Bulleidia extructa gen. nov., sp. nov., isolated from the oral cavity.

Five strains of anaerobic non-sporing Gram-positive bacilli isolated from advanced periodontitis (four strains) and a dentoalveolar abscess (one strain) that did not correspond to existing species were subjected to phenotypic and genetic characterization. Following 16S rDNA sequence analysis, they were found to constitute a novel branch of the low G+C Gram-positive division of the phylogenetic tree related to Erysipelothrix rhusiopathiae and Holdemania filiformis. A new genus Bulleidia, and the species Bulleidia extructa, are proposed. Growth of B. extructa in broth media was poor but was enhanced by the addition of fructose, glucose or maltose together with Tween 80. Glucose and maltose were fermented and arginine was hydrolysed. Acetate, lactate and trace amounts of succinate were the end products of glucose fermentation. The G+C content of the DNA of the type strain is 38 mol%. The type strain of Bulleidia extructa is DSM 13220T.

Three subtypes of the tet(M) gene identified in bacterial isolates from periodontal pockets.

The tet(M) genes were characterized from 84 isolates of 10 different bacterial species isolated from the periodontal pockets of 16 patients with periodontal disease. A 740 bp polymerase chain reaction product from the hypervariable region of the tet(M) structural gene was cleaved with the restriction enzymes AluI and HinfI. Three different restriction patterns were identified for each of the two enzymes. By DNA sequencing, using a direct solid-phase automated sequencing method, the isolates could be grouped into 3 different clusters of tet(M) subtypes. The internal DNA homology within each subtype was 98-100%; the homology between clusters was 89-94%. Two different subtypes were identified in 9 of 10 bacterial species, and the remaining species had 3 different subtypes. One of the subtypes (M3) was seen mainly in the anaerobic isolates. This subtype was different from all earlier sequenced structural tet(M) genes present in the Genbank. Most patients had two different subtypes of tet(M), and a third subtype was seen in the 3 patients who exhibited the greatest variety of tetracycline-resistant bacterial species. It appears that the presence of one subtype of the tet(M) gene within a patient or bacterial species does not prevent the acquisition of another subtype of the same gene. This study identified a new subtype of the tet(M) gene and grouped it into 3 distinct yet highly homologous genetic subtypes.

Tetracycline resistance in Prevotella isolates from periodontally diseased patients is due to the tet(Q) gene.

Tetracycline-resistance in gram-negative periodontal bacteria is often due to the presence of the tet(Q) gene. In the present study the polymerase chain reaction (PCR) was used to examine 54 isolates of gram-negative anaerobic rods (Prevotella intermedia, Prevotella nigrescens and related or Bacteroides-like species) for the presence of the tet(Q) gene. The isolates were recovered from 42 patients with periodontal disease living in northern Europe and North America. An 814 base-pair segment of the tet(Q) gene was amplified from all 41 isolates resistant to tetracycline with minimal inhibitory concentrations of 4 micrograms/ml and above. The presence of the tet(Q) gene was verified using hybridization with a specific oligonucleotide internal to the amplified region and restriction endonuclease digestion with DdeI. A PCR product of the same size was also amplified from one tetracycline susceptible isolate (minimal inhibitory concentration = 0.5 microgram/ml). However, this isolate and the one isolate that was resistant to tetracycline at 4 micrograms/ml showed a weaker signal than the remaining isolates when hybridized with the internal probe. Typing of the PCR products using restriction endonuclease digests with AluI and HpaII revealed two clusters of distinct electrophoresis patterns, indicating that two different subtypes of the tet(Q) gene were present in this material. A control strain containing the tet(Q) gene from Bacteroides thetaiotaomicron had a different electrophoresis pattern for AluI. This study indicated that subtypes of the tet(Q) gene in tetracycline-resistant gram-negative periodontal bacteria exist both within the same patient and within the same species.

Tetracycline-resistant micro-organisms recovered from patients with refractory periodontal disease.

