Hans-Peter Horz

Identification and Quantification of Archaea Involved in Primary Endodontic Infections

ABSTRACT Members of the domain Archaea, one of the three domains of life, are a highly diverse group of prokaryotes, distinct from bacteria and eukaryotes. Despite their abundance and ubiquity on earth, including their close association with humans, animals, and plants, so far no pathogenic archaea have been described. As some archaea live in close proximity to anaerobic bacteria, for instance, in the human gut system and in periodontal pockets, the aim of our study was to assess whether archaea might possibly be involved in human endodontic infections, which are commonly polymicrobial. We analyzed 20 necrotic uniradicular teeth with radiographic evidence of apical periodontitis and with no previous endodontic treatment. Using real-time quantitative PCR based on the functional gene mcrA (encoding the methyl coenzyme M reductase, specific to methanogenic archaea) and on archaeal 16S rRNA genes, we found five cases to be positive. Direct sequencing of PCR products from both genes showed that the archaeal community was dominated by a Methanobrevibacter oralis-like phylotype. The size of the archaeal population at the diseased sites ranged from 1.3 × 105 to 6.8 × 105 16S rRNA gene target molecule numbers and accounted for up to 2.5% of the total prokaryotic community (i.e., bacteria plus archaea). Our findings show that archaea can be intimately connected with infectious diseases and thus support the hypothesis that members of the domain Archaea may have a role as human pathogens.

Evaluation of Universal Probes and Primer Sets for Assessing Total Bacterial Load in Clinical Samples: General Implications and Practical Use in Endodontic Antimicrobial Therapy

ABSTRACT By reexamining 10 previously published “universal” PCR assays using the ARB phylogenetic software package and database with 41,000 16S rRNA gene sequences, we found that they differed considerably in their coverage of the domain Bacteria. We evaluated the broadest-range real-time quantitative PCR protocol for its efficacy in measuring the antimicrobial effects of endodontic treatments.

Viruses versus bacteria—novel approaches to phage therapy as a tool against multidrug-resistant pathogens

Bacteriophage therapy (the application of phages to treat bacterial infections) has a tradition dating back almost a century, but interest in phage therapy slowed down in the West when antibiotics were discovered. With the emerging threat of infections caused by multidrug-resistant bacteria and scarce prospects of newly introduced antibiotics in the future, phages are currently being reconsidered as alternative therapeutics. Conventional phage therapy uses lytic bacteriophages for treatment and recent human clinical trials have revealed encouraging results. In addition, several other modern approaches to phages as therapeutics have been made in vitro and in animal models. Dual therapy with phages and antibiotics has resulted in significant reductions in the number of bacterial pathogens. Bioengineered phages have overcome many of the problems of conventional phage therapy, enabled targeted drug delivery or reversed the resistance of drug-resistant bacteria. The use of enzymes derived from phages, such as endolysin, as therapeutic agents has been efficient in the elimination of Gram-positive pathogens. This review presents novel strategies for phage-related therapies and describes our current knowledge of natural bacteriophages within the human microbiome. Our aim is to provide an overview of the high number of different methodological concepts, thereby encouraging further research on this topic, with the ultimate goal of using phages as therapeutic or preventative medicines in daily clinical practice.

Synergistes Group Organisms of Human Origin

ABSTRACT The bacterial division Synergistes represents a poorly characterized phylotype of which only a few isolates have been cultured, primarily from natural environments. Recent detection of Synergistes-like sequence types in periodontal pockets and caries lesions of humans prompted us to search the R. M. Alden culture collection (Santa Monica, Calif.) for biochemically unidentifiable, slow-growing, obligately anaerobic gram-negative bacilli. Here we report on five clinical isolates cultured from peritoneal fluid and two isolates from soft-tissue infections that together constitute three separate evolutionary lineages within the phylogenetic radiation of the division Synergistes. One of these clusters was formed by the peritoneal isolates and had an 85% similarity to Synergistes jonesii, the first described Synergistes species, which was isolated from the rumen of a goat. The isolates from soft-tissue infections, on the other hand, formed two distinct lineages moderately related to each other with a similarity of approximately 78%. In addition, by using a newly designed 16S rRNA gene-based PCR assay with intended target specificity for Synergistes, we found that the dominant phylotype from a fecal sample was nearly identical to that of the strains obtained from peritonitis. Conversely, sequence types detected in periodontal pockets formed a separate cluster that shared a similarity of only 80% with the soft-tissue isolates. These findings suggest a high diversity of medically important Synergistes clades that apparently are unique to individual ecological niches in the human body. In conclusion, we now have available the first characterized human isolates of the division Synergistes which are colonizing, and probably infecting, several sites in the human body.

