Evaluation of the bactericidal effect of cold atmospheric pressure plasma on contaminated human bone: an in vitro study.

The use of cold atmospheric pressure plasma (CAPP) as a bacterial decontaminant for chronic wounds has shown good results. The purpose of this in vitro study was to evaluate the bactericidal effects of CAPP on the cancellous area of the bone. Sterile glass slides and processed sterile human bone allografts 1, 2, 3, and 4mm thick were used for initial contamination and further CAPP treatment. Each block was contaminated with Staphylococcus aureus suspension on one side. Each slide was turned 180° and treated on the reverse side. The bacterial count in colony-forming units (CFU) was then measured and compared with that of a control group, and the bactericidal effects of CAPP in relation to bone density evaluated. A significant reduction in count was measured between treated and untreated groups (groups A-D: p<0.01 and group E: p=0.04). A strong positive linear relation was found between bone density and the S aureus count (r=0.844, p=0.156). Treatment with CAPP had a bactericidal effect on bone structures with a penetration depth of up to 4mm. It might be used for all diseases involving infected bone, and so extends the existing range of treatments.

Experimental peri-implant mucositis around titanium and zirconia implants in comparison to a natural tooth: part 2-clinical and microbiological parameters.

The aim of this study was to assess the clinical and microbiological parameters around dental zirconia and titanium implants compared with natural teeth during experimental plaque accumulation. Clinical parameters were evaluated (gingival index, plaque index, bleeding on probing, and probing pocket depth). Microbiological samples were analyzed for total bacterial cell counts, as well as Tannerella forsythia and Prevotella intermedia counts. A statistically significant difference over time was observed in the groups in terms of the gingival index (P<0.001), plaque index (P<0.001), and bleeding on probing (P=0.039). The lowest mean total number of bacterial cells was measured around the teeth, followed by the zirconia implants; the highest values were found around the titanium implants. T. forsythia and P. intermedia values showed significant changes over time and sessions around the titanium implants. Compared to the soft tissues around zirconia implants and the teeth, those around titanium implants developed a stronger inflammatory response to experimental plaque accumulation in terms of the total number of bacterial cells and T. forsythia and P. intermedia values.

Relationship between methanogenic archaea and subgingival microbial complexes in human periodontitis.

We compared the amounts of methanogenic archaea with ten of the most important periodontal pathogens in 125 clinical samples. Correlation analysis suggests that the support of the periodontitis-associated bacterial consortium by methanogenic archaea may be driven through direct or indirect interactions with Prevotella intermedia.

T-RFLP-based differences in oral microbial communities as risk factor for development of oral diseases under stress.

The human oral microbiome is comprised of approximately 800 different bacterial species many of which are as yet uncultivated. Their dynamics and variability in relation to health and disease are still poorly understood. Here we tested the hypothesis that the emergence of stress-induced periodontal diseases is predictable based on the composition of the initial microbiota. As a model, we analysed 58 individuals performing a challenging expedition (exposure to various stress-factors due to changes in diet, hygiene, temperature, physical and mental stress) in remote regions of the Himalayans (Annapurna Himal). Plaque samples were taken at start (Bhulbule) and destination (3000 meter difference in altitude) seven days later (Manang). Twenty-eight individuals remained symptom-free (Group I) while 30 participants developed periodontal problems, mostly gingivitis (Group II). The microbiota was monitored via T-RFLP-analysis of amplified 16S rRNA genes directly from the plaque samples. Based on the Additive-Main-Effects-Multiplicative-Interactions-model (AMMI) using the T-Rex software variation from T-RF main effects was at least 95%, indicating that most variation was due to inherent differences in microbial communities among individuals. However, an interaction signal up to 3% was consistently observed between groups I and II but not between the two time points of sampling regardless of selected analytical parameters. The data, supported by heterogeneity, diversity and similarity indices indicated marked differences between groups I and II already prior the onset of clinical symptoms. These differences may provide the basis for using ecological parameters of oral microbial communities as early diagnostic marker for the onset of oral disorders and infections.

SH3BP2-encoding exons involved in cherubism are not associated with central giant cell granuloma.

