Visualization of an extracellular mucoid layer of Treponema denticola ATCC 35405 and surface sugar lectin analysis of some Treponema species.

Slime layers and capsules are common amongst medically relevant bacteria. We herein report that Treponema denticola, which has been associated with periodontitis, synthesizes or acquires an extracellular polysaccharide layer that we have observed through electron microscopy using the polysaccharide-specific dye Alcian blue and phosphotungstate. We have also visualized this extracellular layer by dark-field microscopy of Alcian blue-stained spirochete cells. A representative strain of each of the oral spirochete species T. denticola, Treponema vincentii and Treponema socranskii were differentiated by concanavalin A, phaseolus, lotus A and arachis lectins in a microtiter plate immunoassay for the detection of surface sugars.

An inexpensive solid medium for obtaining colony-forming units of oral spirochetes.

A method for the enumeration of colony-forming units of oral anaerobic spirochetes in new oral spirochete agarose (NOS-A) medium was described recently. However, the high cost of agarose limits the extent to which large assays can be carried out. Accordingly, a search for an inexpensive gelling agent that remains molten at 37 degrees C and gels at 25 degrees C was undertaken. Varying amounts of Noble agar or Bacto agar (0.5 to 1.5%, w/v) were mixed with varying amounts of gelatin (0.5 to 1.0%, w/v) in NOS medium. NOS medium containing 0.5% gelatin-0.5% Noble agar (NOS-GN) or 0.5% gelatin-0.5% Bacto agar (NOS-GB) met the above criteria. NOS-GN and NOS-GB media yielded higher colony-forming units with Treponema denticola than NOS-A medium in that order. However, all three media, NOS-GN, NOS-GB and NOS-A, performed equally well in the recovery of viable counts of T. vincentii. The NOS-GN medium was not liquefied by subgingival bacteria or two gelatinase-producing species of bacteria, Bacillus subtilis and Staphylococcus aureus. Thus NOS-GN medium is the recommended medium both in cost and performance for obtaining colony counts of spirochetes.

Characterization of a 4.2-kb plasmid isolated from periodontopathic spirochetes.

Oral anaerobic treponemes are assoicated with active periodontal disease and may comprise up to 57% of the microbiota in periodontal pockets. Four treponeme strains (designated U2a, U2b, U9b, and U9c) isolated from clincial cases were found to harbor a new 4.2-kb plasmid when plasmid DNA was extracted and purified employing the Qiagen Plasmid Kit. This plasmid differs from the smaller plasmids (2.0-, 2.6-, and 2.7-Kb) reported previously by others in Treponema denticola. The newly discovered 4.2-kb plasmid was found to be the same in all four treponeme strains by restriction endonuclease analysis. It is a circular plasmid since restriction with PstI, Pvu II, Sma I, Xma I, Ava 1 or Bam HI produced a single band of the same size. Bacterial strain U2b was shown to be Treponema socranskii and U9c to be T. denticola. The plasmid is designated "pTS1". The presence of the same plasmid in different species of the treponemes isolated from the same patient suggests the possibility of a naturally occurring genetic transfer system within the oral spirochetes or their ability to take up and maintain mobilizable plasmids.

Iron acquisition by oral hemolytic spirochetes: isolation of a hemin-binding protein and identification of iron reductase activity.

Oral anaerobic spirochetes (OAS) have been implicated in the etiology of periodontal disease. To adapt to the environment of the subgingiva, OAS must be able to acquire iron from limited sources. OAS have previously been shown not to produce siderophores but are beta-hemolytic and can bind hemin via a proteinaceous 47-kDa outer membrane sheath (OMS) receptor. Present studies show that [3H]hemin is not transported into the cytoplasm, that hemin and ferric ammonium citrate, as the sole iron sources, can support the growth of OAS and that protoporphyrin IX and Congo red are inhibitory, thereby implying an important in vivo role for hemin as an iron source. Treponema denticola ATCC 35405 produces an iron reductase. The iron reductase can reduce the central ferric iron moiety of hemin. The 47-kDa OMS hemin-binding protein has been purified to apparent homogeneity by methanol-chloroform extraction of cellular lipoproteins and the use of a hemin-agarose bead affinity column. A model of iron acquisition by OAS is presented.

An extracellular enzyme with hyaluronidase and chondroitinase activities from some oral anaerobic spirochaetes.

Treponema denticola, Treponema vincentii and Treponema socranskii produce an enzyme that hydrolyses hyaluronic acid (HA) and chondroitin sulphate (CS). The secreted enzyme is specifically inhibited by gold sodium thiomalate and anti-bee-venom antibodies. The use of saturated substrate (HA or CS) transblots allowed the visualization of active enzyme directly from culture supernatants and is a useful tool in clarification of complex polysaccharide-degrading enzyme specificities. The affinity-purified extracellular enzyme of T. denticola contains a single molecular species with a molecular mass of 59 kDa. Since it hydrolyses both HA and CS, it can more appropriately be termed a hyaluronoglucosaminidase (HGase). The HGase has been localized at the cell surface by electron microscopy and may play an active role in the degradation of connective tissue ground substance in the initiation and progression of periodontal disease.

