Assessment model blending formative and summative assessments using the SOLO taxonomy.

INTRODUCTION: Formative assessment with emphasis on feedback has been linked to developmental purposes of assessment, whilst summative assessment is assumed to focus on judgemental and quality assurance purposes. This dichotomy is questioned but designs to blend formative and summative assessments in constructive ways are rare in health care education. MATERIALS AND METHODS: We have designed an assessment model blending formative and summative assessments. In the formative assessment at the end of a course, students' responses to real-life scenarios with questions demanding responses at the relational level of understanding were assessed at three levels of understanding (incorrect, descriptive and relational) modified after the SOLO taxonomy. Students were presented with individual feedback for each response. At the summative assessment of a subsequent course, the students' new responses were assessed underpinning a final judgement of students' performance. The assessment model was justified across three student cohorts. RESULTS: Both formative and summative assessment events of the model provided information about the levels of understanding, unique to each student. A comparison of results from the assessments demonstrated that most responses developed to a higher level of understanding. With the summative assessment, it was possible to make judgements about whether or not individual students passed the pre-set standards. CONCLUSIONS: We argue that the current assessment model presents real interdependence between formative and summative assessments and can provide information that meets the needs of students as learners, education institutes and health care organisations. The SOLO taxonomy can be used to emphasise the importance of developing and assessing cognitive complexity.

Streptococcus gordonii Poised for Glycan Feeding through a MUC5B-Discriminating, Lipoteichoic Acid-Mediated Outside-In Signaling Circuit.

Many oral bacteria employ cell wall-anchored adhesins to bind to the salivary films coating the teeth and mucosal surfaces. Surface binding prevents clearance and facilitates catabolism of salivary film glycoproteins. We asked whether Streptococcus gordonii adhesin expression changes in response to surface salivary cues using a eukaryote-like, outside-in recognition and signaling circuit. To determine whether the cues were discriminated, S. gordonii was tested during cell adhesion and biofilm formation on a MUC5B-rich or lower-molecular-mass salivary fraction or an uncoated abiotic surface. Cells were recovered and analyzed for differences in gene expression and proteins in cell wall fractions. In salivary-free conditions, planktonic S. gordonii presented three prominent cell wall LPXTG-motif proteins, SGO_1487, SGO_0890, and MbpA (mucin-binding protein A; SGO_0707). During biofilm formation on MUC5B-coated surfaces, MbpA, a MUC5B-binding protein, and key genes in the tagatose and quorum-sensing pathways were strongly promoted. The response to MUC5B required the two-component system (TCS), streptococcal regulator of adhesins sensor and regulator (SraSR, SGO_1180/81), lipoteichoic acid (LTA), and the homologous paired adhesins, SspA and SspB (SspAB). LTA appears to link the outside signal (MUC5B) to intramembrane SraSR. Tagatose pathway gene expression may poise cells to metabolize MUC5B glycans and, with a quorum-sensing gene (luxS), may direct formation of a consortium to facilitate glycan cross-feeding by S. gordonii. We now show that a Gram-positive bacterium discriminates specific surface environmental cues using an outside-in signaling mechanism to apparently optimize colonization of saliva-coated surfaces. IMPORTANCE All organisms throughout the tree of life sense and respond to their surface environments. To discriminate among mucosal surface environmental cues, we report that Streptococcus gordonii recognizes a high-molecular-weight mucin glycoprotein, MUC5B, using the paired adhesins SspAB and lipoteichoic acid; the latter bridges the outside signal to an intramembrane two-component system to transcriptionally regulate a MUC5B-specific adhesin and genes that may facilitate glycan catabolism.

Polymicrobial synergy stimulates Porphyromonas gingivalis survival and gingipain expression in a multi-species subgingival community.

BACKGROUND: Dysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment-modelling that in the subgingival pocket. METHODS: Growth and proteolytic activity of three Porphyromonas gingivalis strains in nutrient broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants of P. gingivalis was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR. RESULTS: The P. gingivalis strains showed different growth rates in nutrient broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. Together in the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that community growth was facilitated by Rgp gingipain from P. gingivalis. CONCLUSIONS: In the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex protein substrates in serum. Whereas they are constitutively expressed by P. gingivalis in nutrient broth, gingipain expression in the model periodontal pocket environment (serum) appeared to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which then promoted growth of the whole community.

