AP-PCR法鉴别中间普里沃菌和变黑普里沃菌

目的　用引物OPA - 13的随机引物聚合酶链法 (arbitrarilyprimedpolymerasechainreaction ,AP -PCR)区分中间普里沃菌 (Prevotellaintermedia ,Pi)和变黑普里沃菌 (Prevotellanigrescens ,Pn)。 方法　用引物OPA - 13,采用AP -PCR法分析 97株来自 2 5例成年人牙周炎 (adultperiodontitis,AP)的Pi和 4 9株来自 2 1例AP的Pn的基因型。结果　 97株Pi中仅有 1株没有 90 0bp的特异性片段 ,4 9株Pn中也只有 1株没有产生 1.3kb的特异性片段。牙龈卟啉单胞菌 (Pg)ATCC 332 77,PgW 83,产黑色素普里沃氏菌ATCC 2 5 84 5 ,不解糖卟啉单胞菌ATCC 2 5 2 6 0 ,躯体普里沃氏菌ATCC 335 4 7,小齿普里沃氏菌ATCC 33185均不产生相关片段。结论　用引物OPA - 13的AP -PCR法可以区分Pi和Pn。     

Hydrolytic and depolymerising enzyme activity of Prevotella intermedia and Prevotella nigrescens

OBJECTIVE: Prevotella intermedia has been reported to be associated with periodontal disease whilst P. nigrescens has predominantly been isolated from more specific conditions and healthy sites. The aim of the present study was to compare the enzyme activity of these species.
MATERIALS AND METHODS Nine strains of P. intermedio and 12 strains of P. nigrescens were studied. Lipolytic. saccharolytic, nucleolytic and proteolytic activity was determined by traditional microbiological and chromo-genic substrate methods.
RESULTS: All strains hydrolysed gelatine, casein. DNA and RNA. Lipase activity was produced by all strains except P. nigrescens ATCC 33563^T. Lipolytic activity of P. nigrescens strains decreased as the environmental glucose concentration was increased. Only two strains, both P. intermedia , hydrolysed benzyl-arg-p-nitroanilide. All strains hydrolysed alkaline pnitrophenolphosphate (except P. intermedia DAL 100). produced glycylprolyl dipeptidase activity and demonstrated elastase-like activity. All but three strains (2 P. intermedia and I P. nigrescens) hydrolysed suc-ala-ala-pro-phe-p-nitroanilide. Overall, no qualitatively analysed enzyme activity was exclusive to all strains of either species. Quantitatively analysed activity exhibited a high degree of variability both within and between species.
CONCLUSIONS: P. intermedia and P. nigrescens degrade natural and synthetic substrates, but intra- and interspec-ies activity is variable.     

Characterization of immunoglobulin G-degrading proteases of Prevotella intermedia and Prevotella nigrescens

Degradation of immunoglobulins is thought to be an important factor in the causation of periodontal diseases by hindering local host defenses and by providing nutrients to the periodontal microflora. In this study, we characterized the proteolytic activity against human immunoglobulin G (IgG) of 20 strains of Prevotella intermedia and Prevotella nigrescens isolated from periodontal pockets and oral abscesses. IgG degradation was studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. All strains degraded IgG within 48 h after growth in trypticase-yeast extract medium (TY) supplemented with 0.3% IgG. Incorporating IgG in TY broth enhanced bacterial growth. Protease profiles (zymography), which revealed the presence of 1-4 IgG-degrading proteolytic bands in bacterial cell extracts, became more complex after growth in the presence of IgG. A 38-kDa protease capable of degrading IgG nonspecifically was present in almost all strains. The proteolytic activity was mainly located on the surface of the cell envelope. Two strains of P. intermedia and P. nigrescens ATCC 33563 were selected for further studies. Bacterial cell suspensions in phosphate-buffered saline completely degraded human IgG, IgA and IgM within 24 h. This activity depended on reducing conditions and was inhibited at temperatures above 50°C. The pH optimum of immunoglobulin degradation was at pH 7. Strains cultured at 42°C showed a markedly reduced capacity to degrade IgG. Inhibition studies revealed that breakdown of IgG was caused by a cysteine protease(s). The capacity of P. intermedia and P. nigrescens to degrade immunoglobulins may explain their association with polymicrobial oral diseases.     