Central giant cell granuloma (CGCG) is a benign lesion with unpredictable biological behaviour ranging from a slow-growing asymptomatic swelling to an aggressive lesion associated with pain, bone and root resorption and also tooth displacement. The aetiology of the disease is unclear with controversies in the literature on whether it is mainly of reactional, inflammatory, infectious, neoplasic or genetic origin. To test the hypothesis that mutations in the SH3BP2 gene, as the principal cause of cherubism, are also responsible for, or at least associated with, giant cell lesions, 30 patients with CGCG were recruited for this study and subjected to analysis of germ line and/or somatic alterations. In the blood samples of nine patients, one codon alteration in exon 4 was found, but this alteration did not lead to changes at the amino acid level. In conclusion, if a primary genetic defect is the cause for CGCG it is either located in SH3BP2 gene exons not yet related to cherubism or in a different gene.

The discussion goes on: What is the role of Euryarchaeota in humans?

The human body (primarily the intestinal tract, the oral cavity, and the skin) harbours approximately 1,000 different bacterial species. However, the number of archaeal species known to colonize man seems to be confined to a handful of organisms within the class Euryarchaeota (including Methanobrevibacter smithii, M. oralis, and Methanosphaera stadtmanae). In contrast to this conspicuously low diversity of Archaea in humans their unique physiology in conjunction with the growing number of reports regarding their occurrence at sites of infection has made this issue an emerging field of study. While previous review articles in recent years have addressed the putative role of particularly methanogenic archaea for human health and disease, this paper compiles novel experimental data that have been reported since then. The aim of this paper is to inspire the scientific community of "Archaea experts" for those unique archaeal organisms that have successfully participated in the human-microbe coevolution.

T-RFLP-based mcrA gene analysis of methanogenic archaea in association with oral infections and evidence of a novel Methanobrevibacter phylotype.

INTRODUCTION: Increasing evidence suggests a role for methanogenic archaea (methanogens) in human health and disease via syntrophic interactions with bacteria. Here we assessed the prevalence and distribution of methanogens and possible associations with bacteria in oral biofilms. METHODS: Forty-four periodontal and 32 endodontic samples from necrotic teeth with radiographic evidence of apical periodontitis were analysed. Terminal restriction fragment length polymorphism analysis based on the mcrA gene, specific to methanogens, was applied. The prevalence and amounts of methanogens in endodontic samples were compared with those of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema spp. and Synergistes spp. based on real-time quantitative polymerase chain reactions. RESULTS: Besides dominance of the mcrA gene corresponding to Methanobrevibacter oralis, one mcrA gene type, for which no cultivated member has been reported previously, was identified in five periodontal samples and one endodontic sample. Rates of non-synonymous vs. synonymous nucleotide substitutions suggest that this mcrA gene type codes for a functionally active methyl-coenzyme M reductase. Methanobrevibacter smithii, the prominent methanogen in the human gut system, was not detected. Mean proportions of methanogens were comparable to Synergistes spp. ranging from 0.5 to 1.0% of the total microbial community. Treponema spp. dominated with a mean proportion of 10%, while the mean proportions of the other endodontic pathogens were below 0.1%. A positive association between methanogens and Synergistes spp. was found. CONCLUSION: Our data provide evidence of a novel, as yet uncultured methanogenic phylotype in association with oral infections, and indicate possible interactions between methanogens and Synergistes spp., the nature of which deserves further investigation.

Comparative analysis of endodontic pathogens using checkerboard hybridization in relation to culture.

