Analysis of relative bacterial activity and lactate dehydrogenase gene expression of caries-associated bacteria in a site-specific natural biofilm: an ex vivo study.

OBJECTIVES: Detecting bacterial activity is considered a promising approach to monitor shifts from symbiosis to dysbiosis in oral microbiome. The present study aimed at investigating both the relative bacterial activity and the lactate dehydrogenase (ldh) gene expression of caries-associated bacteria in a site-specific natural biofilm. MATERIAL AND METHODS: Sixty subjects (age, mean ± SE: 30.1 ± 1.4) were allocated to two groups: caries-free subjects (CF) or caries-active subjects (CA). CF presented one sound surface (CFS, n = 30). CA presented two donor sites: a cavitated caries lesion (CAC, n = 30) and a sound reference surface (CAS, n = 30). Real-time quantitative PCR (q-PCR) on species or genus level and total bacteria was performed targeting the 16S gene, the 16S rRNA, the ldh gene, and the ldh mRNA (increasing 16S ribosomal RNA copy numbers can function as an indicator of increased energy metabolism). As the 16S rRNA abundance represents the number of ribosomes, while the 16S gene abundance represents the number of genomes, the quotient of the relative abundances functions as a measure for the relative bacterial activity (%). RESULTS: Both lactobacilli and S. mutans showed the highest relative bacterial activity in CAC ((mean ± SE) 218 ± 60% and 61 ± 16%, respectively) and the lowest values for both sound reference surfaces (69 ± 48%; 8 ± 3%). Significant differences were found between CAC and CAS as well as between CAC and CFS for both lactobacilli and S. mutans (p < 0.05). The ldh gene expression of lactobacilli and S. mutans only showed moderate values in CAC (1.90E+03 ± 2.11E+03; 2.08E+04 ± 4.44E+04 transcripts/µl) and CFS (2.04E+03 ± 2.74E+03; 8.16E+03 ± 6.64E+03 transcripts/µl); consequently no significant differences were detected. CONCLUSION AND CLINICAL RELEVANCE: Caries-associated bacteria (lactobacilli and S. mutans) showed the highest relative bacterial activity in plaque of cavitated lesions, the lowest in sound surfaces, allowing the detection of a significant activity shift in health and disease for caries-active patients. However, no significant differences in ldh gene expression could be determined.

Correlation between relative bacterial activity and lactate dehydrogenase gene expression of co-cultures in vitro.

OBJECTIVES: The present study aims at correlating the relative bacterial activity with the H+ concentration and the ldh expression of caries-associated bacteria in co-cultures. MATERIALS AND METHODS: Well plates were prepared with BHI medium and cultures of Lactobacillus paracasei and Fusobacterium nucleatum. Bacterial growth at 37 °C was measured using a microplate-photometer before and after adding sucrose to the samples. Samples of co-cultures (n = 12) and single-species cultures (n = 3) were taken and pH was assessed. Real-time quantitative PCRs were applied targeting the 16S-gene, the 16S-rRNA, the ldh-gene, and the ldh-mRNA. RESULTS: For L. paracasei with sucrose, an increase in relative bacterial activity (62.8% ± 23.5% [mean, SE]) was observed, while F. nucleatum showed a clear decrease in relative bacterial activity (- 35.0% ± 9.6%). Simultaneously, the H+ concentration increased (1.15E-05 mol*l-1 ± 4.61E-07 mol*l-1). Consequently, a significant positive correlation was found between L. paracasei's relative bacterial activity and H+ concentration (Spearman rank correlation, r = 0.638; p = 0.002), while F. nucleatum exhibited a negative correlation (r = - 0.741; p ≤ 0.001). Furthermore L. paracasei with sucrose showed a moderate, but significant positive correlation between relative bacterial activity and ldh-expression (r = 0.307; p = 0.024). CONCLUSIONS AND CLINICAL RELEVANCE: The relative bacterial activity after sucrose pulse showed a significant correlation not only to the acid production (H+ concentration) but also to ldh expression of L. paracasei. However, further research is required to confirm these findings in a mature biofilm in vivo.

Age-related changes in immune function (immune senescence) in caries and periodontal diseases: a systematic review.

