Isolation, characterization, and fibroblast uptake of bacterial extracellular vesicles from Porphyromonas gingivalis strains.

Periodontitis is an inflammatory condition caused by bacteria and represents a serious health problem worldwide as the inflammation damages the supporting tissues of the teeth and may predispose to systemic diseases. Porphyromonas gingivalis is considered a keystone periodontal pathogen that releases bacterial extracellular vesicles (bEVs) containing virulence factors, such as gingipains, that may contribute to the pathogenesis of periodontitis. This study aimed to isolate and characterize bEVs from three strains of P. gingivalis, investigate putative bEV uptake into human oral fibroblasts, and determine the gingipain activity of the bEVs. bEVs from three bacterial strains, ATCC 33277, A7A1-28, and W83, were isolated through ultrafiltration and size-exclusion chromatography. Vesicle size distribution was measured by nano-tracking analysis (NTA). Transmission electron microscopy was used for bEV visualization. Flow cytometry was used to detect bEVs and gingipain activity was measured with an enzyme assay using a substrate specific for arg-gingipain. The uptake of bEVs into oral fibroblasts was visualized using confocal microscopy. NTA showed bEV concentrations from 108 to 1011 particles/mL and bEV diameters from 42 to 356 nm. TEM pictures demonstrated vesicle-like structures. bEV-gingipains were detected both by flow cytometry and enzyme assay. Fibroblasts incubated with bEVs labeled with fluorescent dye displayed intracellular localization consistent with bEV internalization. In conclusion, bEVs from P. gingivalis were successfully isolated and characterized, and their uptake into human oral fibroblasts was documented. The bEVs displayed active gingipains demonstrating their origin from P. gingivalis and the potential role of bEVs in periodontitis.

Identification and susceptibility testing of oral candidiasis in advanced cancer patients.

BACKGROUND: Patients with advanced cancer are prone to develop different opportunistic oral infection due to anti-cancer treatment or the malignancies themselves. Studies of oral fungal samples show an increased prevalence of non-Candida albicans species in mixed oral infections with Candida albicans. Non-C. albicans and C. albicans are associated with varying degrees of resistance to azoles, which may have implications for treatment. This study aimed to assess the diversity and antifungal susceptibility of Candida species detected in the oral cavity. METHODS: An observational study with microbiological analysis was conducted. Clinical fungal isolates were collected from patients in a hospice unit in 2014-2016. Isolates were re-grown on chromID® Candida plates in 2020. Single colony of each species was re-cultivated and prepared for biochemical identification with a VITEK2® system and verified by gene sequencing. Etest was performed on RPMI agar, and the antifungals fluconazole, amphotericin B, anidulafungin and nystatin were applied. RESULTS: Fifty-six isolates from 45 patients were identified. Seven different Candida species and one Saccharomyces species were detected. The results of biochemical identification were confirmed with sequencing analysis. Thirty-six patients had mono infection, and nine out of 45 patients had 2-3 different species detected. Of C. albicans strains, 39 out of 40 were susceptible to fluconazole. Two non-C. albicans species were resistant to fluconazole, one to amphotericin B and three to anidulafungin. CONCLUSION: C. albicans was the predominant species, with a high susceptibility to antifungal agents. Different Candida species occur in both mono and mixed infections. Identification and susceptibility testing may therefore lead to more effective treatment and may prevent the development of resistance among patients with advanced cancer. TRAIL REGISTRATION: The study Oral Health in Advanced Cancer was registered at ClinicalTrials.gov (#NCT02067572) in 20/02/2014.

Salivary IgA and IgG Antibody Responses against Periodontitis-Associated Bacteria in Crohn's Disease.

