Gérard Aboudharam

Methanogenic archaea in subgingival sites: a review

Archaea are non‐bacterial prokaryotes associated with oral microbiota in humans, but their roles in oral pathologies remain controversial. Several studies reported the molecular detection of methanogenic archaea from periodontitis, but the significance of this association has not been confirmed yet. An electronic search was therefore conducted in MEDLINE‐Pubmed to identify all papers published in English connecting archaea and periodontal infections. Data analysis of the selected studies showed that five genera of methanogenic archaea have been detected in the subgingival microbiota, Methanobrevibacter oralis being the most frequently detected species in 41% of periodontitis patients and 55% of periodontal pockets compared to 6% of healthy subjects and 5% of periodontally‐healthy sites (p < 10−5, Chi‐squared test). Based on the five determination‐criteria proposed by Socransky (association with disease, elimination of the organism, host response, animal pathogenicity and mechanisms of pathogenicity), M. oralis is a periodontal pathogen. The methanogenic archaea load correlating with periodontitis severity further supports the pathogenic role of methanogenic archaea in periodontitis. Therefore, detection and quantification of M. oralis in periodontal pockets could help the laboratory diagnosis and follow‐up of periodontitis. Determining the origin, diversity and pathogenesis of archaea in periodontal infections warrants further investigations.

Real-time PCR quantification of Methanobrevibacter oralis in periodontitis

ABSTRACT A real-time PCR assay developed to quantify Methanobrevibacter oralis indicated that its inoculum significantly correlated with periodontitis severity (P = 0.003), despite a nonsignificant difference in prevalence between controls (3/10) and patients (12/22) (P = 0.2, Fisher test). The M. oralis load can be used as a biomarker for periodontitis.

Peri-implantitis: from diagnosis to therapeutics

Peri-implantitis is an infection of the tissue around an implant, resulting in the loss of supporting bone. Risk factors for peri-implantitis consist of a history of periodontitis, dental plaque, poor oral hygiene, smoking, alcohol consumption and diabetes. A clinical diagnosis indicates inflammatory signs including bleeding on probing with or without suppuration and a peri-implant pocket depth ≥5 mm. A radiograph shows images of marginal bone loss ≥2 mm. A differential diagnosis of peri-implant mucositis, occlusal overload, retrograde peri-implantitis and inflammatory implant periapical lesions suggests the appropriate treatment in each case. The non-surgical treatment of peri-implantitis, including a mechanical treatment alone or combined with antiseptics or antibiotics can improve clinical parameters in the short term but residual defects may still persist. Surgical treatment such as guided bone regeneration results in a gain of clinical attachment level and bone reconstruction in the long term. The limited effect of laser-assisted therapy needs to be further evaluated. The concept of prevention based on early detection and regular maintenance plays a principal role in reducing the occurrence of peri-implantitis.

Heat degradation of eukaryotic and bacterial DNA: an experimental model for paleomicrobiology

BackgroundTheoretical models suggest that DNA degradation would sharply limit the PCR-based detection of both eukaryotic and prokaryotic DNA within ancient specimens. However, the relative extent of decay of eukaryote and prokaryote DNA over time is a matter of debate. In this study, the murine macrophage cell line J774, alone or infected with Mycobacterium smegmatis bacteria, were killed after exposure to 90°C dry heat for intervals ranging from 1 to 48 h in order to compare eukaryotic cells, extracellular bacteria and intracellular bacteria. The sizes of the resulting mycobacterial rpo B and murine rpb 2 homologous gene fragments were then determined by real-time PCR and fluorescent probing.FindingsThe cycle threshold (Ct) values of PCR-amplified DNA fragments from J774 cells and the M. smegmatis negative controls (without heat exposure) varied from 26–33 for the J774 rpb 2 gene fragments and from 24–29 for M. smegmatis rpo B fragments. After 90°C dry heat incubation for up to 48 h, the Ct values of test samples increased relative to those of the controls for each amplicon size. For each dry heat exposure time, the Ct values of the 146-149-bp fragments were lower than those of 746-747-bp fragments. During the 4- to 24-h dry heat incubation, the non-infected J774 cell DNA was degraded into 597-bp rpb 2 fragments. After 48 h, however, only 450-bp rpb 2 fragments of both non-infected and infected J774 cells could be amplified. In contrast, the 746-bp rpo B fragments of M. smegmatis DNA could be amplified after the 48-h dry heat exposure in all experiments. Infected and non-infected J774 cell DNA was degraded more rapidly than M. smegmatis DNA after dry heat exposure (ANOVA test, pu2009<u20090.05).ConclusionIn this study, mycobacterial DNA was more resistant to dry-heat stress than eukaryotic DNA. Therefore, the detection of large, experimental, ancient mycobacterial DNA fragments is a suitable approach for paleomicrobiological studies.

