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The affinity of [ 3 H]benzylpenicillin for penicillin-binding protein (PBP) 3A was reduced in 25 clinical isolates of ␤-lactamase-negative ampicillin (AMP)-resistant (BLNAR)Haemophilus influenzae for which the AMP MIC was >1.0 g/ml. The affinities of PBP 3B and PBP 4 were also reduced in some strains. The sequences of the ftsI gene encoding the transpeptidase domain of PBP 3A and/or PBP 3B and of the dacB gene encoding PBP 4 were determined for these strains and compared to those of AMP-susceptible Rd strains. The BLNAR strains were classified into three groups on the basis of deduced amino acid substitutions in the ftsI gene, which is thought to be involved in septal peptidoglycan synthesis. His-517, near the conserved Lys-ThrGly (KTG) motif, was substituted for Arg-517 in group I strains (n ‫؍‬ 9), and Lys-526 was substituted for Asn-526 in group II strains (n ‫؍‬ 12). In group III strains (n ‫؍‬ 4), three residues (Met-377, Ser-385, and Leu-389), positioned near the conserved Ser-Ser-Asn (SSN) motif, were replaced with Ile, Thr, and Phe, respectively, in addition to the replacement with Lys-526. The MICs of cephem antibiotics with relatively high affinities for PBP 3A and PBP 3B were higher than those of AMP and meropenem for group III strains. The MICs of ␤-lactams for H. influenzae transformants into which the ftsI gene from BLNAR strains was introduced were as high as those for the donors, and PBP 3A and PBP 3B showed decreased affinities for ␤-lactams. There was no clear relationship between 7-bp deletions in the dacB gene and AMP susceptibility. Even though mutations in another gene(s) may be involved in ␤-lactam resistance, these data indicate that mutations in the ftsI gene are the most important for development of resistance to ␤-lactams in BLNAR strains.Haemophilus influenzae is one of the important pathogens causing respiratory tract infection (RTI), acute otitis media (AOM), pneumonia, and purulent meningitis. Ampicillin (AMP) resistance in this organism is due to two well-known mechanisms. One is resistance mediated by the production of TEM-1 (20) and ROB-1 (13) ␤-lactamases, and the other is decreasing affinity of AMP for penicillin-binding proteins (PBPs) involved in peptidoglycan synthesis (14)(15)(16)19). Strains with resistance due to the latter mechanism are termed ␤-lactamase-negative AMP-resistant (BLNAR) H. influenzae.In surveillance studies conducted in the United States the incidence of ␤-lactamase-producing AMP-resistant (BLPAR) H. influenzae has gradually increased (6, 7, 10) and accounted for 36.4% of all isolates (5) in 1994 and 1995. In contrast, BLNAR isolates remain extremely uncommon in the United States.In Japan, according to nation wide surveillance studies conducted in 1997 and 1998, the proportion of clinical isolates supposed to be BLNAR has rapidly increased to 28.8% in parallel with the increased prevalence of penicillin (PEN)-resistant Streptcoccus pneumoniae. The characteristic of resistant H. influenzae isolates is that the AMP MIC is Ն1 g/ml, whereas the MIC for susce...

A continuous noninvasive method of systolic blood pressure estimation is described. Systolic blood pressure is estimated by combining two separately obtained components: a higher frequency component obtained by extracting a specific frequency band of pulse arrival time and a lower frequency component obtained from the intermittently acquired systolic blood pressure measurements with an auscultatory or oscillometric system. The pulse arrival time was determined by the time interval from QRS apex in electrocardiogram to the onset of photoplethysmogram in a fingertip beat-by-beat via an oximetric sensor. The method was examined in 20 patients during cardiovascular surgery. The estimated values of systolic blood pressure were compared with those measured invasively using a radial arterial catheter. The results showed that the correlation coefficients between estimated values and invasively obtained systolic blood pressure reached 0.97 +/- 0.02 (mean +/- SD), and the error remained within +/- 10% in 97.8% of the monitoring period. By using a system with automatic cuff inflation and deflation to acquire intermittent systolic blood pressure values, this method can be applicable for the continuous noninvasive monitoring of systolic blood pressure.