Intracellular Ser/Thr/Tyr phosphoproteome of the oral commensal Streptococcus gordonii DL1.

BACKGROUND: To respond and adapt to environmental challenges, prokaryotes regulate cellular processes rapidly and reversibly through protein phosphorylation and dephosphorylation. This study investigates the intracellular proteome and Ser/Thr/Tyr phosphoproteome of the oral commensal Streptococcus gordonii. Intracellular proteins from planktonic cells of S. gordonii DL1 were extracted and subjected to 2D-gel electrophoresis. Proteins in general were visualized using Coomassie Brilliant Blue and T-Rex staining. Phosphorylated proteins were visualized with Pro-Q Diamond Phosphoprotein Gel Stain. Proteins were identified by LC-MS/MS and sequence analysis. RESULTS: In total, sixty-one intracellular proteins were identified in S. gordonii DL1, many of which occurred at multiple isoelectric points. Nineteen of these proteins were present as one or more Ser/Thr/Tyr phosphorylated form. The identified phosphoproteins turned out to be involved in a variety of cellular processes. CONCLUSION: Nineteen phosphoproteins involved in various cellular functions were identified in S. gordonii. This is the first time the global intracellular Ser/Thr/Tyr phosphorylation profile has been analysed in an oral streptococcus. Comparison with phosphoproteomes of other species from previous studies showed many similarities. Proteins that are consistently found in a phosphorylated state across several species and growth conditions may represent a core phosphoproteome profile shared by many bacteria.

Parvimonas micra stimulates expression of gingipains from Porphyromonas gingivalis in multi-species communities.

Dental biofilms are complex ecosystems containing many bacterial species that live in mutualistic relationships. These interactions can profoundly affect the virulence properties of the community. In this study we investigated how the production of gingipains, virulence factors from Porphyromonas gingivalis important in periodontal disease, was affected by other commonly found members of the sub-gingival microbiome. To mimic the subgingival microbiome, multispecies consortia (P. gingivalis, Fusobacterium nucleatum, Actinomyces naeslundii, Streptococus oralis, Streptococcus mitis, Streptococcus gordonii and Streptococcus cristatus, with or without Parvimonas micra) as well as dual species consortia (P. gingivalis with P. micra, S. oralis or F. nucleatum) were constructed and maintained anaerobically in 10% serum for up to seven days. The number of P. gingivalis was determined by plating on Brucella agar and the gingipain specific fluorogenic substrate BikKam-10 was used to investigate gingipain activity. The effect of secreted products from P. micra on gingipain activity was investigated by adding supernatants from P. micra to P. gingivalis cultures. The most prominent secreted proteins in the supernatant were identified using mass spectrometry. P. gingivalis was unable to grow in serum, either alone or in the presence of S. oralis or F. nucleatum. In contrast, with P. micra growth was significantly enhanced and this was associated with an increase in gingipain activity. In the multi-species consortia, the presence of P. micra caused a 13-fold increase in gingipain activity. Exposure of P. gingivalis to supernatants from P. micra for 24 h caused a 3-fold increase in gingipain activity. This effect was reduced by 43% after heat-treatment of the supernatant. Two dimensional gel electrophoresis revealed that several of the most prominent proteins in the P. micra supernatant were glycolytic enzymes. The results from this study suggests that gingipains are produced in response to a P. micra derived signalling molecule that is most likely a protein. This is the first time it has been shown that P. micra can affect P. gingivalis virulence properties. This is likely to be of significance for the development of be of periodontitis since these two microorganisms are often found together in the subgingival biofilm.

Threshold values affect predictive accuracy of caries risk assessment.