Elevated serum immunoglobulin (Ig) antibody levels are observed in Crohn's disease patients. The aim of this study was to evaluate the salivary IgA and IgG antibody levels against Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia in Crohn's disease patients. Eighty-eight participants (47 Crohn's disease patients and 41 systemically healthy age- and gender-matched controls) were included in the study. Oral and medical health statuses were recorded and salivary samples were collected. Salivary P. gingivalis, T. forsythia, A. actinomycetemcomitans, and P. intermedia carriage were analyzed with DNA sequencing technique, salivary levels of IgG1, IgG2, IgG3, IgG4, and IgM were measured with the Luminex® xMAP™ technique, and salivary IgA and IgG antibody levels against P. gingivalis, T. forsythia, A. actinomycetemcomitans, and P. intermedia were detected by ELISA. As result, higher salivary IgG2 (p = 0.011) and IgG3 (p = 0.006), P. gingivalis IgA (p < 0.001), A. actinomycetemcomitans IgG (p = 0.001), and P. intermedia IgG (p < 0.001) antibody levels were detected in the Crohn's disease group compared to the controls. Salivary P. gingivalis carriage was lower in the Crohn's disease group in comparison to the controls (p = 0.024). In conclusion, salivary IgA antibody responses against P. gingivalis and IgG antibody responses against P. intermedia have independent associations with Crohn's disease.

The blood microbiome and its association to cardiovascular disease mortality: case-cohort study.

BACKGROUND: Little is known about the association between bacterial DNA in human blood and the risk of cardiovascular disease (CVD) mortality. METHODS: A case-cohort study was performed based on a 9 ½ year follow-up of the Oslo II study from 2000. Eligible for this analysis were men born in 1923 and from 1926 to 1932. The cases were men (n = 227) who had died from CVD, and the controls were randomly selected participants from the same cohort (n = 178). Analysis of the bacterial microbiome was performed on stored frozen blood samples for both cases and controls. Association analyses for CVD mortality were performed by Cox proportional hazard regression adapted to the case-cohort design. We used the Bonferroni correction due to the many bacterial genera that were identified. RESULTS: Bacterial DNA was identified in 372 (82%) of the blood samples and included 78 bacterial genera from six phyla. Three genera were significantly associated with CVD mortality. The genera Kocuria (adjusted hazard ratio (HR) 8.50, 95% confidence interval (CI) (4.05, 17.84)) and Enhydrobacter (HR 3.30 (2.01, 5.57)) indicate an association with CVD mortality with increasing levels. The genera Paracoccus (HR 0.29 (0.15, 0.57)) was inversely related. Significant predictors of CVD mortality were: the feeling of bad health; and the consumption of more than three cups of coffee per day. The following registered factors were borderline significant, namely: a history of heart failure; increased systolic blood pressure; and currently taking antihypertensive drugs now, versus previously. CONCLUSIONS: The increasing levels of two bacterial genera Kocuria (skin and oral) and Enhydrobacter (skin) and low levels of Paracoccus (soil) were associated with CVD mortality independent of known risk factors for CVD.

Child saliva microbiota and caries: a randomized controlled maternal education trial in rural Uganda.

Undernutrition is a public health challenge in sub-Saharan countries, including Uganda. In a previous randomized controlled trial (RCT) with a nutrition, hygiene and stimulation education intervention among mothers of 6 months' old children, we found less caries in the intervention group when the children were 36 months of age. We now examined the effects of (i) the intervention on the microbiota, (ii) microbiota on caries, and (iii) the intervention and microbiota on caries. The original RCT comprised 511 mother/child pairs whereas in the current study we had access to data from 344/511 (67%) children aged 36 months. The saliva microbiota was determined using 16S rRNA gene sequencing. Carious lesions (a proxy for dental health) were identified using close-up intra-oral photographs of the upper front teeth. Statistical models were used to determine host-microbiota associations. The intervention had a significant effect on the microbiota, e.g. an increase in Streptococcus abundance and decreases in Alloprevotella and Tannerella. Significant associations between the microbiota and dental caries were identified: Positive associations of Capnocytophaga and Tannerella suggest that these taxa may be deleterious to dental health while negative associations of Granulicatella, Fusobacterium, and Abiotrophia suggest taxa potentially beneficial or benign contributors to dental health. Based on taxonomic profiles, the effects of the intervention and microbiota on dental health may be independent of one another. Educational interventions with emphasis on nutrition and oral hygiene may provide a feasible strategy to decrease progression of childhood caries in low-resource settings.

A 12-month randomized controlled trial evaluating erythritol air-polishing versus curette/ultrasonic debridement of mandibular furcations in supportive periodontal therapy.

