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.

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.

Clinical isolates of anaerobic gram-negative rods with a formate-fumarate energy metabolism: Bacteroides corrodens, Vibrio succionogenes, and unidentified strains.

Strains of anaerobic, gram-negative bacteria, isolated from human clinical specimens and from studies of human normal flora, that have energy metabolism similar to Vibrio succinogenes are described. Included are four human isolates of V. succinogenes, five similar strains of motile straight rods, three strains of Bacteroides corrodens, and two unidentified strains. All strains studied grew poorly in usual anaerobic broth media but produced good turbidity in overnight broth cultures in media containing fromate and fumarate, indicating that all have an energy metabolism similar to V. succinogenes: they gain energy by the transfer of electrons from formate or hydrogen to fumarate.

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.

Pyruvate oxidation by the Reiter strain of Treponema phagedenis.

Spectrophotometric assays of pyruvate oxidation catalyzed by extracts of the Reiter strain of Treponema phagedenis indicated that viologen dyes, flavin nucleotides, and a ferric iron chelate, but not pyridine nucleotides, were utilized as electron acceptors. Benzyl viologen-linked activity partially sedimented during ultracentrifugation and appeared similar to clostridial pyruvate:ferredoxin oxidoreductase with respect to the spectral properties of the enzyme chromophore. Electron carrier activity in treponemal extracts was quantitated by a metronidazole-linked assay in which the oxidation of pyruvate by carrier-depleted extracts led to the reduction of electron carrier in the crude extracts which then reduced metronidazole. The rate of metronidazole reduction was proportional to the amount of electron carrier present in the assay. Electron carrier activity in Triton X-100-solubilized, crude extracts partially purified by DEAE-cellulose chromatography and gel filtration was attributed to a protein possessing the spectral and physical properties of a ferredoxin. A similar protein appeared to be present in extracts of Treponema denticola ST10.

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.

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.

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.

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.

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.

Lipid catabolism of cultivated treponemes.

Treponema require long-chain fatty acids for growth in vitro. Serum, added to culture media, provides a source of long-chain fatty acids. These fatty acids, however, are esterified to triglycerides, phospholipids, and cholesterol. In this study, the major pathways of complex lipid catabolism in T. phagedenis, T. denticola, T. refringens, T. minutum, and T. vincentii were investigated. Lipase activity was demonstrated in five Treponema species using four lipid substrates. Chromatographic data demonstrated that, during growth, treponemes completely utilized lysophosphatidylcholine, present in serum-supplemented culture media, while phosphatidylcholine and phosphatidylinositol were not utilized. Phospholipase B and glycerophosphorylcholine diesterase activities were demonstrated in the five species of Treponema studied. Treponema phagedenis and T. denticola had phosphatase activity, while T. refringens, T. minutum, and T. vincentii did not have an acid phosphatase activity. Phospholipase A, C, and D and alkaline phosphatase activities were not found in five species of Treponema. Based on the enzymes demonstrated in this study, two pathways of phospholipid catabolism are proposed.

Fatty acid requirement of Treponema denticola and Treponema vincentii.

Treponema denticola and Treponema vincentii were cultured in a medium supplemented with either 0.2 or 0.4% (w/v) alpha globulin in place of serum. The active factor(s) in alpha globulin was stable at pH 7.0 to autoclaving and was nondialyzable. Extraction of lipids from alpha globulin showed that both protein and lipid, supplied by the alpha globulin, were required for maximal growth of these two oral treponemes. The lipid component was investigated by adding sodium salts of long-chain fatty acids to the basal medium supplemented with 0.4% delipified alpha globulin. The lipid component of alpha globulin was replaced by either oleic acid (cis-18:1(9)) or by elaidic acid (trans- 18:1 (9)0. No other saturated or unsaturated fatty acid tested could support good growth. Tween 80 (polysorbitan monooleate) was the only Tween compound able to support maximal growth of T. denticola. The cellular lipids of T. denticola, grown with oleate in broth supplemented with 0.4% delipified alpha globulin, were extracted and analyzed by gas chromatography. The principle fatty acids were myristic, pentadecanoic, and palmitic acids. Lesser amounts of oleic acid, eicosadienoic acid, and an unidentified fatty acid (retention time, 88 min) were also detected. Treponema denticola appears to be capable of limited synthesis of cellular fatty acids such as myristic, pentadecanoic, and palmitic acids from oleic acid.

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.

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.

Studies of the microaerophilic nature of Campylobacter fetus subsp. jejuni. II. Role of exogenous superoxide anions and hydrogen peroxide.

The addition of bovine superoxide dismutase to Brucella broth or Brucellar agar greatly echanced the oxygen tolerance of Campylobacter fetus subsp. jejuni strain H840 (ATCC 29428). Catalase also enhanced oxygen tolerance, but to a lesser extent. These enzymes must act externally to the bacteria. All of the diverse compounds which enhance oxygen tolerance of C. fetus, including nor-epinephrine and a combination of ferrous sulfate, sodium metabisulfite, and sodium pyruvate, share the ability to quench either superoxide anions or hydrogen peroxide. On the basis of these and other data, we propose that C. fetus is more sensitive to exogenous superoxide anions and hydrogen peroxide than are aerotolerant bacteria, despite the occurrence of superoxide dismutase and catalse activities in C. fetus. Compounds that enhance oxygen tolerance in C. fetus appear to act by quenching superoxide anions and hydrogen peroxide which occur spontaneously in the culture medium.

Improved media for growth and aerotolerance of Campylobacter fetus.

The microaerophilic nature of Campylobacter fetus has complicated its recovery from human and animal sources. In this study, modifications of brucella agar and broth were tested for enhancement of growth and aerotolerance of 64 strains of C. fetus, representing each subspecies. Brucella agar supplemented with 0.025% each FeSO4 7H2O, sodium metabisulfite, and sodium pyruvate, supported growth of 98, 77, and 63% of the strains at 6% O2, 17% O2, and 21% O2, respectively. Unsupplemented brucella agar supported growth of 94, 48, and 20% of the strains. Brucella broth supplemented with 0.2% FeSO4.7H2O, 0.025% sodium metabisulfite, and 0.05% sodium pyruvate supported growth of 98% of the strains at 21% O2, compared to 75% with unsupplemented brucella broth. With both the supplemented agar and broth, growth responses occurred 1 to 2 days earlier than usual. Growth and aerotolerance of three strains of Campylobacter sputorum subsp. bubulus were not enhanced by the supplements.

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.

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.