OBJECTIVE: To evaluate effects of thresholds on estimates of predictive accuracy of methods for caries risk assessment. MATERIAL AND METHODS: Adolescents, aged 12 visiting two dental clinics, were examined by visual/tactile examination and bitewing radiography at baseline and after one year. Three methods for caries risk assessment were applied: previous caries experience, dentists' risk assessment according to set criteria (presence or absence of caries lesion) and acid tolerance of dental biofilm. The measure for validity (the reference standard) comprised caries lesion progression at 1 year. Predictive accuracy estimates were calculated for several thresholds. RESULTS: Accuracy estimates changed with threshold values of the methods and the reference standard. Patient spectrum differed between the clinics, which resulted in different accuracy estimates for the two samples. Generally, negative predictive values were high while positive ones were low indicating that these methods were more efficient in finding individuals who are at low risk of developing caries lesions than those with increased risk. CONCLUSIONS: As thresholds and patient spectrum affected predictive accuracy, it may be difficult to design a universal model with set thresholds for caries risk assessment. Foremost, a model should consider the level of aspiration for prediction and clinical decisions that will be made based on the risk assessment in the actual clinical setting.

Titanium granules pre-treated with hydrogen peroxide inhibit growth of bacteria associated with post-operative infections in spine surgery.

PURPOSE: Post-operative infections are relatively common after posterior spine surgery, and there are several observations reflecting different infection complications related to various metals implanted. Here, we selected an array of different bacterial species that are often found in infections associated with orthopaedic implants and tested for inhibition by hydrogen peroxide-treated titanium (Ti-peroxy). METHODS: To study the possibility of using Ti-peroxy as an antimicrobial prophylaxis, we developed a protocol for standardized susceptibility testing of bacteria. RESULTS: Importantly, we found that the resulting Ti-peroxy was highly antimicrobial against all aerobic species tested, among others, Staphylococcus aureus and Pseudomonas aeruginosa. Proteus mirabilis was slightly more resistant than, for example, Klebsiella pneumoniae and enterococci. In contrast, anaerobic bacteria Cutibacterium acnes and Parvimonas micra were equally susceptible compared to staphylococci. CONCLUSIONS: Our findings suggest that the Ti-peroxy is a promising perioperative antimicrobial strategy that may be highly effective for prevention of post-operative infections. We therefore suggest application of hydrogen peroxide to implants prior to implantation. These slides can be retrieved under Electronic supplementary material.

The proteins of Fusobacterium spp. involved in hydrogen sulfide production from L-cysteine.

BACKGROUND: Hydrogen sulfide (H2S) is a toxic foul-smelling gas produced by subgingival biofilms in patients with periodontal disease and is suggested to be part of the pathogenesis of the disease. We studied the H2S-producing protein expression of bacterial strains associated with periodontal disease. Further, we examined the effect of a cysteine-rich growth environment on the synthesis of intracellular enzymes in F. nucleatum polymorphum ATCC 10953. The proteins were subjected to one-dimensional (1DE) and two-dimensional (2DE) gel electrophoresis An in-gel activity assay was used to detect the H2S-producing enzymes; Sulfide from H2S, produced by the enzymes in the gel, reacted with bismuth forming bismuth sulfide, illustrated as brown bands (1D) or spots (2D) in the gel. The discovered proteins were identified with liquid chromatography - tandem mass spectrometry (LC-MS/MS). RESULTS: Cysteine synthase and proteins involved in the production of the coenzyme pyridoxal 5'phosphate (that catalyzes the production of H2S) were frequently found among the discovered enzymes. Interestingly, a higher expression of H2S-producing enzymes was detected from bacteria incubated without cysteine prior to the experiment. CONCLUSIONS: Numerous enzymes, identified as cysteine synthase, were involved in the production of H2S from cysteine and the expression varied among Fusobacterium spp. and strains. No enzymes were detected with the in-gel activity assay among the other periodontitis-associated bacteria tested. The expression of the H2S-producing enzymes was dependent on environmental conditions such as cysteine concentration and pH but less dependent on the presence of serum and hemin.

Crystalline anatase-rich titanium can reduce adherence of oral streptococci.