AIM: To systematically review the evidence regarding immune senescence in the pathogenesis of periodontitis and dental caries. METHODS: A systematic search of electronic databases utilizing medical subject headings (MeSH terms) supplemented by screening of review articles and other relevant texts was undertaken. RESULTS: Seventy-three articles were included (43 for periodontitis, 30 for caries). Study results were found to be generally heterogeneous. Regarding periodontitis, human studies suggest evidence for altered neutrophil function and increased production of pro-inflammatory mediators (e.g. interleukin-1ß, interleukin-6 and prostaglandin E2 ) in older compared to younger subjects, and animal experiments suggest increased expression of genes that contribute to a pro-inflammatory state in older compared to younger animals. Regarding dental caries, research relating to changes in immune functioning and the impact of ageing is in its infancy. A small number of studies have reported components of innate and adaptive immunity that affect the composition of saliva and dental biofilms with possible impacts on caries progression. CONCLUSION: There is evidence that immune functioning related to periodontitis and (less investigated) dental caries alters with increasing age. In both conditions, age-associated mechanistic changes in immune functioning are complex and incompletely understood and it is not clear how these relate to disease susceptibility.

Analysis of Bacterial Activity in Sound and Cariogenic Biofilm: A Pilot in vivo Study.

Dental caries is a multifactorial disease with many associated microbial taxa, but only a few are notably contributing to acidogenicity. The ribosome number and the corresponding 16S ribosomal RNA (rRNA) concentration are considered a molecular indicator for general metabolic activity of bacteria, as they are elevated with increased anabolic and catabolic activities. We hypothesize that the activity of aciduric/acidogenic bacterial taxa, reflected by a rise in ribosomal counts, could resolve differences between plaque biofilm from sound surfaces and caries lesions. The included subjects were allocated to two groups: caries-free (CF) or caries-active (CA). CF subjects presented one donor site, namely one sound surface (CFS, n = 10), whereas CA subjects presented two donor sites: a cavitated lesion with an ICDAS score of 5-6 (CAC, n = 13), and a sound reference surface (CAS, n = 13). Four aciduric/acidogenic bacterial taxa (Streptococcus mutans, lactobacilli, Bifidobacterium dentium, and Scardovia wiggsiae) and one asaccharolytic taxon (fusobacteria) as a contrast were selected. 16S rRNA and 16S rRNA genes were quantified by quantitative PCR. Based on these parameters, bacterial and ribosomal counts, as well as relative activities were calculated as the quotient of relative ribosomal abundance and relative genome abundance. Caries-associated bacteria showed the highest relative activity in caries lesions (e.g. lactobacilli CAC: 177.5 ± 46.0%) and lower activities on sound surfaces (e.g. lactobacilli CAS: 96.3 ± 31.5%), whereas asaccharolytic fusobacteria were most active on sound surfaces and less active in caries lesions (CFS: 275.7 ± 171.1%; CAS: 205.8 ± 114.3%; CAC: 51.1 ± 19.0%). Thus, the present study suggests different activity patterns for biofilms from CF and CA individuals.

Trends in antibiotic use and microbial diagnostics in periodontal treatment: comparing surveys of German dentists in a ten-year period.

OBJECTIVES: The use of antibiotics and microbial tests in periodontal treatment among German dental practitioners was investigated in 2012-2013 and compared with 2002-2003 data. MATERIALS AND METHODS: One thousand four hundred representative German practitioners received a postal questionnaire requesting their prescribing habits concerning type, dose, frequency, and sequence of antibiotics adjunctive to mechanical debridement. Additionally, the use of local antimicrobials and microbial tests were recorded. RESULTS: The response rate was 29.1 % (407 reports). Drug combinations, especially amoxicillin plus metronidazole, were prescribed most frequently (32.8 %) with an increase of 7.4 % during the past decade, followed by clindamycin (29.3 %). Amoxicillin monotherapy was used unexpectedly frequently (17.0 %) and doxycycline (2.8 %) very infrequently. Then, 24.7 % prescribed antibiotics prior to mechanical therapy, while most dentists followed the recommended sequence. The use of local antimicrobials increased by 6.2 % and of microbial diagnostics by 20.8 %. CONCLUSIONS: Positive trends regarding position-paper-conform prescribing habits including the scheduling of systemic antibiotics and increasing use of local antimicrobials and microbial tests were observed. However, deficits and malpractice still exist in German practices. Unexpected is the widespread and increasing use of clindamycin. Continuing educational campaigns and strictly expressed real guidelines are needed. CLINICAL RELEVANCE: Indication and choice of antibiotic agents in causal periodontal therapy among German dentists have changed between 2003 and 2013 toward a more position-paper-based concept, but inappropriate prescriptions of second choice antibiotics still remain conspicuous.