BACKGROUND: Due to complex morphology and limited access, the cleaning of the furcation area is extremely challenging. Therefore, novel therapeutic approaches need to be tested to potentially overcome debridement limitations. The aim of the present prospective 12-month study was to compare clinical and microbiological effects following erythritol air-polishing versus conventional mechanical debridement of furcation defects in a cohort of periodontal maintenance patients. METHODS: Twenty patients with grade II mandibular molar furcation defects volunteered to enroll in this single-centre, examiner masked, randomized controlled trial. In a split-mouth study design, two furcation sites in each patient were randomly assigned to either receive subgingival debridement using erythritol air-polishing (test) or conventional ultrasonic/curette debridement (control) at baseline, and at 3, 6, 9 and 12 months. Probing depth, clinical attachment level and bleeding on probing were recorded at 3-month intervals. Subgingival microbiological samples obtained at baseline, 6 and 12 months were analyzed using checkerboard DNA-DNA hybridization. Discomfort from treatment was scored at 12 months using a visual analogue scale. The differences between treatments, and time-points, were tested using multilevel analysis (mixed effect models and robust variance estimates). RESULTS: A significant reduction in probing depth took place following both treatments (p < 0.001). Control sites experienced a significant mean gain in clinical attachment level of 0.5 mm (± 0.2) (p = 0.004), whereas a non-significant gain of 0.4 mm (± 0.3) was observed at test sites (p = 0.119). At 6 months, a significant between-treatment difference of 0.8 mm (± 0.4) was observed in favor of the control (p = 0.032). No significant between-treatment differences were observed in microbial load or composition. Notably, at 12 months patients experienced significantly less discomfort following air-polishing compared with control (p = 0.001). CONCLUSIONS: The 12-month observations indicate that erythritol air-polishing and conventional mechanical debridement both support clinical improvements. A significant between-treatment difference in clinical attachment level was, however, detected in favour of control debridement at 6 months. In terms of patient comfort, erythritol air-polishing is superior. TRIAL REGISTRATION: The clinical trial was retrospectively registered in ClinicalTrial.gov with registration NCT04493398 (07/28/2020).

Outcome of Endodontic Retreatment Using 2 Root Canal Irrigants and Influence of Infection on Healing as Determined by a Molecular Method: A Randomized Clinical Trial.

INTRODUCTION: This study compared the clinical and radiographic outcome of endodontic retreatment of teeth with apical periodontitis using either 1% sodium hypochlorite (NaOCl) or 2% chlorhexidine digluconate (CHX) as the irrigant. The influence of residual infection detected by a molecular method on the outcome was also examined. METHODS: Fifty-two root-filled teeth with apical periodontitis were randomly assigned into 2 groups according to the irrigant used during retreatment. Root canal microbiological samples taken before (S1) and after (S2) preparation using either NaOCl or CHX irrigation and after calcium hydroxide medication (S3) were subjected to 16S ribosomal RNA gene-based real-time quantitative polymerase chain reaction (qPCR) to quantify total bacteria. The periapical status was scored using the periapical index and dichotomized as healed (<3) or not healed (≥3) at the 1- and 4-year follow-up. RESULTS: Forty-five (NaOCl, n = 20; CHX, n = 25) and 33 teeth (NaOCl, n = 16; CHX, n = 17) were available at the 1- and 4-year follow-up, respectively. After 1 year, 65% in the NaOCl group and 64% in the CHX group healed, with no differences between them (P > .05). At the later follow-up, the corresponding figures were 81% and 82%, respectively (P > .05). Canals that yielded qPCR-negative results in S3 had a higher healing rate (79%) than qPCR-positive canals (45%, P < .05). The mean bacterial load increased from S2 to S3 in half of the unhealed cases (P < .05). All S3-positive canals containing <3.12 × 103 bacterial cell counts healed. Increasing the apical level of the root canal filling influenced the outcome (P < .05). CONCLUSIONS: No significant differences in the clinical outcome between 1% NaOCl and 2% CHX were found. Bacterial persistence at the time of filling as detected by qPCR significantly affected the outcome.

Maintenance of the aseptic working field during endodontic treatment.