The prevention of dental caries and periodontal diseases is targeted at the control of dental plaque. In this context, chemical agents could represent a valuable complement to mechanical plaque control. The active agents should prevent biofilm formation without affecting the biological equilibrium within the oral cavity. Depending on the goals of the preventive measures, various strategies may be considered. Anti-plaque agents with properties other than bactericidal or bacteriostatic activities may be used in primary prevention. In this approach, a modest antiplaque effect may be sufficient or even desirable, as it would decrease the side effects of the active agent. Antimicrobial agents are best indicated in secondary and tertiary prevention, as the objectives are to restore health and to prevent disease recurrence. The rational is to prevent or delay subgingival recolonization by pathogenic micro-organisms. The development of in vitro oral biofilm models certainly represents a major advance for studying and testing oral anti-plaque agents in recent years. The results of these studies have shown that chlorhexidine, hexetidine, delmopinol, amine fluoride/stannous fluoride, triclosan, phenolic compounds, among others, may inhibit biofilm development and maturation as well as affect bacterial metabolism.

The oral microbiota change dramatically with each part of the oral cavity, even within the same mouth. Nevertheless, the microbiota associated with peri-implantitis and periodontitis have been considered the same. To improve our knowledge of the different communities of complex oral microbiota, we compared the microbial features between peri-implantitis and periodontitis in 20 patients with both diseases. Although the clinical symptoms of peri-implantitis were similar to those of periodontitis, the core microbiota of the diseases differed. Correlation analysis revealed the specific microbial co-occurrence patterns and found some of the species were associated with the clinical parameters in a disease-specific manner. The proportion of Prevotella nigrescens was significantly higher in peri-implantitis than in periodontitis, while the proportions of Peptostreptococcaceae sp. and Desulfomicrobium orale were significantly higher in periodontitis than in peri-implantitis. The severity of the peri-implantitis was also species-associated, including with an uncultured Treponema sp. that correlated to 4 clinical parameters. These results indicate that peri-implantitis and periodontitis are both polymicrobial infections with different causative pathogens. Our study provides a framework for the ecologically different bacterial communities between peri-implantitis and periodontitis, and it will be useful for further studies to understand the complex microbiota and pathogenic mechanisms of oral polymicrobial diseases.

Aim: The microbial differences between peri-implantitis and periodontitis in the same subjects were examined using 16S rRNA gene clone library analysis and real-time polymerase chain reaction. Materials and methods: Subgingival plaque samples were taken from the deepest pockets of peri-implantitis and periodontitis sites in six subjects. The prevalence of bacteria was analysed using a 16S rRNA gene clone library and real-time polymerase chain reaction. Results: A total of 333 different taxa were identified from 799 sequenced clones; 231 (69%) were uncultivated phylotypes, of which 75 were novel. The numbers of bacterial taxa identified at the sites of peri-implantitis and periodontitis were 192 and 148 respectively. The microbial composition of peri-implantitis was more diverse when compared with that of periodontitis. Fusobacterium spp. and Streptococcus spp. were predominant in both peri-implantitis and periodontitis, while bacteria such as Parvimonas micra were only detected in peri-implantitis. The prevalence of periodontopathic bacteria was not high, while quantitative evaluation revealed that, in most cases, prevalence was higher at peri-implantitis sites than at periodontitis sites. Conclusions: The biofilm in peri-implantitis showed a more complex microbial composition when compared with periodontitis. Common periodontopathic bacteria showed low prevalence, and several bacteria were identified as candidate pathogens in peri-implantitis.

