, 4 and 1 μg/ml, respectively) as the selective agents, plus 5% defibrinated horse blood in Fastidious Anaerobe Agar Base (Lab M). This formula allowed luxuriant growth of all 82 strains (eight recognized species) of fusobacteria tested, while significantly inhibiting 51/51 (100%) strains of facultative anaerobes and 45/51 (88%) strains of other obligate anaerobes. JVN medium allowed the successful isolation of strains of Fusobacterium naviforme, F. nucleatum and F. necrophorum from the gingivae of 9/16 healthy volunteers, and strains of F. varium and F. mortiferum from faecal suspensions seeded with these organisms.     

Fluorescence based measurements of Fusobacterium nucleatum coaggregation and of fusobacterial attachment to mammalian cells

Fusobacterium nucleatum is a common oral anaerobe associated with gingivitis, periodontal disease and preterm deliveries. Coaggregation among oral bacteria is considered to be a significant factor in dental plaque development. Adhesion to host cells was suggested to be important for the F. nucleatum virulence associated with oral inflammation and with preterm births. An uncharacterized fusobacterial galactose inhibitible adhesin mediates coaggregation of F. nucleatum 12230 and F. nucleatum PK1594 with the periodontal pathogen Porphyromonas gingivalis. This adhesin is also involved with the attachment of both fusobacterial strains to host cells. However, it has been suggested that additional unidentified fusobacterial adhesins are involved in F. nucleatum virulence associated with preterm births. In this study, a fluorescence-based high throughput sensitive and reproducible method was developed for measuring bacterial coaggregation and bacterial attachment to mammalian cells. Using this method we found that coaggregation of F. nucleatum 4H with P. gingivalis and its attachment to murine macrophages is less inhibitible by galactose than that of F. nucleatum PK1594. These findings suggest that F. nucleatum 4H can serve as a model organism for identifying nongalactose inhibitible F. nucleatum adhesins considered to be involved in fusobacterial attachment to mammalian cells.     

Microbiological and clinical effects of a 1% chlorhexidine-gel in untreated periodontal pockets from adult periodontitis patients.

The aim of this study was to report the microbiological and clinical effects of repeated subgingival administration of a 1% Chlorhexidine-gel in periodontal pockets from 10 patients with adult periodontitis. Results showed that the experimental treatment significantly improved clinical parameters (Plaque Index, Gingival Bleeding Index, and Pocket Probing Depth). Direct subgingival administration of Chlorhexidine-gel also produced a remarkable modification in the proportions of putative periodontopathic microorganisms, such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Veillonella parvula, Fusobacterium nucleatum, and Peptostreptococcus micros, in subgingival bacterial plaque from periodontitis patients.     

Coaggregation of Porphyromonas gingivalis and Fusobacterium nucleatum PK 1594 is mediated by capsular polysaccharide and lipopolysaccharide

Previous reports have shown that coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum, two important periodontopathogens, is mediated by a galactoside on the surface of P. gingivalis and a lectin on F. nucleatum. In the present study, purified capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of P. gingivalis PK 1924 (serotype K5) were found to be able to bind to F. nucleatum cells and to inhibit binding of F. nucleatum to P. gingivalis serotype K5. Sugar binding studies showed that the requirements for binding of P. gingivalis serotype K5 CPS and LPS to the F. nucleatum lectin are: the presence of a metal divalent ion, an axial free hydroxyl group at position 4 and free equatorial hydroxyl groups at position 3 and 6 of d-galactose. These data suggest that P. gingivalis serotype K5- CPS and LPS act as receptors mediating coaggregation between P. gingivalis and fusobacteria.     

Identification of a methioninase inhibitor, myrsinoic acid B, from Myrsine seguinii Lév., and its inhibitory activities

A methioninase inhibitor from Myrsine seguinii was purified and identified as myrsinoic acid B. Its inhibitory activities as to crude methioninase from periodontal bacteria such as Fusobacterium nucleatum, Porphyromonas gingivalis, and Treponema denticola were determined. The IC50 values were 10.5, 82.4, and 30.3 microM respectively.     

Intracellular degradation of Fusobacterium nucleatum in human gingival epithelial cells

The role of Fusobacterium nucleatum in oral health and disease is controversial. We have previously shown that F. nucleatum invades gingival epithelial cells. However, the destiny of the internalized F. nucleatum is not clear. In the present study, the intracellular destiny of F. nucleatum and its cytopathic effect on gingival epithelial cells were studied. The ability of F. nucleatum and seven other oral bacterial species to invade immortalized human gingival epithelial (HOK-16B) cells were compared by confocal microscopy and flow cytometry. F. nucleatum had the highest invasive capacity, comparable to that of Porphyromonas gingivalis, a periodontal pathogen. Confocal microscopic examination revealed colocalization of internalized F. nucleatum with endosomes and lysosomes. Examination by transmission electron microscopy revealed that most intracellular F. nucleatum was located within vesicular structures with single enclosed membranes. Furthermore, F. nucleatum could not survive within gingival epithelial cells and had no cytopathic effects on host cells. Interestingly, endosomal maturation played a role in induction of the antimicrobial peptides human beta defensin (HBD)-2 and -3 by F. nucleatum from gingival epithelial cells. F. nucleatum is destined to enter an endocytic degradation pathway after invasion and has no cytopathic effect on gingival epithelial cells, which may cast new light on the role of F. nucleatum in the pathogenesis of periodontitis.     