Dental implant abutments that emerge through the mucosa are rapidly covered with a salivary protein pellicle to which bacteria bind, initiating biofilm formation. In this study, adherence of early colonizing streptococci, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis and Streptococcus sanguinis to two saliva-coated anodically oxidized surfaces was compared with that on commercially pure titanium (CpTi). Near edge X-ray absorption (NEXAFS) showed crystalline anatase was more pronounced on the anodically oxidized surfaces than on the CpTi. As revealed by fluorescence microscopy, a four-species mixture, as well as individual bacterial species, exhibited lower adherence after 2 h to the saliva-coated, anatase-rich surfaces than to CpTi. Since wettability did not differ between the saliva-coated surfaces, differences in the concentration and/or configuration of salivary proteins on the anatase-rich surfaces may explain the reduced bacterial binding effect. Anatase-rich surfaces could thus contribute to reduced overall biofilm formation on dental implant abutments through diminished adherence of early colonizers.

Adherence of human oral keratinocytes and gingival fibroblasts to nano-structured titanium surfaces.

BACKGROUND: A key element for long-term success of dental implants is integration of the implant surface with the surrounding host tissues. Modification of titanium implant surfaces can enhance osteoblast activity but their effects on soft-tissue cells are unclear. Adherence of human keratinocytes and gingival fibroblasts to control commercially pure titanium (CpTi) and two surfaces prepared by anodic oxidation was therefore investigated. Since implant abutments are exposed to a bacteria-rich environment in vivo, the effect of oral bacteria on keratinocyte adhesion was also evaluated. METHODS: The surfaces were characterized using scanning electron microscopy (SEM). The number of adhered cells and binding strength, as well as vitality of fibroblasts and keratinocytes were evaluated using confocal scanning laser microscopy after staining with Live/Dead Baclight. To evaluate the effect of bacteria on adherence and vitality, keratinocytes were co-cultured with a four-species streptococcal consortium. RESULTS: SEM analysis showed the two anodically oxidized surfaces to be nano-structured with differing degrees of pore-density. Over 24 hours, both fibroblasts and keratinocytes adhered well to the nano-structured surfaces, although to a somewhat lesser degree than to CpTi (range 42-89% of the levels on CpTi). The strength of keratinocyte adhesion was greater than that of the fibroblasts but no differences in adhesion strength could be observed between the two nano-structured surfaces and the CpTi. The consortium of commensal streptococci markedly reduced keratinocyte adherence on all the surfaces as well as compromising membrane integrity of the adhered cells. CONCLUSION: Both the vitality and level of adherence of soft-tissue cells to the nano-structured surfaces was similar to that on CpTi. Co-culture with streptococci reduced the number of keratinocytes on all the surfaces to approximately the same level and caused cell damage, suggesting that commensal bacteria could affect adherence of soft-tissue cells to abutment surfaces in vivo.

Human PRH1, PRH2 susceptibility and resistance and Streptococcus mutans virulence phenotypes specify different microbial profiles in caries.

BACKGROUND: Lifestyle- and sucrose-dependent polymicrobial ecological shifts are a primary cause of caries in populations with high caries prevalence. In populations with low prevalence, PRH1, PRH2 susceptibility and resistance phenotypes may interact with the Streptococcus mutans adhesin cariogenicity phenotype to affect caries progression, but studies are lacking on how these factors affect the microbial profile of caries. METHODS: We analysed how the residency and infection profiles of S. mutans adhesin (SpaP A/B/C and Cnm/Cbm) phenotypes and commensal streptococci and lactobacilli influenced caries progression in a prospective case-referent sample of 452 Swedish adolescents with high (P4a), moderate (P6), and low (P1) caries PRH1, PRH2 phenotypes. Isolates of S. mutans from participants were analysed for adhesin expression and glycosylation and in vitro and in situ mechanisms related to caries activity. FINDINGS: Among adolescents with the resistant (P1) phenotype, infection with S. mutans high-virulence phenotypes was required for caries progression. In contrast, with highly (P4a) or moderately (P6) susceptible phenotypes, caries developed from a broader polymicrobial flora that included moderately cariogenic oral commensal streptococci and lactobacilli and S. mutans phenotypes. High virulence involved unstable residency and fluctuating SpaP ABC, B-1, or Cnm expression/glycosylation phenotypes, whereas low/moderate virulence involved SpaP A phenotypes with stable residency. Adhesin phenotypes did not display changes in individual host residency but were paired within individuals and geographic regions. INTERPRETATION: These results suggest that receptor PRH1, PRH2 susceptibility and resistance and S. mutans adhesin virulence phenotypes specify different microbial profiles in caries. FUNDING: Swedish Research Council and funding bodies listed in the acknowledgement section.