Evidence for two distinct locomotory phenotypes of Treponema denticola ATCC 35405.

Using a two-layer system, a bottom layer containing treponema cells suspended in NOS (New Oral Spirochete)-Noble agar medium or NOS-Bacto agar medium and overlaid with cell-free NOS-agarose medium resulted in the spirochete cells migrating into the top layer. However, if the positions of the medium layers were reversed with the cells inoculated into the bottom layer containing NOS-agarose, there was no migration into the upper layer. This suggests migration of the spirochetes away from Bacto and Noble agars. Using a 3-layer system in which cells were inoculated into a middle layer consisting of NOS-agarose medium and sandwiched between cell-free NOS-agarose medium layers, cells remained within the middle layer. If the cells were inoculated into a middle layer consisting of NOS-Bacto agar medium while the upper and lower layers remained unchanged, cells migrated into both upper and lower layers. If cells that had migrated into the upper layer were transferred into a middle layer, they virtually all migrated into the upper layer repeatedly. Cells that had migrated into the lower layer and transferred to the middle layer migrated repeatedly into the lower layer. These results suggest the possible existence of two distinct locomotory phenotypes within this strain of treponeme.

Distribution and characterization of plasmids in oral anaerobic spirochetes.

Fifteen oral spirochete strains belonging to the species Treponema denticola, Treponema vencentii and Treponema socranskii as well as 9 fresh clinical isolates were screened for the presence of extrachromosomal plasmid DNA by a modified alkaline lysis procedure. A 2.6-kb plasmid was detected in both T. denticola ATCC 33520 and T. denticola e'. The 2.6-kb plasmid from T. denticola e' was shown to be similar to pTD1, previously reported by Ivic et al. in T. denticola ATCC 33520 on the basis of molecular weight, restriction endonuclease profile and DNA:DNA hybridization. T. denticola ATCC 33520 and T. denticola e' share 65% DNA homology and belong to different serological groups. This dissimilarity has been reconfirmed by specific immunofluorescence using polyclonal and monoclonal antibodies. A plasmid-free T. denticola ATCC 33520 was identified. Comparative studies have shown no antigenic, morphological, or genetic differences between the plasmid-bearing and the plasmid-free strain. In addition, screening of fresh clinical isolates of spirochetes revealed the presence of a 4.2-kb plasmid in 4 of these strains.

Enumeration of viable oral spirochetes from periodontal pockets.

We recently developed a successful method for quantifying oral anaerobic spirochetes in pure culture by a viable count. New oral spirochete medium was used with low temperature-gelling agarose in polystyrene tissue-culture flasks. We have extended the use of this method to determine the viable count of spirochetes from periodontal pockets. Sixteen subgingival plaque samples were obtained by insertion of sterile paper points into deep periodontal pockets. The points were placed into reduced transport medium at chairside, vortexed in the microbiology laboratory and aliquots of the medium inoculated into molten new oral spirochete-agarose medium (37 degrees C) containing rifampin (20 micrograms/ml) in a flask. Subsequent dilutions were made from this initial flask to other flasks containing selective medium in sequence. All flasks were incubated anaerobically. Most other subgingival bacteria were selectively inhibited by rifampin. Spirochete colonies were typically spherical and were either dense or cottony. Their identities were checked by darkfield examination. Counts of colony-forming units of cultivable spirochetes ranged from 12.5% to 28.2% of the total cultivable anaerobic flora by the method described.

Viscosity-dependent locomotion of oral spirochetes.

Spirochetes are thought to remain motile in environments (such as intercellular spaces) that immobilize extracellularly flagellated eubacteria. This attribute suggests that the viscosity of the milieu is of importance to locomotion. We sought to determine the interdependence of oral spirochete locomotion with media viscosity. Video time-lapse microscopy using darkfield optics was used. The motility of the spirochetes in media of different viscosities (various concentrations of Noble agar) was measured. Treponema denticola exhibited the fastest speed (18.7 +/- 4.4 microns/min) at a viscosity of 30 mPa.s. The highest speeds for Treponema vincentii and Treponema socranskii were 41.9 +/- 14.9 and 33.4 +/- 13.2 microns/min, respectively, at 88 mPa.s. These data show that optimal migration of spirochetes is viscosity-dependent. The results support the hypothesis that such viscosity-dependent locomotion could be a virulence factor that enables oral spirochetes to initiate and sustain periodontal disease.

Binding of hemin and congo red by oral hemolytic spirochetes.