Aciduric microbial taxa including Scardovia wiggsiae and Bifidobacterium spp. in caries and caries free subjects.

Actinobacteria came into focus of being potential caries-associated pathogens and could, together with the established Streptococcus mutans and lactobacilli thus function as caries indicator species. Here we analyzed the role and diagnostic predictive value of the acidogenic-aciduric species Scardovia wiggsiae and Bifidobacterium dentium together with S. mutans, lactobacilli and bifidobacteria in biofilm of non-cavitated (n = 20) and cavitated (n = 6) caries lesions versus controls (n = 30). For the genus Bifidobacterium and for B. dentium new sets of primers were designed. Based on real-time quantitative PCR and confirmed by DNA sequencing we found a higher prevalence (61.5%) of S. wiggsiae in caries lesions than in controls (40%). However, among the controls we found three individuals with both the highest absolute and relative S. wiggsiae numbers. Testing for S. mutans revealed the same prevalence as S. wiggsiae in caries lesions (61.5%) but in controls its prevalence was only 10%. B. dentium was never found in healthy plaque but in 30.8% of clinical cases, with the highest numbers in cavitated lesions. The Bifidobacterium-genus specific PCR had less discriminative power as more control samples were positive. We calculated the relative abundances and applied receiver operating characteristic analyses. The top results of specificity (93% and 87%) and sensitivity (100% and 88%) were found when the constraint set was "Lactobacillus relative abundance ≥0.02%" and "two aciduric species with a relative abundance of each ≥0.007%". Combinatory measurement of several aciduric taxa may be useful to reveal caries activity or even to predict caries progression.

Global analysis of saliva as a source of bacterial genes for insights into human population structure and migration studies.

BACKGROUND: The genetic diversity of the human microbiome holds great potential for shedding light on the history of our ancestors. Helicobacter pylori is the most prominent example as its analysis allowed a fine-scale resolution of past migration patterns including some that could not be distinguished using human genetic markers. However studies of H. pylori require stomach biopsies, which severely limits the number of samples that can be analysed. By focussing on the house-keeping gene gdh (coding for the glucose-6-phosphate dehydrogenase), on the virulence gene gtf (coding for the glucosyltransferase) of mitis-streptococci and on the 16S-23S rRNA internal transcribed spacer (ITS) region of the Fusobacterium nucleatum/periodonticum-group we here tested the hypothesis that bacterial genes from human saliva have the potential for distinguishing human populations. RESULTS: Analysis of 10 individuals from each of seven geographic regions, encompassing Africa, Asia and Europe, revealed that the genes gdh and ITS exhibited the highest number of polymorphic sites (59% and 79%, respectively) and most OTUs (defined at 99% identity) were unique to a given country. In contrast, the gene gtf had the lowest number of polymorphic sites (21%), and most OTUs were shared among countries. Most of the variation in the gdh and ITS genes was explained by the high clonal diversity within individuals (around 80%) followed by inter-individual variation of around 20%, leaving the geographic region as providing virtually no source of sequence variation. Conversely, for gtf the variation within individuals accounted for 32%, between individuals for 57% and among geographic regions for 11%. This geographic signature persisted upon extension of the analysis to four additional locations from the American continent. Pearson correlation analysis, pairwise Fst-cluster analysis as well as UniFrac analyses consistently supported a tree structure in which the European countries clustered tightly together and branched with American countries and South Africa, to the exclusion of Asian countries and the Congo. CONCLUSION: This study shows that saliva harbours protein-coding bacterial genes that are geographically structured, and which could potentially be used for addressing previously unresolved human migration events.

Analysis of structure and composition of bacterial core communities in mature drinking water biofilms and bulk water of a citywide network in Germany.