Characterization of MdpS: an in-depth analysis of a MUC5B-degrading protease from Streptococcus oralis.

Oral biofilms, comprising hundreds of bacteria and other microorganisms on oral mucosal and dental surfaces, play a central role in oral health and disease dynamics. Streptococcus oralis, a key constituent of these biofilms, contributes significantly to the formation of which, serving as an early colonizer and microcolony scaffold. The interaction between S. oralis and the orally predominant mucin, MUC5B, is pivotal in biofilm development, yet the mechanism underlying MUC5B degradation remains poorly understood. This study introduces MdpS (Mucin Degrading Protease from Streptococcus oralis), a protease that extensively hydrolyses MUC5B and offers an insight into its evolutionary conservation, physicochemical properties, and substrate- and amino acid specificity. MdpS exhibits high sequence conservation within the species and also explicitly among early biofilm colonizing streptococci. It is a calcium or magnesium dependent serine protease with strict physicochemical preferences, including narrow pH and temperature tolerance, and high sensitivity to increasing concentrations of sodium chloride and reducing agents. Furthermore, MdpS primarily hydrolyzes proteins with O-glycans, but also shows activity toward immunoglobulins IgA1/2 and IgM, suggesting potential immunomodulatory effects. Significantly, MdpS extensively degrades MUC5B in the N- and C-terminal domains, emphasizing its role in mucin degradation, with implications for carbon and nitrogen sequestration for S. oralis or oral biofilm cross-feeding. Moreover, depending on substrate glycosylation, the amino acids serine, threonine or cysteine triggers the enzymatic action. Understanding the interplay between S. oralis and MUC5B, facilitated by MdpS, has significant implications for the management of a healthy eubiotic oral microenvironment, offering potential targets for interventions aimed at modulating oral biofilm composition and succession. Additionally, since MdpS does not rely on O-glycan removal prior to extensive peptide backbone hydrolysis, the MdpS data challenges the current model of MUC5B degradation. These findings emphasize the necessity for further research in this field.

Salivary pellicles on titanium and their effect on metabolic activity in Streptococcus oralis.

BACKGROUND: Titanium implants in the oral cavity are covered with a saliva-derived pellicle to which early colonizing microorganisms such as Streptococcus oralis can bind. The protein profiles of salivary pellicles on titanium have not been well characterized and the proteins of importance for binding are thus unknown. Biofilm bacteria exhibit different phenotypes from their planktonic counterparts and contact with salivary proteins may be one factor contributing to the induction of changes in physiology. We have characterized salivary pellicles from titanium surfaces and investigated how contact with uncoated and saliva-coated titanium surfaces affects metabolic activity in adherent cells of S. oralis. METHODS: Salivary pellicles on smooth titanium surfaces were desorbed and these, as well as purified human saliva, were subjected to two-dimensional gel electrophoresis and mass spectroscopy. A parallel plate flow-cell model was used to study binding of a fresh isolate of S. oralis to uncoated and saliva-coated titanium surfaces. Metabolic activity was assessed using the BacLight CTC Vitality Kit and confocal scanning laser microscopy. Experiments were carried out in triplicate and the results analyzed using Student's t-test or ANOVA. RESULTS: Secretory IgA, α-amylase and cystatins were identified as dominant proteins in the salivary pellicles. Selective adsorption of proteins was demonstrated by the enrichment of prolactin-inducible protein and absence of zinc-α2-glycoprotein relative to saliva. Adherence of S. oralis to titanium led to an up-regulation of metabolic activity in the population after 2 hours. In the presence of a salivary pellicle, this effect was enhanced and sustained over the following 22 hour period. CONCLUSIONS: We have shown that adherence to smooth titanium surfaces under flow causes an up-regulation of metabolic activity in the early oral colonizer S. oralis, most likely as part of an adaptation to the biofilm mode of life. The effect was enhanced by a salivary pellicle containing sIgA, α-amylase, cystatins and prolactin-inducible protein which was, for the first time, identified as an abundant component of salivary pellicles on titanium. Further studies are needed to clarify the mechanisms underlying the effect of surface contact on metabolic activity as well as to identify the salivary proteins responsible for enhancing the effect.

