Détection Et Culture Des Archaea Associées Aux Muqueuses

AIX-MARSEILLE UNIVERSITE

FACULTE DE MEDECINE DE MARSEILLE

ECOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE

THESE DE DOCTORAT

Présentée par

Saber KHELAIFIA

En vue de l'obtention du grade de Docteur de l'Université Aix-Marseille

Spécialité: Maladies Transmissibles et Pathologies Tropicales

Détection et culture des archaea associées aux muqueuses

intestinale et orale humaines

Soutenue le 07 Juin 2013

COMPOSITION DU JURY

Mr le Professeur Jean-Louis Mège Président du jury

Mr le Docteur Bruno Franzetti Rapporteur

Mr le Professeur Max Maurin Rapporteur

Mr le Professeur Michel Drancourt Directeur de thèse

Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UMR CNRS6236

Prof – Didier Raoult

2013 Avant propos

Le format de présentation de cette thèse correspond à une recommandation de la spécialité

Maladies Infectieuses et Microbiologie du Master des Sciences de la Vie et de la Santé qui dépend de l’Ecole Doctorale des Sciences de la Vie de Marseille. Le candidat est amené à respecter des règles qui lui sont imposées et qui comportent un format de thèse utilisé dans le

Nord de l’Europe et qui permet un meilleur rangement que les thèses traditionnelles. Par ailleurs, la partie introduction et bibliographie est remplacée par une revue publiée dans un journal scientifique afin de permettre une évaluation extérieure de la qualité de la revue et de permettre à l’étudiant de commencer le plus tôt possible une bibliographie exhaustive sur le domaine de cette thèse. Par ailleurs, la thèse est présentée sur article publié, accepté ou soumis associé d’un bref commentaire donnant le sens général du travail. Cette forme de présentation a paru plus en adéquation avec les exigences de la compétition internationale et permet de se concentrer sur des travaux qui bénéficieront d’une diffusion internationale.

Prof. Didier Raoult

Sommaire

Résumé ...... …..p.01

Abstract ...... ….p.05

Introduction ...... p.08

Chapitre.1: Methanogenic archaea in subgingival sites: a review…...... …....p.13

Chapitre.2: Susceptibility of Archaea to antimicrobial agents: applications to clinical microbiology...... ……...p.26

Chapitre.3: A semi-automated protocol for Archaea DNA extraction from stools……….p.36

Chapitre.4: Real-time PCR quantification of Methanobrevibacter oralis in periodontitis...... p.5

Chapitre.5: Tungsten-enhanced growth of Methanosphaera stadtmanae….…….....……..p.63

Chapitre.6: A versatile medium for cultivating methanogenic archaea…...... p.69

Chapitre.7: Culturomics reveals hidden intestinal archaea...... ……..p.76

Chapitre.8: Imidazole derivatives hydrophobicity correlates with improved activity against human methanogenic archaea ……………...…………………………………………...... p.89

Chapitre.9: In-vitro archaeacidal activity of biocides against human-associated archaea...... p.95

Conclusion et perspectives...... p.101

Références ...... …....p.104

______Résumé

Résumé

Les archaea constituent l’un des quatre domaines connus du vivant. Contrairement à ce que leur nom laisse supposer, elles ont colonisé tous les écosystèmes et les microbiotes de certains hôtes dont l’Homme. Chez l’homme, certaines espèces d’archaea méthanogènes ont

été associées aux muqueuses orale, intestinale et vaginale. Ces archaea méthanogènes sont des procaryotes anaérobies stricts et leurs conditions de culture restent fastidieuses et très mal connues. Quatre archaea methanogènes seulement ont été isolées à partir de prélèvements humains y compris dans le microbiote digestif Methanobrevibacter smithii détectée dans

95,7% des individus, Methanosphaera stadtmanae retrouvée chez environ un tiers des individus et plus récemment dans notre laboratoire Methanomassilicoccus luminyensis détectée en moyenne chez 4% des individus avec une prévalence liée à l'âge ; et dans le microbiote orale Methanobrevibacter oralis isolée à partir de la plaque dentaire.

Après l’isolement de M. luminyensis, nous nous sommes intéressés à l’importance des

éléments nutritifs et des facteurs de croissance dans la culture des archaea méthanogènes de l’homme et notre travail de these a porté sur les techniques d’isolement et de culture de ces microorganismes fastidieux, et leur sensibilité aux biocides et aux agents antimicrobiens.

Nous avons testé l’effet d’une solution de tungstate/sélénate sur une culture de M. stadtmanae qui présente un métabolisme similaire à celui de M. luminyensis. Toutes les deux produisent du méthane en réduisant le méthanol en présence d’hydrogène comme donneur d’électrons.

Dans ces conditions, nous avons observé une accélération de la vitesse de croissance de M. stadtmanae par un facteur 3.

Les observations citées précédemment nous ont incités à nous intéresser à l’optimisation d’un milieu de culture polyvalent avec pour objectif de cultiver toutes les archaea méthanogènes de l’Homme en utilisant un seul milieu. Les résultats obtenus ont

______Résumé montré que non seulement il était possible d’utiliser un seul milieu de culture polyvalent pour cultiver toutes les archaea méthanogènes de l’Homme mais que ce milieu améliorait la croissance de toutes les souches testées en réduisant leur temps de génération.

