A new method for routine isolation of oral treponemes using U-tubes and pectin medium.

Determination of the composition of the oral microflora has traditionally been based on cultivation. Treponemes are prevalent in many oral infections but, unfortunately, are not regularly cultured. In this study a new method was established for routine isolation of oral treponemes from clinical samples. Bacterial samples from 47 periodontal pockets and 4 endodontic infections were incubated anaerobically under nitrogen atmosphere at 37 degrees C in U-tubes containing pectin medium. In the U-tube a 'bacterial sample side' and a 'sterile medium side' were established on separate sides of a membrane filter and an agar plug. Using this method we were able to isolate viable treponemes from all bacterial samples. This was in contrast to previously established methods such as the agar dilution technique, the technique involving the membrane filter placed on the surface of solid agar media and the well in agar plate technique. We believe that the 'U-tube method' is a valuable supplement to previously described techniques in routine isolation of treponemes from clinical samples.

Periodontal microflora of HIV positive subjects with gingivitis or adult periodontitis.

The subgingival microflora of 39 HIV+ subjects with gingivitis or adult periodontitis was cultured quantitatively anaerobically for bacteria, spirochetes, and mycoplasma and aerobically for yeasts. Isolates were characterized by conventional biochemical tests, polyacrylamide gel electrophoresis of soluble proteins, cellular fatty acid profiles, immunofluorescence, and immunodiffusion. In general, the same types of bacteria were isolated from the subgingival crevice of HIV+ subjects as we previously had isolated from the subgingival crevice of non-HIV subjects. A statistically significant difference was found between the composition of the flora of HIV+ subjects with adult periodontitis (AP) and concurrent studies of a non-HIV+ AP population. Mycoplasma salivarium was significantly elevated in the HIV+ subjects examined. Yeasts were isolated from only 10% of the samples and from 13% of the HIV-positive subjects at 0.05 to 0.0002% of the total cultivable count when present.

Cytochrome composition and oxygen-dependent respiration-driven proton translocation in Wolinella curva, Wolinella recta, Bacteroides ureolyticus, and Bacteroides gracilis.

The membrane fractions of the microaerobically grown type strains of Wolinella curva, Wolinella recta, Bacteroides ureolyticus, and Bacteroides gracilis contained membrane-bound cytochrome b, cytochrome c, and CO-binding cytochrome c. Soluble cytochrome c and CO-binding cytochrome c were also present. Although B. gracilis is oxidase negative, it possessed cytochrome c. With H2 or formate as the electron donor, proton efflux from anaerobic cells occurred upon addition of a pulse of oxygen. With formate as the electron donor, the H+/O ratios of W. curva, W. recta, B. ureolyticus, and B. gracilis were 0.75, 1.66, 2.06, and 2.04, respectively. With H2 as the electron donor, the H+/O ratios of W. curva, B. ureolyticus, and B. gracilis were 1.25, 1.97, and 2.36, respectively. Proton translocation was inhibited by the protonophore carbonylcyanide m-chlorophenylhydrazone. The results confirm that the organisms are not anaerobes but are microaerophiles capable of respiring with oxygen.

The microflora of periodontal sites showing active destructive progression.

20 adult periodontitis (AP) subjects were examined every 2 to 4 months and microbiological samples were collected and cultured when 2 mm or more loss of attachment (active sites) was detected by 2 examiners. Similar sites in which no progressive destruction was observed (control sites) also were sampled in the same subjects. By lambda-analysis, there was no statistically significant difference in floras of active (42 sites from 12 subjects) and control (36 sites from 12 subjects) sites or between the floras of the active and control sites and of 63 samples from 22 AP subjects that were examined previously in a cross-sectional study. By paired t test, no microbial species had a significantly greater association with active than with control sites. The only species that were detected in one or more samples from all subjects with active sites were Wolinella recta, Fusobacterium nucleatum, and Peptostreptococcus micros. Porphyromonas gingivalis and 9 other taxa were isolated from one-half or more of the persons with active sites. The composition of microbiological floras of all periodontitis samples was statistically significantly different from that of subjects with healthy gingiva. The composition of microfloras of sites in subjects with naturally-occurring gingivitis was intermediate between that of subjects with healthy gingiva and that of active and control sites in AP subjects.

Wolinella recta, Wolinella curva, Bacteroides ureolyticus, and Bacteroides gracilis are microaerophiles, not anaerobes.