Objective: The aim of this clinical quality study was to determine whether the aseptic working field is maintained during the endodontic procedure. Materials and methods: Bacterial samples were collected from the rubber dam of 27 patients during endodontic treatment performed by postgraduate students at the Department of Endodontics, University of Oslo. A bacterial sample was first obtained immediately after disinfection of the working field (A), and the second sample was collected just before obturation or dressing with calcium hydroxide cement (B). Aerobic cultivation technique and PCR were used for detection of bacterial growth and species. Results: All samples were negative on culturing except in one case, which showed positive results with cultivation in both sample A and B. Specie detected with cultivation technique were Streptococcus mitis. With PCR technique, 6 samples in 5 patients (11%), showed positive results. Species detected with PCR technique were Bacteroidales spp. Propionibacterium spp., Bacteroidetes spp., Prevotella nigrescens, Haemophilus parainfluenzae, Neisseria elongata, Alloprevotella tannerae, Capnocytophaga granulosa, Cardiobacterium hominis, Fusobacterium nucleatum and Streptococcus mitis. Conclusion: The present study showed that an aseptic working field was maintained throughout the endodontic procedure in 81% (22/27) of the cases after disinfection of the rubber dam.

Microbial Analysis of Endodontic Infections in Root-filled Teeth with Apical Periodontitis before and after Irrigation Using Pyrosequencing.

INTRODUCTION: The root canal microbiota in root-filled teeth with post-treatment apical periodontitis before and after chemomechanical instrumentation and irrigation with either 1% sodium hypochlorite (NaOCl) or 2% chlorhexidine digluconate were analyzed by using the pyrosequencing method. METHODS: Samples from 10 root-filled teeth with apical periodontitis undergoing retreatment were taken before (S1) and after (S2) preparation using irrigation with either NaOCl (n = 5) or 2% chlorhexidine digluconate (n = 5). DNA was extracted, and the 16S rRNA gene (V3-V5) variable regions were amplified and subjected to pyrosequencing (GS junior 454) to determine the bacterial composition. RESULTS: Pyrosequencing yielded 43,797 sequence reads in S1 and 9196 in S2 samples. Overall, 125 bacterial species belonging to 68 genera (S1, 59; S2, 38) and 9 phyla were found. The most abundant and prevalent phyla in S1 and S2 samples were Firmicutes, Fusobacteria, Bacteroidetes, and Actinobacteria. The most represented, abundant, and prevalent genera in S1 and S2 samples were Streptococcus and Fusobacterium. The most prevalent species in S1 and S2 samples were Fusobacterium nucleatum ss. vincentii, Streptococcus oralis/mitis, Streptococcus intermedius, and Streptococcus gordonii. The mean number of species per root canal was 20 (range, 4-37) in S1 and 9 (range, 4-15) in S2, respectively. CONCLUSIONS: A high interindividual diversity was observed in both S1 and S2 samples, with no difference between the two irrigation groups. F. nucleatum ss. vincentii and some Streptococcus species were the most prevalent species in pre-preparation and post-preparation samples during retreatment of root-filled teeth with infection.

Antimicrobial Effects of Three Different Treatment Modalities on Dental Implant Surfaces.

Resolution of peri-implant inflammation and re-osseointegration of peri-implantitis affected dental implants seem to be dependent on bacterial decontamination. The aims of the study were to evaluate the antimicrobial effects of 3 different instrumentations on a micro-textured dental implant surface contaminated with an avirulent or a virulent Porphyromonas gingivalis strain and to determine alterations to the implant surface following instrumentation. Forty-five dental implants (Straumann SLA) were allocated to 3 treatment groups: Er:YAG laser, chitosan brush, and titanium curette (10 implants each) and a positive (10 implants) and a negative (5 implants) control. Each treatment group and the positive control were split into subgroups of 5 implants subsequently contaminated with either the avirulent or virulent P. gingivalis strain. The antimicrobial effect of instrumentation was evaluated using checkerboard DNA-DNA hybridization. Implant surface alterations were determined using a light interferometer. Instrumentation significantly reduced the number of attached P. gingivalis ( P < .001) with no significant differences among groups ( P = .310). A significant overall higher median score was found for virulent compared with avirulent P. gingivalis strains ( P = .007); the Er:YAG laser uniquely effective removing both bacterial strains. The titanium curette significantly altered the implant surface micro-texture. Neither the Er:YAG laser nor the chitosan brush significantly altered the implant surface. The 3 instrumentations appear to have a similar potential to remove P. gingivalis. The titanium curette significantly altered the microstructure of the implant surface.

