Genome-Wide Identification and Mapping of NBS-Encoding Resistance Genes in Solanum tuberosum Group Phureja

The majority of disease resistance (R) genes identified to date in plants encode a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain containing protein. Additional domains such as coiled-coil (CC) and TOLL/interleukin-1 receptor (TIR) domains can also be present. In the recently sequenced Solanum tuberosum group phureja genome we used HMM models and manual curation to annotate 435 NBS-encoding R gene homologs and 142 NBS-derived genes that lack the NBS domain. Highly similar homologs for most previously documented Solanaceae R genes were identified. A surprising ∼41% (179) of the 435 NBS-encoding genes are pseudogenes primarily caused by premature stop codons or frameshift mutations. Alignment of 81.80% of the 577 homologs to S. tuberosum group phureja pseudomolecules revealed non-random distribution of the R-genes; 362 of 470 genes were found in high density clusters on 11 chromosomes

Growth and treatment of oral biofilms

From the ecological perspective, preserving symbiotic biofilms should be a keystone in the prevention of oral diseases. The results of in vitro and in vivo studies of this thesis have shown that the use of an oxygenating agent (AX) showed selective inhibition of oral bacteria which may lead to a healthier ecosystem. The results indicate that AX containing mouthwash may contribute to this demanding balance and deserves further investigation. The development of a novel subgingival biofilm model led to the growth of bacteria known to be associated with periodontal disease such as Porphyromonas gingivalis, Parvimonas micra, Peptostreptococcus, Fusobacterium, Filifactor, Phocaeicola, Anaeroglobus, and Mogibacterium among other 47 genera. An important finding was that statistically significant differences in bacterial composition were consistently found in biofilms derived from different inoculum donors. This finding reflects the large variability in the subgingival microbial composition in vivo among periodontitis patients and healthy donors. The development of a peri-implantitis biofilm model needs to be further investigated. Periodontal disease is associated with alterations of the complex microbial community rather than dominance of a single pathogenic species. The analysis of specific subgingival bacterial profiles for the decision to prescribe antibiotics in the treatment of periodontitis is currently not recommend. Authors also highlighted the need for alternative therapies based on ecological approaches. Instead of aiming to kill “pathogens”, the search to restore the symbiotic biofilm by other means than using antibiotics are of interest

Does routine analysis of subgingival microbiota in periodontitis contribute to patient benefit?

In clinical periodontology it is common practice to sample subgingival plaque from periodontitis patients and to search for the presence of alleged periodontal pathogens using routine laboratory techniques such as culture, DNA-DNA hybridization or real-time PCR. Usually, special attention is given to the recognition of 'red complex' microorganisms and to Aggregatibacter actinomycetemcomitans. Recently, molecular open-ended techniques have been introduced which are distinct from the more 'classical' approaches in that they do not preselect for certain species. In this study, we investigated to what extent the outcome of these techniques has changed our insight into the composition of the subgingival microbiota and whether this has consequences on clinical decision making. The open-ended approaches showed that the composition of subgingival plaque is much more complex than previously thought. Next to the 'classical' putative periodontal pathogens, several non-culturable and fastidious species are now recognized as being associated with periodontitis, thus enlarging the group of suspected periodontal pathogens. We conclude that routine analyses of subgingival plaque in the clinic are not necessarily of benefit to the patient

Effect of mouthwashes on the composition and metabolic activity of oral biofilms grown in vitro

Objective: The aim of this study was to determine the effect of an oxygenating mouthwash compared to two other established mouthwash products on bacterial composition and metabolic activity of oral biofilms in vitro. Material and methods: Twelve healthy subjects participated as donors. Plaque-saliva mixture inoculated biofilms were grown and treated with 3 different chemotherapeutic mouthwashes [amine fluoride/stannous fluoride (MD), oxygenating agent (AX), chlorhexidine 0.12 % (PA), and water (W)]. Effects of treatments were assessed on biofilm composition (16S rRNA gene amplicon sequencing), production of organic acids (formate, acetate, lactate, propionate, butyrate using capillary electrophoresis), and viability of the remaining biofilm (CFUs). Results: Microbial profiles of biofilms clustered per inoculum donor and were dominated by the genera Veillonella, Streptococcus, and Prevotella. Microbial diversity was only reduced after PA treatment. Significant changes in composition occurred after treatment with AX, resulting in lower proportions of Veillonella and higher proportions of non-mutans streptococci. Production of all organic acids after PA and lactate after MD was significantly lower as compared to W. AX resulted in reduction of acetate, butyrate, and propionate and increase in lactate production (p < 0.05). Viable counts were significantly lower after PA and AX treatments compared to W, while no significant reduction was observed after MD. Conclusions: All studied mouthwashes affected the in vitro biofilms differently. The effects of the AX treatment were the most prominent which resulted in changes of the bacterial composition and metabolism

A reproducible microcosm biofilm model of subgingival microbial communities

Objective: To develop a reproducible subgingival microcosm biofilm model. Material and Methods: Subgingival plaque samples were collected from four deep pockets (probing pocket depth ≥6 mm) in each of seven patients with periodontitis and from shallow pockets (probing pocket depth ≤3 mm) in two periodontally healthy donors. An active attachment model and a peptone medium (Thompson et. al., Appl Environ Microbiol 2015;81:8307–8314) supplemented with 30% serum was used. Biofilms were harvested at 2 and 4 weeks. DNA of dead cells was blocked for amplification by propidium monoazide treatment. Composition was analyzed using 16S rRNA gene amplicon pyrosequencing. Similarities between the biofilm samples were assessed by non-metric multidimensional scaling using the Bray-Curtis similarity index and similarity percentage analysis. Data from duplicate experiments, different biofilm sources and different biofilm age were compared. Results: The non-metric multidimensional scaling revealed a strong clustering by the inoculum source, the donor and their periodontal status. Statistically significant differences were found between the sources of inoculum (P=.0001) and biofilm age (P=.0016). Furthermore, periodontitis biofilms (P) were distinct in composition from health-derived biofilms (H) by genera: Porphyromonas (P=19%; H=0%), Filifactor (P=10%; H=0%), Anaeroglobus (P=3%; H=0%), Phocaeicola (P=1.5%; H=0%), Parvimonas (P=19%; H=14%), Fusobacterium (P=2%; H=26%), Peptostreptococcus (P=20%; H=30%), Veillonella (P=7%; H=8%) and 57 other genera. Similarity distances (Bray-Curtis) (mean 0.73, SD 0.15) and the Shannon diversity index (mean 2, SD 0.2) revealed no differences between duplicate experiments (P=.121). Conclusion: This biofilm model allows reproducible production of complex subgingival microbial communities