Colony-forming units or cells in suspension of oral anaerobic spirochetes (Treponema denticola, Treponema vincentii and Treponema socranskii) bind hemin and Congo red. Hemin or Congo red binds to a hydrophobic polypeptide receptor that is located in the outer membrane of the bacterial cells and it has a relative molecular mass of 47 kDa. These oral spirochetes also lyse sheep erythrocytes to produce beta-hemolytic zones around colony-forming units. The oral spirochetes may acquire iron for growth when they lyse erythrocytes and bind heme from which they may sequester and transport iron into the cells.

Treponema denticola (ex Brumpt 1925) sp. nov., nom. rev., and identification of new spirochete isolates from periodontal pockets.

Standard growth and isolation methods were used to obtain five new treponema strains in pure culture from deep periodontal pockets. The strains were identified to the species level by various methods, including agglutination, immunofluorescence, a dot blot immunoassay, electron microscopy, gas-liquid chromatography of metabolic volatile fatty acids, and DNA hybridization. Two isolates were strains of Treponema socranskii; the other three were strains of "Treponema denticola," a species described in 1925 by Brumpt. Because no type strain was designated for this species previously, the name was not included on the Approved Lists of Bacterial Names and has no current nomenclatural standing. We propose that Treponema denticola (ex Brumpt) sp. nov., nom. rev. is a valid and distinct species of the genus Treponema and designate strain ATCC 35405 as the type strain and strains ATCC 33520 and ATCC 35404 as reference strains. T. denticola appears to be the species that is most frequently isolated from periodontal pockets. Unless new isolation and cultivation techniques are introduced, it appears that present technology can yield only isolates belonging to the currently described oral anaerobic spirochete species and that there is little chance of isolating the larger treponemes.

A successful method for quantifying viable oral anaerobic spirochetes.

Spirochetes are markedly prevalent in periodontal disease but are not included as predominant cultivable organisms because of the inability to quantify them by viable count. A successful method was developed for enumerating viable oral spirochetes as colony-forming units (CFU) in an agarose-based medium. Treponema denticola, Treponema vincentii and Treponema socranskii in log-phase growth in new oral spirochete (NOS) broth were used for evaluation of the method. Critical components of the method include enzyme-free low temperature-gelling (37 degrees C) agarose in NOS medium in small tissue-culture flasks into which the spirochetes were seeded and diluted. The flasks were anaerobically incubated in a glove-box. Reliable, consistent and reproducible viable counts of pure spirochete cultures were obtained. The injurious effects of spirochete temperature-sensitivity were averted by using molten agarose at 37 degrees C. Distinctive colony morphologies of spirochete species could be compared from pure cultures. Addition of rifampin into the medium showed no decrease in spirochete CFU count. The method as described allows for selection of mutants and detection of biochemical activity and is potentially useful for enumeration of spirochetes from periodontal pockets as members of the predominant cultivable flora.

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.

Clinical documentation and occurrence of putative periodontopathic bacteria in human immunodeficiency virus-associated periodontal disease.

Human immunodeficiency virus (HIV)-associated gingivitis (HIV-G) and HIV-associated periodontitis (HIV-P) are two intraoral lesions manifested by patients with HIV infection. Periodontal indices were measured for 87 subjects in 5 study groups: HIV-seropositive patients with healthy periodontium (HIV-H), with HIV-G, or with HIV-P; and non-HIV-infected subjects with healthy periodontium (H) or with adult chronic periodontitis (P). The quantitative clinical parameters were compared and statistically significant intergroup differences were noted. The mean scores on PI and PD do not discriminate between HIV-seropositive and non-HIV-infected seronegative cohorts, but a significant difference in the GI between HIV-H and H was noted. When categories of PD and AL are examined, some differences become apparent. Generally, the PD and AL of HIV-P are not as great as those of P. PI correlates well with GI (r = 0.86) in P, but does not (r = 0.33) in HIV-P. In addition, the occurrence of selected putative periodontopathic bacteria (Porphyromonas gingivalis, spirochetes, and motile eubacteria) in these lesions was determined by brightfield (after staining), darkfield and immunofluorescent microscopy. No difference in microbiological profile in the bacterial groups monitored was found between P and HIV-P. Spirochetes were found to be more abundant than P. gingivalis in the lesions of P and HIV-P. In marked contrast, P. gingivalis was found to be in highest numbers in samples from the gingival crevice of H as determined by indirect immunofluorescence.

Purification of phospholipase C by hydrophobic interaction affinity chromatography.

A simple procedure is described for the purification of phosphatidylcholine-hydrolyzing phospholipase C(PLC). Lecithin, the substrate for PLC, was ligated hydrophobically to octyl-Sepharose in 2 M (NH4)2SO4. The washed lecithin-conjugated resin was then used to purify PLC from crude preparations by affinity chromatography. PLC binds to the lecithin moiety in the presence of Zn2+ and is eluted with an acidic buffer containing EDTA. PLC activity was recovered in the eluate. Both sodium dodecyl sulphate polyacrylamide gel electrophoresis and pI electrofocusing showed that the eluate contained a single monomeric protein with an apparent molecular mass of 66 kDa and a pI of 5.5.

