Untersuchungen zur Populationsstruktur und Virulenz von Porphyromonas gingivalis anhand intraspezifischer Diversitätsanalysen ausgewählter Virulenzloci

Die Nukleinsäurestruktur der Virulenzfaktoren fimA, rgpA, rgpB, kgp, prtT und tpr wurden mit Hilfe von Sequenz- und Restriktionsanalysen identifiziert. So konnten zum Teil schon bekannte Subtypen nachgewiesen, aber auch neue Allele identifiziert werden. Bei 108 analysierten Isolaten konnten 82 verschiedene Allelkombinationen nachgewiesen werden. Die Berechnung der „Coverage“ zeigte in Übereinstimmung mit der Rarefaction-Analyse, dass die Gesamtdiversität der untersuchten Loci damit erst zu circa. 46% erfasst wurde. Um eine Einschätzung der Populationsstruktur von P. gingivalis vornehmen zu können wurde der standardisierte Assoziationsindex berechnet, der mit einem Wert von 0,0079 nahe an Null liegt. Dies bedeutet, dass die untersuchte Population nahezu das „linkage equilibrium“ erreicht. Ebenso wie die große Anzahl verschiedener Genotypen ist dies ein Indiz für eine fehlende Kopplung der untersuchten Genloci und eine eher panmiktisch geprägte Populationsstruktur bei P. gingivalis

Gene expression in periodontal tissues following treatment

<p>Abstract</p> <p>Background</p> <p>In periodontitis, treatment aimed at controlling the periodontal biofilm infection results in a resolution of the clinical and histological signs of inflammation. Although the cell types found in periodontal tissues following treatment have been well described, information on gene expression is limited to few candidate genes. Therefore, the aim of the study was to determine the expression profiles of immune and inflammatory genes in periodontal tissues from sites with severe chronic periodontitis following periodontal therapy in order to identify genes involved in tissue homeostasis.</p> <p>Gingival biopsies from 12 patients with severe chronic periodontitis were taken six to eight weeks following non-surgical periodontal therapy, and from 11 healthy controls. As internal standard, RNA of an immortalized human keratinocyte line (HaCaT) was used. Total RNA was subjected to gene expression profiling using a commercially available microarray system focusing on inflammation-related genes. Post-hoc confirmation of selected genes was done by Realtime-PCR.</p> <p>Results</p> <p>Out of the 136 genes analyzed, the 5% most strongly expressed genes compared to healthy controls were Interleukin-12A (IL-12A), Versican (CSPG-2), Matrixmetalloproteinase-1 (MMP-1), Down syndrome critical region protein-1 (DSCR-1), Macrophage inflammatory protein-2β (Cxcl-3), Inhibitor of apoptosis protein-1 (BIRC-1), Cluster of differentiation antigen 38 (CD38), Regulator of G-protein signalling-1 (RGS-1), and Finkel-Biskis-Jinkins murine osteosarcoma virus oncogene (C-FOS); the 5% least strongly expressed genes were Receptor-interacting Serine/Threonine Kinase-2 (RIP-2), Complement component 3 (C3), Prostaglandin-endoperoxide synthase-2 (COX-2), Interleukin-8 (IL-8), Endothelin-1 (EDN-1), Plasminogen activator inhibitor type-2 (PAI-2), Matrix-metalloproteinase-14 (MMP-14), and Interferon regulating factor-7 (IRF-7).</p> <p>Conclusion</p> <p>Gene expression profiles found in periodontal tissues following therapy indicate activation of pathways that regulate tissue damage and repair.</p

Significant Short-Term Shifts in the Microbiomes of Smokers With Periodontitis After Periodontal Therapy With Amoxicillin & Metronidazole as Revealed by 16S rDNA Amplicon Next Generation Sequencing

The aim of this follow-up study was, to compare the effects of mechanical periodontal therapy with or without adjunctive amoxicillin and metronidazole on the subgingival microbiome of smokers with periodontitis using 16S rDNA amplicon next generation sequencing. Fifty-four periodontitis patients that smoke received either non-surgical periodontal therapy with adjunctive amoxicillin and metronidazole (n = 27) or with placebos (n = 27). Subgingival plaque samples were taken before and two months after therapy. Bacterial genomic DNA was isolated and the V4 hypervariable region of the bacterial 16S rRNA genes was amplified. Up to 96 libraries were normalized and pooled for Illumina MiSeq paired-end sequencing with almost fully overlapping 250 base pairs reads. Exact ribosomal sequence variants (RSVs) were inferred with DADA2. Microbial diversity and changes on the genus and RSV level were analyzed with non-parametric tests and a negative binomial regression model, respectively. Before therapy, the demographic, clinical, and microbial parameters were not significantly different between the placebo and antibiotic groups. Two months after the therapy, clinical parameters improved and there was a significantly increased dissimilarity of microbiomes between the two groups. In the antibiotic group, there was a significant reduction of genera classified as Porphyromonas, Tannerella, and Treponema, and 22 other genera also decreased significantly, while Selenomonas, Capnocytophaga, Actinomycetes, and five other genera significantly increased. In the placebo group, however, there was not a significant decrease in periodontal pathogens after therapy and only five other genera decreased, while Veillonella and nine other genera increased. We conclude that in periodontitis patients who smoke, microbial shifts occurred two months after periodontal therapy with either antibiotics or placebo, but genera including periodontal pathogens decreased significantly only with adjunctive antibiotics

