Analysis of genotypic variation in genes associated with virulence in Aggregatibacter actinomycetemcomitans clinical isolates.

BACKGROUND AND OBJECTIVE: Although certain serotypes of Aggregatibacter actinomycetemcomitans are associated more with aggressive periodontitis than are other serotypes, the correlation between distinct lineages and virulence traits in this species is poorly understood. This study aimed to evaluate the polymorphism of genes encoding putative virulence factors of clinical isolates, and to correlate these findings with A. actinomycetemcomitans serotypes, genotypes and periodontal status of the hosts. MATERIAL AND METHODS: Twenty-six clinical isolates from diverse geographic populations with different periodontal conditions were evaluated. Genotyping was performed using pulse-field gel electrophoresis. Polymorphisms in the genes encoding leukotoxin, Aae, ApaH and determinants for serotype-specific O polysaccharide were investigated. RESULTS: The isolates were classified into serotypes a-f, and exhibited three apaH genotypes, five aae alleles and 25 macrorestriction profiles. Two serotype b isolates (7.7%), obtained from Brazilian patients with aggressive periodontitis, were associated with the highly leukotoxic genotype; these isolates showed identical fingerprint patterns and aae and apaH genotypes. Serotype c, obtained from various periodontal conditions, was the most prevalent among Brazilian isolates, and isolates were distributed in two aae alleles, but formed a genetically distinct group based on apaH analysis. Cluster analysis showed a close relationship between fingerprinting genotypes and serotypes/apaH genotypes, but not with aae genotypes. CONCLUSION: Apart from the deletion in the ltx promoter region, no disease-associated markers were identified. Non-JP2-like strains recovered from individuals with periodontal disease exhibited considerable genetic variation regarding aae/apaH genotypes, serotypes and XhoI DNA fingerprints.

Genetic diversity and toxic activity of Aggregatibacter actinomycetemcomitans isolates.

INTRODUCTION: Very little is known of the diversity and expression of virulence factors of serotypes of Aggregatibacter actinomycetemcomitans. Toxic activity on Chinese hamster ovary (CHO) cells and cdt and ltx genotyping were evaluated in A. actinomycetemcomitans serotypes. METHODS: Forty-one A. actinomycetemcomitans isolates were analysed for CHO cell growth inhibition. Genotyping was performed by polymerase chain reactions specific to the ltx promoter region, serotype-specific and cdt region and by sequencing of cdtB. RESULTS: cdtABC was detected in 40 strains. Analysis of the cdtA upstream region revealed 10 cdt genotypes. Toxicity to CHO cells was detected for 92.7% of the isolates; however, no correlation between the toxic activity and the cdt genotype was detected. Serotype c was more prevalent among Brazilian samples (68.0%). Four serotype b isolates from subjects with aggressive periodontitis were associated with high leukotoxin production and exhibited moderate to strong toxic activity in CHO cells, but were classified in different cdt genotypes. High levels of toxicity in CHO cells were not associated with a particular serotype; 57.1% of serotype a isolates presented low toxicity to CHO cells whereas the highly toxic strains belonged to serotypes b and c. Sequencing of cdtB revealed a single nucleotide polymorphism of amino acid 281 but this was not related to the toxic activity in CHO cells. CONCLUSION: Differences in prevalence of the low and highly cytotoxic strains among serotypes reinforce the hypothesis that serotype b and c isolates of A. actinomycetemcomitans are more virulent than serotype a strains.

Microflora in oral ecosystems in subjects with radiation-induced hyposalivation.

AIM: To analyse the microbial flora in specific oral sites in 13 dentate subjects, 6-8 months after completed radiation therapy (RT group) and in 13 matched controls. MATERIAL AND METHODS: The microflora on the tongue, buccal mucosa, vestibulum, supragingival plaque and subgingival region was analysed using duplicate sampling and cultivation technique. A clinical examination was also performed. RESULTS: Candida albicans was found in one or more sites in 54% of the RT subjects and in 15% of the controls. In three RT subjects, C. albicans was found at all four sites analysed. An unexpected finding was that enterococci were found in all RT subjects and in high number in 38%. None of the controls harboured enterococci. In supragingival plaque, Lactobacillus spp. were detected in 92% of the RT subjects and the number and proportion of Lactobacillus spp. were extremely high compared with the controls. Mutans streptococci were detected in high numbers in 31% of the RT subjects, while they were not detected in 23%. CONCLUSION: The microbial results explain why some RT subjects have an increased susceptibility to oral diseases and stress that site-specific microbial analysis is an important diagnostic tool when planning oral health preventive care for RT subjects.