BACKGROUND/AIM: The purpose of this study was to detect bacterial species and to quantify the total number of bacteria from samples of infected root canals before (S1) and after chemo-mechanical preparation using 2% chlorhexidine (CHX) gel as auxiliary chemical substance (S2) and after 7 days of intracanal dressing (S3) to compare microbial changes. METHOD: Twenty-four teeth were selected for this study. Chemo-mechanical preparation was performed using 2% CHX gel, then three different intracanal medicaments [M1: Ca(OH)(2) paste; M2: 2% CHX gel; and M3: Ca(OH)(2) paste plus 2% CHX gel] were used for 7 days. Checkerboard DNA-DNA hybridization was performed to detect 40 bacterial species. Aerobic and anaerobic culture techniques were used to determine the bacterial community by counting the colony-forming units (CFU). RESULTS: The species most frequently identified by checkerboard in S1 were: Fusobacterium nucleatum ssp. polymorphum, Treponema socranskii ssp. socranskii, Parvimonas micra and Enterococcus faecalis. In S2 and S3 a total of eight different species were identified; and only one of them was gram-positive (E. faecalis). Microorganisms were not identified after use of M2 for 7 days. The quantification obtained on agar plates ranged from 4 x 10(5) to 2.6 x 10(6) CFU/ml in S1, mean CFU was reduced by 99.96% in S2, and there was no statistical difference between the CFU in S2 and S3. CONCLUSION: The antibacterial effect of the mechanical preparation supplemented by the use of an antibacterial auxiliary substance greatly reduced the microorganisms in the main root canal.

Quantitative analysis of three hydrogenotrophic microbial groups, methanogenic archaea, sulfate-reducing bacteria, and acetogenic bacteria, within plaque biofilms associated with human periodontal disease.

Human subgingival plaque biofilms are highly complex microbial ecosystems that may depend on H(2)-metabolizing processes. Here we investigated the ubiquity and proportions of methanogenic archaea, sulfate reducers, and acetogens in plaque samples from 102 periodontitis patients. In contrast to the case for 65 healthy control subjects, hydrogenotrophic groups were almost consistently detected in periodontal pockets, with the proportions of methanogens and sulfate reducers being significantly elevated in severe cases. In addition, antagonistic interactions among the three microbial groups indicated that they may function as alternative syntrophic partners of secondary fermenting periodontal pathogens.

Effect of root canal procedures on endotoxins and endodontic pathogens.

BACKGROUND/AIMS: The purpose of this study was to determine the amount of endotoxin (lipopolysaccharide) and cultivable bacteria in human necrotic root canals before (S1) and after chemo-mechanical preparation using chlorhexidine (CHX) gel as auxiliary chemical substance (S2), and after 7 days of intracanal dressing (S3) in order to evaluate the anti-endotoxin and antimicrobial effects of endodontic procedures. METHOD: Twenty-four teeth were selected for the present study. Chemo-mechanical preparation was performed using 2% CHX gel and three different intracanal medicaments [CaOH2 paste; 2% CHX gel; and CaOH2 + 2% CHX gel]. A quantitative chromogenic Limulus amoebocyte lysate assay was used to measure the amount of endotoxin. Aerobic and anaerobic techniques were used to isolate and identify bacteria, and to determine the bacterial reduction by counting colony-forming units (CFU). RESULTS: Endotoxins and bacteria were present in 100% of the initial samples, with endotoxin concentration ranging from 62.93 to 214.56 UE/ml and CFU ranging from 4 x 10(5) to 2.6 x 10(6). After chemo-mechanical preparation a mean endotoxin reduction of 44.4% was found. Eight (33.3%) root canals were still positive by culture analysis with a mean reduction of bacteria (CFU) of 99.96%. After 7 days of intracanal dressing, endotoxin concentration decreased by only 1.4% compared with S2, and residual bacteria were recovered by culture analysis in 13 cases (54.1%). No significant difference was found among different intracanal medicaments. CONCLUSION: Relatively high values of endotoxin were still present in the root canal after chemo-mechanical preparation although the majority of bacteria were eliminated. No improvement was achieved by 7 days of intracanal dressing.

Quantification and characterization of Synergistes in endodontic infections.