The Predictive Role of Tooth Extractions, Oral Infections, and hs-C-Reactive Protein for Mortality in Individuals with and without Diabetes: A Prospective Cohort Study of a 12 1/2-Year Follow-Up.

The predictive role of high-sensitivity C-reactive protein (hs-CRP), number of tooth extractions, and oral infections for mortality in people with and without diabetes is unclear. This prospective cohort study is a 12 1/2-year follow-up of the Oslo II study, a health survey in 2000. In all, 12,764 men were invited. Health information was retrieved from 6434 elderly men through questionnaire information, serum measurements, and anthropometric and blood pressure measurements. Diabetes was reported by 425 men. Distinct differences were observed in baseline characteristics in individuals with and without diabetes. In the diabetes group, age and hs-CRP were statistically significant whereas in the nondiabetes group, age, hs-CRP, number of tooth extractions, tooth extractions for infections and oral infections combined, nonfasting glucose, systolic blood pressure, total cholesterol, regular alcohol drinking, daily smoking, and level of education were independent risk factors. The number of tooth extractions <5 was inversely related whereas more extractions increased the risk. Multivariate analyses showed that hs-CRP was a significant predictor in persons with diabetes and tooth extractions and oral infections combined; the number of teeth extracted and hs-CRP were for persons without diabetes. Infection and inflammation were associated with mortality in individuals both with and without diabetes.

Influence of the Apical Preparation Size and the Irrigant Type on Bacterial Reduction in Root Canal-treated Teeth with Apical Periodontitis.

INTRODUCTION: This clinical study evaluated the influence of the apical preparation size using nickel-titanium rotary instrumentation and the effect of a disinfectant on bacterial reduction in root canal-treated teeth with apical periodontitis. METHODS: Forty-three teeth with posttreatment apical periodontitis were selected for retreatment. Teeth were randomly divided into 2 groups according to the irrigant used (2.5% sodium hypochlorite [NaOCl], n = 22; saline, n = 21). Canals were prepared with the Twisted File Adaptive (TFA) system (SybronEndo, Orange, CA). Bacteriological samples were taken before preparation (S1), after using the first instrument (S2), and then after the third instrument of the TFA system (S3). In the saline group, an additional sample was taken after final irrigation with 1% NaOCl (S4). DNA was extracted from the clinical samples and subjected to quantitative real-time polymerase chain reaction to evaluate the levels of total bacteria and streptococci. RESULTS: S1 from all teeth were positive for bacteria. Preparation to the first and third instruments from the TFA system showed a highly significant intracanal bacterial reduction regardless of the irrigant (P < .01). Apical enlargement to the third instrument caused a significantly higher decrease in bacterial counts than the first instrument (P < .01). Intergroup comparison revealed no significant difference between NaOCl and saline after the first instrument (P > .05). NaOCl was significantly better than saline after using the largest instrument in the series (P < .01). CONCLUSIONS: Irrespective of the type of irrigant, an increase in the apical preparation size significantly enhanced root canal disinfection. The disinfecting benefit of NaOCl over saline was significant at large apical preparation sizes.

Bacterial diversity in medication-related osteonecrosis of the jaw.

OBJECTIVE: The aim was to study the association between microflora and medication-related osteonecrosis of the jaw (MRONJ) by using culture-independent molecular techniques to detect bacteria in necrotic bone lesions. STUDY DESIGN: Included were 18 consecutive patients with MRONJ, 10 with osteoporosis and 8 cancer patients. Bone biopsies were retrieved from the center of the necrotic bone and from visually healthy bone, and 16 S rRNA gene fragments from bacterial DNA were amplified with polymerase chain reaction. RESULTS: The study revealed a diversity of bacteria represented by 16 S rRNA sequences in all the necrotic bone samples and in 60% of the visually healthy bone. Eight dominating taxa groups were identified at the genus level: Porphyromonas, Lactobacillus, Tannerella, Prevotella, Actinomyces, Treponema, Streptococcus, and Fusobacterium. CONCLUSIONS: The necrotic bone lesions contained mainly anaerobic bacteria, representative of periodontal microflora, suggesting that a periodontal infection in combination with antiresorptive treatment could initiate osteonecrosis.