Dental plaque pH and micro-organisms during hyposalivation.

We have previously reported that minor gland and whole saliva flow rates and salivary proteins showed differences in individuals with primary Sjögren's syndrome or head and neck radiation therapy, compared with controls (Eliasson et al., 2005). We now hypothesize that pH and number of acidogenic micro-organisms in dental plaque as well as saliva buffering capacity also differ in these individuals. Plaque pH was measured by the microtouch method up to 60 min after a sucrose rinse. Plaque collected from the same sites was analyzed for counts of total and acidic micro-organisms. Compared with their controls, the irradiated group but not the Sjögren's syndrome group displayed significantly lower plaque pH, increased numbers of lactobacilli and Candida species, as well as reduced buffering capacity. Stepwise regression tests suggested that the buccal minor-salivary-gland secretion rate in the test groups and counts of mutans streptococci in the controls were of significant importance for dental plaque pH.

Proton-translocating cytochrome c oxidase in artificial phospholipid vesicles.

The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated. Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H+ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K+. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons. The proton ejection is the result of true translocation of H+ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system. Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a H+/e- quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions. It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H+.

Calorimetric studies of cytochrome oxidase-phospholipid interactions.

Thermotropic phase transitions in phospholipid vesicles reconstituted with mitochondrial cytochrome oxidase (EC 1.9.3.1) were studied using differential scanning calorimetry. Both dimyristoylphosphatidylcholine (DMPC) and mixtures of DMPC and cardiolipin were used at different lipid-to-protein ratios. The incorporated protein reduces the energy absorbed during phase transitions of DMPC vesicles, and causes a small decrease in the transition temperature (tm). delta H depends on the amount of protein in the vesicles. This dependence indicates that about 72 DMPC molecules are influenced per cytochrome alpha alpha 3 monomer. The transition parameters remain unaffected by changes in ionic strength or by reduction of the enzyme. Incorporation of cytochrome oxidase depleted of subunit III into DMPC liposomes resulted in a larger decrease of tm, but the amount of perturbed phospholipids remains similar to that in the case of the intact enzyme. Incorporation of cytochrome oxidase into DMPC/cardiolipin vesicles counteracts the effect of cardiolipin in decreasing the enthalpy of the DMPC transition. Thus cytochrome oxidase segregates the phospholipids by attracting cardiolipin from the bulk lipid. Cytochrome c does not significantly affect this apparent cardiolipin 'shell' around membranous cytochrome oxidase.

A novel antiporter activity catalyzing sodium and potassium transport from right-side-out vesicles of E. coli.

Downhill sodium efflux from right-side-out E. coli membrane vesicles was found to be stimulated by negative electric potential, as has been reported earlier [Bassilana et al., Biochemistry 23 (1984) 1015-1022], and in agreement with the concept of electrogenic Na+/nH+ antiporters with n > 1. However, sodium efflux was much more accelerated by positive electric potential, indicating the operation of another sodium transport system. delta pH (alkaline inside), created by a pH shift from 8.5 to 6.8 in the medium was found to drive sodium efflux against its concentration gradient, but only when the vesicles had been loaded with both Na+ and K+. Efflux of K+ against the concentration gradient was also observed under these conditions. When the vesicles were loaded separately with sodium tricine or potassium tricine, no K+ efflux and insignificant Na+ efflux were observed. We propose that there are at least two different mechanisms responsible for Na+ efflux in E. coli vesicles. One is the Na+/nH+ antiporter previously described, and the other is a novel Na+,K+/mH+ antiporter.

Oral microflora in subjects with reduced salivary secretion.