Genetic heterogeneity in Prevotella intermedia, Prevotella nigrescens, Prevotella corporis and related species isolated from oral and nonoral sites

Prevotella intermedia (43 isolates), Prevotella nigrescens (55) and Prevotella corporis (8) from oral and nonoral sites were distinguished by species-specific DNA fragments, after hybridization of DNA fragments with ribosomal RNA (ribotyping). Eight strains previously identified as P. intermedia did not have these specific fragments. P. nigrescens, P. intermedia and P. corporis formed separate clusters in dendrograms constructed using clustering with an unweighted pair group method with arithmetic averages of similarity values derived from ribotype patterns, with 10 subclusters in P. intermedia isolates and 26 in P. nigrescens. Nine groups of P. intermedia isolates and 6 of P. nigrescens shared identical patterns. Specific ribotypes or species were not associated with particular diseases when all isolates were analyzed. However, results from organisms isolated by one laboratory using consistent clinical reporting indicated that P. intermedia was associated with more severe forms of periodontitis and P. nigrescens with mild to moderate disease.     

β-lactamase-producing strains in the species Prevotella intermedia and Prevotella nigrescens

A total of 96 strains were collected that included laboratory strains and clinical isolates classified Prevotella intermedia sensu lato and the type strains of the species P. intermedia sensu stricto and Prevotella nigrescens. Susceptibility to amoxicillin and amoxicillin-clavulanic acid was determined by the Etest. PCRDNA probe assays were used to speciate each strain as P. intermedia sensu stricto or P. nigrescens. By Etest, 71 strains (74%) were susceptible to both amoxicillin and amoxicillin-clavulanic acid with minimum inhibitory concentrations in the 0.016–0.064 μg/ml range. In contrast, amoxicillin minimum inhibitory concentrations of 25 strains (26%) were in the range of 1.5–96 μg/ml with concomitant amoxicillin-clavulanic acid minimum inhibitory concentrations in the low range 0.016–0.38 μg/ml, indicating a production of β-lactamase as confirmed by nitrocefin tests. Of these β-lactamase-producing strains, 20% (5/25) were identified as P. intermedia sensu stricto by the PCR-DNA probe assay and 72% (18/25) as P. nigrescens. Our results provide support for the major role of P. nigrescens in the failure of therapy using β-lactam antibiotics.     

Characterization of Prevotella intermedia and Prevotella nigrescens by enzyme production, restriction endonuclease and ribosomal RNA gene restriction analyses

Restriction endonuclease analysis, rRNA gene restriction analysis (ribotyping), multilocus enzyme electrophoresis and lipase production were investigated for their potential to differentiate isolates belonging to the closely-related species Prevotella intermedia and Prevotella nigrescens . Of 122 strains identified originally as P. intermedia , 52 were assigned to P. intermedia and 68 to P. nigrescens using multilocus enzyme electrophoresis. All 39 P. intermedia and 52 out of 53 P. nigrescens tested produced lipase. Restriction endonuclease analysis identified clonal variants, but did not facilitate the differentiation of strains into species. Tag I ribotyping of 99 strains revealed that all P. intermedia demonstrated a species-specific fragment of 0.40 kbp, which was always associated with a second fragment of 0.57 kbp, and all P. nigrescens tested shared a species-specific fragment of 2.21 kbp. Two strains atypical by multilocus enzyme electrophoresis had none of the above species-specific fragments. Thus, lipase production and restriction endonuclease analysis did not distinguish between P. intermedia and P. nigrescens , but Tag I ribotyping did and also allowed the characterization of individual strains.     

Dipeptide utilization by the periodontal pathogens Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum

Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum, which can frequently be isolated from periodontal pockets, preferentially utilize proteins and peptides as growth substrates. In this study, we determined the size of peptide that is preferentially utilized as a source of energy and material for cell growth by P. gingivalis, P. intermedia, P. nigrescens and F. nucleatum using various sizes of poly amino acids consisting of two to approximately 100 molecules of aspartate or glutamate. Resting cells of P. gingivalis, P. intermedia and P. nigrescens utilized aspartylaspartate, while cells of P. gingivalis and F. nucleatum utilized glutamylglutamate. The addition of aspartylaspartate to the culture medium increased the growth of P. gingivalis, P. intermedia and P. nigrescens, while the addition of glutamylglutamate promoted the growth of P. gingivalis and F. nucleatum. These results clearly indicate that dipeptides such as aspartylaspartate and glutamylglutamate can be utilized as growth substrates for P. gingivalis, P. intermedia, P. nigrescens and F. nucleatum.     