T-RFLP-based mcrA gene analysis of methanogenic archaea in association with oral infections and evidence of a novel Methanobrevibacter phylotype

INTRODUCTION Increasing evidence suggests a role for methanogenic archaea (methanogens) in human health and disease via syntrophic interactions with bacteria. Here we assessed the prevalence and distribution of methanogens and possible associations with bacteria in oral biofilms. METHODS Forty-four periodontal and 32 endodontic samples from necrotic teeth with radiographic evidence of apical periodontitis were analysed. Terminal restriction fragment length polymorphism analysis based on the mcrA gene, specific to methanogens, was applied. The prevalence and amounts of methanogens in endodontic samples were compared with those of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema spp. and Synergistes spp. based on real-time quantitative polymerase chain reactions. RESULTS Besides dominance of the mcrA gene corresponding to Methanobrevibacter oralis, one mcrA gene type, for which no cultivated member has been reported previously, was identified in five periodontal samples and one endodontic sample. Rates of non-synonymous vs. synonymous nucleotide substitutions suggest that this mcrA gene type codes for a functionally active methyl-coenzyme M reductase. Methanobrevibacter smithii, the prominent methanogen in the human gut system, was not detected. Mean proportions of methanogens were comparable to Synergistes spp. ranging from 0.5 to 1.0% of the total microbial community. Treponema spp. dominated with a mean proportion of 10%, while the mean proportions of the other endodontic pathogens were below 0.1%. A positive association between methanogens and Synergistes spp. was found. CONCLUSION Our data provide evidence of a novel, as yet uncultured methanogenic phylotype in association with oral infections, and indicate possible interactions between methanogens and Synergistes spp., the nature of which deserves further investigation.

New methods for selective isolation of bacterial DNA from human clinical specimens

Separation of bacterial DNA from human DNA in clinical samples may have an important impact on downstream applications, involving microbial diagnostic systems. We evaluated two commercially available reagents (MolYsis), Molzym GmbH & Co. KG, Bremen and Pureprove, SIRS-Lab GmbH, Jena, both Germany) for their potential to isolate and purify bacterial DNA from human DNA. We chose oral samples, which usually contain very high amounts of both human and bacterial cells. Three different DNA preparations each were made from eight caries and eight periodontal specimens using the two reagents above and a conventional DNA extraction strategy as reference. Based on target-specific real-time-quantitative PCR assays we compared the reduction of human DNA versus loss of bacterial DNA. Human DNA was monitored by targeting the beta-2-microglobulin gene, while bacteria were monitored by targeting 16S rDNA (total bacteria and Porphyromonas gingivalis) or the glycosyltransferase gene (Streptococcus mutans). We found that in most cases at least 90% of human DNA could successfully be removed, with complete removal in eight of 16 cases using MolYsis, and two (of 16) cases using Pureprove. Conversely, detection of bacterial DNA was possible in all cases with a recovery rate generally ranging from 35% to 50%. In conclusion, both strategies have the potential to reduce background interference from the host DNA which may be of remarkable value for nucleic-acid based microbial diagnostic systems.