The repertoire of archaea cultivated from severe periodontitis.

In previous studies, the abundance and diversity of methanogenic archaea in the dental microbiota have been analysed by the detection of specific DNA sequences by PCR-based investigations and metagenomic studies. Few data issued regarding methanogens actually living in dental plaque. We collected dental plaque specimens in 15 control individuals and 65 periodontitis patients. Dental plaque specimens were cultured in an anoxic liquid medium for methanogens in the presence of negative control tubes. Dental plaque methanogens were cultured from 1/15 (6.67%) control and 36/65 (55.38%) periodontitis patient samples (p<0.001). The cultures yielded Methanobrevibacter oralis in one control and thirty-one patients, Methanobrevibacter smithii in two patients and a potential new species named Methanobrevibacter sp. strain N13 in three patients with severe periodontitis. Our observations of living methanogens, strengthen previous observations made on DNA-based studies regarding the role of methanogens, in periodontitis.

Diversity of Human-Associated Methanobrevibacter smithii Isolates Revealed by Multispacer Sequence Typing

Methanobrevibacter smithii is the main archaea in human, detoxifying molecular hydrogen resulting from anaerobic bacteria fermentations into gaseous methane. Its identification relies on gene sequencing, but no method is available to discriminate among genetic variants of M. smithii. Here, we developed a multispacer sequence typing (MST) for genotyping the genetic variants of M. smithii. Four intergenic spacers recovered from the M. smithii reference genome were PCR amplified and sequenced in three M. smithii reference strains and in a collection of 22 M. smithii isolates from the oral cavity in two individuals and the gut of 10 additional individuals. Sequencing yielded 216 genetic polymorphisms including 89 single nucleotide polymorphisms (41.2xa0%), 83 insertions (38.4xa0%), and 44 deletions (20.4xa0%). Combining these genetic polymorphisms yielded 15 genotypes with an index of discrimination of 0.942 (confidence interval 0.9–0.984; Pxa0<xa00.05). Five M. smithii isolates made from the oral cavity yielded five different genotypes; seven gut isolates yielded nine different genotypes; genotypes MST5 and MST6 were found both in the oral cavity and the gut. Multiple genotypes were identified in some individuals at the same anatomical site. MST is a sequencing-based method which discriminates several genetic variants within M. smithii. Individuals may harbor several contemporary genetic variants of M. smithii in the oral cavity and gut. MST will allow studying population dynamics of M. smithii and tracing its circulation between individuals and their environment.

Bacteria and archaea paleomicrobiology of the dental calculus: a review.

Dental calculus, a material observed in the majority of adults worldwide, emerged as a source for correlating paleomicrobiology with human health and diet. This mini review of 48 articles on the paleomicrobiology of dental calculus over 7550xa0years discloses a secular core microbiota comprising nine bacterial phyla - Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, TM7, Synergistetes, Chloroflexi, Fusobacteria, Spirochetes - and one archaeal phylum Euryarchaeota; and some accessory microbiota that appear and disappear according to time frame. The diet residues and oral microbes, including bacteria, archaea, viruses and fungi, consisting of harmless organisms and pathogens associated with local and systemic infections have been found trapped in ancient dental calculus by morphological approaches, immunolabeling techniques, isotope analyses, fluorescent inxa0situ hybridization, DNA-based approaches, and protein-based approaches. These observations led to correlation of paleomicrobiology, particularly Streptococcus mutans and archaea, with past human health and diet.

Paleomicrobiology of Bartonella infections.