Although the nonfermentative, asaccharolytic, putative anaerobes Wolinella curva, Wolinella recta, Bacteroides ureolyticus, and Bacteroides gracilis are phylogenetically related to the true campylobacters, the type strains of these species exhibited O2-dependent microaerophilic growth in brucella broth and on brucella agar. The optimum O2 levels for growth of these strains ranged from 4 to 14% in brucella broth and from 2 to 8% on brucella agar, when H2 was provided as the electron donor. No growth occurred under 21% O2, and scant or no growth occurred under anaerobic conditions unless fumarate or nitrate was provided as a terminal electron acceptor. Aspartate, asparagine, and malate also served as apparent electron acceptors. The organisms were catalase negative and, except for B. gracilis, oxidase positive. Catalase added to brucella broth enhanced growth. O2 uptake by all species was inhibited by cyanide and 2-heptyl-4-hydroxyquinoline N-oxide. We concluded that these organisms are not anaerobes but instead are microaerophiles, like their campylobacter relatives.

Catalase activity in Campylobacter jejuni: comparison of a wild-type strain with an aerotolerant variant.

A comparison of Campylobacter jejuni VPI strain H840 (ATCC 29428), which can grow at O2 levels up to 15%, with variant strain MC711-01 (which can grow at O2 levels up to 21-26%) indicated that the specific activity of catalase in crude cell extracts was higher in the variant by a factor of 1.6 to 2.5, depending on cultural conditions. Smaller differences occurred with superoxide dismutase activity, while peroxidase activities were invariably lower in the variant strain. The variant strain was much more resistant than the wild type to the bactericidal effects of H2O2. The results suggest that catalase activity might be one of the factors associated with the greater tolerance of O2 by the variant strain. However, both strains became more susceptible to H2O2 when cultures were initially grown at 6% O2 and then shifted to 21% O2; thus the role of catalase in the oxygen tolerance of C. jejuni is probably minor.

Effect of incubation temperature, ageing, and bisulfite content of unsupplemented Brucella agar on aerotolerance of Campylobacter jejuni.

A mutant strain of Campylobacter jejuni ATCC 29428 was isolated that grows on unsupplemented Brucella agar at O2 levels as high as 21% at 37 degrees C. While measuring the degree of aerotolerance of this mutant on unsupplemented Brucella medium and comparing it with that of the wild type, we found considerable variation among our estimates. As measured by colony counts on unsupplemented Brucella agar incubated at various oxygen levels, the degree of aerotolerance was affected by incubation temperature and the age of the medium. Aerotolerance was consistently higher on plates incubated at 42 degrees C than at 37 degrees C. When the commercial dehydrated Brucella medium that was used to prepare the Brucella agar was stored in a beaker for 2.5 months, the degree of aerotolerance of C. jejuni was decreased. Addition of 0.01% sodium bisulfite reversed this inhibition. Storage of bottles of hydrated Brucella agar for 1.5 months also resulted in a decreased aerotolerance; again, in addition of 0.01% bisulfite reversed the effect. Aerotolerance was greatly decreased when Brucella agar was prepared from all its individual components except 0.01% bisulfite. The results indicate that the bisulfite component of Brucella agar deteriorates during storage of the dehydrated and hydrated media, and that this deterioration can affect measurements of aerotolerance.

Bacteriology of human gingivitis.

The subgingival bacterial floras of naturally occurring gingivitis in adults and children were characterized and compared with the floras of other periodontal conditions previously studied. The composition of the gingivitis floras was found to be distinct from that of floras associated with health or with moderate, severe, or juvenile periodontitis. There were no major differences between the floras of naturally-occurring gingivitis and the floras of the human experimental gingivitis model. Data indicated that the flora of healthy sites within a mouth is influenced by the number of inflamed sites, which argues against independence of sites bacteriologically. Proportions of ten bacterial species increased in both gingivitis and periodontitis, as compared with health, in both adults and children. These species were found in both affected and unaffected sites of people with gingivitis. The numbers of five other cultivable species and the "large treponeme", which was not cultivated, increased in gingivitis and periodontitis of adults only. Significant differences in non-spirochetal floras between children and adults were not found, although they were in the experimental gingivitis model studied previously. Cultivable spirochetes did differ between children and adults. Children had fewer samples positive for spirochetes, and children's positive samples contained greater proportions of T. socranskii subsp. paredis. Some species that predominate in periodontitis, but which are absent from healthy gingivae, were found as a small percentage of the flora in gingivitis. This suggests that increased serum and blood in the gingival crevice encourage species that relate to periodontitis.

DNA homology studies of the catalase-negative campylobacters and "Campylobacter fecalis," an emended description of Campylobacter sputorum, and proposal of the neotype strain of Campylobacter sputorum.