Comparative analysis of endodontic pathogens using checkerboard hybridization in relation to culture

BACKGROUND/AIM The purpose of this study was to detect bacterial species and to quantify the total number of bacteria from samples of infected root canals before (S1) and after chemo-mechanical preparation using 2% chlorhexidine (CHX) gel as auxiliary chemical substance (S2) and after 7 days of intracanal dressing (S3) to compare microbial changes. METHOD Twenty-four teeth were selected for this study. Chemo-mechanical preparation was performed using 2% CHX gel, then three different intracanal medicaments [M1: Ca(OH)(2) paste; M2: 2% CHX gel; and M3: Ca(OH)(2) paste plus 2% CHX gel] were used for 7 days. Checkerboard DNA-DNA hybridization was performed to detect 40 bacterial species. Aerobic and anaerobic culture techniques were used to determine the bacterial community by counting the colony-forming units (CFU). RESULTS The species most frequently identified by checkerboard in S1 were: Fusobacterium nucleatum ssp. polymorphum, Treponema socranskii ssp. socranskii, Parvimonas micra and Enterococcus faecalis. In S2 and S3 a total of eight different species were identified; and only one of them was gram-positive (E. faecalis). Microorganisms were not identified after use of M2 for 7 days. The quantification obtained on agar plates ranged from 4 x 10(5) to 2.6 x 10(6) CFU/ml in S1, mean CFU was reduced by 99.96% in S2, and there was no statistical difference between the CFU in S2 and S3. CONCLUSION The antibacterial effect of the mechanical preparation supplemented by the use of an antibacterial auxiliary substance greatly reduced the microorganisms in the main root canal.

McrA and 16S rRNA gene analysis suggests a novel lineage of Archaea phylogenetically affiliated with Thermoplasmatales in human subgingival plaque.

Based on the molecular analysis of human subgingival plaque samples from 30 periodontitis patients a novel lineage of Archaea within the phylogenetic radiation of Thermoplasmatales was identified in 10% of cases. Co-occurrence of unique 16S rRNA gene and mcrA gene sequences suggests that this lineage corresponds to a hitherto unknown group of methanogens.

Diagnosis and anti-infective therapy of periodontitis

Periodontal diseases (gingivitis and periodontitis) are chronic bacterial infections with a remarkably high prevalence and morbidity. Periodontitis, in contrast to gingivitis, is not reversible, is associated with certain bacterial species and affects all of the soft tissue and bone that support teeth. Among the periodontal pathogens, species, such as Aggregatibacter (Actinobacillus) actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and several forms of uncultivable spirochetes play the major role in the pathogenesis. In severe chronic, recurrent and especially aggressive forms of periodontitis, diagnosis of the species involved and, whenever possible, an optimized evidence-based antimicrobial treatment is indicated. In order to monitor alarming bacterial changes in the periodontal pocket, several techniques, namely microscopy, culture, immunoassays, enzyme tests and DNA-based techniques, have been established and the methods are described in the first part of this review. In the second part, the selection and use of locally delivered (topical) and systemic antibiotics used adjunctively in periodontal therapy are discussed.

The saliva microbiome of Pan and Homo

BackgroundIt is increasingly recognized that the bacteria that live in and on the human body (the microbiome) can play an important role in health and disease. The composition of the microbiome is potentially influenced by both internal factors (such as phylogeny and host physiology) and external factors (such as diet and local environment), and interspecific comparisons can aid in understanding the importance of these factors.ResultsTo gain insights into the relative importance of these factors on saliva microbiome diversity, we here analyze the saliva microbiomes of chimpanzees (Pan troglodytes) and bonobos (Pan paniscus) from two sanctuaries in Africa, and from human workers at each sanctuary. The saliva microbiomes of the two Pan species are more similar to one another, and the saliva microbiomes of the two human groups are more similar to one another, than are the saliva microbiomes of human workers and apes from the same sanctuary. We also looked for the existence of a core microbiome and find no evidence for a taxon-based core saliva microbiome for Homo or Pan. In addition, we studied the saliva microbiome from apes from the Leipzig Zoo, and found an extraordinary diversity in the zoo ape saliva microbiomes that is not found in the saliva microbiomes of the sanctuary animals.ConclusionsThe greater similarity of the saliva microbiomes of the two Pan species to one another, and of the two human groups to one another, are in accordance with both the phylogenetic relationships of the hosts as well as with host physiology. Moreover, the results from the zoo animals suggest that novel environments can have a large impact on the microbiome, and that microbiome analyses based on captive animals should be viewed with caution as they may not reflect the microbiome of animals in the wild.