The bacterial core communities of bulk water and corresponding biofilms of a more than 20-year-old drinking water network were compared using 16S rRNA single-strand confirmation polymorphism (SSCP) fingerprints based on extracted DNA and RNA. The structure and composition of the bacterial core community in the bulk water was highly similar (>70%) across the city of Braunschweig, Germany, whereas all biofilm samples contained a unique community with no overlapping phylotypes from bulk water. Biofilm samples consisted mainly of Alphaproteobacteria (26% of all phylotypes), Gammaproteobacteria (11%), candidate division TM6 (11%), Chlamydiales (9%), and Betaproteobacteria (9%). The bulk water community consisted primarily of Bacteroidetes (25%), Betaproteobacteria (20%), Actinobacteria (16%), and Alphaproteobacteria (11%). All biofilm communities showed higher relative abundances of single phylotypes and a reduced richness compared to bulk water. Only biofilm communities sampled at nearby sampling points showed similar communities irrespective of support materials. In all of our bulk water studies, the community composition determined from 16S rRNA was completely different from the 16S rRNA gene-based community composition, whereas in biofilms both molecular fractions resulted in community compositions that were similar to each other. We hypothesize that a higher fraction of active bacterial phylotypes and a better protection from oxidative stress in drinking water biofilms are responsible for this higher similarity.

Assessing the viability of bacterial species in drinking water by combined cellular and molecular analyses.

The question which bacterial species are present in water and if they are viable is essential for drinking water safety but also of general relevance in aquatic ecology. To approach this question we combined propidium iodide/SYTO9 staining ("live/dead staining" indicating membrane integrity), fluorescence-activated cell sorting (FACS) and community fingerprinting for the analysis of a set of tap water samples. Live/dead staining revealed that about half of the bacteria in the tap water had intact membranes. Molecular analysis using 16S rRNA and 16S rRNA gene-based single-strand conformation polymorphism (SSCP) fingerprints and sequencing of drinking water bacteria before and after FACS sorting revealed: (1) the DNA- and RNA-based overall community structure differed substantially, (2) the community retrieved from RNA and DNA reflected different bacterial species, classified as 53 phylotypes (with only two common phylotypes), (3) the percentage of phylotypes with intact membranes or damaged cells were comparable for RNA- and DNA-based analyses, and (4) the retrieved species were primarily of aquatic origin. The pronounced difference between phylotypes obtained from DNA extracts (dominated by Betaproteobacteria, Bacteroidetes, and Actinobacteria) and from RNA extracts (dominated by Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria) demonstrate the relevance of concomitant RNA and DNA analyses for drinking water studies. Unexpected was that a comparable fraction (about 21%) of phylotypes with membrane-injured cells was observed for DNA- and RNA-based analyses, contradicting the current understanding that RNA-based analyses represent the actively growing fraction of the bacterial community. Overall, we think that this combined approach provides an interesting tool for a concomitant phylogenetic and viability analysis of bacterial species of drinking water.

High-resolution in situ genotyping of Legionella pneumophila populations in drinking water by multiple-locus variable-number tandem-repeat analysis using environmental DNA.

Central to the understanding of infections by the waterborne pathogen Legionella pneumophila is its detection at the clonal level. Currently, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) of L. pneumophila isolates can be used as a tool for high-resolution genotyping. Since L. pneumophila is difficult to isolate, the isolation of outbreak strains often fails due to a viable but nonculturable (VBNC) state of the respective environmental population. Therefore, we developed a cultivation-independent approach to detect single clones in drinking water. This approach is based on the extraction of DNA from drinking water followed by PCR using a set of eight VNTR primer pairs necessary for MLVA genotyping of L. pneumophila. The PCR amplicons were analyzed by single-strand conformation polymorphism (SSCP) and capillary electrophoresis to obtain the respective MLVA profiles. Parallel to the high-resolution analysis, we used the same environmental DNA to quantify the number of L. pneumophila cells in drinking water using real-time PCR with 16S rRNA gene-targeted primers. We used a set of drinking water samples from a small-scale drinking water network to test our approach. With these samples we demonstrated that the developed approach was directly applicable to DNA obtained from drinking water. We were able to detect more L. pneumophila MLVA genotypes in drinking water than we could detect by isolation. Our approach could be a valuable tool to identify outbreak strains even after the outbreak has occurred and has the potential to be applied directly to clinical material.