Surface-associated MUC5B mucins promote protease activity in Lactobacillus fermentum biofilms.

BACKGROUND: Mucosal surfaces are coated with layers of mucus gel that protect the underlying tissues and promote colonization by members of the commensal microflora. Lactobacillus fermentum is a common inhabitant of the oral cavity, gastrointestinal and reproductive tracts and is one of the most important lactic acid bacteria contributing to the formation of a healthy intestinal microflora. We have investigated the proteolytic activity in L. fermentum in response to interactions with the MUC5B mucin, which is a major component of mucus gels at sites colonized by this micro-organism. METHODS: Biofilms of Lactobacillus fermentum were established in mini-flow cells in the presence or absence of human salivary MUC5B. The proteolytic activity of biofilm cells was examined in a confocal scanning laser microscope with a fluorescent protease substrate. Degradation of MUC5B by L. fermentum was analysed using SDS-PAGE followed by Western blotting with antisera raised against the MUC5B peptide. Cell surface proteins differentialy expressed in a MUC5B-rich environment were identified with the aid of comparative two-dimensional electrophoresis followed by LC-MS/MS. RESULTS: Lactobacillus fermentum adhered well to surfaces coated with MUC5B mucin and in biofilms of L. fermentum formed in a MUC5B environment, the proportion of proteolytically-active cells (47 ± 0.6% of the population), as shown by cleavage of a fluorescent casein substrate, was significantly greater (p < 0.01) than that in biofilms formed in nutrient broth (0.4 ± 0.04% of the population). Thus, the presence of MUC5B mucins enhanced bacterial protease activity. This effect was mainly attributable to contact with surface-associated mucins rather than those present in the fluid phase. Biofilms of L. fermentum were capable of degrading MUC5B mucins suggesting that this complex glycoprotein can be exploited as a nutrient source by the bacteria.Comparison of the surface proteomes of biofilm cells of L. fermentum in a MUC5B environment with those in nutrient broth using two-dimensional electrophoresis and mass spectroscopy, showed that the enhanced proteolytic activity was associated with increased expression of a glycoprotease; O-sialoglycoprotein endopeptidase, as well as chaperone proteins such as DnaK and trigger factor. CONCLUSIONS: Adhesion to mucin-coated surfaces leads to a shift towards a more protease-active phenotype within L. fermentum biofilms and proteases produced within the biofilms can degrade MUC5B mucins. The enhanced proteolytic activity was associated with an increase in O-sialoglycoprotein endopeptidase on the cell surface. We propose that the upregulation of chaperone proteins in the mucin environment may contribute to the protease-active phenotype through activation of the glycopeptidase. This would represent one way for commensal lactobacilli e.g. L. fermentum to exploit complex substrates in their local environment in order to survive on mucosal surfaces.

Characterization of a highly conserved MUC5B-degrading protease, MdpL, from Limosilactobacillus fermentum.

MUC5B is the predominant glycoprotein in saliva and is instrumental in the establishment and maintenance of multi-species eubiotic biofilms in the oral cavity. Investigations of the aciduric Lactobacillaceae family, and its role in biofilms emphasizes the diversity across different genera of the proteolytic systems involved in the nutritional utilization of mucins. We have characterized a protease from Limosilactobacillus fermentum, MdpL (Mucin degrading protease from Limosilactobacillus) with a high protein backbone similarity with commensals that exploit mucins for attachment and nutrition. MdpL was shown to be associated with the bacterial cell surface, in close proximity to MUC5B, which was sequentially degraded into low molecular weight fragments. Mapping the substrate preference revealed multiple hydrolytic sites of proteins with a high O-glycan occurrence, although hydrolysis was not dependent on the presence of O-glycans. However, since proteolysis of immunoglobulins was absent, and general protease activity was low, a preference for glycoproteins similar to MUC5B in terms of glycosylation and structure is suggested. MdpL preferentially hydrolyzed C-terminally located hydrophobic residues in peptides larger than 20 amino acids, which hinted at a limited sequence preference. To secure proper enzyme folding and optimal conditions for activity, L. fermentum incorporates a complex system that establishes a reducing environment. The importance of overall reducing conditions was confirmed by the activity boosting effect of the added reducing agents L-cysteine and DTT. High activity was retained in low to neutral pH 5.5-7.0, but the enzyme was completely inhibited in the presence of Zn2+. Here we have characterized a highly conserved mucin degrading protease from L. fermentum. MdpL, that together with the recently discovered O-glycanase and O-glycoprotease enzyme groups, increases our understanding of mucin degradation and complex biofilm dynamics.