En outre, basée sur la détection moléculaire par PCR, d’autres espèces d’archaea ont

été détectées chez l’Homme. Methanobrevibacter arboriphilicus et Methanosaeta concilii ont

été détectées dans des biopsies de la muqueuse intestinale en association avec certaines archaea halophiles, mais aucune souche n’a été cultivée. Les connaissances actuelles sur la diversité des archaea méthanogènes associées à l’homme et le rôle potentiel qu’elles ont sur la santé humaine restent limitées aux seules informations apportées par les techniques de détection moléculaire par PCR. Ces techniques fondées sur la détection de l’ADN ribosomal

16S et du gène mcrA codant la sous-unité alpha du methyl-coenzyme M reductase, ont montré que M. smithii colonisait le tube digestif de 95,7% des individus et M. stadtmanae de 29,4 % des individus. Ces techniques ne reflètent pas la diversité des archaea qui colonisent le tube digestif et la cavité buccale, suggérant ainsi la mise au point de nouvelles méthodes de détection moléculaire et de culture adaptées aux caractéristiques des parois de ces organismes fastidieux. Dans ce travail, nous nous sommes fixés comme objectif de mettre au point une méthode de détection moléculaire basée sur l’extraction et la détection par PCR de l’ADN ribosomal 16S à partir des selles en prenant comme témoin d’extraction M. smithii présente chez tous les individus. Ce protocole nous a permis d’obtenir des résultats comparables à ceux déjà publiés en termes de prévalence mais aussi il nous a permis d’augmenter considérablement le rendement d'extraction de l'ADN d’archaea à partir des selles humaines et de diminuer la charge de travail. En utilisant ce protocole et moyennant une approche moléculaire basée sur une PCR universelle ciblant l’ADN ribosomal 16S, le séquençage et le clonage, nous avons également détecté Methanobrevibacter arboriphilicus,

______Résumé

Methanobrevibacter oralis et Methanobrevibacter millerae pour la première fois dans des selles humaines.

Nous avons procédé par la suite à l’isolement de ces souches par les techniques d’anaérobie mises au point par Hungate en 1969 sous une atmosphère composée de 80% H2 et

20% CO2 nécessaire à la croissance des archaea méthanogènes. Ce travail a permis d’élargir de trois à sept le nombre des archaea cultivées du tube digestif humain.

Concernant la muqueuse orale, les archaea méthanogènes ont été impliquées dans la parodontite depuis plus de deux décennies et M. oralis est considérée comme l'archaea méthanogène dominante au cours de cette infection. Cependant, les études antérieures n’avaient pas quantifié avec précision M. oralis dans les poches parodontales et la corrélation de M. oralis avec la sévérité de la parodontite n’était pas démontrée. Dans cette étude, nous avons cherché à corréler la sévérité de la parodontite avec la charge de M. oralis quantifiée par PCR en temps réel dans des prélèvements de plaques sous-gingivale de patients atteints de parodontites et de sujets sains avec la sévérité de la parodontite. Les résultats obtenus indiquent qu'il est possible de mesurer la charge de M. oralis dans la plaque sous-gingivale à l'aide de PCR en temps réel et de corréler cette charge avec la sévérité de la parodontite. Cette

étude a en outre établi le concept que la charge de M. oralis dans les poches parodontales correspond à un score standard de sévérité de la parodontite. La charge accrue chez les malades peut permettre son utilisation comme biomarqueur du microbiote altéré en surveillant la charge de M. oralis.

Dans une dernière partie de notre travail, nous avons étudié l’effet de certains agents antimicrobiens sur des cultures d’archaea méthanogènes de l’Homme. Les archaea méthanogènes sont caractérisées par leur large spectre de résistance aux agents antimicrobiens. Seule une sensibilité au metronidazole, à la squalamine ainsi qu’à la fucidine ont été décrites. Dans cette étude, nous avons testé 10 dérivés imidazole pour leur cytotoxicité

______Résumé in-vitro ainsi que leur activité anti-archaea contre six archaea méthanogènes, y compris quatre méthanogènes isolées chez l’Homme. Ces résultats indiquent un index thérapeutique de 20-

400 pour ces composés par rapport au métronidazole. Ces composés sont donc des molécules prometteuses pour le traitement des infections liées au archaea méthanogènes.

Dans cette même perspective, nous avons aussi testé 10 dérivés de la squalamine dans un but de décontamination du matériel médical qui rentre en contact avec les archaea méthanogènes lors d’un examen coloscopique. Ces résultats indiquent que la squalamine et ses dérivés, en particulier le dérivé S1 sont des biocides prometteurs pour la décontamination des instruments en milieu hospitalier.

Mots-clés : Archaea méthanogènes, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanomassilicoccus luminyensis, Methanobrevibacter oralis,

Methanobrevibacter arboriphilicus, Methanobrevibacter millerae, microbiote humain, culture anaérobie, parodontite, agents anti-archaea, tests de sensibilité, activité in-vitro, metronidazole, biocides, squalamine.

______Abstract

Abstract

Archaea are unicellular microorganisms known to colonize essentially extreme environments.

In human, some methanogenic archaea have been associated with the intestinal, vaginal and oral mucosa. These organisms are strictly anaerobic and their culture conditions are fastidious and poorly known.

So far, only four methanogenic archaea were isolated from human including

Methanobrevibacter smithii detected in 95.7% of individuals Methanosphaera stadtmanae found in about one third of individuals and more recently Methanomassilicoccus luminyensis in average 4% individuals with an age-dependent prevalence and Methanobrevibacter oralis isolated from dental plaque. After the isolation of M. luminyensis, we are interested to the importance of nutrients and growth factors in the human methanogenic archaea culture. In this context, we tested the effect of a tungstate / selenate solution on a culture of M. stadtmanae.

M. stadtmanae has a similar metabolism to that of M. luminyensis, both reduce methanol to methane using hydrogen as an electron donor. We observed an acceleration of the growth rate of M. stadtmanae by a factor of 3.

These observations prompted us to take an interest in the optimization of a versatile culture medium that will allow cultivating all human methanogenic archaea in this same medium. The results showed that it was possible to cultivate human methanogenic archaea in a single versatile culture medium and this medium also improves the growth of all strains tested by reducing their doubling time.

Moreover, based on the molecular detection by PCR, other species of archaea were detected in humans. Methanobrevibacter arboriphilicus and Methanosaeta concilii were detected in biopsies of the intestinal mucosa in association with some halophilic archaea, but no strain was cultivated. Current knowledge about the diversity of human-associated

______Abstract methanogens thier potential role in human diseases remained limited to the only information provided by the molecular-based detection by PCR. These techniques based on 16S ribosomal

DNA and mcrA gene (encoding the alpha subunit of methyl coenzyme-M-reductase, a key enzyme in methanogenesis process) detection, showed that M. smithii colonized 95.7% of individuals and of M. stadtmanae in 29.4%. These techniques can not reflect the diversity of archaea that colonize the human gut and its oral cavity, suggesting the development of new molecular detection methods and culture adapted to the characteristics of the walls of these fastidious organisms.