Sex-specific differences in the occurrence of Fusobacterium nucleatum subspecies and Fusobacterium periodonticum in the oral cavity.

The periodontitis-associated species Fusobacterium nucleatum (FN) has been implicated in several extra-oral diseases, including preterm birth and colorectal cancer. Due to its genetic and phenotypic heterogeneity, FN is classified in four subspecies which may differ in their disease potential. Here we compared the prevalence of FN subspecies and the close relative F. periodonticum (FP) via 16S rRNA gene analysis in saliva from 100 healthy individuals (60 females, and 40 males) from eleven countries spanning five continents. By focusing on the most abundant sequence types (i.e. analysis of approximately ten clone sequences each) the average number of FN/FP subspecies per individual differed significantly between females and males, i.e. 2.93 versus 2.5, respectively (P = 0.043). FN subsp. fusiforme/vincentii was significantly more prevalent in females vs males, with 2.85 vs. 1.68 sequence reads per individual, respectively (P = 0.012). A significant age-related difference was observed in females but not in males, i.e. 2.6 subspecies on average in females ≤ 30 years vs. 3.2 in females > 30 (P = 0.0076). Given the link between FN and systemic disorders our findings highlight the need for microbial studies at the subspecies level to further characterize the role of periodontal pathogens in diseases that affect females and males differently, e.g. colorectal cancer.

Streptococcus tigurinus is frequent among gtfR-negative Streptococcus oralis isolates and in the human oral cavity, but highly virulent strains are uncommon.

Streptococcus tigurinus is a new member of the Mitis group and is associated with infective endocarditis. Low and high virulent variants have been described. A search was made in the national reference collection of endocarditis isolates for S. tigurinus-like strains by sequencing housekeeping genes (16S rRNA-gene, gdh, groEL, sodA). The strains were further profiled by polymerase chain reaction (PCR) targeting a choice of virulence genes (rib-like, cshA-like, gtfR, int, pitA, hylA). To study the prevalence and abundance of S. tigurinus in the saliva and on the mucosal membranes of 35 healthy adults, PCRs detecting two variants of the 16S operon and virulence genes were applied. Among the endocarditis isolates, eight strains (all gtfR-negative and former S. oralis) holding the specific S. tigurinus 16S motif were found, but the pattern of genes related to high virulence found in the S. tigurinus type strain could not be detected in any of these strains. A close phylogenetic proximity between S. tigurinus and S. oralis was observed, with intersectional hybrid strains formed. This was supported by concatenated housekeeping sequences, in silico DNA-DNA hybridization, pathogenomic profiling, and multidimensional scaling. In the oral samples, S. tigurinus could be detected frequently, especially in the most common operon variant, but none of the type strain-related virulence factors were found. Low virulent S. tigurinus-like strains can be found frequently and in high prevalence (66%) and abundance (12.5%) in the oral cavity of healthy adults. In strain collections, they are among the formerly known gtfR-negative S. oralis. Highly virulent strains seem to be uncommon. Though closely related, S. oralis and S. tigurinus can be separated by the presence or absence of gtfR and dextran production. Hybrids of both species can be found. The variable arsenal of virulence genes found in this study emphasizes the genetic plasticity of Mitis group streptococci.

Cloning of an agr homologue of Staphylococcus saprophyticus.

An agr homologue of Staphylococcus saprophyticus was identified, cloned and sequenced. The gene locus shows homologies to other staphylococcal agr systems, especially to those of S. epidermidis and S. lugdunensis. A putative RNAIII was identified and found to be differentially expressed during the growth phases. In contrast to the RNAIII molecules of S. epidermidis and S. aureus it does not contain an open reading frame that codes for a protein with homologies to the delta-toxin. Using PCR, the agr was found to be present in clinical isolates of S. saprophyticus.

Shifts in Campylobacter species abundance may reflect general microbial community shifts in periodontitis progression.