INTRODUCTION: Bacterial species belonging to the poorly characterized division Synergistes have recently been reported in endodontic infections, and therefore may be part of the etiology of periradicular diseases. The objective of this study was to characterize and quantify the predominant Synergistes phylotypes in infected root canals. METHODS: We analyzed 32 necrotic teeth, each from a different patient, with radiographic evidence of apical periodontitis and with primary endodontic infections. RESULTS: Using real-time quantitative polymerase chain reaction based on Synergistes-specific primers, seven of the 32 cases were found to be positive. Comparative sequence analysis showed that each of the seven samples was infected by one numerically dominant phylotype. Diversity among phylotypes was such that they could be grouped into three major evolutionary branches within the Synergistes division. The size of the total Synergistes population ranged from 4.5 x 10(4) to 1.5 x 10(6) 16S rRNA gene copies, and the median proportion accounted for 0.79% of the total bacterial community. For comparison, we also quantified such recognized endodontic pathogens as Prevotella intermedia, Porphyromonas gingivalis, and Treponema. The first two species were found in five and nine cases, respectively, with a median proportion below 0.01%, while Treponema was found in 18 cases with a median proportion of 1.48%. CONCLUSION: Thus, the prevalence and quantity of Synergistes was clearly within the range of the other analyzed pathogens, suggesting their clinical relevance in endodontic infections. Furthermore, the diversity of Synergistes found at the diseased sites designates infected root canals as an important human ecosystem providing several unique micro-niches for this novel group of bacteria.

Distribution and persistence of probiotic Streptococcus salivarius K12 in the human oral cavity as determined by real-time quantitative polymerase chain reaction.

The bacteriocin producer Streptococcus salivarius K12 is used as a probiotic targeting the oral cavity, so our study aimed to assess whether its dispersal and persistence could be monitored using real-time quantitative polymerase chain reaction. To this end, we designed polymerase chain reaction primers and a hybridization probe specifically targeting salA, which encodes for the prepropeptide of salivaricin A. Using a single individual as our subject, we administered four lozenges of K12 Throat Guard per day over 3 days, then measured salA gene levels for 16 different oral sites at six different intervals over 35 days. Four samples each from gingival sulci and from teeth all remained negative. In contrast, in saliva and at all mucosal membranes K12 was detected, but with varying amounts and time profiles. Relatively high salA gene copy numbers, calibrated on the basis of colony-forming units, were seen on the tongue (maximum 4.6 x 10(4)/swab at day 4), in stimulated saliva (2.4 x 10(4)/ml, day 4) and on buccal membranes (1.3 x 10(4)/swab, day 8). K12 was present on both sides of the pharynx but asymmetrically in both quantity and duration. In conclusion, we have developed a real-time quantitative-polymerase chain reaction for counting S. salivarius K12 at various sites in the oral cavity. In the individual studied, K12 could be detected at the mucosal membranes for as long as 3 weeks, but with steadily decreasing numbers after day 8. Thus, K12 may have the potential to control oral bacterial infections only when the uptake is repeated frequently.

In vivo evaluation of microbial reduction after chemo-mechanical preparation of human root canals containing necrotic pulp tissue.

AIM: To determine in vivo, the degree of microbial reduction after chemo-mechanical preparation of human root canals containing necrotic pulp tissue when using two endodontic irrigating reagents, sodium hypochlorite (NaOCl) or chlorhexidine gel (CHX). METHODOLOGY: Thirty-two single rooted teeth with necrotic pulp were divided into two groups. One group (n=16) was irrigated with 2.5% NaOCl, whilst the other group (n=16) was irrigated with 2% CHX gel. Assessment of the bacterial load was accomplished by use of real-time quantitative-polymerase chain reaction (RTQ-PCR) directed against the small subunit ribosomal DNA using the SYBRGreen and TaqMan formats. Statistical analysis was performed using the Mann-Whitney test. For contrast, bacterial load was also determined by traditional culture techniques. RESULTS: The bacterial load was reduced substantially in both groups (over 96%). However, using RTQ-PCR the bacterial load before and after chemo-mechanical preparation was greater when compared with evaluation using colony forming units (CFU). Furthermore, as measured by RTQ-PCR, the bacterial reduction in the NaOCl-group (SYBRGreen 99.99%; TaqMan: 99.63%) was significantly greater (P<0.01) than in the CHX-group (SYBRGreen 96.62%; TaqMan: 96.60%). According to culture technique 75% of cases were free of bacteria after chemo-mechanical preparation in the NaOCl-group, whilst 50% of cases were bacteria free in the CHX-group. CONCLUSION: NaOCl has not only a higher capacity to kill microorganisms but is also more able to remove cells from the root canal.