Microcontact-Imprinted Optical Sensors for Virulence Factors of Periodontal Disease.

Periodontitis (gum disease) is a common biofilm-mediated oral condition, with around 7% of the adult population suffering from severe disease with risk for tooth loss. Moreover, periodontitis virulence markers have been found in atherosclerotic plaque and brain tissue, suggesting a link to cardiovascular and Alzheimer's diseases. The lack of accurate, fast, and sensitive clinical methods to identify patients at risk leads, on the one hand, to patients being undiagnosed until the onset of severe disease and, on the other hand, to overtreatment of individuals with mild disease, diverting resources from those patients most in need. The periodontitis-associated bacterium, Porphyromonas gingivalis, secrete gingipains which are highly active proteases recognized as key virulence factors during disease progression. This makes them interesting candidates as predictive biomarkers, but currently, there are no methods in clinical use for monitoring them. Quantifying the levels or proteolytic activity of gingipains in the periodontal pocket surrounding the teeth could enable early-stage disease diagnosis. Here, we report on a monitoring approach based on high-affinity microcontact imprinted polymer-based receptors for the Arg and Lys specific gingipains Rgp and Kgp and their combination with surface plasmon resonance (SPR)-based biosensor technology for quantifying gingipain levels in biofluids and patient samples. Therefore, Rgp and Kgp were immobilized on glass coverslips followed by microcontact imprinting of poly-acrylamide based films anchored to gold sensor chips. The monomers selected were N-isopropyl acrylamide (NIPAM), N-hydroxyethyl acrylamide (HEAA) and N-methacryloyl-4-aminobenzamidine hydrochloride (BAM), with N,N'-methylene bis(acrylamide) (BIS) as the crosslinker. This resulted in imprinted surfaces exhibiting selectivity towards their templates high affinity and selectivity for the templated proteins with dissociation constants (K d) of 159 and 299 nM for the Rgp- and Kgp-imprinted, surfaces respectively. The former surface displayed even higher affinity (K d = 71 nM) when tested in dilute cell culture supernatants. Calculated limits of detection for the sensors were 110 and 90 nM corresponding to levels below clinically relevant concentrations.

Formation and Analysis of Mono-species and Polymicrobial Oral Biofilms in Flow-Cell Models.

The oral microbiota, which is known to include at least 600 different bacterial species, is found on the teeth and mucosal surfaces as multi-species communities or biofilms. The oral surfaces are covered with a pellicle of proteins absorbed from saliva, and biofilm formation is initiated when primary colonizers, which express surface adhesins that bind to specific salivary components, attach to the oral tissues. Further development then proceeds through co-aggregation of additional species. Over time, the composition of oral biofilms, which varies between different sites throughout the oral cavity, is determined by a combination of environmental factors such as the properties of the underlying surface, nutrient availability and oxygen levels, and bacterial interactions within the community. A complex equilibrium between biofilm communities and the host is responsible for the maintenance of a healthy biofilm phenotype (eubiosis). In the face of sustained environmental perturbation, however, biofilm homeostasis can break down giving rise to dysbiosis, which is associated with the development of oral diseases such as caries and periodontitis.In vitro models have an important part to play in increasing our understanding of the complex processes involved in biofilm development in oral health and disease, and the requirements for experimental system, microbial complexity, and analysis techniques will necessarily vary depending on the question posed. In this chapter we describe some current and well-established methods used in our laboratory for studying oral bacteria in biofilm models which can be adapted to suit the needs of individual users.