Peptostreptococcus micros coaggregates with Fusobacterium nucleatum and non-encapsulated Porphyromonas gingivalis

Coaggregation is one of the potential colonization strategies of oral microorganisms, often involving fimbrial structures in the interactions. In this study, the coaggregation characteristics of the rough and smooth genotypes of the periodontal pathogen Peptostreptococcus micros were compared to investigate the role of the fibril-like structures of the rough genotype in coaggregation. Of the 11 oral species tested, only Fusobacterium nucleatum strains and non-encapsulated Porphyromonas gingivalis strains coaggregated with P. micros. No differences in coaggregation between the smooth type (Sm), the rough type (Rg) and the smooth variant of the Rg type (Rg(Sm)) of P. micros were observed. Heat-stable, periodate-sensitive structures on P. micros appeared to interact with heat- and protease-sensitive structures on F. nucleatum and P. gingivalis. These data indicate that these unimodal coaggregations are not mediated by the proteinaceous fibril-like structures of the Rg genotype, but by carbohydrates present on both genotypes of P. micros.     

A selective medium for Fusobacterium spp

A new selective medium (JVN) for the isolation of Fusobacterium spp. from clinical material is described. The medium incorporates josamycin, vancomycin and norfloxacin (at 3, 4 and 1 microgram/ml, respectively) as the selective agents, plus 5% defibrinated horse blood in Fastidious Anaerobe Agar Base (Lab M). This formula allowed luxuriant growth of all 82 strains (eight recognized species) of fusobacteria tested, while significantly inhibiting 51/51 (100%) strains of facultative anaerobes and 45/51 (88%) strains of other obligate anaerobes. JVN medium allowed the successful isolation of strains of Fusobacterium naviforme, F. nucleatum and F. necrophorum from the gingivae of 9/16 healthy volunteers, and strains of F. varium and F. mortiferum from faecal suspensions seeded with these organisms.     

Development of strain-specific PCR primers based on a DNA probe Fu12 for the identification of fusobacterium nucleatum subsp. nucleatum ATCC 25586T

The objective of this study was to assess the strain-specificity of a DNA probe, Fu12, for Fusobacterium nucleatum subsp. nucleatum ATCC 25586T (F. nucleatum ATCC 25586T), and to develop sets of strain-specific polymerase chain reaction (PCR) primers. Strain-specificity was tested against 16 strains of F. nucleatum and 3 strains of distinct Fusobacterium species. Southern blot hybridization revealed that the Fu12 reacted exclusively with the HindIII-digested genomic DNA of F. nucleatum ATCC 25586T. The results of PCR revealed that three pairs of PCR primers, based on the nucleotide sequence of Fu12, generated the strain-specific amplicons from F. nucleatum ATCC 25586T. These results suggest that the DNA probe Fu12 and the three pairs of PCR primers could be useful in the identification of F. nucleatum ATCC 25586T, especially with regard to the determination of the authenticity of the strain.     

三种根管充填材料对 乳牙根尖周炎常见细菌的抑菌性

目的评估和比较3种乳牙根管充填材料对乳牙根尖周炎常见细菌的抗菌功效。方法在体外实验中,采用纸片扩散法检测并观察3种乳牙根管充填材料及蒸馏水作用于粪肠球菌、牙龈卟啉单胞菌和具核梭杆菌后形成的抑菌环直径(mm),用SPSS 24.0软件进行统计分析。结果三重抗生素糊剂对乳牙根尖周炎根管中3种细菌均显示出最强的抗菌潜力,其次是Vitapex?、氢氧化钙糊剂,结果差异具有统计学意义(P0.05);蒸馏水对3种细菌是非抑制性的。粪肠球菌对3种药物反应最敏感,其次是牙龈卟啉单胞菌、具核梭杆菌,结果差异具有统计学意义(P0.05)。结论三重抗生素糊剂是最推荐用于治疗乳牙根尖周炎的根管充填材料。     

Localization of the Fusobacterium nucleatum T18 adhesin activity mediating coaggregation with Porphyromonas gingivalis T22.

Adherence of pathogenic bacteria is often an essential first step in the infectious process. The ability of bacteria to adhere to one another, or to coaggregate, may be an important factor in their ability to colonize and function as pathogens in the periodontal pocket. Previously, a strong and specific coaggregation was demonstrated between two putative periodontal pathogens, Fusobacterium nucleatum and Porphyromonas gingivalis. The interaction appeared to be mediated by a protein adhesin on the F. nucleatum cells and a carbohydrate receptor on the P. gingivalis cells. In this investigation, we have localized the adhesin activity of F. nucleatum T18 to the outer membrane on the basis of the ability of F. nucleatum T18 vesicles to coaggregate with whole cells of P. gingivalis T22 and the ability of the outer membrane fraction of F. nucleatum T18 to inhibit coaggregation between whole cells of F. nucleatum T18 and P. gingivalis T22. Proteolytic pretreatment of the F. nucleatum T18 outer membrane fraction resulted in a loss of coaggregation inhibition, confirming the proteinaceous nature of the adhesin. The F. nucleatum T18 outer membrane fraction was found to be enriched for several proteins, including a 42-kDa major outer membrane protein which appeared to be exposed on the bacterial cell surface. Fab fragments prepared from antiserum raised to the 42-kDa outer membrane protein were found to partially but specifically block coaggregation. These data support the conclusion that the 42-kDa major outer membrane protein of F. nucleatum T18 plays a role in mediating coaggregation with P. gingivalis T22.