Twenty-three strains of catalase-negative campylobacters and five strains of "Campylobacter fecalis," which is catalase-positive, were examined by DNA hybridization experiments. These organisms formed four distinct DNA homology groups corresponding to Campylobacter sputorum, Campylobacter mucosalis, Campylobacter concisus, and a currently unnamed group referred to as the "catalase-negative or weak" (CNW) strains. The strains were further characterized to determine which phenotypic characteristics provide the most reliable identification at the species level. Campylobacter sputorum ssp. sputorum, C. sputorum ssp. bubulus, and "C. fecalis" could not be distinguished by DNA homology; consequently, it is proposed that these three taxa be considered as biovars of C. sputorum. The description of C. sputorum is emended accordingly. ATCC strain 35980 is proposed as the neotype strain of C. sputorum.

Comparative bacteriology of juvenile periodontitis.

Statistical comparisons of the floras associated with juvenile periodontitis, severe periodontitis, and moderate periodontitis indicated that differences in the bacterial compositions of affected sites in these populations were not statistically significant. The subgingival flora of affected juvenile periodontitis sites was statistically significantly different from the adjacent supragingival flora and from the subgingival floras of people with healthy gingiva and of children with developing (experimental) gingivitis. However, the subgingival flora of affected juvenile periodontitis sites was not significantly different from the flora of sites with gingival index scores of 1 or 2 in adults with developing (experimental) gingivitis. Of 357 bacterial taxa among over 18,000 isolates, 54 non-treponemal species, 2 treponemal species, and mycoplasma were most associated with diseased periodontal sulci. These species comprised an increasing proportion of the flora during developing gingivitis and constituted over half of the cultivable flora of diseased sites.

Improved biotyping schemes for Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni (20 strains) and Campylobacter coli (12 strains) were assigned to four biovars for each species based on phenotypic tests that were easy to perform and interpret. The resulting biotyping schemes offer a greater degree of distinction among C. jejuni and C. coli strains than any of the other biotyping schemes previously described for these organisms.

Bacteriology of experimental gingivitis in children.

Children are more resistant to gingivitis than are adults. To determine possible differences in their periodontal floras, an experimental gingivitis study, identical in design to one reported earlier with young adults, was conducted with four 4- to 6-year-old children. The incidence of sites that developed gingival index scores of 2 in children was less than one-third of the incidence observed in adults. The composition of the flora of each child was statistically significantly different from that of any other child or adult. The floras of the children as a group were statistically significantly different from those of the adults. Children had 3-fold greater proportions of Leptotrichia species, 2.5-fold greater proportions of Capnocytophaga species, 2.3-fold greater proportions of Selenomonas species, 2-fold greater proportions of bacterial species that require formate and fumarate, and 1.5-fold greater proportions of Bacteroides species. Adults had greater proportions of Fusobacterium, Eubacterium, and Lactobacillus species. Fusobacterium nucleatum, Actinomyces WVa 963, Selenomonas D04, and Treponema socranskii were predominant species that correlated with increasing gingival index scores in both children and adults.

Campylobacter laridis causing bacteremia in an immunosuppressed patient.

An unusual species, Campylobacter laridis , belonging to the group of nalidixic acid resistant thermophilic Campylobacter species, was isolated from the blood of a 71-year-old man with multiple myeloma, hyperviscosity syndrome, and renal failure. The organism was first recognized in the laboratory by gram-stain reaction and resistance to nalidixic acid. The organism differs from C. jejuni and C. coli by its resistance to nalidixic acid, whereas anaerobic growth in the presence of trimethylamine N-oxide hydrochloride differentiates this organism from other Campylobacter species. Biochemical characterization and DNA homology studies confirmed the identity of this species as being C. laridis . To our knowledge, this is the first recorded case of bacteremia due to C. laridis in humans.

Differential characteristics of catalase-positive campylobacters correlated with DNA homology groups.

Eighty-four strains of catalase-positive campylobacters could be placed into seven distinct DNA homology groups (species), corresponding to Campylobacter fetus, "C. hyointestinalis," C. jejuni, C. coli, "C. laridis," "C. fecalis," and aerotolerant campylobacters. The biochemical and physiological characteristics of the strains were examined for their correlation with the homology groups. The characterization tests that provided the most reliable differentiation at the species and subspecies level were growth at 25 and 42 degrees C, sensitivity to cephalothin and nalidixic acid, growth in semisolid media containing 1% glycine and 3.5% NaCl, growth on plates containing 1.5% NaCl, growth in a semisolid minimal medium, anaerobic growth in the presence of 0.1% trimethylamine-N-oxide, hydrogen sulfide production in SIM medium and triple-sugar iron agar, hippurate hydrolysis, nitrite reduction, and growth on plates under an air atmosphere.

Bacteriology of moderate (chronic) periodontitis in mature adult humans.