Multivariable prediction models of caries increment: a systematic review and critical appraisal.

BACKGROUND: Multivariable prediction models are used in oral health care to identify individuals with an increased likelihood of caries increment. The outcomes of the models should help to manage individualized interventions and to determine the periodicity of service. The objective was to review and critically appraise studies of multivariable prediction models of caries increment. METHODS: Longitudinal studies that developed or validated prediction models of caries and expressed caries increment as a function of at least three predictors were included. PubMed, Cochrane Library, and Web of Science supplemented with reference lists of included studies were searched. Two reviewers independently extracted data using CHARMS (Critical Appraisal and Data Extraction for Systematic Reviews of Prediction Modelling Studies) and assessed risk of bias and concern regarding applicability using PROBAST (Prediction model Risk Of Bias ASessment Tool). Predictors were analysed and model performance was recalculated as estimated positive (LR +) and negative likelihood ratios (LR -) based on sensitivity and specificity presented in the studies included. RESULTS: Among the 765 reports identified, 21 studies providing 66 prediction models fulfilled the inclusion criteria. Over 150 candidate predictors were considered, and 31 predictors remained in studies of final developmental models: caries experience, mutans streptococci in saliva, fluoride supplements, and visible dental plaque being the most common predictors. Predictive performances varied, providing LR + and LR - ranges of 0.78-10.3 and 0.0-1.1, respectively. Only four models of coronal caries and one root caries model scored LR + values of at least 5. All studies were assessed as having high risk of bias, generally due to insufficient number of outcomes in relation to candidate predictors and considerable uncertainty regarding predictor thresholds and measurements. Concern regarding applicability was low overall. CONCLUSIONS: The review calls attention to several methodological deficiencies and the significant heterogeneity observed across the studies ruled out meta-analyses. Flawed or distorted study estimates lead to uncertainty about the prediction, which limits the models' usefulness in clinical decision-making. The modest performance of most models implies that alternative predictors should be considered, such as bacteria with acid tolerant properties. TRIAL REGISTRATION: PROSPERO CRD#152,467 April 28, 2020.

Synergistic metabolism of salivary MUC5B in oral commensal bacteria during early biofilm formation.

IMPORTANCE: The study of bacterial interactions and salivary-mediated regulation of early dental biofilm activity is of interest for understanding oral microbial adaptation to environmental cues and biofilm maturation. Findings in oral commensals can prove useful from the perspectives of both oral and systemic health of the host, as well as the understanding of general microbial biofilm physiology. The knowledge may provide a basis for the development of prognostic biomarkers, or development of new treatment strategies, related to oral health and disease and possibly also to other biofilm-induced conditions. The study is also an important step toward developing the methodology for similar studies in other species and/or growth conditions.

Effects of saliva or serum coating on adherence of Streptococcus oralis strains to titanium.

The use of dental implants to treat tooth loss has increased rapidly over recent years. 'Smooth' implants showing high long-term success rates have successively been replaced by implants with rougher surfaces, designed to stimulate rapid osseointegration and promote tissue healing. If exposed in the oral cavity, rougher surfaces may promote bacterial adhesion leading to formation of microbial biofilms which can induce peri-implant inflammation. Streptococcus oralis is an early colonizer of oral surfaces and has been recovered from titanium surfaces in vivo. The purpose of this study was to examine the adherence of clinical strains of S. oralis to titanium with smooth or moderately rough surface topography and to determine the effect of a saliva- or serum-derived coating on this process. Adherence was studied using a flow-cell system with confocal laser scanning microscopy, while putative adhesins were analysed using proteomics of bacterial cell wall proteins. This showed that adherence to moderately rough surfaces was greater than to smooth surfaces. Serum did not promote binding of any of the studied S. oralis strains to titanium, whereas a saliva coating increased adherence in two of three strains tested. The higher level of adherence to the moderately rough surfaces was maintained even in the presence of a saliva coating. The S. oralis strains that bound to saliva expressed an LPXTG-linked protein which was not present in the non-adherent strain. Thus strains of S. oralis differ in their capacity to bind to saliva-coated titanium and we propose that this is due to differential expression of a novel adhesin.