It is generally assumed that a decreased salivary secretion rate will promote plaque accumulation and increase the risk for caries, gingival inflammation, and mucosal infections. In this study, the effect of hyposalivation on the oral microflora was examined. The following micro-organisms were analyzed in rinsing samples from 14 subjects with hyposalivation: the total number of anaerobically growing micro-organisms, alpha-hemolytic streptococci, mutans streptococci, lactobacilli, Fusobacterium nucleatum, Prevotella intermedia/Prevotella nigrescens, Staphylococcus aureus, Candida albicans, and enterics. The study group, age 53 +/- 7 years, had no history of radiation therapy and showed no signs of inflammation in their salivary glands on biopsy. All were dentate with a mean of 24 +/- 3 teeth. Their salivary secretion rates were 0.03 +/- 0.02 mL/min (unstimulated) and 0.84 +/- 0.65 mL/min (stimulated). The control group was matched to the hyposalivation group according to age, sex, and number of teeth. There was a significantly increased number of lactobacilli, and a tendency, not statistically significant and with large variations within the groups, toward a higher proportion of mutans streptococci and a lower proportion of alpha-hemolytic streptococci in the hyposalivation group. The presence of micro-organisms associated with gingival inflammation and mucosal infections was comparable with that in the healthy controls. The results indicated that a low salivary secretion rate mainly promotes a flora associated with the development of caries.

Production of histolytic enzymes by a combination of oral bacteria with known pathogenicity.

Eight oral bacterial strains, isolated from an infected root canal, have been investigated for their capacity to produce histolytic enzymes. The determination was performed using methods espoused by two different principles. Eleven out of 12 enzymes examined were demonstrated in the "eight-strain collection". In no single bacterial strain were all enzymes revealed. It was suggested that the pathogenicity of the bacterial strains, singly or in combination, was not solely dependent on the production of these enzymes. The histolytic enzymes may have a potentiating role on other pathogenic factors in infectious diseases.

Move of adults with mental retardation from institutions to community-based living: changes in the oral microbiological flora.

In the Western world, the policy of deinstitutionalization and integration of individuals with mental retardation is generally accepted. We tested the hypothesis that de-institutionalization may lead to changes of habits with a potential to influence oral health. When 57 adults with mental retardation moved from an institution to community-based living, their oral hygiene habits, gingival bleeding, and a three-day food record were registered one month before and 9 and 21 months after the move. Mutans streptococci and lactobacilli in saliva, P. intermedia/P. nigrescens, P. gingivalis, and A. actinomycetemcomitans in supragingival plaque, and C. albicans on mucous membranes were analyzed. After 21 months of community-based living, fewer persons showed high classes of mutans streptococci, growth of P. intermedia/P. nigrescens, and high frequency of sucrose intake, and more subjects showed growth of C. albicans. In a short perspective, the indicators of oral diseases suggest an unchanged or lower risk of oral diseases after the de-institutionalization of individuals with moderate or severe mental retardation.

Treatment of periodontal disease based on microbiological diagnosis. Relation between microbiological and clinical parameters during 5 years.

The purpose of this study was to assess the clinical effect of treatment aimed to suppress Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis below detection level and Prevotella intermedia below 5% of the cultivable periodontal pocket flora. Sixteen patients and a total of 111 periodontal pockets with probing depth > or = 6 mm were included in the study. Twelve patients and a total of 77 sites completed the 5-year study. The results demonstrated clinical improvement of probing depth and gain of clinical attachment level of 3.4 mm and 1.2 mm, respectively. Treatment to eliminate indicator bacteria continued for 3 years before the aim was fulfilled. In order to eliminate A. actinomycetemcomitans from a majority of the sites, a combination of surgery and generalized tetracycline treatment was performed. A recolonization or regrowth of the indicator bacteria exceeding detection levels took place in several sites. The presence of A. actinomycetemcomitans. P. gingivalis, and P. intermedia, alone or in combination, correlated with attachment level change on the individual level. No such correlation was obtained by using presence of plaque, bleeding on probing, or three other bacteria (Campylobacter rectus, Eikenella corrodens, and Fusobacterium nucleatum) not used as treatment goal markers.

Treatment of periodontal disease based on microbiological diagnosis. A 5-year follow-up on individual patterns.

Sixteen patients with advanced periodontitis (more than 3 sites with a probing depth > or = 6 mm) were treated with the aim of eliminating Actinobacillus actinomyce-temcomitans and Porphyromonas gingivalis and reducing Prevotella intermedia below 5% of the total viable count. The patients were followed clinically and microbiologically for over 5 years and showed a highly individual pattern with respect to presence of indicator bacteria, type of treatment needed to accomplish treatment goals, clinical response, and bacterial and disease recurrencies. The results of this study suggest that presence of the indicator bacteria in microbial samples taken after treatment may identify patients at risk for recurrent periodontitis.