Synthesis and secretion of phospholipase C by oral spirochetes.

Four strains of oral treponemes and Treponema phagedenis Reiter synthesize and secrete phospholipase C (PLC), which was detected by the hydrolysis of p-nitrophenylphosphorylcholine. PLC was detected in gingival crevicular fluid from diseased but not from healthy sulci. The initiation and progression of periodontal lesions may begin with the hydrolysis of membrane phospholipids by PLC.

Coexistence of rheumatoid arthritis, ankylosing spondylitis and dermatomyositis in a patient with diabetes mellitus and the associated linked HLA antigens.

A patient is described who had insulin-dependent diabetes mellitus for 2 years, prior to developing rheumatoid arthritis and then subsequently ankylosing spondylitis and dermatomyositis. Diagnostic criteria for all diseases are fulfilled. HLA typing revealed the presence of HLA A2, A9, B8, B27, DR3 and DR4 antigens. The concomitant coexistence of diabetes mellitus, rheumatoid arthritis, ankylosing spondylitis and dermatomyositis appears to have occurred in an individual genetically susceptible to these diseases.

HLA associations in an Arab type 1 diabetic population.

We have studied HLA-A, -B, -C, -DR, and -DQ antigen frequencies in 63 Type 1 diabetic Arab patients resident in Kuwait. Both HLA-DR3 (relative risk (RR) = 5.80) and -DR4 (RR = 2.87) showed positive associations with Type I diabetes mellitus in these patients whilst -DR2 (RR 0.16) and -DR5 (RR = 0.15) were negatively associated. The strong positive association with both HLA-DR3 and -DR4 was confirmed in Non-Gulf Arabs (RR = 12.55 and 4.29, respectively) whereas the Gulf Arabs had a significant positive association with HLA-DR3 (RR = 4.41) only. The disease was negatively associated with HLA-DR2 (RR = 0.05) in Gulf Arab patients only and with HLA-DR5 (RR = 0.10) in Non-Gulf Arabs only. HLA-DRw52 and -DRw53 were increased in Non-Gulf Arabs only (RR = 3.14 and 4.63, respectively). In both groups there was strong association with HLA-DQ3 (Gulf, RR = 28.11; Non-Gulf, RR = 6.25). Amongst HLA-A, -B, and -C loci, there was a positive association with HLA-B8 (RR = 19.06).

Counterimmunoelectrophoresis using an arc 5 antigen for the rapid diagnosis of hydatidosis and comparison with the indirect hemagglutination test.

A modified counterimmunoelectrophoresis and a conventional indirect hemagglutination test were compared for routine diagnosis of human hydatid disease in an endemic area in the Middle East. Counterimmunoelectrophoresis was performed on a cellulose acetate membrane with dilutions of a commercially available antigen which interacts with sera of patients with confirmed hydatid disease to produce the arc 5 precipitin line. The test was performed with unconcentrated human sera and the lines stained in an aqueous solution of Ponceau red. Sensitivity (95.5% vs. 93.2%) and specificity (99.2% vs. 89.9%) were higher with counterimmunoelectrophoresis than with indirect hemagglutination. Cross-reactivity with sera of patients with other parasitic infections was noted with indirect hemagglutination but not with counterimmunoelectrophoresis. There was no cross-reactivity with sera of patients with autoimmune disorders by either test.

Induced malaria and antibody titres in acute infections and in blood donors in Kuwait.

Concurrent with the increase in the number of imported cases of malaria into non-endemic Kuwait during the past 5 years, induced infections have been identified for the first time. We report 10 such cases over a 4-year-period. Of 8 transfusion-induced infections, 4 were due to Plasmodium falciparum and 4 to P. vivax. The mean incubation period for P. falciparum patients was 13 d and for P. vivax, 17 d. An accidental syringe-needle transmission and a congenital infection were due to P. falciparum and P. vivax respectively. Malarial antibody levels were assayed on commercially-available cultured P. falciparum schizonts by the indirect fluorescent antibody (IFA) test. To establish a base line, the sera of patients with blood film-confirmed P. falciparum and P. vivax were assayed. 96% of the P. falciparum sera were positive, the geometric mean titre (GMT) being 10,280. However, all sera from P. vivax patients were reactive but the GMT was lower at 505. 28% of sera from Kuwaitis and 45% of sera of a consecutive group of blood donors were also reactive, the respective GMTs being 38 and 51. The risk of transfusion malaria was calculated as 79 per million units drawn, an unacceptably high figure for a non-endemic country. We suggest a revised blood donor policy.