A total of 171 taxa was represented among 1,900 bacterial isolates from 60 samples of sites affected with moderate periodontitis in 22 mature adult humans. The composition of the subgingival sulcus flora was statistically significantly different from that of the adjacent supragingival flora and the subgingival flora of 14 people with healthy gingiva, but was not significantly different from that of sulci affected with severe periodontitis in 21 young human adults. The sulcus floras of moderate periodontitis and severe periodontitis shared many of their predominant bacterial species, but there were differences in the relative proportions of some of these species. Similar relationships were found for seven taxa of treponemes that were cultured from the samples.

Albumin requirement of Treponema denticola and Treponema vincentii.

Treponema denticola and Treponema vincentii were found to require albumin, oleic acid, and thiamine pyrophosphate (TPP) for growth. Previous studies indicated that commercial human alpha globulin, which is 50% albumin, was the only serum fraction that supported growth of these two oral treponemes. The alpha-globulin proteins were separated from the contaminating albumin with Affi-Gel Blue affinity chromatography. Both the albumin fraction and one of the alpha-globulin fractions were required for growth of T. denticola. Oleic acid was supplied by the alpha-globulin fraction and the albumin functioned as a chelator to maintain a low level of free fatty acid in the medium. Purified serum albumin (bovine or human) could substitute for the alpha-globulin fraction that contained albumin. Optimal growth of T. denticola and T. vincentii was in a medium supplemented with 0.4% (w/v) delipified albumin, 0.08 mg/mL of sodium oleate, and 25 micrograms/mL of TPP. Serum albumin tightly bound TPP (0.5 microgram of TPP per milligram of albumin). Optimal growth of T. denticola was only in an albumin-oleate supplemented medium with sufficient TPP to saturate the albumin binding sites and provide excess free TPP. Albumin bound long-chain fatty acids and thus detoxified the medium. Neither starch- nor charcoal-treated Tween 80 (polysorbitan monooleate) replaced albumin for optimal growth. Short-chain fatty acids supported only limited growth of T. denticola when added to a medium with TPP or to a medium that contained 0.4% delipified albumin and TPP.

Bacteriology of severe periodontitis in young adult humans.

A total of 78 bacteriological samples were taken from the supragingival tooth surface after superficial cleaning with toothpicks or from the periodontal sulci of 42 affected sites in 21 adolescents or young adults with severe generalized periodontitis. Of 190 bacterial species, subspecies, or serotypes detected among 2,723 isolates, 11 species exceeded 1% of the subgingival flora and were most closely associated with the diseased sulci. Eleven others were also sufficiently frequent to be suspect agents of tissue destruction. Many of these species are known pathogens of other body sites. In addition, 10 species of Treponema were isolated. One of these and the "large treponeme" were also more closely associated with severe periodontitis than they were with healthy sites or gingivitis. There were highly significant differences between the composition of the flora of the affected sulci and the flora of (i) the adjacent supragingival tooth surface, (ii) the gingival crevice of periodontally healthy people, and (iii) sites with a gingival index score of 0 or 2 in experimental gingivitis studies. The floras of different individuals were also significantly different. There was no statistically detectable effect of sampling per se upon the composition of the flora of subsequent samples from the same sites. The composition of the supragingival flora of the patients with severe generalized periodontitis that had serum antibody to Actinobacillus actinomycetemcomitans was significantly different from the supragingival flora of patients without this serum antibody. However, there was no statistically significant difference in the composition of their subgingival floras.

Fumarate reduction and product formation by the Reiter strain of Treponema phagedenis.

The catabolic pathways for butyrate, acetate, succinate, and ethanol formation by the Reiter strain of Treponema phagedenis were investigated. Enzyme activities were demonstrated for glucose catabolism to pyruvate by the Embden-Meyerhof-Parnas pathway. Butyrate formation from acetyl-coenzyme A (acetyl-CoA) does not generate ATP by substrate level phosphorylation and involves NAD+-dependent 3-hydroxybutyryl-CoA dehydrogenase and NAD(P)+-independent butyryl-CoA dehydrogenase activities. Butyrate is formed from butyryl-CoA in a CoA transphorase reaction. Phosphate acetyltransferase and acetate kinase activities convert acetyl-CoA to acetate. An NADP+-dependent alcohol dehydrogenase participates in ethanol formation; however, the manner in which acetyl-CoA is reduced to acetaldehyde is unclear. A membrane-associated fumarate reductase was found which utilized reduced ferredoxin or flavin nucleotides as physiological electron donors. Additional electron carriers may also be involved in electron transfer for fumarate reduction. Strains of Treponema denticola, T. vincentii, and T. minutum utilized fumarate without succinate formation, whereas strains of T. phagedenis and T. refringens formed succinate from exogenously supplied fumarate.