High Interlaboratory Reproducibility and Accuracy of Next-Generation-Sequencing- Based Bacterial Genotyping in a Ring Trial

Today, next-generation whole-genome sequencing (WGS) is increasingly used to determine the genetic relationships of bacteria on a nearly whole-genome level for infection control purposes and molecular surveillance. Here, we conducted a multicenter ring trial comprising five laboratories to determine the reproducibility and accuracy of WGS-based typing. The participating laboratories sequenced 20 blind-coded Staphylococcus aureus DNA samples using 250-bp paired-end chemistry for library preparation in a single sequencing run on an Illumina MiSeq sequencer. The run acceptance criteria were sequencing outputs >5.6 Gb and Q30 read quality scores of > 75%. Subsequently, spa typing, multilocus sequence typing (MLST), ribosomal MLST, and core genome MLST (cgMLST) were performed by the participants. Moreover, discrepancies in cgMLST target sequences in comparisons with the included and also published sequence of the quality control strain ATCC 25923 were resolved using Sanger sequencing. All five laboratories fulfilled the run acceptance criteria in a single sequencing run without any repetition. Of the 400 total possible typing results, 394 of the reported spa types, sequence types (STs), ribosomal STs (rSTs), and cgMLST cluster types were correct and identical among all laboratories; only six typing results were missing. An analysis of cgMLST allelic profiles corroborated this high reproducibility; only 3 of 183,927 (0.0016%) cgMLST allele calls were wrong. Sanger sequencing confirmed all 12 discrepancies of the ring trial results in comparison with the published sequence of ATCC 25923. In summary, this ring trial demonstrated the high reproducibility and accuracy of current next-generation sequencing-based bacterial typing for molecular surveillance when done with nearly completely locked-down methods

Prospective Genomic Characterization of the German Enterohemorrhagic Escherichia coli O104:H4 Outbreak by Rapid Next Generation Sequencing Technology

An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak

Specific antibiotics in the treatment of periodontitis - A proposed strategy

Background: The purpose of the present study was to propose a strategy for the selection of antibiotics that specifically target complexes of periodontal pathogens present in patients with periodontitis. Methods: Seven hundred seventy-four (774) patients with various forms of periodontitis were included in the study. Subgingival plaque samples were taken from the deepest periodontal pockets in each quadrant using a sterile curet, and pooled. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Eikenella corrodens, Tannerella forsythensis, Prevotella intermedia, and Prevotella nigrescens were identified by polymerase chain reaction, and the prevalence of combinations of these pathogens was determined. To each pathogen complex (PC), i.e., combination of pathogens, those antibiotics were assigned that were most specific according to the published minimum inhibitory concentration (MIC90) values and the gingival crevicular fluid (GCF) concentrations achievable in vivo. Antibiotic GCF concentrations had to be at least 10 times the MIC90 values, and the narrowest spectrum was selected with respect to the assessed periodontal pathogens. Results: Nine major PCs (each ≥3% of all patients) were found in 73.4% of all patients, whereas 38 minor PCs (each <3% of all patients) were distributed in 26.6% of all patients. Ten different antibiotic regimens were found to be specific for the total of 46 PCs; i.e., metronidazole and amoxicillin in 11 PCs (55.0% of all patients), metronidazole and amoxicillin/clavulanic acid or metronidazole and ciprofloxacin in 13 PCs (18.9%), amoxicillin in 4 PCs (8.3%), doxycycline in 2 PCs (6.1%), metronidazole in 8 PCs (4.1%), amoxicillin/clavulanic acid in 3 PCs (2.9%), clindamycin in 2 PCs (1.5%), ciprofloxacin in 2 PCs (0.4%), and tetracycline in 1 PC (0.3%). Conclusion: The results of the study indicate that there are at least 46 different combinations of the assessed periodontal pathogens in subjects with periodontitis, and at least 10 different antibiotic regimens might be required to specifically target the various pathogen complexes.link_to_subscribed_fulltex

Development and evaluation of a core genome multilocus typing scheme for whole-genome sequence-based typing of Acinetobacter baumannii

We have employed whole genome sequencing to define and evaluate a core genome multi-locus sequence typing (cgMLST) scheme for Acinetobacter baumannii. To define a core genome we downloaded a total of 1,573 putative A. baumannii genomes from NCBI as well as representative isolates belonging to the eight previously described international A. baumannii clonal lineages. The core genome was then employed against a total of fifty-three carbapenem-resistant A. baumannii isolates that were previously typed by PFGE and linked to hospital outbreaks in eight German cities. We defined a core genome of 2,390 genes of which an average 98.4% were called successfully from 1,339 A. baumannii genomes, while Acinetobacter nosocomialis, Acinetobacter pittii, and Acinetobacter calcoaceticus resulted in 71.2%, 33.3%, and 23.2% good targets, respectively. When tested against the previously identified outbreak strains, we found good correlation between PFGE and cgMLST clustering, with 0-8 allelic differences within a pulsotype, and 40-2,166 differences between pulso-types. The highest number of allelic differences was between the isolates representing the international clones. This typing scheme was highly discriminatory and identified separate A. baumannii outbreaks. Moreover, because a standardised cgMLST nomenclature is used, the system will allow inter-laboratory exchange of data