Oral, intestinal, and skin bacteria in ventral hernia mesh implants.

BACKGROUND: In ventral hernia surgery, mesh implants are used to reduce recurrence. Infection after mesh implantation can be a problem and rates around 6-10% have been reported. Bacterial colonization of mesh implants in patients without clinical signs of infection has not been thoroughly investigated. Molecular techniques have proven effective in demonstrating bacterial diversity in various environments and are able to identify bacteria on a gene-specific level. OBJECTIVE: The purpose of this study was to detect bacterial biofilm in mesh implants, analyze its bacterial diversity, and look for possible resemblance with bacterial biofilm from the periodontal pocket. METHODS: Thirty patients referred to our hospital for recurrence after former ventral hernia mesh repair, were examined for periodontitis in advance of new surgical hernia repair. Oral examination included periapical radiographs, periodontal probing, and subgingival plaque collection. A piece of mesh (1×1 cm) from the abdominal wall was harvested during the new surgical hernia repair and analyzed for bacteria by PCR and 16S rRNA gene sequencing. From patients with positive PCR mesh samples, subgingival plaque samples were analyzed with the same techniques. RESULTS: A great variety of taxa were detected in 20 (66.7%) mesh samples, including typical oral commensals and periodontopathogens, enterics, and skin bacteria. Mesh and periodontal bacteria were further analyzed for similarity in 16S rRNA gene sequences. In 17 sequences, the level of resemblance between mesh and subgingival bacterial colonization was 98-100% suggesting, but not proving, a transfer of oral bacteria to the mesh. CONCLUSION: The results show great bacterial diversity on mesh implants from the anterior abdominal wall including oral commensals and periodontopathogens. Mesh can be reached by bacteria in several ways including hematogenous spread from an oral site. However, other sites such as gut and skin may also serve as sources for the mesh biofilm.

Antibacterial Effectiveness of 2 Root Canal Irrigants in Root-filled Teeth with Infection: A Randomized Clinical Trial.

INTRODUCTION: This study compared the antibacterial effects of 1% sodium hypochlorite (NaOCl) and 2% chlorhexidine digluconate (CHX) during retreatment of teeth with apical periodontitis. METHODS: Root canal-treated teeth with apical periodontitis were randomly distributed into 2 groups. Bacteriological samples were taken from the canals before (S1) and after (S2) preparation using either NaOCl or CHX irrigation and after calcium hydroxide medication (S3); 16S ribosomal RNA gene-based real-time quantitative polymerase chain reaction was performed to quantify total bacteria, streptococci, and Enterococcus faecalis. RESULTS: Forty-nine teeth were available for analysis (NaOCl, n = 20; CHX, n = 29). Bacterial DNA occurred in all S1 samples, streptococci in 57% and E. faecalis in 6%. The total bacterial counts decreased from S1 to S2 in both groups (P < .01) but were higher in S3 than S2 (P < .01). Thirty-five percent of the teeth in the NaOCl group were positive in S2, decreasing to 20% in S3. In the CHX group, 41% were positive in S2, decreasing to 31% in S3. The bacterial load in S1 influenced the incidence of bacteria in S2 (P < .01). Streptococci were significantly reduced in both groups, and E. faecalis was found in only 1 S2 sample and not in S3. No significant difference between NaOCl and CHX was found. CONCLUSIONS: NaOCl and CHX both reduced bacterial counts and the number of infected canals. Intracanal medication with calcium hydroxide reduced the number of canals with persistent infection but resulted in overall larger bacterial counts in the cases positive for bacteria. The effectiveness of antimicrobial treatment can be influenced by the initial bacterial load.

Diversity and antifungal susceptibility of Norwegian Candida glabrata clinical isolates.