Identification and quantification of archaea involved in primary endodontic infections.

Members of the domain Archaea, one of the three domains of life, are a highly diverse group of prokaryotes, distinct from bacteria and eukaryotes. Despite their abundance and ubiquity on earth, including their close association with humans, animals, and plants, so far no pathogenic archaea have been described. As some archaea live in close proximity to anaerobic bacteria, for instance, in the human gut system and in periodontal pockets, the aim of our study was to assess whether archaea might possibly be involved in human endodontic infections, which are commonly polymicrobial. We analyzed 20 necrotic uniradicular teeth with radiographic evidence of apical periodontitis and with no previous endodontic treatment. Using real-time quantitative PCR based on the functional gene mcrA (encoding the methyl coenzyme M reductase, specific to methanogenic archaea) and on archaeal 16S rRNA genes, we found five cases to be positive. Direct sequencing of PCR products from both genes showed that the archaeal community was dominated by a Methanobrevibacter oralis-like phylotype. The size of the archaeal population at the diseased sites ranged from 1.3 x 10(5) to 6.8 x 10(5) 16S rRNA gene target molecule numbers and accounted for up to 2.5% of the total prokaryotic community (i.e., bacteria plus archaea). Our findings show that archaea can be intimately connected with infectious diseases and thus support the hypothesis that members of the domain Archaea may have a role as human pathogens.

Evaluation of universal probes and primer sets for assessing total bacterial load in clinical samples: general implications and practical use in endodontic antimicrobial therapy.

By re-examining 10 previously published "universal" PCR assays using the ARB phylogenetic software package and database with 41,000 16S rRNA gene sequences, we found that they differed considerably in their coverage of the domain Bacteria. We evaluated the broadest-range real-time quantitative PCR protocol for its efficacy in measuring the antimicrobial effects of endodontic treatments.

Microarrays complement culture methods for identification of bacteria in endodontic infections.

The aim of this study was to investigate the microbial composition of necrotic root canals using culture methods and microarray technology. Twenty uniradicular teeth with radiographic evidence of periapical bone loss and with no previous endodontic treatment were selected for this study. For molecular diagnosis a DNA chip with 20 different species-specific, 16S-rDNA-directed catcher probes was used. The microorganisms most frequently detected by the DNA chip were: Micromonas micros, Fusobacterium nucleatum ssp., Tannerella forsythia, Treponema denticola, Veillonella parvula, Eubacterium nodatum, Porphyromonas gingivalis, Actinomyces odontolyticus, and Streptococcus constellatus. As expected, additional important bacterial taxa were found by culture analysis, but microorganisms such as T. forsythia and T. denticola could not be identified. In conclusion, microarrays may provide significant additional information regarding the endodontic microbiota by detecting bacterial species that are otherwise difficult or impossible to culture.

In vitro activities of ABT-773, a new ketolide, against aerobic and anaerobic pathogens isolated from antral sinus puncture specimens from patients with sinusitis.

The comparative in vitro activities of ABT-773 against 207 aerobic and 162 anaerobic antral sinus puncture isolates showed that erythromycin-resistant pneumococcal strains were susceptible to ABT-773 (< or =0.125 microg/ml); the MIC at which 90% of the isolates tested were inhibited for Haemophilus influenzae and other Haemophilus spp. was 4 microg/ml; and all Moraxella spp. and beta-lactamase-producing Prevotella species strains were inhibited by < or =0.125 microg/ml. Among the anaerobes tested, only fusobacteria (45%) required > or =4 microg of ABT-773/ml for inhibition. ABT-773 may offer a therapeutic alternative for sinus infections.

Diode laser radiation and its bactericidal effect in root canal wall dentin.