Abstract:The quantity of periodontopathic bacteria in plaque samples is an important determinant for understanding the etiologic role of bacteria. The real-time PCR method was used to detect and quantify periodontopathic bacteria, such as Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii, in saliva and subgingival plaque samples. There was good agreement between the results of conventional PCR and real-time PCR methods. Using the LighrCycler" system we were able to determine the amount of periodontopathic bacteria within an hour. The real-time PCR method was linear for samples containing from IlY to more than lOS cells (r=O.999). The application of the real-time PCR method should be useful in the rapid detection and quantification of periodontopathic bacteria in clinical samples.Key words: Periodontopathic bacteria, Periodontitis, Quantitative PCR Periodontal disease, a polymicrobial mixed infection, is one of the major oral diseases. It is caused by several microbial species, such as Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii. Proper diagnosis of the disease depends on early detection of the above bacteria. Currently, the l6S rRNA-based polymerase chain reaction (PCR) method seems to be the most sensitive and rapid method for determining the prevalence of such microorganisms (l, 6, 10). Accurate quantification of periodontal pathogens in subgingival plaque is needed for understanding the etiologic role of these bacteria. The conventional PCR (endpoint PCR) method detects the plateau phase of the reaction, however, is difficult to quantify. Recently, a real-time PCR using the Lightf'ycler" system (Roche Molecular Biochemicals, Mannheim, Germany) that allows monitoring of the exponential phase has been used to detect Borrelia burgdorferi in tissue samples (5). This method allows rapid detection and quantification of the above bacteria in clinical samples. In addition, a real-time PCR using the TaqMan system (PElABI) has been used to quantitate B. forsythus (7) givalis (3) in subgingival plaque. This system, however, requires more time for analysis than the Lightf'ycler" system.In this study, we compared three methods, the conventional PCR method, the real-time PCR method using the Lightf'ycler" system, and the culture method, for the detection and quantification of periodontopathic bacteria including A. actinomycetemcomitans, B. forsythus, P. gingivalis, T. denticola, and T. socranskii in saliva and subgingival plaque. Materials and MethodsSample collection and nucleic acid extraction. Saliva samples were collected in sterile plastic tubes from five patients [three subjects with rapidly progressive periodontitis (RP), mean age ± standard deviation (SD) of 32 ± 8.7 years, and two subjects with adult periodontitis (AP), 60±5.7 years]. The saliva samples were washed four times with sterile distilled water. After the final wash, bacterial cell p...

BackgroundPeri-implantitis (PI) is an inflammatory disease which leads to the destruction of soft and hard tissues around osseointegrated implants. The subgingival microbiota appears to be responsible for peri-implant lesions and although the complexity of the microbiota has been reported in PI, the microbiota responsible for PI has not been identified.ObjectiveThe purpose of this study was to identify the microbiota in subjects who have PI, clinically healthy implants, and periodontitis-affected teeth using 16S rRNA gene clone library analysis to clarify the microbial differences.DesignThree subjects participated in this study. The conditions around the teeth and implants were evaluated based on clinical and radiographic examinations and diseased implants, clinically healthy implants, and periodontally diseased teeth were selected. Subgingival plaque samples were taken from the deepest pockets using sterile paper points. Prevalence and identity of bacteria was analyzed using a 16S rRNA gene clone library technique.ResultsA total of 112 different species were identified from 335 clones sequenced. Among the 112 species, 51 (46%) were uncultivated phylotypes, of which 22 were novel phylotypes. The numbers of bacterial species identified at the sites of PI, periodontitis, and periodontally healthy implants were 77, 57, and 12, respectively. Microbiota in PI mainly included Gram-negative species and the composition was more diverse when compared to that of the healthy implant and periodontitis. The phyla Chloroflexi, Tenericutes, and Synergistetes were only detected at PI sites, as were Parvimonas micra, Peptostreptococcus stomatis, Pseudoramibacter alactolyticus, and Solobacterium moorei. Low levels of periodontopathic bacteria, such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, were seen in peri-implant lesions.ConclusionsThe biofilm in PI showed a more complex microbiota when compared to periodontitis and periodontally healthy teeth, and it was mainly composed of Gram-negative anaerobic bacteria. Common periodontopathic bacteria showed low prevalence, and several bacteria were identified as candidate pathogens in PI.