In this work, we set the objective to develop a molecular detection method based on the extraction and detection by PCR of 16S ribosomal DNA from human. This protocol allowed obtaining similar results to those already published in terms of prevalence but it also significantly increase the efficiency of DNA extraction of archaea from human feces and reduce workload. Using this protocol and with a molecular approach based on PCR Universal

16S ribosomal DNA detection, sequencing and cloning, we also detected M. arboriphilicus,

M. oralis and M. millerae for the first time in human feces.

We proceeded the isolation of these strains using the anaerobic techniques developed by Hungate in 1969 under an atmosphere of 80% H2 and 20% CO2 required for the growth of methanogenic archaea. This work allowed broadening the knowledge about human-associated archaea diversity limited to the four cultivated species.

Methanogenic archaea have been implicated in periodontitis for over two decades and

M. oralis is the dominant methanogenic archaea in such an infection. It has recently attracted attention because of new increasing evidence of their involvement in human periodontal disease. Previous studies of methanogenic archaea in periodontitis did not accurately quantify

M. oralis or did not specify M. oralis. Therefore, the correlation of M. oralis with the severity of periodontitis remained problematic. In this study, we sought to correlate the of M. oralis

______Abstract charge quantified by real-time PCR in subgingival plaque with the severity of periodontitis.

The results indicate that it is possible to measure the charge of M. oralis in subgingival plaque using real-time PCR. This study has also established the concept that the M. oralis charge in periodontal pockets corresponding to a standard score of severity of periodontitis. Increased load in patients may indicate its use as a biomarker of altered microbiota by monitoring M. oralis load.

We then studied the effect of some antimicrobial agents on human methanogenic archaea cultures. Methanogenic archaea are characterized by their broad spectrum of antimicrobial resistance. Only sensitivity to metronidazole, squalamine and the fucidin has been described. In this study, we synthesized 10 imidazole derivatives and tested their in-vitro cytotoxicity and their anti-archaea activity against six archaea strains, including the four human-assiciated methanogenic. These results indicate a therapeutic index of 20-400 for these compounds compared to metronidazole. These compounds are promising compounds for the treatment of infections related to methanogenic archaea.

In the same perspective, we also synthesized 10 derivatives of squalamine in the interests of decontamination of medical device that comes into contact with methanogenic archaea during a colonoscopic examination. These results suggest that squalamine and its derivatives such as derived S1 are promising biocides for decontamination of archaea in the hospital setting.

Keywords: methanogenic archaea, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanomassilicoccus luminyensis, Methanobrevibacter oralis, human microbiota, anaerobic culture, DNA extraction, periodontitis, anti-archaea, sensitivity tests, in vitro activity, metronidazole, biocides, squalamine.

______Introduction

Introduction

Les archaea sont des micro-organismes unicellulaires initialement détectées dans des environnements extrêmes, dont on pensait qu’ils mimaient les conditions d’environnement au moment de l’apparition de la vie sur terre [1]. Bien que la taille et la forme des archaea soient similaires à celles des bactéries, certaines archaea présentent une morphologie inhabituelle telle que Ignicoccus hospitalis qui ressemble à un eucaryote unicellulaire de petite taille [2] et

Haloquadra walsbyi qui possède une forme carrée [3] (Figure 1).

Figure 1 : Photographies en microscopie électronique et optique illustrant certaines morphologies des archaea.

(A) Image en microscopie électronique à transmission d’une coupe ultra-fine de

Ignicoccus hospitalis [2]. La barre d'échelle représente 1 µm.

(B) Image en microscopie optique de Haloquadra walsbyi [3]. La barre d'échelle

représente 5 µm.

Les archaea, anciennement nommées les archaebactéries, ont été classées en premier lieu parmi les bactéries dans le groupe des procaryotes. Néanmoins, certains caractères ont amené

à reconsidérer ce classement. D’une part, la structure lipidique de leur membrane est complètement différente de celle des autres organismes; les lipides membranaires des archaea

______Introduction consistent en de longues chaînes d'alcool isopréniques attachées au glycérol par des liaisons

éther, alors que les autres organismes fabriquent les lipides de leurs membranes en assemblant deux chaînes d'acides gras avec une molécule de glycérol par l'intermédiaire d'une liaison ester [4] (Tableau 1). D’autre part, la transcription et la traduction sont plus proches chez les archaea de celles des eucaryotes que de celles des bactéries.

Tableau 1 : Les différences et similitudes entre les archaea et les bactéries.

______Introduction

Figure 2 : Arbre phylogénétique indiquant la position des séquences du gène codant l’ARN ribosomal 16S (ARNr) des archaea dont le G+C% est répertorié dans GOLD genomes on line data base (http://www.genomesonline.org/cgi-bin/GOLD/Search.cgi). En vert les espèces dont le

G+C% est inférieur ou égal à 35%, en bleu les espèces dont le G+C% est compris entre 36% et 45%, en rose les espèces dont le G+C% est compris entre 46% et 65% et en rouge les espèces dont le G+C% est supérieur à 65%

______Introduction

La classification phylogénétique basée sur la séquence de l’ADN ribosomal 16S a confirmé l’appartenance des archaea à un domaine de la vie différent des bactéries, des eucaryotes et des virus géants à ADN [5,6]. Ce domaine a été divisé en quatre phyla :

Euryarchaeota et Crenarchaeota qui incluent la plupart des archaea cultivées, ainsi que deux nouveau phyla Nanoarchaeota et Korarchaeota qui comporte chacun une seule espèce cultivée (Figure 2). Dans cette grande diversité, les archaea méthanogènes qui appartiennent au phylum Euryarchaeota sont associées à des hôtes, y compris les mammifères dont l’Homme. Nous avons remarqué à la suite des travaux de Miller [7] que certaines de ces archaea ont un G+C % ≤ 38% (Figure 2).

Les archaea méthanogènes font partie de plusieurs microbiotes humains et ont été impliquées dans quelques pathologies telles que la parodontite [8–10] et l'obésité [11,12].

