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High-throughput sequencing has helped to reveal the close relationship between Prevotella and periodontal disease, but the roles of subspecies diversity and genomic variation within this genus in periodontal diseases still need to be investigated. We performed a comparative genome analysis of 48 Prevotella intermedia and Prevotella nigrescens isolates that from the same cohort of subjects to identify the main drivers of their pathogenicity and adaptation to different environments. The comparisons were done between two species and between disease and health based on pooled sequences. The results showed that both P. intermedia and P. nigrescens have highly dynamic genomes and can take up various exogenous factors through horizontal gene transfer. The major differences between disease-derived and health-derived samples of P. intermedia and P. nigrescens were factors related to genome modification and recombination, indicating that the Prevotella isolates from disease sites may be more capable of genomic reconstruction. We also identified genetic elements specific to each sample, and found that disease groups had more unique virulence factors related to capsule and lipopolysaccharide synthesis, secretion systems, proteinases, and toxins, suggesting that strains from disease sites may have more specific virulence, particularly for P. intermedia. The differentially represented pathways between samples from disease and health were related to energy metabolism, carbohydrate and lipid metabolism, and amino acid metabolism, consistent with data from the whole subgingival microbiome in periodontal disease and health. Disease-derived samples had gained or lost several metabolic genes compared to healthy-derived samples, which could be linked with the difference in virulence performance between diseased and healthy sample groups. Our findings suggest that P. intermedia and P. nigrescens may serve as "crucial substances" in subgingival plaque, which may reflect changes in microbial and environmental dynamics in subgingival microbial ecosystems. This provides insight into the potential of P. intermedia and P. nigrescens as new predictive biomarkers and targets for effective interventions in periodontal disease.

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SummaryRecurrent hemorrhage has been reported in humans as a result of acquired antibody inhibitors which interfere with the crosslinking of fibrin by factor XIII. One type of these inhibitors (Type III) prevents activated factor XIII from acting on fibrin. We have generated an anti- fibrin monoclonal antibody, called mAb 4A5, which binds to a peptide sequence at the carboxyl-terminus of human fibrinogen γ-chains. MAb 4A5 acts like a Type III inhibitor and prevents proper factor XHI-mediated crosslinking. Pre-incubation of fibrinogen or pooled human plasma with mAb 4A5, but not mAb D2 (specific for the carboxyl terminus of fibrin α-chains), resulted in clots which are soluble in either 5 M urea or 1% monochloroacetic acid. SDS-PAGE and immunoblotting analysis of these clots confirmed that mAb 4A5 inhibited γ-chain crosslinking in plasma clots and fibrin clots. Results from a factor XIII activity assay demonstrated that biotinylcadaverine cross-linking into fibrin by factor XIII could be inhibited by mAb 4A5 but not mAb D2, arguing that mAb 4A5 acted by binding the crosslinking site of factor XIII. Studies of the immunoreactivity of these mAbs with 12 different animal species showed that the γ-chain epitope recognized by mAb 4A5 was more conserved than the α-chain epitope recognized by mAb D2. The species fibrinogens, recognized by mAb 4A5 in binding assays, also showed impaired crosslinking when mAb 4A5 was present during the clotting reaction.

Interactions among bacteria can affect biofilm properties. Method: Here, we investigated the role of different bacteria in functional dysbiosis of an in vitro polymicrobial subgingival plaque model using both 16S rRNA and metatranscriptomic sequencing. Results: We found that high-virulence Porphyromonas gingivalis W83 had greater effects on the symbiotic species than the low-virulence P. gingivalis ATCC33277, and that Prevotella intermedia exacerbated the effects of W83. P. gingivalis significantly influenced the expression of genes related to metabolic pathways and quorum sensing of commensal oral species in a strain-specific manner. P. intermedia exerted synergistic effects with P. gingivalis W83 but antagonistic effects with strain ATCC33277, which may regulate the expression of virulence factors of P. gingivalis through the clp regulator. Discussion: The interaction networks indicated that the strongest correlation was between Fusobacterium nucleatum and Streptococcus mitis, which demonstrated their bridge and cornerstone roles in biofilm. Changes in the expression of genes relating to outer membrane proteins in F. nucleatum indicated that the addition of different bacteria can interfere with the co-adherence among F. nucleatum and other partners. Conclusion: We report here the existence of strain-specific interactions in subgingival plaque, which may enhance our understanding of periodontal micro-ecology and facilitate the development of improved plaque control strategies.

Early childhood caries (ECC) is a considerable pediatric and public health problem worldwide. Preceding studies have focused primarily on bacterial diversity at the taxonomic level. Although these studies have provided significant information regarding the connection between dental caries and oral microbiomes, further comprehension of this microbial community’s ecological relevance is limited. This study identified the carbon source metabolic differences in dental plaque between children with and without ECC. We compared the microbial community functional diversity in 18 caries-free subjects with 18 severe ECC patients based on sole carbon source usage using a Biolog assay. The anaerobic microbial community in the ECC patients displayed greater metabolic activity than that of the control group. Specific carbon source metabolism differed significantly between the two groups. Subjects from the two groups were well distinguished by cluster and principal component analyses based on discriminative carbon sources. Our results implied that the microbial functional diversity between the ECC patients and healthy subjects differed significantly. In addition, the Biolog assay furthered our understanding of oral microbiomes as a composite of functional abilities, thus enabling us to identify the ecologically relevant functional differences among oral microbial communities.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been presented as a superior method for the detection of microorganisms in body fluid samples (e.g., blood, saliva, pus, etc.) However, the performance of MALDI-TOF MS in routine identification of caries-related Lactobacillus isolates from saliva of adult patients with caries has not been determined. In the present study, we introduced a new MALDI-TOF MS system for identification of lactobacilli. Saliva samples were collected from 120 subjects with caries. Bacteria were isolated and cultured, and each isolate was identified by both 16S rRNA sequencing and MALDI-TOF MS. The identification results obtained by MALDI-TOF MS were concordant at the genus level with those of conventional 16S rRNA-based sequencing for 88.6% of lactobacilli (62/70) and 95.5% of non-lactobacilli (21/22). Up to 96 results could be obtained in parallel on a single MALDI target, suggesting that this is a reliable high-throughput approach for routine identification of lactobacilli. However, additional reference strains are necessary to increase the sensitivity and specificity of species-level identification.

SummaryBy deriving an anti-peptide monoclonal antibody, mAb 7A4, we characterized the relatively unstudied carboxyl-terminal end of the α-chain of human factor XIII, the plasma transglutaminase. MAb 7A4 was directed against the last eight amino acids (Gln-Ile-Gln-Arg-Arg-Pro-Ser-Met) and bound with a dissociation constant of 3.4 × 10−8 M. In a solid assay format, mAb 7A4 bound equally well to factor XIII obtained from human plasma, platelets or placenta. However, in a solution-phase assay format, the epitope was largely unavailable but could be readily exposed by heat denaturation. Tmmunoblotting showed that this epitope is conserved among all species of plasma factor XIII tested except rabbit suggesting that the carboxyl-terminus might be an important structural element. Other competitive binding experiments with synthetic peptides as inhibitors pointed toward the final carboxyl-terminal amino acid, Met-731, as an immunochemically important determinant. This was used advantageously to confirm the finding that the caiboxyl-leiminal Mel-731 is largely absent from placental factor XIII (1) as compared to platelet or plasma factor XIII.