Studying ancient infectious diseases is a challenge, as written contemporary descriptions, when available, are often imprecise and do not allow for accurate discrimination among the pathogens endemic at that time. Paleomicrobiology offers a unique access to the history of these infections by identifying precisely the causative agents. Body louse-transmitted infections are amongst the most epidemic diseases in history, especially in war and famine periods. Of these, Bartonella quintana was detected by suicide PCR in 4000-year-old human remains, thus representing the oldest evidence to date of an arthropod-transmitted infection to human beings. This species has also been detected in human specimens from the 11th to 15th, 18th and 19th centuries. In addition, Bartonella henselae, a cat- and flea-associated pathogen, was detected in cat specimens from the 13th to 18th centuries, therefore demonstrating an association of the bacterium and its reservoir for over 800 years. Therefore, pathogenic Bartonella species have been involved in several outbreaks in the past millennia and should systematically be investigated in human remains from suspected epidemics.

Historical and geographical parallelism between the incidence of dental caries, Streptococcus mutans and sugar intake

Currently, the distribution of dental caries in patients over 35xa0years can be super-imposed to that of the countries where the food industry has developed, with, in particular the addition of sugar in the food diet. Thus, countries with a very high incidence are Western Europe, North America, Australia, Brazil, Chile and Peru. However, in Africa, South and East Asia the incidence is much lower [1]. This will tend to also self correct as preventive measures against dental caries have been taken in children. n nInterestingly, one can note that dental caries are associated with a particular oral microbiota associated with said cariogenic bacteria, including Streptococcus mutans. We have been working for many years on paleomicrobiology which is the study of the diseases of the past by identifying antigens or DNA trapped bacteria within the dental pulp [2]. A recent work of paleo-microbiology from another team has demonstrated the increased presence of cariogenic bacteria over time [3]. Indeed, the work has focused on the systematic analysis of tartar and dental plaque in skeletons dating from the Mesolithic in our time through the Neolithic, Bronze Age and the Middle-Ages. By studying the PCR amplified sequences of 16S rDNA, a microbiote complex preserved until the nineteenth century was identified. From the nineteenth century, a very sharp decline in the biodiversity of dental bacteria has been observed as well as the onset of the oral microbiota associated with the presence of caries with a predominance of S. mutans. This is contemporary to the industrial age and to the sharp increase in the consumption of sugar and sugary drinks, of industrialized countries. n nWe have reanalyzed the teeth that we have received for paleomicrobiological analysis, especially that of the eighteenth and early nineteenth century. When comparing the oral health status of these skeletons we will find that during the pre-industrial period teeth were in a better condition as out of 110 adults died of the plague in Martigues near Marseille, in a small town in 1720 during the great Plague of Marseille, 12.6xa0% presented a carious lesion. Out of 113 adults died of the plague in Marseilles in 1,722, 11.69xa0% of the teeth had carious lesions. Finally, among 146 adults, male only, soldiers of the Grand Army and died in Vilnius in 1,812 only, 8.1xa0% had at least one carious lesion. In these three cases, the prevalence of caries was lower than it is now recognized in France in patients over 35xa0years. Another study showed that England was the first country to industrialize the production of sugar, was the first country to experience an increase in the proportion of dental caries [4]. Thus, in the early nineteenth century the English had more dental caries than the American. n nUnder such conditions, there is an obvious historical link between the dramatic increase in the prevalence of dental caries, and that of S. mutans. The latter appears as the consequence of food globalization and industrialization which translated into an increase in the consumption of added sugar in foods. This resulted in an increase in caries in the nineteenth century in industrialized countries. In the twentieth and twenty first century, the number of caries is increasing in the countries and regions affected most recently by globalization [5]. This should be an element adding to the debate on the prevention of excessive sugar intake in children to prevent both obesity and dental caries. n nFinally, anthropological studies as well as paleomicrobiological works appear to contribute to explaining the current public health problems by analyzing changes in our microbiota, alimentation, bone and teeth lesions.

Tracing back ancient oral microbiomes and oral pathogens using dental pulps from ancient teeth

Ancient dental pulps are highly precious samples because they conserve DNA from humans and blood-borne pathogens for ages. However, little is known about the microbial communities present in dental pulps. Here, we analyzed ancient and modern dental pulp samples from different time periods and geographic regions and found that they are colonized by distinct microbial communities, which can be differentiated from other oral cavity samples. We found that despite the presence of environmental bacteria, ancient dental pulps conserve a clear and well-conserved record of oral microbes. We were able to detect several different oral pathogens in ancient and modern dental pulps, which are commonly associated with periodontal diseases. We thus showed that ancient dental pulps are not only valuable sources of DNA from humans and systemic infections, but also an open window for the study of ancient oral microbiomes.