BACKGROUND: Oral Campylobacter species have been found to be associated with periodontitis progression. While the etiological significance of Campylobacter rectus is quite established, the association of C. gracilis, C. concisus, and C. curvus with health or disease remains contradictory. OBJECTIVES: This study hypothesizes that the proportion of species within the Campylobacter genus rather than the absolute abundance of a single species is a suitable indicator for periodontitis progression. DESIGN: Subgingival plaque from 90 periodontitis patients and gingival sulcus fluid of 32 healthy individuals were subjected to a newly developed nested PCR approach, in which all Campylobacter spp. were amplified simultaneously. The resulting mixture of 16S-rRNA-gene-amplicons were separated by single-stranded conformation polymorphism (SSCP) gel electrophoresis, followed by sequencing and identification of excised bands and relative quantification of band intensities. In all samples, the abundance of selected periodontitis marker species was determined based on DNA hybridization on a microarray. RESULTS: The highly prevalent Campylobacter community was composed of varying proportions of C. rectus, C. gracilis, C. concisus, and C. curvus. Cluster analysis based on SSCP-banding pattern resulted in distinct groups which in turn coincided with significant differences in abundance of established periodontitis marker species (Tannerella forsythia, Porphyromonas gingivalis, and Fusobacterium nucleatum) and progression. CONCLUSIONS: The shift in the Campylobacter community composition seems to display the general microbial community shift during clinical progression in a simplified manner. The focus on members of the Campylobacter in this study suggests that this genus can be an indicator of ecological changes in the subgingival oral microflora.

Comparing the cariogenic species Streptococcus sobrinus and S. mutans on whole genome level.

BACKGROUND: Two closely related species of mutans streptococci, namely Streptococcus mutans and Streptococcus sobrinus, are associated with dental caries in humans. Their acidogenic and aciduric capacity is directly associated with the cariogenic potential of these bacteria. To survive acidic and temporarily harsh conditions in the human oral cavity with hundreds of other microbial co-colonizers as competitors, both species have developed numerous mechanisms for adaptation. OBJECTIVES: The recently published novel genome information for both species is used to elucidate genetic similarities but especially differences and to discuss the impact on cariogenicity of the corresponding phenotypic properties including adhesion, carbohydrate uptake and fermentation, acid tolerance, signaling by two component systems, competence, and oxidative stress resistance. CONCLUSIONS: S. sobrinus can down-regulate the SpaA-mediated adherence to the pellicle. It has a smaller number of two-component signaling systems and bacteriocin-related genes than S. mutans, but all or even more immunity proteins. It lacks the central competence genes comC, comS, and comR. There are more genes coding for glucosyltransferases and a novel energy production pathway formed by lactate oxidase, which is not found in S. mutans. Both species show considerable differences in the regulation of fructan catabolism. However, both S. mutans and S. sobrinus share most of these traits and should therefore be considered as equally virulent with regard to dental caries.

Seasonal dynamics of bacterial community structure and composition in cold and hot drinking water derived from surface water reservoirs.

In temperate regions, seasonal variability of environmental factors affects the bacterial community in source water and finished drinking water. Therefore, the bacterial core community and its seasonal variability in cold and the respective hot drinking water was investigated. The bacterial core community was studied by 16S rRNA-based SSCP fingerprint analyses and band sequencing of DNA and RNA extracts of cold and hot water (60 °C). The bacterial communities of cold and hot drinking water showed a highly different structure and phylogenetic composition both for RNA and DNA extracts. For cold drinking water substantial seasonal dynamics of the bacterial community was observed related to environmental factors such as temperature and precipitation affecting source and drinking water. Phylogenetic analyses of the cold water community indicated that the majority of phylotypes were very closely affiliated with those detected in former studies of the same drinking water supply system (DWSS) in the preceding 6 years, indicating a high stability over time. The hot water community was very stable over time and seasons and highly distinct from the cold water with respect to structure and composition. The hot water community displayed a lower diversity and its phylotypes were mostly affiliated with bacteria of high temperature habitats with high growth rates indicated by their high RNA content. The conversion of the cold to the hot water bacterial community is considered as occurring within a few hours by the following two processes, i) by decay of most of the cold water bacteria due to heating, and ii) rapid growth of the high temperature adapted bacteria present in the hot water (co-heated with the cold water in the same device) using the nutrients released from the decaying cold water bacteria. The high temperature adapted bacteria originated partially from low abundant but beforehand detected members of the cold water; additionally, the rare members ("seed bank ") of the cold water are considered as a source.