Sequence variations in rgpA and rgpB of Porphyromonas gingivalis in periodontitis

Objective: The aim of the present study was to determine sequence variations in the active centre of the Arg-X-specific protease encoding genes rgpA and rgpB of clinical Porphyromonas gingivalis isolates and to analyse their prevalence in periodontitis patients before and 3 months after mechanical periodontal therapy. Background: Genetic diversity at nucleotides 281, 283, 286 and 331 has been shown to result in amino acid substitutions in the catalytic domain of RgpA and RgpB that affect the substrate specificity and thus may influence the efficacy of Arg-X-protease specific inhibitors. Methods: Sequence analysis of rgpA and rgpB genes in clinical P. gingivalis strains isolated from subgingival plaque samples of 82 periodontitis patients before and 3 months after mechanical supra- and subgingival debridement was performed. Results: No specific variation within the rgpA sequence was observed. However, the rgpB sequence in the region of the active centre showed five different rgpB genotypes, which were named NYPN, NSSN, NSSK, NYPK and DYPN according to the derived amino acid substitution. Porphyromonas gingivalis genotype NYPN was detected in 27 patients (32.9%) before and in 8 patients (9.8%) after therapy, NSSN in 26 (31.7%) and 10 (12.2%), NSSK in 22 (26.8%) and 2 (2.4%), NYPK in 5 (6.2%) and 1 (1.2%), and DYPN in 1 patient (1.2%) and 0 patients (0%), respectively. Only one patient (1.2%) harboured two P. gingivalis rgpB genotypes (NSSK/NYPN) before treatment; these were no longer detected after therapy. Conclusion: The results indicate that five rgpB genotypes are maintained in natural populations of P. gingivalis. These data may be of importance with regard to the development of specific rgpB inhibitors. © Blackwell Munksgaard 2005.link_to_subscribed_fulltex

Prevalence of Porphyromonas gingivalis fimA genotypes in Caucasians

The aim of the present study was to determine the prevalence of Porphyromonas gingivalis fimA genotypes in Caucasian patients with periodontitis. A total of 102 patients harboring P. gingivalis subgingivally were enrolled into the study. Pooled subgingival plaque samples of the six most severely affected sites were taken and analysed by fimA-specific polymerase chain reaction (PCR) and restriction analysis. Moreover, 26 P. gingivalis isolates were analysed by sequence analysis of the fimA gene. Sequence analysis revealed five major fimA genotypes (fimA types I-V) and allowed further subtyping of fimA genotypes II and IV into two subgroups each. The overall prevalences of fimA genotypes as assessed by PCR and restriction analysis among the P. gingivalis-positive patients with periodontitis were: type I, 25.5%; type II, 38.2%; type III, 4.9%; type IV, 18.6%; type V, 3.9%; and non-typable, 6.9%. Two patients were colonized by both type II and type IV, or type III and type IV fimA genotypes, respectively. Patients harboring different fimA genotypes showed no significant difference in severity of periodontal disease, as assessed by pocket probing depth and bleeding on probing following adjustment for smoking habit and age. The results indicate that predominant fimA genotypes in Caucasian periodontitis patients are types I, II, and IV. However, there was no difference in the association of the various fimA genotypes with disease severity.link_to_subscribed_fulltex

Updating benchtop sequencing performance comparison

In April 2012, your journal published a study by Loman et al.1 that systematically compared desktop next-generation sequencers (NGS) from three instrument providers. Using the custom scripts supplied by the authors, the same software and the same draft genome (with 153 remaining gaps within several scaffolds) as the reference, we reproduced their results with their data of the enterohemorrhagic Escherichia coli (EHEC) strain found in the 2011 outbreak in Germany. However, we wish to bring readers’ attention to some shortcomings in the report from Loman et al.1, focusing particularly on its discussion of read-level error analysis. NGS is a rapidly changing market, which clearly complicates the comparisons such as that made by Loman et al. Since the original study1, Illumina (San Diego) has launched the MiSeq sequencer officially and has released Nextera library construction kits and 2 × 250–base-pair (250-bp) paired-end (PE) sequencing chemistry. Furthermore, Life Technologies (Carlsbad, California), has made 200-bp and 300-bp kits available for the Ion Torrent Personal Genome Machine (PGM). Roche (Basel, Switzerland) has updated the Sequencing System software for its 454 GS Junior (GSJ) from version 2.6 to 2.7. In this report, we provide an up-to-date snapshot of how benchtop platforms have evolved since the previous study1