Nils-Erik Fiehn

Resistance of Streptococcus sanguis biofilms to antimicrobial agents

Bacteria living in biofilms as dental plaque on tooth surfaces are generally more resistant to antimicrobial agents than bacteria in batch culture normally used for in vitro susceptibility testing. In order to compare the resistance of free‐living and surface‐grown oral bacteria, the MIC of Streptococcus sanguis 804 and ATCC 10556 to amoxicillin, doxycycline and chlorhexidine was determined by a broth dilution method. Subsequently, S. sanguis biofilms established in an in vitro flow model were perfused with the antimicrobial agents for 48 h at concentrations equal to and up to 500 times the MIC, and biofilm cell number was determined during this period. The antibiotics at the MIC did not affect the cell number of S. sanguis biofilms compared to the starting point, and only after 48 h at 500 times the MIC were the biofilm bacteria eliminated. At intermediate concentrations biofilm cell number gradually decreased. Chlorhexidine also gradually reduced biofilm cell number, but was inhibitory at concentrations closer to the MIC than was the case for the antibiotics. Thus S. sanguis in biofilms survived up to 500 times the MIC found in batch culture for up to 48 h.

Oral infections and systemic diseases

An association between periodontal infection and CVD has been revealed in some epidemiologic studies, whereas other studies were unable to demonstrate such an association. A link between the two diseases may be explained by shared established or nonestablished risk factors. Future studies with extended control of confounding factors and intervention studies may add to the understanding of a possible relationship between the diseases. In some cases, IE is caused by dental plaque bacteria. Several studies are suggestive of oral bacteria causing respiratory infection. The pathogenesis and course of a number of other diseases including DM and rheumatoid arthritis have been associated wish periodontitis, but more research is necessary to elucidate possible pathogenic interactions.

Copenhagen Aging and Midlife Biobank (CAMB): an introduction

Since the early 1980s, a number of large-scale, population-based, epidemiologic studies have focused on the development of disability, cognitive impairment, dementia, long-term care, and other health issues with particular relevance to older adults. With few exceptions, these health issues have been investigated in older populations, usually defined on the basis of chronological age (e.g., older than 65). Examples are the Established Populations for Epidemiologic Studies of the Elderly (EPESE), Women’s Health and Aging Studies (WHAS), and several other American, Australian, Canadian, and

Coaggregation between probiotic bacteria and caries-associated strains: an in vitro study.

Objective. To evaluate the in vitro abilities of probiotic bacteria derived from consumer products to coaggregate with caries-associated mutans streptococci. Material andMethods. Six lactobacillus strains (L. acidophilus (CCUG 5917), L. plantarum 299v, L. rhamnosus GG and LB21, L. paracasei F19, L. reuteri PTA5289) were cultivated under anaerobic conditions at 37°C in Man Rogosa Sharpe (MSB) broth for 24 h. Four strains of human streptococci (S. mutans Ingbritt, S. mutans (ATCC 25175), S. mutans GS-5, S. sobrinus (ATCC 33478) were similarly grown in Brain Heart Infusion (BHI) broth. A gastrointestinal pathogen (Escherichia coli) was aerobically cultivated on BHI broth as a positive control. After incubation, the bacteria were aerobically harvested, washed, and suspended in 10 mmol/l phosphate-buffered saline (pH 7.2). The probiotic strains were characterized with the API 50 CH system to confirm their identity. Coaggregation was determined by spectrophotometry in mixtures and bacterial suspensions alone after 1, 2, 4, and 24 h and expressed as the aggregation ratio (%). Results. All probiotic strains showed coaggregation abilities with the oral pathogens and the results were strain specific and dependent on time. S. mutans GS-5 exhibited a significantly higher ability to coaggregate with all the probiotic strains than the other mutans streptococci and E. coli. The differences among the probiotic strains were modest with L. acidophilus being the most prone and L. rhamnosus LB21 the least prone to coaggregate with the oral streptococci. Conclusions. The results demonstrated different abilities of lactobacilli-derived probiotic bacteria to coaggregate with selected oral streptococci. Aggregation assays may be a useful complement for screening of probiotic candidates with possible anti-caries properties.

Bacterial profiles of saliva in relation to diet, lifestyle factors, and socioeconomic status.