Pathogenicity of Prevotella intermedia and Prevotella nigrescens isolates in a wound chamber model in rabbits

The pathogenicity of 14 isolates identified as Prevotella intermedia or Prevotella nigrescens by serogrouping using monoclonal antibodies was compared in a tissue cage model in rabbits. Seven strains from periodontal abscesses, 5 strains from deep periodontal pockets and 2 strains from gingivitis were tested in the animal model comprising 6 Teflon tissue cages implanted on the back each of 34 rabbits. A total of 105–108 cells of P. intermedia or P. nigrescens strains were inoculated alone or together with either Actinobacillus actinomycetemcomitans or Streptococcus mitis. Five strains of Porphyromonas gingivalis were used as a reference. The infectivity was recorded as pus formation and log viable count in aspirated material for 3, 7 and 14 days. None of the Prevotella strains inoculated in monoculture survived more than 3 days, and they had no capacity to produce abscess. P. intermedia or P. nigrescens strains in combination with A. actinomycetemcomitans produced abscesses in 33–100% and with S. mitis in 42–100%. No difference in abscess formation or log viable count in samples after 14 days was recorded between serogroup I ( P. intermedia ) and serogroup II and III ( P. nigrescens ). The infectivity of P. intermedia or P. nigrescens strains did not differ whether they were isolated from periodontal abscess, periodontal pocket or gingivitis. P. intermedia and P. nigrescens strains produced abscesses in combination with a facultative anaerobic strain and appears to have a similar pathogenicity in the wound chamber model in rabbits.     

Similarity of salivary and subgingival Prevotella intermedia and Prevotella nigrescens isolates by arbitrarily primed polymerase chain reaction

The distribution and the genetic similarity of Prevotella intermedia and Prevotella nigrescens in saliva and in subgingival samples recovered from the same subject were studied in 16 subjects with different periodontal status. The isolates (4 salivary and 4 subgingival P. intermedia/nigrescens group isolates per subject) were identified to species level by hybridization with species-specific oligonucleotide probes, and the clonal analysis was performed using arbitrarily primed polymerase chain reaction (AP-PCR) (all isolates) and ribotyping (isolates from 5 subjects). In addition, the applicability of AP-PCR in differentiating between P. intermedia and P. nigrescens species was tested using 18 P. intermedia and 20 P. nigrescens isolates from 34 subjects. P. intermedia was detected in 7 and P. nigrescens in 14 of the 16 subjects. In all subjects the same species was found both in saliva and in subgingival plaque. In 15 of the 16 subjects, similar AP-PCR types of P. intermedia and/or P. nigrescens between salivary and subgingival samples were found. The salivary and subgingival isolates that were similar by AP-PCR were indistinguishable also by ribotyping. The AP-PCR analysis revealed a P. intermedia or P. nigrescens species-specific AP-PCR product in most isolates. This study indicates that both P. intermedia and P. nigrescens were found both in salivary and in subgingival samples, and both sampling sites within the same individual were usually colonized with identical AP-PCR types of the species. Thus, in addition to a subgingival sample a salivary sample seems to be suitable for detection and clonal analysis of these species. The AP-PCR method proved to be a simple method applicable for differentiation and clonal analysis of P. intermedia and P. nigrescens.     

β-lactamase-producing strains in the species Prevotella intermedia and Prevotella nigrescens

A total of 96 strains were collected that included laboratory strains and clinical isolates classified Prevotella intermedia sensu lato and the type strains of the species P. intermedia sensu stricto and Prevotella nigrescens. Susceptibility to amoxicillin and amoxicillin-clavulanic acid was determined by the Etest. PCRDNA probe assays were used to speciate each strain as P. intermedia sensu stricto or P. nigrescens. By Etest, 71 strains (74%) were susceptible to both amoxicillin and amoxicillin-clavulanic acid with minimum inhibitory concentrations in the 0.016–0.064 μg/ml range. In contrast, amoxicillin minimum inhibitory concentrations of 25 strains (26%) were in the range of 1.5–96 μg/ml with concomitant amoxicillin-clavulanic acid minimum inhibitory concentrations in the low range 0.016–0.38 μg/ml, indicating a production of β-lactamase as confirmed by nitrocefin tests. Of these β-lactamase-producing strains, 20% (5/25) were identified as P. intermedia sensu stricto by the PCR-DNA probe assay and 72% (18/25) as P. nigrescens. Our results provide support for the major role of P. nigrescens in the failure of therapy using β-lactam antibiotics.     