BACKGROUND: Increasing numbers of immunocompromised patients have resulted in greater incidence of invasive fungal infections with high mortality. Candida albicans infections dominate, but during the last decade, Candida glabrata has become the second highest cause of candidemia in the United States and Northern Europe. Reliable and early diagnosis, together with appropriate choice of antifungal treatment, is needed to combat these challenging infections. OBJECTIVES: To confirm the identity of 183 Candida glabrata isolates from different human body sites using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and VITEK(®)2, and to analyze isolate protein profiles and antifungal susceptibility. The minimum inhibitory concentration (MIC) of seven antifungal drugs was determined for the isolates to elucidate susceptibility. DESIGN: A total of 183 C. glabrata isolates obtained between 2002 and 2012 from Norwegian health-care units were analyzed. For species verification and differentiation, biochemical characterization (VITEK(®)2) and mass spectrometry (MALDI-TOF) were used. MIC determination for seven antifungal drugs was undertaken using E-tests(®). RESULTS: Using VITEK(®)2, 92.9% of isolates were identified as C. glabrata, while all isolates (100%) were identified as C. glabrata using MALDI-TOF. Variation in protein spectra occurred for all identified C. glabrata isolates. The majority of isolates had low MICs to amphotericin B (≤1 mg/L for 99.5%) and anidulafungin (≤0.06 mg/L for 98.9%). For fluconazole, 18% of isolates had MICs >32 mg/L and 82% had MICs in the range ≥0.016 mg/L to ≤32 mg/L. CONCLUSIONS: Protein profiles and antifungal susceptibility characteristics of the C. glabrata isolates were diverse. Clustering of protein profiles indicated that many azole resistant isolates were closely related. In most cases, isolates had highest susceptibility to amphotericin B and anidulafungin. The results confirmed previous observations of high MICs to fluconazole and flucytosine. MALDI-TOF was more definitive than VITEK(®)2 for C. glabrata identification.

Site-specific treatment outcome in smokers following non-surgical and surgical periodontal therapy.

AIM: To evaluate the effect of smoking at patient, tooth, and site level following non-surgical and surgical periodontal therapy. MATERIAL AND METHODS: Eighty chronic periodontitis patients, 40 smokers and 40 non-smokers, were recruited to this single-arm clinical trial. Smoking status was validated by measuring serum cotinine levels. Periodontal examinations were performed at baseline (T0) and 3 months following non-surgical and surgical periodontal therapy (T1). At T0 and T1, subgingival plaque samples were collected from the deepest periodontal pocket in each patient and analysed using checkerboard DNA-DNA hybridization. Probing depth (PD) ≥ 5 mm with bleeding on probing (BoP) was defined as the primary outcome. Unadjusted and adjusted logistic regression analyses, corrected for clustered observations within patients and teeth, were conducted comparing smokers with non-smokers. RESULTS: Clinical parameters significantly improved in both groups (p < 0.001). An association was revealed between smoking and PD ≥ 5 mm with BoP (OR= 1.90, CI: 1.14, 3.15, p = 0.013), especially for plaque-positive sites (OR= 4.14, CI: 2.16, 7.96, p < 0.001). A significant reduction of red complex microbiota was observed for non-smokers only (p = 0.010). CONCLUSION: Smokers respond less favourably to non-surgical and surgical periodontal therapy compared with non-smokers, in particular at plaque-positive sites.

Association of the rgpB gingipain genotype to the major fimbriae (fimA) genotype in clinical isolates of the periodontal pathogen Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis produces outer membrane-attached proteins that include the virulence-associated cysteine proteinases RgpA and RgpB (Arg-gingipains) and Kgp (Lys-gingipain). The gingipains provide P. gingivalis with a general proteolytic tool for degradation of proteinaceous nutrients for growth. They are also essential for the processing and maturation of the major fimbriae (FimA) which are important in facilitating bacterial adhesion to host tissues. FimA has been characterized as an important virulence factor for P. gingivalis, and many studies, both animal experiments and clinical investigations, have characterized fimA genotypes II, Ib, and IV to be associated with disease (periodontitis and cardiovascular disease) while genotypes I, III, and V represent avirulent strains. The relationship between virulence and gene variation of the rgpB gene has not been investigated extensively. However, nucleotide variations of the rgpB gene result in four amino acid substitutions in the catalytic domain identifying five different rgpB genotypes. They are named according to the different amino acid sequences in the primary protein structure of RGPB and named NYPN, NSSN, NSSK, NYPK, and DYPN (Beikler et al. 2005). AIM: The aim of the present study is to elucidate a possible relationship between fimA virulent and avirulent genotypes to the corresponding rgpB genotypes. METHODS: The total length of the rgpB (2212 bp) and fimA genes (1140 bp) from 20 clinical isolates was sequenced. Primers for pcr and sequencing of the genes were selected according to earlier investigations. RESULTS: The rgpB genotypes, NSSN and NYPN (n=2 and 6, respectively), were detected in the fimA II genotypes of P. gingivalis, while NSSK was detected in one fimA Ib genotype in the population. For the eight fimA IV genotypes, five NSSK and three NSSN rgpB genotypes were detected. In two fima I genotypes were detected one DYPN and one NYPN of the rgpB variants, while one fimA III genotype was associated with NSSK. CONCLUSION: The rgpB genotypes NSSN, NYPN, NSSK of P. gingivalis may be associated with the virulent fimA genotypes II, Ib and IV, indicating a possible connection to their virulent properties.