Background and objective The bacterial profile of saliva is composed of bacteria from different oral surfaces. The objective of this study was to determine whether different diet intake, lifestyle, or socioeconomic status is associated with characteristic bacterial saliva profiles. Design Stimulated saliva samples from 292 participants with low levels of dental caries and periodontitis, enrolled in the Danish Health Examination Survey (DANHES), were analyzed for the presence of approximately 300 bacterial species by means of the Human Oral Microbe Identification Microarray (HOMIM). Using presence and levels (mean HOMIM-value) of bacterial probes as endpoints, the influence of diet intake, lifestyle, and socioeconomic status on the bacterial saliva profile was analyzed by Mann–Whitney tests with Benjamini–Hochbergs correction for multiple comparisons and principal component analysis. Results Targets for 131 different probes were identified in 292 samples, with Streptococcus and Veillonella being the most predominant genera identified. Two bacterial taxa (Streptococcus sobrinus and Eubacterium [11][G-3] brachy) were more associated with smokers than non-smokers (adjusted p-value<0.01). Stratification of the group based on extreme ends of the parameters age, gender, alcohol consumption, body mass index (BMI), and diet intake had no statistical influence on the composition of the bacterial profile of saliva. Conversely, differences in socioeconomic status were reflected by the bacterial profiles of saliva. Conclusions The bacterial profile of saliva seems independent of diet intake, but influenced by smoking and maybe socioeconomic status.

Development of a flow method for susceptibility testing of oral biofilms in vitro

Bacteria in biofilms are known to be more resistant than bacteria in batch cultures to antimicrobial agents. The purpose of the present study was to develop a flow method for formation of oral biofilms permitting susceptibility testing of plaque bacteria. A brain heart infusion (BHI) Streptococcus sanguis 804 culture was pumped through a modified Robbins Device (MRD) with 25 exchangeable silicone disks at 40 ml/h. After 24–48 h disks were removed and biofilm cells dispersed by vortex mixing and low‐output ultrasonication. Colony forming units (cfu)/cm2 were determined after aerobic incubation on blood agar plates. Optimal biofilm formation was found after growth for 48 h at 37°C in BHI + 1% sucrose, using saliva‐coated silicone disks in inverted MRDs, yielding on average 4.4 × 105 cfu/cm2. Similar results were obtained for S. sanguis ATCC 10556 and five clinical isolates. Testing the susceptibility of S. sanguis to chlorhexidine gluconate showed increased resistance of biofilms compared to batch culture. Thus an appropriate biofilm model for susceptibility testing of oral microorganisms has been established.

Temporal Stability of the Salivary Microbiota in Oral Health

Objectives Saliva is a biological fluid suitable for biomarker analysis, and differences in the salivary microbiota in oral health and disease have been reported. For such comparative analyses, time of sampling is critical since the bacterial composition may vary throughout the day, i.e., diurnal variation. The purpose of this study is to compare the salivary microbiome over time to determine the optimal time for sampling. Design Stimulated saliva samples were collected from 5 orally healthy individuals in 4 h intervals for 24 h, and collection was repeated 7 days later (number of samples per person, n = 12, total number of samples, n = 60). Salivary microbiota was analyzed using the Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS), and statistical analysis was performed using the Kruskal-Wallis test with Benjamini-Hochberg’s correction for multiple comparisons, cluster analysis, principal component analysis and correspondence analysis. Results From a total of 60 saliva samples, 477 probe targets were collectively identified with a mean number of probes per sample of 207 (range: 153–307). Little or no variation in microbial profiles within subjects was observed over time. Conclusions Although there was considerable variation between subjects, microbial profiles within subjects were stable throughout a 24 hour period and after 1 week. Since there is little or no evidence of diurnal variation of the salivary microbiome, time of sampling of saliva is not critical for perturbation or other microbial studies.

Findings from the oral health study of the Danish Health Examination Survey 2007-2008.