Tetracycline in combination with scaling and root planing is frequently used to treat refractory periodontal disease. This study examined tetracycline resistance in bacteria recovered from periodontal pockets of patients with refractory periodontitis. Bacterial isolates resistant to 10 micrograms/ml of tetracycline were isolated from plaque samples of 17 patients, of whom 6 had received tetracycline within 8 weeks prior to sampling. Minimal inhibitory concentrations (MICs) of tetracycline and minocycline were determined by agar dilution. In the 6 patients who had received tetracycline, a mean of 22.9% (+/- 38.2) of the total cultivable subgingival flora were resistant to tetracycline, compared with a mean of 7.2% (+/- 8.5) in the untreated group. Although various organisms were isolated, in most patients, the tetracycline-resistant organisms were dominated by Streptococcus spp. Overgrowth of Candida was found in one patient, and of Enterobacteriaceae in another patient, while small numbers of yeast or Staphylococcus spp. were isolated from the plaque samples of 9 others. 3 out of 4 patients who did not respond to tetracycline treatment had a variety of tetracycline-resistant anaerobic Gram-negative rods present. No correlation was found between increased proportions of tetracycline resistance in the whole bacterial sample and the presence of resistant periodontal pathogens.

Detection of tet(M) and tet(O) using the polymerase chain reaction in bacteria isolated from patients with periodontal disease.

The polymerase chain reaction was used to examine 114 tetracycline-resistant anaerobic and facultative anaerobic bacterial isolates from patients with periodontal disease for the tet(M) and tet(O) genes. A 740-base-pair fragment of the tet(M) gene was amplified from 84 of 114 isolates, and a 519-base-pair fragment of the tet(O) gene was amplified from 13 streptococcal isolates. Six of 7 tetracycline-resistant isolates of Veillonella spp. and tetracycline-resistant isolates of Eubacterium spp. (n = 3), Eubacterium saburreum (n = 1), Streptococcus intermedius (n = 5) and Gemella morbillorum (n = 2) all harbored the tet(M) gene. The tet(M) and tet(O) negative as well as selected positive isolates were tested for the tet(K) and tet(L) genes using DNA probes. All isolates of Staphylococcus spp. (n = 11) hybridized with the tet(K) probe. None of the isolates tested hybridized with the probe for tet(L). This is the first report of the tet(M) gene in the facultative bacterium G. morbillorum and in E. saburreum.

The tet(Q) gene in bacteria isolated from patients with refractory periodontal disease.

Twenty-two tetracycline-resistant (tetr) anaerobic and facultative anaerobic bacteria isolated from periodontal pockets of 12 patients with refractory periodontitis were examined for the presence of the Tet Q determinant by DNA-DNA hybridization. Dot blots of bacterial DNA were tested with an intragenic digoxigenin-labelled tet(Q) probe consisting of a 1.45 kb EcoRI/PvuII fragment from plasmid pNFD13-2. Southern blots of chromosomal DNA digested with the restriction enzyme EcoRI were also examined. The tet(Q) probe hybridized with DNA from 8 of the 22 tetr strains, including 2 Prevotella intermedia strains and one strain each of Prevotella nigrescens, Prevotella loescheii, Prevotella veroralis and Prevotella melaninogenica. The tetr strains of Mitsuokella dentalis and Capnocytophaga ochracea also hybridized with the probe. The lack of discernible plasmid DNA in all the probe-positive isolates suggests that these tetracycline-resistance genes were chromosomally encoded. The probe hybridized with a different size fragment in all the isolates. This study extends the number of species that carry the tet(Q) gene to include several outside the genera Prevotella and Bacteroides.

Tetracycline resistance in periodontal pathogens.

Antimicrobial agents are used in combination with debridement to eliminate putative periodontal pathogens, including Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans, from diseased tissues. The most frequently used antimicrobial agents are the tetracyclines. However, these agents are not effective in some patients. This lack of efficacy may be due to antimicrobial resistance. As many as 75% of the bacteria in the subgingival flora may be resistant to tetracycline after long-term, low-dose treatment. Tetracycline resistance is mediated by the tet(M) determinant in some isolates of Veillonella species and Fusobacterium nucleatum, while a DNA probe to the tet(Q) determinant hybridizes to isolates of Prevotella denticola and P. intermedia. The mechanism of tetracycline resistance for most periodontal organisms, however, has yet to be determined. Before tetracycline is used as adjunctive therapy for refractory periodontitis, the subgingival bacterial flora should be tested for susceptibility.

[Tetracycline resistance in oral microorganisms in patients with periodontal disease]. / Tetracyklin og tetracyklinresistens hos orale bakterier i forbindelse med behandling av periodontitt.