Lorsque nous avons débuté notre travail de thèse, le microbiote intestinal humain ne comptait que trois archaea méthanogènes cultivées, Methanobrevibacter smithii [13], Methanosphaera stadtmanae [14] et plus récemment dans notre laboratoire, Methanomassilicoccus luminyensis le premier représentant d’un nouveau genre d’archaea méthanogène [15]. Une autre archaea méthanogène Methanobrevibacter oralis [16] avait été isolée à partir de la plaque dentaire. En outre, par PCR, Methanobrevibacter arboriphilicus [17] et Methanosaeta concilii [18] avaient

été détectées dans des biopsies de la muqueuse intestinale en association avec certaines archaea halophiles [19], mais aucune souche n’avait été cultivée chez l’Homme. Récemment, de nouvelles espèces d’archaea méthanogènes phylogénétiquement affiliées aux

Thermoplasmatales ont été détectées dans des plaques sous-gingivales humaines [20].

L'isolement des archaea méthanogènes à partir des microbiotes humains reste une tâche fastidieuse en raison de la croissance lente de telles archaea et leur intolérance extrême à l'oxygène [1]. En conséquence, une atmosphère constituée de 80% H2 et 20% de CO2 est l'une des exigences spécifiques pour la croissance optimale de ces archaea [15]. Cependant, la

______Introduction maîtrise des techniques de culture anaérobie ne semble pas suffisant pour isoler et cultiver de tels microorganismes, des connaissances supplémentaires sur les besoins nutritifs ainsi que des facteurs de croissance sont utiles pour la conception de nouveaux milieux de culture polyvalents afin d’isoler et de cultiver de nouvelles espèces d’archaea méthanogènes [21–26].

Quant à leur rôle dans les pathologies, plusieurs éléments impliquent les archaea dans les parodontites [12,20,27]. Par conséquent, nos investigations récentes ont montré une relation significative entre la sévérité de la parodontite chronique et l'abondance relative de

Methanobrevibacter oralis dans le microbiote sous-gingival [9].

Etant donné que le rôle potentiel des archaea en pathologie humaine, il est utile de déterminer leur sensibilité aux biocides et aux agents antimicrobiens. En effet, les archaea sont caractérisées par leur large spectre de résistance aux agents antimicrobiens à des concentrations supérieures à 100 mg/L [28]. Le métronidazole et la squalamine sont parmi les seuls agents antimicrobiens ayant démontré une activité anti-archaea [28]. Ces composés sont

également actifs contre les bactéries anaérobies, y compris les bactéries anaérobies du microbiote sous-gingival [10,29].

L'environnement est une source possible de l’acquisition des archaea méthanogènes par l’homme; néanmoins, leurs niches écologiques précises, les voies d'acquisition et leur rôle potentiel dans les pathologies humaines demeurent inconnus. Le développement de nouvelles méthodes d'identification et de culture de ces organismes particuliers et exigeants à partir d'échantillons cliniques est donc nécessaire. Cela permettra d’isoler de nouvelles espèces pour les caractériser phénotypiquement, d'explorer leur génome par séquençage et d’étudier la dynamique des populations notamment au cours des pathologies pour préciser leur rôle exact au sein des flores complexes associées aux muqueuses de l'homme.

______Chapitre 1

Chapitre 1

Methanogenic archaea in subgingival sites: a review

Tung Nguyen-Hieu 1,2, Saber Khelaifia1, Gérard Aboudharam 1,2, Michel Drancourt 1

1 Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UMR CNRS

6236, IRD198, Méditerranée Infection, Aix-Marseille Université, 27 boulevard Jean Moulin,

13005 Marseille, France.

2 UFR Odontologie, Université de la Méditerranée, 27 boulevard Jean Moulin, 13005

Marseille, France.

Key words: Methanogenic archaea; diversity; periodontitis; severity; periodontal

APMIS (2012)

______Chapitre 1

Chapitre 1 : Préambule

Au cours de cette thèse, nous avons fait le point des connaissances concernant l’implication des archaea méthanogènes dans les pathologies orales. Les archaea méthanogènes constituent une partie intégrante de la flore buccale chez l’homme, mais leur rôle dans les pathologies orales restent controversé. Plusieurs travaux dans la littérature ont rapporté la détection moléculaire des archaea méthanogènes dans les parodontites, mais cette association n'a pas été confirmée. Une recherche bibliographique a donc été effectuée dans

MEDLINE et Pubmed pour réunir l’ensemble des travaux publiés sur les archaea et les infections parodontales. L'analyse des données des études sélectionnées a montré que cinq genres d’archaea méthanogènes ont été détectés dans le microbiote sous-gingival,

Methanobrevibacter oralis étant l'espèce la plus fréquemment détectées chez 41% des patients atteints de parodontite et 55% des poches parodontales, contre 6% chez les sujets sains et 5 % dans des sites parodontaux sains (p <105). La corrélation entre la charge en archaea méthanogènes et la sévérité de la parodontite soutient en outre le rôle pathogène des archaea méthanogènes dans les parodontites. Par conséquent, la détection et la quantification de M. oralis dans les poches parodontales pourrait aider au diagnostic de laboratoire et le suivi de la parodontite.

Methanogenic archaea in subgingival sites: a review

TUNG NGUYEN-HIEU,1,2 SABER KHELAIFIA,1 GERARD ABOUDHARAM1,2 and MICHEL DRANCOURT1

1URMITE, UMR63, CNRS 7278, IRD 198, Inserm 1095, Aix-Marseille Universite´, Marseille; and 2UFR Odontologie, Aix-Marseille Universite´, Marseille, France

Nguyen-Hieu T, Khelaifia S, Aboudharam G, Drancourt M. Methanogenic archaea in subgingival sites: a review. APMIS 2012. 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 < 10À5, 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. Key words: Methanogenic archaea; diversity; periodontitis; severity; periodontal pathogen. Michel Drancourt, Unite´des Rickettsies, Faculte´de Me´decine, 27, Boulevard Jean Moulin, 13385, Marseille Cedex 05, France. e-mail: [email protected]