Association of the FAM46A gene VNTRs and BAG6 rs3117582 SNP with non small cell lung cancer (NSCLC) in Croatian and Norwegian populations.

We analyzed for associations between a variable number of tandem repeat (VNTR) polymorphism in the Family with sequence similarity 46, member A (FAM46A) gene and a single nucleotide polymorphism (rs3117582) in the BCL2-Associated Athanogene 6 (BAG6) with non small cell lung cancer in Croatian and Norwegian subjects. A total of 503 (262 Croatian and 241Norwegian) non small cell lung cancer patients and 897 controls (568 Croatian and 329 Norwegian) were analyzed. We found that the frequency of allele b (three VNTR repeats) of FAM46A gene was significantly increased in the patients compared to the healthy controls in the Croatian and the combined Croatian and Norwegian subjects. Genotype frequencies of cd (four and five VNTR repeats) and cc (four VNTR repeats homozygote) of the FAM46A gene were significantly decreased in the patients compared to the healthy controls in the Croatian and Norwegian subjects, respectively. Logistic regression analyses revealed FAM46A genotype cc to be an independent predictive factor for non small cell lung cancer risk in the Norwegian subjects after adjustment for age, gender and smoking status. This is the first study to suggest an association between the FAM46A gene VNTR polymorphisms and non small cell lung cancer. We found also that BAG6 rs3117582 SNP was associated with non small cell lung cancer in the Norwegian subjects and the combined Croatian-Norwegian subjects corroborating the earlier finding that BAG6 rs3117582 SNP was associated with lung cancer in Europeans. Logistic regression analyses revealed that genotypes and alleles of BAG6 were independent predictive factor for non small cell lung cancer risk in the Norwegian and combined Croatian-Norwegian subjects, after adjustment for age and gender.

Porphyromonas gingivalis fimbriae.

Marginal periodontitis is not a homogeneous disease but is rather influenced by an intricate set of host susceptibility differences as well as diversities in virulence among the harbored organisms. It is likely that clonal heterogeneity of subpopulations with both high and low levels of pathogenicity exists among organisms harbored by individuals with negligible, slight, or even severe periodontal destruction. Therefore, specific virulent clones of periodontal pathogens may cause advanced and/or aggressive periodontitis. Porphyromonas gingivalis is a predominant periodontal pathogen that expresses a number of potential virulence factors involved in the pathogenesis of periodontitis, and accumulated evidence shows that its expression of heterogenic virulence properties is dependent on clonal diversity. Fimbriae are considered to be critical factors that mediate bacterial interactions with and invasion of host tissues, with P. gingivalis shown to express two distinct fimbria-molecules, long and short fimbriae, on the cell surface, both of which seem to be involved in development of periodontitis. Long fimbriae are classified into six types (I to V and Ib) based on the diversity of fimA genes encoding FimA (a subunit of long fimbriae). Studies of clones with type II fimA have revealed their significantly greater adhesive and invasive capabilities as compared to other fimA type clones. Long and short fimbriae induce various cytokine expressions such as IL-1α, IL-ß, IL-6, and TNF-α, which result in alveolar bone resorption. Although the clonal diversity of short fimbriae is unclear, distinct short fimbria-molecules have been found in different strains. These fimbriae variations likely influence the development of periodontal disease.