Optimized oligonucleotides for the differentiation of Prevotella intermedia and Prevotella nigrescens

The gram-negative, anaerobic bacterium Prevotella intermedia plays an important role in the progression of periodontitis, whereas the etiological role of the closely related but phenotypically indistinguishable species Prevotella nigrescens is controversial. To differentiate between these species properly, 16S rDNA/RNA directed, computer-optimized oligonucleotides were designed and tested with 26 P. intermedia , 26 P. nigrescens and a number of closely and more distantly related strains. The oligonucleotides were used as primers in a polymerase chain reaction and could be demonstrated to be species specific with a detection limit of 50 bacterial cells, which could also be detected when diluted 1:1⁵ with different plaque bacteria. In addition, the described oligonucleotides were digoxigenin-labeled at the 3' end and used as DNA probes in a dot blot hybridization assay. This assay, although slightly less sensitive than the polymerase chain reaction-based method, gave species-specific reactions and also allowed, (semi-)quantification of bacterial cells in clinical specimens.     

Pathways for amino acid metabolism by Prevotella intermedia and Prevotella nigrescens

Pathways for amino acid metabolism by Prevotella intermedia and Prevotella nigrescens were investigated. Prevotella strains grew anaerobically in tryptone-based medium and their growth increased upon the addition of aspartate to the medium. Washed cells of tryptone-grown strains metabolized aspartate to succinate, acetate, fumarate, malate, formate and ammonia, while from tryptone they produced isobutyrate and isovalerate in addition to the end products from aspartate. Cell extracts obtained from the tryptone-grown cells had aspartate ammonia-lyase for the conversion of aspartate to fumarate. Methylviologen-dependent fumarate reductase was found to reduce fumarate to succinate. A series of enzymatic activities, including fumarase, NAD-dependent malate dehydrogenase, oxaloacetate decarboxylase, methylviologen-dependent pyruvate oxidoreductase, phosphotransacetylase and acetate kinase, was detected for the oxidative conversion of fumarate to acetate. Pyruvate formate-lyase and NAD-dependent formate dehydrogenase were also found for the production and consumption of formate, respectively. Methylviologen: NAD(P) oxidoreductase was found to be responsible for linkage between these reductive and oxidative pathways. Furthermore, the cell extracts had branched-chain amino acid aminotransferase and methylviologen-dependent branched-chain 2-oxoacid oxidoreductase, concomitantly with NAD-dependent glutamate dehydrogenase. Valine and leucine could be converted to isobutyryl CoA and isovaleryl CoA, respectively, through the sequential catalyses of these enzymes, and consequently to isobutyrate and isovalerate, respectively.     

Intra-familial transmission and distribution of Prevotella intermedia and Prevotella nigrescens

The periodontal bacteria Prevotella intermedia and Prevotella nigrescens have been recently separated from each other. The purpose of this study was to investigate the distribution and routes of transmission of these bacteria among family members. Seven patients with moderate to severe periodontitis were selected. These probands, their spouses and 14 of their children were investigated. The presence of Pr. Intermedia and Pr. nigrescens was determined by culture techniques in pooled subgingival plaque samples, in the saliva, on the tongue, tonsils and buccal mucosa. Differentiation of Pr. intermedia and Pr. nigrescens was performed by enzyme electrophoretic mobility. From all 7 patients, as well as 4 spouses and 3 of the children, Pr. intermedia could be isolated. Pr. Nigrescens was found in 2 of the 7 patients, in 5 of the spouses and in 5 of the 6 children aged 5–10 yr. In the 8 children aged 0–4 yr both species were seldom isolated. These data are in accordance with earlier findings that Pr. Intermedia is associated with periodontitis and Pr. Nigrescens with a relatively healthy periodontal condition. Ribotyping of bacteria was performed by hybridization of Hindlll restriction endonuclease digests of chromosomal DNA with ribosomal DNA. Isolates from unrelated individuals always had distinct ribotypes. Indistinguishable ribotypes of Pr. intermedia and Pr. Nigrescens were found both among married couples and among parents and children. This indicates that intrafamilial transmission of Pr. intermedia and Pr. Nigrescens is possible both between adults and between parents and children.     