OBJECTIVES: The aim of this study was to investigate the antibacterial effect of a diode laser in deep root canal dentin. BACKGROUND DATA: The microbial colonization of root canal dentin can lead to failures in conventional endodontic treatment if an inadequate bacterial reduction only is achieved through canal treatment and chemical disinfection. METHODS: 100 microm, 300 microm and 500 microm bovine dentin slices obtained by longitudinal sections were sterilized and inoculated on one side with an Enterococcus faecalis suspension. Laser radiation was performed on the opposite side with the diode laser (810 nm) at a setting of 3 W in continuous mode (CW). Radiation was performed using a 400-microm tapered fiber tip at an angle of approximately 5 degrees to the surface over a period of 30 seconds. The output power at the distal end of the tip was 0.6 W. The bacteria were then eluted through vibration and cultured on blood agar plates. The colony count reflected the antibacterial effect of laser radiation as a function of the layer thickness. RESULTS: A mean bacterial reduction of 74% was achieved even with a 500-microm thick slice. CONCLUSION: This investigation indicates that the diode laser radiation reduces the number of bacteria in deep layers of infected root canal wall dentin.

The bactericidal effect of Ho:YAG laser irradiation within contaminated root dentinal samples.

OBJECTIVE: This in vitro study investigates the bactericidal effect of pulsed Ho:YAG laser irradiation in the depth of contaminated dentin specimens. BACKGROUND DATA: Previous studies have shown the effectiveness of laser irradiation in bacterial reduction of infected root canal. METHODS: Root dentin of bovine teeth were sliced longitudinally in 180 samples of 100 microm, 300 microm, and 500 microm thickness, sterilized, dried, and inoculated on one side, with 1 microL of Enterococcus faecalis suspension. The opposite side's were irradiated four times for 5 seconds each with Ho:YAG laser irradiation, a wavelength of 2.10 microm, using four different energy settings: 1 W/5 Hz; 1 W/10 Hz; 1.5 W/5 Hz, and 2.0 W/5 Hz through a 320-microm quartz fiber at an angle of approximately 5 degrees. In addition, two control groups were investigated, the first was inoculated and not submitted to any treatment, the second was inoculated and treated with NaOCl and H2O2. The remaining bacteria from each dentin sample in a transport media were removed by vibration, serially diluted, and plated out on culture dishes selective for Enterococcus faecalis. RESULTS: When compared with the untreated control group or even with the group treated with NaOCl plus H2O2, counting of colonies forming units (CFU) from the laser-treated samples revealed a high significant bacterial elimination with a maximum of 98.46% and a minimum of 83.65%. CONCLUSIONS: Our findings demonstrate a significant decrease of the bacterial population in depth, suggesting that the Ho:YAG laser irradiation could be effective to eliminate the microorganisms harbored within dentin or contaminated canals.

Sampling of periodontal pathogens by paper points: evaluation of basic parameters.

Paper points are widely established for the collection of subgingival plaque or other oral samples to analyze the microbiota, especially the presence of periodontal pathogenic bacteria such as Actinobacillus actinomycetemcomitans or Porphyromonas gingivalis. In contrast to the high frequency of usage of paper points in oral sampling, very few data are available about the parameters influencing the sampling process. Therefore, in four different in vitro experiments (6-9 repeats), we inoculated paper points with standardized suspensions (2 x 10(9) colony-forming units/ml) of A. actinomycetemcomitans and P. gingivalis to test the influence of the origin (kind) of paper point ("manufacturer"), size (according to the International Organization for Standardization ISO 25-80), sampling time (5 to 60 s), and elution time (5 to 60 s). Sampled bacteria were detected and (semi-) quantified using 16S rRNA/DNA-directed oligonucleotide probes. The bacterial load was categorized and calculations performed with index values ranging between 0 (below the detection limit of 10(2) to 10(3) colony-forming units) and 9 (> 10(6) colony-forming units). We found statistically significant differences in the efficiency of bacterial sampling between the 5 paper point manufacturers tested, expressed in a mean bacterial index between 4.4 and 7.8. ISO 45 Paper points were statistically proven to work most efficiently. According to our results, a sampling time of 60 s seems to be optimum. However, shorter times between 5 and 30 s did not significantly reduce the sampling efficiency. We found an interval of 20 s best to elute bacteria from the paper points by vortexing. The evaluation of basic parameters for subgingival plaque sampling by paper points might help to optimize microbiologically based diagnostics in periodontal diseases.