Abstract Objective. The aims of the oral part of the Danish Health Examination Survey (DANHES 2007–2008) were (1) to establish an oral health database for adult Danes and (2) to explore the influence of general diseases and lifestyle on oral health. This paper presents the study population, examination methods, questionnaire and baseline results. Materials and methods. The study population comprised 4402 subjects, aged 18–96, consecutively enrolled from 18 065 DANHES participants from 13 municipalities in Denmark. The oral part consisted of a validated questionnaire and a clinical examination, carried out in mobile units by three trained and calibrated dental hygienists. The data were processed with descriptive statistics and mono- and bivariate analyses. Results. The mean age was 54.1 years and 60% were women. The mean number of natural teeth was 26.6; the mean DMFT/DMFS values were 18.9 and 61.0, and varied with age (DMFT 8.7–24.3). A higher proportion of females suffered from dental erosion in the younger age groups. Forty per cent of all subjects had a mean clinical attachment loss ≥ 3 mm, varying from 4% among those aged 18–34 to 80% in those over 75. A sub-optimal saliva secretion rate was more common among females than males (17.7% vs 10.4%) and this was reflected by the reported frequency of dry mouth. Conclusion. This extensive cross-sectional study provides a platform for obtaining future knowledge of the impact of health- and lifestyle-related factors on oral diseases. The validated questionnaire and the clinical characteristics enable robust analyses, although the conclusions may be hampered by limited external validity.

Altered bacterial profiles in saliva from adults with caries lesions: a case-cohort study.

The aim of this study was to learn whether presence of caries in an adult population was associated with a salivary bacterial profile different from that of individuals without untreated caries. Stimulated saliva samples from 621 participants of the Danish Health Examination Survey were analyzed using the Human Oral Microbe Identification Microarray technology. Samples from 174 individuals with dental caries and 447 from a control cohort were compared using frequency and levels of identified bacterial taxa/clusters as endpoints. Differences at taxon/cluster level were analyzed using Mann-Whitneys test with Benjamini-Hochberg correction for multiple comparisons. Principal component analysis was used to visualize bacterial community profiles. A reduced bacterial diversity was observed in samples from subjects with dental caries. Five bacterial taxa (Veillonella parvula, Veillonella atypica, Megasphaera micronuciformis, Fusobacterium periodontium and Achromobacter xylosoxidans) and one bacterial cluster (Leptotrichia sp. clones C3MKM102 and GT018_ot417/462) were less frequently found in the caries group (adjusted p value <0.01) while two bacterial taxa (Solobacterium moorei and Streptococcus salivarius) and three bacterial clusters (Streptococcus parasanguinis I and II and sp. clone BE024_ot057/411/721, Streptococcus parasanguinis I and II and sinensis_ot411/721/767, Streptococcus salivarius and sp. clone FO042_ot067/755) were present at significantly higher levels (adjusted p value <0.01). The principal component analysis displayed a marked difference in the bacterial community profiles between groups. Presence of manifest caries was associated with a reduced diversity and an altered salivary bacterial community profile. Our data support recent theories that ecological stress-induced changes of commensal microbial communities are involved in the shift from oral health to tooth decay.

Salivary bacterial fingerprints of established oral disease revealed by the Human Oral Microbe Identification using Next Generation Sequencing (HOMI N G S ) technique

Background and objective The composition of the salivary microbiota, as determined using various molecular methods, has been reported to differentiate oral health from diseases. Thus, the purpose of this study was to utilize the newly developed molecular technique HOMINGS (Human Oral Microbe Identification using Next Generation Sequencing) for comparison of the salivary microbiota in patients with periodontitis, patients with dental caries, and orally healthy individuals. The hypothesis was that this method could add on to the existing knowledge on salivary bacterial profiles in oral health and disease. Design Stimulated saliva samples (n=30) were collected from 10 patients with untreated periodontitis, 10 patients with untreated dental caries, and 10 orally healthy individuals. Salivary microbiota was analyzed using HOMINGS and statistical analysis was performed using Kruskal–Wallis test with Benjamini–Hochbergs correction. Results From a total of 30 saliva samples, a mean number of probe targets of 205 (range 120–353) were identified, and a statistically significant higher mean number of targets was registered in samples from patients with periodontitis (mean 220, range 143–306) and dental caries (mean 221, range 165–353) as compared to orally healthy individuals (mean 174, range 120–260) (p=0.04 and p=0.04). Nine probe targets were identified with a different relative abundance between groups (p<0.05). Conclusions Cross-sectional comparison of salivary bacterial profiles by means of HOMINGS analysis showed that different salivary bacterial profiles were associated with oral health and disease. Future large-scale prospective studies are needed to evaluate if saliva-based screening for disease-associated oral bacterial profiles may be used for identification of patients at risk of acquiring periodontitis and dental caries.