Archaea were recognized as a separate group isolation of a first human intestinal methano- of prokaryotes after ribosomal RNA (rRNA) genic archaea Methanobrevibacter smithii (6). gene sequencing (1). Unlike bacteria, archaea Two other species Methanosphaera stadtmanae lack cell wall peptidoglycan and instead con- (7) and Methanomassiliicoccus luminyensis (8) tain pseudopeptidoglycan (2). Bacteria and were further isolated from the human gut mic- eukaryotes have membranes composed mainly robiota. M. smithii is the dominant methanogen of glycerol-ester lipids whereas archaeal mem- in the human gut, detected in 95.7% of individu- branes possess glycerol-ether lipids (3). Also, als whereas M. stadtmanae and M. luminyensis archaea are genetically distinct from bacteria are detected in 29.4% and 4% of individuals and eukaryotes (4). Accordingly, archaea were respectively (9, 10). After unidentified methano- recently classified as one of the four primary genic archaea were cultured from the human domains of life besides Viruses, Bacteria and subgingival plaque (11) and periodontal pock- Eukaryotes (5). The observation that human ets of periodontitis patients (12), Methanobrev- breathing released methane gave way to the ibacter oralis isolated from human subgingival plaque was proposed as a new archaeal species Received 20 July 2012. Accepted 20 September 2012 characterized as a coccobacillary, non-motile,

1 NGUYEN-HIEU et al.

Gram-positive, methane-producing microorgan- or methanogens, samples taken from periodon- ism (13). The introduction of molecular meth- tal pockets or subgingival plaque. Exclusion cri- ods expanded the knowledge of the subgingival teria comprised articles published in non- microbiota and the importance of yet fastidious English, samples collected from endodontic microorganisms such as methanogenic archaea infections or from periapical lesions, but no associated with infected sites (14, 15). This sample taken from subgingival sites. In the sec- increased awareness of a higher diversity and ond phase of selection, full-text of selected complexity of the subgingival flora suggested papers and full-text of papers without abstract the possibility of identifying new pathogenic or with insufficient data in the title and abstract species (16). The role of methanogenic archaea, to allow a previous selection were downloaded chiefly M. oralis in the severity of periodontitis for reading. From references of these papers, remained, however, controversial as discrepant we carried-out a manual search and added data issued from studies using different sam- appropriate articles. We analysed the data of all pling protocols and laboratory protocols for selected papers for assessing the diversity of ar- the detection and identification of this microor- chaea in subgingival sites in periodontitis ganism. According to the Seventh European patients and healthy subjects as well as the Workshop on Periodontology, periodontal dis- association of methanogenic archaea with peri- eases are the pathological manifestations of the odontitis and its severity. Chi-squared test was host response against the bacterial challenge used for comparison of prevalences and the dif- from dental biofilm at the tooth/gingival inter- ference was statistically significant with value face (17). Gingivitis is a chronic inflammatory p < 0.05. response to the accumulation of supragingival biofilm, whereas periodontitis is a chronic inflammatory disease that results from a complex ANALYSIS OF STUDIES polymicrobial infection, leading to tissue destruction (17). Likewise, peri-implant mucositis is There was a total of 16 studies reporting the described as an inflammatory lesion that resides detection of methanogenic archaea in subgingi- in the mucosa, whereas peri-implantitis also val sites (Table 1). These studies included 8– affects the supporting bone (18). The purpose of 167 subjects aged 16–74 years old with male/ this review was to assess the association of meth- female ratio ranging from 1/2.3 to 1.1/1. Peri- anogenic archaea with periodontal diseases. odontal infections included gingivitis, periodontitis, pericoronitis and peri-implantitis. Six studies incorporated a control group of SEARCH AND SELECTION STRATEGY periodontally healthy subjects and six studies classified the severity of periodontitis accord- The literature search was performed using elec- ing to American Dental Association (ADA) tronic databases MEDLINE via Pubmed for periodontal classification and other classifica- studies published up to June 2012. We also used tions based on bleeding on probing (BOP), the Boolean operator AND/OR to combine the clinical attachment loss (CAL) and probing medical subject headings terms for performing depth (PD) (Table 1). Culture followed by gas a sensitive literature search: (archaea OR archa- chromatography measure of methane produc- eal OR methanogen* OR methanogenic) AND tion and microscopic observation, PCR ampli- (subgingival OR periodontal OR periodontitis fication of archaeal 16S rRNA, archaeal small OR ‘subgingival flora’ OR ‘periodontal subunit (SSU) rDNA and methyl-coenzyme M pocket*’ OR ‘periodontal infection*’ OR ‘peri- reductase (mcrA) specific genes followed by odontal disease*’). In the first phase of selec- cloning, sequencing and phylogenetic analysis, tion, we read titles and abstracts to select were usually carried-out for detecting metha- appropriate articles according to inclusion and nogenic archaea. In fact, six studies used cul- exclusion criteria. Inclusion criteria consisted in ture for archaeal detection whereas ten studies original research articles and reviews published used culture-independent molecular methods in English, human and clinical studies, animal including PCR-sequencing or real-time quanti- and experimental studies, detection of archaea tative PCR (Table 1).