Detection of Porphyromonas gingivalis in gingival exudate by a dipeptide-enhanced trypsin-like activity.

Porphyromonas gingivalis in subgingival plaque is an important risk factor for future periodontal attachment loss in susceptible adults. The elimination of P. gingivalis is usually concomitant with a healing process. Therefore, it should be valuable to have an easy chairside method to follow the effect of periodontal treatment on P. gingivalis detection as well as on its eventual reappearance during the maintenance period. We have previously reported the stimulation of amidolytic activity of P. gingivalis by the addition of glycyl-glycine to the assay buffer. In this study we determined the proportions of P. gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Capnocytophaga spp, Campylobacter rectus, and Eikenella corrodens by cultivation technique and the amidolytic activity, using N-benzoyl-L-arginine-p-nitroanilide (BAPNA) as substrate, in gingival exudate before and during a 3-year treatment and maintenance period. P. gingivalis was the only species yielding a high and persistent correlation to stimulated amidolytic activity (P values < or = 0.0001) on both site (r = 0.5) and subject (r = 0.8) level. Testing pure cultures of suggested periodontal pathogens for effect of glycyl-glycine on amidolytic activity, we found that of P. gingivalis to be 5.9-fold increased. The amidolytic activity of Treponema denticola was only slightly stimulated (ratio with/without glycyl-glycine = 1.2) and that of Capnocytophaga slightly inhibited (ratio with/without glycyl-glycine = 0.8). The outcome of this study has the potential to be used for the development of a simple, rapid, and inexpensive assay for a qualitative and quantitative determination of P. gingivalis in gingival crevicular fluid.

Comparative study of subgingival microbiological sampling techniques.

The presence of specific bacteria in subgingival plaque has been used as an indicator of active periodontal disease. The technique of subgingival sampling may conjecturally influence the identification and enumeration of microorganisms reported. In this study, paper point sampling and scaler sampling are compared. Subgingival samples using both methods were taken from three surfaces in each of 12 patients at the following time points: at each of two appointments one week apart before treatment and at each of two appointments 12 and 13 weeks following treatment. Microbiological analyses were undertaken to determine the total number of colony forming units, the proportions of suspected periodontal pathogens, and the number of spirochetes using phase contrast microscopy. Significantly higher numbers of colony forming units and spirochetes were found for paper point sampling both before and after treatment.

Effect of treatment on some periodontopathogens and their antibody levels in periodontal abscesses.

Twenty patients suffering from abscess of periodontal origin were treated and followed clinically and microbiologically for 6 months. Microbiological examination was performed by culture technique. One deep and one shallow periodontal pocket in the same patient were examined concomitantly. Serum was collected and analyzed for specific antibody level using ELISA methodology. Treatment included supragingival scaling, drainage, and irrigation of the periodontal pocket with 0.85% sodium chloride and systemic tetracycline administration, 1 g per day for 2 weeks. At baseline, 90% of the abscesses harbored Porphyromonas gingivalis and/or Prevotella intermedia. After 6 months, abscess sites demonstrated a reduced probing depth, less bleeding on probing, and gain of attachment. Abscess sites showed no P. gingivalis and the proportion of P. intermedia was significantly reduced 6 months after treatment. In deep periodontal pockets a similar pattern was seen. Shallow pockets demonstrated few clinical signs of inflammation and the number of bacteria was generally low. Campylobacter rectus, Capnocytophaga spp, and Fusobacterium nucleatum were frequently seen in low numbers in most sites during the study period, while Actinobacillus actinomycetemcomitans was detected only in a few sites. The IgG levels in patient sera against antigens of homologous bacterial strains remained fairly constant for 6 months. The result of the present study indicates that P. gingivalis and P. intermedia are involved in periodontal abscess formation. Also, treatment where drainage is combined with tetracycline administration promotes healing and reattachment.

Adhesion and invasion to epithelial cells by fimA genotypes of Porphyromonas gingivalis.