Several longitudinal studies have shown that periodontal treatment consisting of surgical and/or nonsurgical debridement of the teeth often is sufficient to prevent continued attachment loss when the patients' oral hygiene measures are good. Nevertheless, some patients will, in spite of excellent oral hygiene measures, continue to show attachment loss. The treatment of these patients has mainly been empirical, and the clinicians have used different kinds of antibiotics for different time intervals to eliminate proposed periodontal pathogens. The preferred antibiotic has been tetracycline. Most bacteria associated with destructive periodontal disease will normally be susceptible to tetracycline at the levels achieved in the gingival crevicular fluid after systemic administration of the antibiotic. Some patients have a microbial flora that will not change enough or be inhibited by these substances. The reason for this may in many cases be the development of antimicrobial resistance. Today 14 genotypes of tetracycline resistance have been found. The resistance is one of three types: active secretion, protection of the ribosomes by proteins and an active breakdown of tetracycline. The third type has recently been discovered as a cryptic gene in the microorganism Bacteroides fragilis. This gene is only expressed after transfer to Eschericia coli. Active breakdown of tetracycline does normally not occur in nature and the global level of the drug is increasing since tetracycline is the second most used antibiotic in the world today. It is therefore of interest to identify tetracycline resistance determinants in the oral cavity and to compare these with tetracycline resistance genes from other sources of human, animal and environmental origin.

Plasmids in Actinobacillus actinomycetemcomitans strains isolated from periodontal lesions of patients with rapidly destructive periodontitis.

Several strains of Actinobacillus actinomycetemcomitans newly isolated from periodontal lesions of patients with rapidly destructive periodontitis were all shown to possess identical plasmid profiles consisting of 4 plasmids. The largest plasmid, 20 MegaDalton (MDa), was also found in reference strains. Two different methods were used for isolation of the plasmids; the large 20 MDa plasmid (pHRP1) was found using the Kado and Liu method only. The 3 small plasmids of 7.0, 5.2 and 4.0 MDa (pHRP2, pHRP3, pHRP4), respectively, were seen using the Birnboim and Doly method. These plasmids are so far to be regarded as cryptic; no phenotypical characters have been linked to their presence. The large 20 MDa plasmid was found in all strains examined, and may be a genotypical marker for the A. actinomycetemcomitans species.

[Prerequisites for bacterial invasion of the periodontal tissues]. / Forutsetninger for bakteriell invasjon av periodontalt bindevev.

Bacterial invasion of the periodontium has recently been described by several authors. The conditions necessary for this to occur in vivo are being discussed. The anatomical relations of the periodontal pocket are described macroscopically and microscopically as well as the postulated mechanisms for colonization, adhesion and penetration. It is necessary for the microorganisms to colonize and adhere in order to be able to invade tissues. The possibility of establishment of microorganisms in the tissues and the importance of the immune system are also being described. The conclusion is that there is a possibility of bacterial invasion of the periodontal connective tissue. However, the human body with help from the immune system will in most cases be able to neutralize and get rid of these microorganisms.

[Periodontal disease--specific or nonspecific infection?]. / Periodontal sykdom--spesifikk eller non-spesifikk infkesjon?

The concept of a non-specific etiology of periodontal disease has been predominant during the last century. It was believed that this disease was caused by the normal oral microbial flora and dependent on the state of the immune system of the host. The specific plaque hypothesis, which is currently discussed, claims that specific oral bacteria cause different forms of periodontal disease. High counts of these bacteria have been found in association with different forms of periodontal disease. These bacteria have also been found to have stronger pathogenic potential than other members of the oral microbiota. It is concluded that there are different forms of periodontal disease and that some of these forms are probably opportunistic infections associated with different microbial etiologies.

The similarity of periodontal microorganisms between husband and wife cohabitants. Association or transmission?

Fourteen married couples were studied to determine the degree of similarity of the periodontal subgingival flora between paired contralateral sites and between couples. Six sites per person were sampled by a subgingival lavage technique and 10 darkfield morphotypes enumerated at each site. Cross-arch matching revealed a positive association for the presence of medium spirochetes, filaments, large motile rods and cocci (P less than 0.05). The odds that medium spirochetes will be detected at a subgingival site are elevated 7-fold, if present at the contralateral site. Similarly, the odds ratio for filaments was 4.0, 1.3 for large motile rods and 1.6 for cocci. Analysis of data, pairing between couples, revealed a positive association for medium spirochetes and filaments (P less than 0.05). No significant associations were found for randomized pairs of couples using matched sites as controls. The odds of medium spirochetes being present in a patient are 3 times greater if the morphotype is present in the spouse and 30% greater for filaments. Although an individual is "at risk" if these morphotypes are present in the spouse, analyses failed to demonstrate that the presence of any morphotype depends upon, or requires the presence of, that morphotype in the spouse.