Table 1. Data analysis of studies Study (ref.) Subjects M/f (age) Samples Diagnosis Methods Results/conclusions [Country] (Pa/HS) (Pa/HS) Brusa et al. 10 (10/0) – (20–35) 10 (10/0) – Culture The first evidence (11) [Italy] of the presence of methanogenic bacteria (Methanobrevibacter genus) in human subgingival plaque. Belay et al. 36 (36/0) – (?) 54 (54/0) ADA classification Culture Immunoenzymatic assay (12) [USA] of periodontitis detecting an antigenic similarity to M. smithii and M. stadtmanae, relationship of methanogenic archaea and type III and IV of periodontitis. Brusa et al. 20 (20/0) 6/14 (28–55) 20 (20/0) Pocket depth: 3–7 mm Culture Detection of M. smithii in (26) [Italy] subgingival plaque and saliva, no correlation of methanogenic archaea presence in faeces with its presence in oral cavity. Ferrari et al. 10 (0/10) – (?) 10 (0/10) Healthy sites Culture, A new species named (13) [Italy] DNA-DNA M. oralis isolated from hybridization subgingival plaque of healthy subjects. Kulik et al. 48 (48/0) – (16–74) 48 (48/0) Juvenile, adult, rapidly PCR (SSU Predominant presence of (27) [Switzeland] progressing and rDNA gene) M. oralis and minor refractory presence of M. smithii periodontitis in subgingival plaque, detection of a novel group of archaea. Robichaux et al. 8 (8/0) – (?) 8 (8/0) ADA classification Culture Methanogenic archaea and (31) [USA] of periodontitis sulfate-reducing bacteria may be etiologically involved in destructive periodontally diseases indirectly. Robichaux 17 (17/0) – (?) 17 (17/0) ADA classification Culture Methanosarcina spp. et al. (28) [USA] of periodontitis isolated from type IV periodontal pocket, methanogenic archaea could increase the microbial activity and indirectly contribute to local tissue damage. Lepp et al. 58 (50/8) 31/27 (34–46) 256 (225/31) Healthy sites, gingivitis, PCR (archaeal 16S Relationship between the (14) [USA] (43.3 ± 13.6) slight, moderate and rRNA gene), severity of periodontal severe periodontitis RTQ-PCR infections and the relative (SSU rDNA gene), abundance of M. oralis FISH -like phylotype in periodontal pockets, periodontal pathogen Treponema denticola are potential hydrogen competitors with methanogens.

(continued)

Table 1. (continued)

Study (ref.) Subjects M/f (age) Samples Diagnosis Methods Results/conclusions [Country] (Pa/HS) (Pa/HS) Yamabe et al. 66 (49/17) – (?) 141 (111/30) Health sites, moderate, PCR (archaeal 16S Antigenic role of archaea (22) [Japan] severe, aggressive and rRNA gene), in the pathogenesis of chronic periodontitis Western Blotting periodontitis, majority of methanogenic archaea in periodontal pockets were M. oralis-like phylotype. Vianna et al. 167 (102/65) 42/60 (50.7 ± 11.2) 167 (102/65) Healthy sites, moderate RTQ-PCR, (mcrA M. oralis mcrA and (21) [Germany] and severe gene), sequencing Desulfovibrio piger periodontitis dsrAB genes were designated as potential biomarkers for progression of periodontitis. Li et al. 56 (41/15) 22/34 (37–54) 56 (41/15) Healthy sites and PCR (archaeal Apart from the (15) [China] generalized chronic 16S rRNA gene) predominant colonizers periodontitis of M. oralis, other archaea such as Thermoplasmata also have opportunities to colonize the periodontal pockets. Vianna et al. 44 (44/0) – (?) 44 (44/0) – PCR and RTQ-PCR M. oralis detected from (30) [Germany] (mcrA and archaeal periodontal pockets; 16S rRNA genes), evidence of a novel, as T-RFLP analysis yet uncultured methanogenic phylotype associated with periodontal infections. Faveri et al. 50 (25/25) 22/28 (46.5–49.4) 125 (75/50) Healthy sites and PCR (archaeal 16S Detection of M. oralis and (23) [Brazil] peri-implantitis rRNA gene) M. curvum/congolense from both healthy sites and peri-implant pockets. Matarazzo et al. 60 (30/30) 27/33 (24.5–26.2) 180 (120/60) Healthy sites and PCR and RTQ-PCR Low diversity of (24) [Brazil] generalized (archaeal 16S methanogenic archaea in aggressive rRNA gene) subgingival plaque, periodontitis M. oralis detected from subgingival sites of both healthy subjects and periodontitis patients, origin of M. mazeii and M. curvum/congolense from the ingestion of contaminated food. Horz et al. 30 (30/0) 16/14 (60 ± 14) 30 (30/0) Chronic periodontitis RTQ-PCR (mcrA and Detection of M. oralis and (29) [Germany] archaeal 16S rRNA Thermoplasmatales from genes), sequencing periodontal pockets, detection of an unknown group of archaea Mansﬁeld et al. 12 (12/0) – (?) 30 (30/0) Healthy incisors, PCR (archaeal 16S M. oralis found from (36) [USA] asymptomatic third rRNA gene) symptomatic third molars, pericoronitis molars and a healthy of the third molars incisor. Pa/HS, Patients/Healthy subjects; M/F, Males/Females; ADA, American Dental Association; PCR, Polymerase chain reaction; RTQ-PCR, Real-time quantitative PCR; FISH, Fluorescence in situ hybridization; T-RFLP, Terminal restriction fragment length polymorphism; SSU, Archaeal small subunit; mcrA, Methyl-Coenzyme M reductase; –/(?), no information.