Adhesion to and invasion of epithelial cells by the periodontopathogen Porphyromonas gingivalis is promoted by the major fimbriae, encoded by fimA. The microorganism can be classified in six genotypes, based on fimA sequence, and genotype II strains are more prevalent than others in periodontitis patients. This study aimed to determine the adhesive and invasive abilities on KB cells of different fimA allelic variants of P. gingivalis isolates. Twenty-two isolates and six reference strains representing the six fimA genotypes and non-typeable strains were screened for their adhesion and invasion abilities on KB cells, using standard methods. All strains were able to adhere and, except for one, to invade KB cells. However, these properties were not homogeneous among strains belonging to the same genotype. There was no correlation between adhesion and invasion efficiencies. Isolate KdII 865 (fimA genotype II) was the most invasive and the second most adhesive strain, whereas reference strain ATCC 33277 (fimA I) showed a low adhesion ability but was highly invasive. These data indicated that fimA genotypes of P. gingivalis are not related to the adhesion and invasion abilities on KB cells, suggesting that the increased prevalence and proportion of certain genotypes may be attributed to other characteristics besides FimA variation.

Occurrence of enteric rods, staphylococci and Candida in subgingival samples.

The frequency and percentage of enteric rods, staphylococci and Candida were determined in 973 subgingival samples collected from 535 patients subjected to different periodontal treatment procedures. The analysis was performed with culture technique using selective and nonselective media. One or more organisms were detected in 65.5% of the samples and in 76.7% of the patients. In most samples enteric rods, staphylococci and/or Candida constituted a small amount of the total microbial viable count. Enteric rods exceeded 10% of the total viable count in 30 samples. Staphylococci occurred in more than 10% in only 3 samples. In these 3 samples, enterics constituted more than 10% of the total viable count. Candida was not found to exceed 10% of the total viable count in any of the samples. No statistically significant correlation was found between the presence of any of the target microorganisms and kind of periodontal treatment procedure received, antibiotic administration or sample transport time.

Ability of oral bacteria to degrade fibronectin.

The fibronectin-degrading ability of 116, mainly oral, strains was assayed by using plasma-derived fibronectin adsorbed to a polystyrene surface. Ability to degrade fibronectin was revealed in strains of Bacteroides gingivalis, Bacteroides intermedius, Bacteroides loeschii, Staphylococcus aureus, Staphylococcus epidermidis, Peptococcus prevotii, Clostridium sporogenes, and Propionibacterium acnes. The fibronectinolytic activity of subgingival bacteriological samples was found to be related to the presence of B. gingivalis and B. intermedius. In addition, strains of the nonoral Bacteroides species B. asaccharolyticus and B. fragilis showed fibronectin-degrading ability. No such ability was detected in the oral strains tested of Streptococcus, Veillonella, Actinomyces, Lactobacillus, Actinobacillus, Capnocytophaga, Fusobacterium, or Haemophilus species.

Decreased lactoferrin content in granulocytes from subjects with Actinobacillus actinomycetemcomitans associated periodontal diseases.

Fourteen subjects were examined for lactoferrin content in PMNs of venous blood. Eight of the subjects were diagnosed localized juvenile periodontitis (LJP) and four adult periodontitis (AP), all having subgingival occurrence of Actinobacillus actinomycetemcomitans (A.a.). Two subjects had healthy gingival conditions and no detectable A.a. Deficiency or low PMN lactoferrin amounts were found in six of the eight subjects with LJP and in two of the subjects with AP. The reduced lactoferrin content in the PMNs was suggested to be depending on a cytotoxic factor produced by A.a. adding to an intrinsic PMN defect.

Quaternary structure of bovine cytochrome oxidase.

A hydrodynamically homogeneous preparation of bovine mitochondrial cytochrome c oxidase can be obtained by anion-exchange chromatography of alkaline-treated enzyme, followed by a gel permeation chromatography step, which further removes some (aggregated) apoprotein. The molecular weight, Mr, of the monodisperse enzyme in Triton X-100 was found to be 210000. This complex is composed of six different polypeptides, with Mr summing up to about 110000 in toto, in a relative one-to-one stoichiometry. Two sets of these subunits constitute the 210000-Mr enzyme complex. In contrast to our earlier report [Saraste, Penttilä, Coggins, and Wikström, FEBS Lett. 114 (1980) 35-38] the 210000-Mr enzyme contains four (and not two) haems A, and therefore represents the dimer of cytochrome aa3. One of the proposed seven subunits, number III, is lacking in this enzyme preparation.