Occurrence of Prevotella intermedia and Prevotella nigrescens in relation to gingivitis and gingival health

AIM: The occurrence of Prevotella intermedia (Pi) and Prevotella nigrescens (Pn) in relation to natural gingivitis, gingival health and 14-day experimental gingivitis was investigated in 25 non-dental students. MATERIALS AND METHODS: Samples were taken from the dorsum of the tongue, the tonsils (or tonsillar area), and the supra- and subgingival plaque. RESULTS: The microbiological results show that 73% of the samples were positive for the bacterial species presumed to be Pi and/or Pn. In natural gingivitis, gingival health and in experimental gingivitis 25, 23 and 25 subjects were found to be positive for Pi and/or Pn, respectively. The results of the 889 isolates that were successfully purified and differentiated, show that almost all subjects were colonized with Pn whereas approximately half of the study population harboured Pi. These 2 species were isolated from both dental plaque and mucosal sites and were found to colonize the oral cavity simultaneously. CONCLUSION: In natural gingivitis, at the start and after 14 days of experimental gingivitis, Pn was the predominant micro-organism.     

变黑普里沃菌基因多态性及其在成人牙周炎患者龈下菌斑中的分布

目的 了解变黑普里沃菌（P.n)的基因多态性及其在成人牙周炎（AP）病变部位与正常部位是否存在基因型的差异。方法 采用随机引物多聚酶链扩增法（APPCR）用引物OPA-03和OPA-13对来自21例AP的47株P.n作基因型分析。结果 同一患者口内有1-3种基因型，以1种和2种为主；同一患者病变部位（P）与正常部位（H）有1-2种基因型，以1种为主；未发现与牙周正常状态相关的特定基因型P.n。结论 变黑普里沃菌是口腔的正常菌群。     

Clonality of Porphy givens intermedia and Preotella nigrescens isolated from periodontally diseased and healthy sites

Black-pigmented anaerobes have been implicated as major pathogens in the aetiology of adult periodontitis but these organisms are also found in healthy sites. This study aimed to examine the relationship between genotypes of black-pigmented anaerobes and disease status of periodontal sites using restriction endonuclease analysis (REA) and ribotyping. The main black-pigmented species recovered from sites were Pot phyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens . Each of the 58 subjects investigated harboured distinct genotypes of these three species. Most subjects appeared to be colonized by a single genotype of P. gingivalis and Pr. intermedia . whereas multiple types of Pr. nigrescens colonized many individuals. Plasmids were only found in a few Pr. nigrescens strains. No association was found between the disease status of sites and any specific or group of genotypes of either species or presence of a plasmid. Since the same genotypes of P. gingivalis, Pr. intermedia and Pr. nigrescens were found at both diseased and non-diseased sites in a subject, adult periodontitis is not explained by the presence of specially virulent clones of these organisms. Their role in periodontitis, therefore, is likely to be opportunistic.     

Purification and partial characterization of a dipeptidyl peptidase from Prevotella intermedia

A peptidase hydrolyzed X-Pro-p-nitroanilide was purified from the cell extract of Prevotella intermedia ATCC 25611 by ion-exchange chromatography and hydrophobic interaction chromatography. The purified enzyme exhibited a molecular size of 74 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the maximum enzyme activity was found between pH 7.0 and pH 7.5. This peptidase was a serine enzyme and hydrolyzed Lys-Pro-p-nitroanilide, Arg-Pro-p-nitroanilide, and Ala-Pro-p-nitroanilide, but Lys-Ala-p-nitroanilide was not split. The enzyme may be classified as a dipeptidyl peptidase IV.     

Characterization of binding and utilization of hemoglobin by Prevotella nigrescens

The ability of Prevotella nigrescens to utilize and bind to hemoglobin was investigated. Growth studies showed that P. nigrescens was able to utilize hemoglobin efficiently as an iron source. Binding of P. nigrescens to hemoglobin was demonstrated by dot blot assay. Heat and trypsin treatments of the bacteria led to a decrease in activity. Globin gave nearly complete inhibition of activity. Additionally, lactoferrin partially inhibited activity. In contrast, transferrin, cytochrome C and catalase exerted little or no inhibitory effect. Although the sugars tested did not affect activity, several of the amino acids tested, including arginine, cysteine, histidine and lysine, inhibited activity. In a solid phase assay, 41-, 56- and 59-kDa proteins of P. nigrescens reacted with hemoglobin. These results suggest that P. nigrescens utilizes hemoglobin for growth and 41-, 56- and 59-kDa proteins may be involved in hemoglobin binding.