The pooled prevalence of these studies showed DIVERSITY OF METHANOGENIC that M. oralis was detected in 41% of peri- ARCHAEA IN SUBGINGIVAL SITES odontitis patients and 6% of healthy subjects (p < 10À5, Chi-squared test). Also, M. oralis Detection of methanogenic archaea in healthy was isolated from 55% of periodontal pock- individuals À ets and 5% of healthy sites (p < 10 5, Obligate anaerobes could be found in subgin- Chi-squared test) (Table 3). Only one study gival plaque of both periodontitis patients and detected M. stadtmanea in periodontal pockets periodontally healthy subjects, with Tannerella by immunological analysis of the enrichment forsythia and Porphyromonas gingivalis belong- cultures, which showed a weak antigenic simi- ing to the ‘red complex’ of periodontal patho- larity to the reference of this methanogenic gens only detected in periodontitis patients archaea (12). M. smithii was detected by the (19, 20). Several studies confirmed the absence same method (12) and by microscopic observa- of methanogenic archaea in the subgingival tion using phase contrast and epifluorescence plaque of healthy subjects (14, 15, 21, 22) microscopy (26). M. smithii was further docu- although some studies reported their detection mented in periodontal pockets by PCR- in 12–86% of healthy subjects (13, 23, 24). sequencing (27). The physiology, morphology Analysing these studies (Table 2) showed that and Gram-staining found a methanogenic four studies reporting the absence of methanoarchaea looking like a Methanosarcina spp. genic archaea in subgingival plaque of healthy from one severe periodontitis pocket (28). subjects used commercial kits and benzyl-alcohol Methanosarcina mazeii detection was after- method for DNA extraction and PCR-based wards confirmed by PCR-sequencing in both detections. Instead, three studies detecting periodontitis patients and healthy subjects archaea in subgingival samples of healthy sub- (24). By using PCR amplifying the archaeal jects used a different method for DNA extrac- 16S rRNA gene followed by cloning, sequencing tion, comprising of proteinase K plus 0.5% and phylogenetic analysis, two studies (15, 29) Tween-20 or 137 MPa pressure for cell lysis and identified Thermoplasmata spp. in periodontal DNA extraction. Accordingly, two of these pockets, but not in healthy subjects (15). PCR- studies detected both M. oralis and Methano- based method detected M. curvum/congolense in bacterium congelense/curvum by direct PCR- periodontitis and peri-implantitis patients as sequencing (23, 24). A previous comparison of well as from pockets and healthy sites (23, 24). DNA extraction protocols for archaea showed Moreover, novel genotypes of methanogenic that adding the mechanical cell lysis step archaea were also detected in some studies increased 100- to 1000-fold the efficiency of (27, 29, 30). Indeed, cloning and sequencing PCR-based detection of archaea (9). The pro- identified a SSU rDNA sequence that had teinase K-resistant cell wall of family Methano- M. oralis and M. smithii signatures in five and bacteriales (25) suggested that the cell lysis step four positions, respectively, and deviated in an was decisive in the efficiency of DNA extrac- additional three positions from both archaea, tion. This may explain differences in the preva- suggesting a novel phylotype of Methanobrevib- lence of methanogenic archaea in healthy acter spp. from human and animal sources (27). subjects according to the various studies. Terminal restriction fragment length polymorphism analysis detected a new mcrA gene tracing to methanogenic archaea. The sequence identity Detection of methanogenic archaea in periodontitis of this new mcrA phylotype was of 88–89% with patients M. oralis and M. smithii mcrA sequences (30). Methanogenic archaea currently reported in This result was afterwards verified using PCR- the subgingival plaque comprise five genera sequencing of the 16S rRNA gene, further including Methanobrevibacter, Methanosphae- showing evidence of a new Methanobrevibacter ra, Methanosarcina, Thermoplasmata and Met- phylotype (30). Although M. oralis detection in hanobacterium, and new, as yet, unidentified periodontal pockets was confirmed in 11 of 16 genotypes (Table 3). M. oralis and M. oralis- studies, evidence for M. stadtmanea, M. smithii, like phylotypes were detected in 11/16 studies. Methanosarcina spp., Thermoplasmata spp. and

Table 2. Studies with and without detection of methanogenic archaea in periodontally healthy subjects No detection of methanogenic archaea Detection of methanogenic archaea Lepp Yamabe Vianna Li Ferrari Faveri Matarazzo et al. (14) et al. (22) et al. (21) et al. (15) et al. (13) et al. (23) et al. (24) Country USA Japan Germany China Italy Brazil Brazil Population age 43.3 ± 13.6 – 50.7 ± 11.2 27–43 – 46.5 ± 11.0 24.5 ± 5.1 Males/females 11/20 ––6/9 – 10/15 13/17 No antibiotherapy 3 months ––3 months – 6 months – Saliva isolation No No Yes No No No No Samples Subgingival Subgingival Subgingival Subgingival Subgingival Subgingival Subgingival plaque plaque plaque plaque plaque biofilm sample Collecting Sterile curette Paper point Paper point Sterile curette Sterile curette Sterile curette Sterile curette instrument Transfer c-Irradiated PBS solution, – Transport Blank dilution, TE buffer TE buffer, condition water, stock stock medium, stock anaerobic stock at À80 °C at À30 °C at À20 °C condition, at À20 °C within 1 h Detecting RTQ-PCR PCR-sequencing RTQ-PCR PCR-sequencing Culture, PCR-sequencing PCR-sequencing method DNA-DNA hybridization Cuture condition ––––37 °C, 20% ––

CO2, 80% H2 for 10 days and 3 months Identification ––––Microscopy, gas –– chromatography DNA Benzyl InstaGene Qiamp DNA Genomic DNA Cells broken at 137 Cell lysis by Cell lysis by extraction alcohol-method Matrix Mini kit Extraction kit Mpa and Marmur Proteinase K Proteinase K (Bio-Rad, (Quiagen, (Tiangen, procedure + Tween-20 + Tween-20 Hercules, Courtaboeuf, Beijing, CA, USA) France) China) Archaeal gene SSU rDNA 16S rRNA mcrA 16S rRNA – 16S rRNA 16S rRNA Negative c-Irradiated E. coli/M. –/M. oralis –– Reaction mix Reaction mix /positive water/archaeal oralis DNAs DNA /methanogenic /methanogenic controls DNA archaea DNA archaea DNA Amplification 50 35 40 35 – 35 35 cycles Prevalence 0/8 (0) 0/17 (0) 0/65 (0) 0/15 (0) 4/10 (40) 3/25 (12) 26/30 (86) in HS (%) Archaeal ––––M. oralis M. oralis, M. oralis, species M. congelense M. congelense /curvum /curvum, M. mazeii HS, Healthy subjects; PCR, Polymerase chain reaction; RTQ-PCR, Real-time quantative PCR; PBS, Phosphate buffered saline; TE, Tris–HCl and Ethylenedi- aminetetraacetic acid; –, no information.

Methanobacterium spp. in periodontitis patients gums begin to appear, teeth begin to shift due was reported in only 1–3 studies, each (Table 3). to bone loss) and type IV advanced periodon- Determining the occurrence and prevalence of titis (bone loss becomes more widespread, new genotypes of methanogenic archaea in sub- shifting of teeth and even loss of teeth can gingival plaque warrants further studies. occur) (32) reported the detection of methanogenic archaea in different degrees of periodontitis. Balay et al. (12) first suggested the METHANOGENIC ARCHAEA AND association of methanogenic archaea and peri- PERIODONTITIS SEVERITY odontitis severity after their detection in 7% of ADA types I and II subgingival speci- Three studies (12, 28, 31) incorporating the mens, in 26% of ADA type III periodontal ADA periodontal classification with type I gin- pockets (p = 0.153, Chi-squared test) and in givitis (swollen and bleeding gums), type II 76% of ADA type IV periodontal pockets early periodontitis (same symptoms as gingivi- (p < 0.001, Chi-squared test). Two studies fur- tis, only more severe, some jawbone loss may ther confirmed the detection of methanogenic also occur), type III moderate periodontitis archaea only in ADA types III and IV peri- (more profound bone loss and pockets in the odontal pockets (28, 31). Another study which

Table 3. Diversity of methanogenic archaea in subgingival plaque Prevalence in periodontitis Prevalence in healthy References patients subjects Archaeal genera/species Methods Patients Pockets HSi Subjects HSi Methanobrevibacter Culture, nt nt nt 4/10 + Ferrari oralis PCR, et al. (13) FISH ++ nt nt nt Kulik et al. (27) 18/50 121/158 0/38 0/8 0/31 Lepp et al. (14) 11/49 15/83 0/28 0/17 0/30 Yamabe et al. (22) 44/102 + nt 0/65 – Vianna et al. (21) ++ nt ––Li et al. (15) 39/44 + nt nt nt Vianna et al. (30) 12/25 12/25 6/48 3/25 5/50 Faveri et al. (23) ++ nt ++Matarazzo et al. (24) 2/30 + nt nt nt Horz et al. (29) 3/12 3/11 1/19 nt nt Mansﬁeld et al. (36) 129/312 151/277 (55)1 7/133 (5) 7/125 (6)1 5/111 (5)1 Total (%) (41)1 Methanosphaera Culture ++ nt nt nt Baley stadtmanea et al. (12) Methanobrevibacter Culture, ++ nt nt nt Baley smithii PCR et al. (12) 9/20 + nt nt nt Brusa et al. (26) ++ nt nt nt Kulik et al. (27) Methanosarcina Culture, 1/17 1/12 nt nt nt Robichaux spp./M. mazeii PCR et al. (28) ++ nt ++Matarazzo et al. (24) Thermoplasmata PCR ++ nt ––Li spp./Thermoplasmatales et al. (15) 3/30 + nt nt nt Horz et al. (29) Methanobacterium PCR 4/25 3/25 2/48 2/25 2/50 Faveri curvum/congolense et al. (23) ++ nt ++Matarazzo et al. (24) Genotypes PCR ++ nt nt nt Kulik et al. (27) 3/44 + nt nt nt Vianna et al. (30) 3/30 + nt nt nt Horz et al. (29) HSi, Healthy sites; +, Detection; –, No detection; nt, no test. 1p < 10À5, Chi-squared test.

© 2012 The Authors APMIS © 2012 APMIS 7 NGUYEN-HIEU et al. classified 4 mm PD 5 mm as moderate periodontal pockets and only 5% of healthy periodontitis and PD 6 mm as severe sites harboured M. oralis (p < 10À5, Chi- periodontitis, also confirmed an absence of squared test) (Table 3). The criterion, ‘elimina- archaea in healthy sites compared to the pres- tion of the organism’ specifies that progression ence of methanogenic archaea in 7% of mod- of periodontitis is arrested after the putative erate periodontitis (p = 0.15, Chi-squared test) pathogen is eliminated by appropriate treat- and 21% of severe periodontitis (p < 0.01, ments (33). Accordingly, one study found a Chi-squared test) (22). Lepp et al. (14) classi- significant decrease (p < 0.001, t-test) in the fied slight periodontitis (BOP, CAL 2–3 mm, relative abundance of M. oralis rDNA in sub- PD 4 mm), moderate periodontitis (BOP, gingival samples obtained 12–18 months after CAL 4–5 mm, PD 4 mm) and severe peri- treatment compared to pre-treatment values odontitis (BOP, CAL 6 mm, PD 4 mm). (14). Moreover, this decrease paralleled a drop This study showed a direct correlation between in the patients’ average CAL, indicating an the relative abundance of the archaeal 16S improvement in disease status. ‘Host response’ rRNA gene and the periodontitis severity: the is an important criterion for causation of peri- archaeal 16S rRNA gene accounted for 1% of odontal damage, which is believed to be due total prokaryotic 16S rRNA gene in samples to host inflammatory responses to the putative from slight periodontitis compared to 7% and pathogen. A cellular and/or humoural immune 19% of those from moderate periodontitis response to a microorganism suggests its possi- and severe periodontitis respectively (p < 0.05, ble aetiological role (33). The IgG antibodies t-test) (14). Likewise, moderate periodontitis against M. oralis detected using Western (PD < 6 mm, CAL < 4 mm) and severe peri- immunoblotting in 72% of patients with severe odontitis (PD 6 mm, CAL 4 mm) were periodontitis and no detection in healthy sub- defined in the study of Vianna et al. (21), which jects supported the potential role of M. oralis indicated that the average proportion of metha- in the pathogenesis of periodontitis (22). Fur- nogenic archaea (rRNA gene copy number) ther study showed that M. oralis chaperonin was significantly increased in severe periodonti- subunits were antigenic molecules that were tis in comparison with moderate periodontitis recognized by serum IgG antibodies in peri- (p = 0.038, Mann–Whitney test). Overall, these odontitis patients (35). The criterion, ‘animal findings support that methanogenic archaea pathogenicity’ is verified by an inoculation of load correlates to periodontitis degrees. How- the suspected pathogen into an experimental ever, because different classifications were used animal for establishing its aetiological role for assessing the clinical status of periodontitis, (33). However, studying M. oralis and peri- a standard for diagnosis of periodontitis sever- odontitis in experimental animal model has ity based on the level of methenogenic archaea not been performed yet for verifying this crite- detected in periodontal pockets was not been rion. The last criterion, ‘mechanisms of patho- established yet. genicity’ refers to virulence factors that promote inflammation and periodontal damage. Virulence factors allow the pathogen to ASSOCIATION OF M. ORALIS AND avoid host defences and contribute to peri- PERIODONTITIS: CLINICAL odontal tissue destruction (33). The optimum RELEVANCE growth conditions of M. oralis are 37 °C and a pH of 6.9–7.4 under H –CO atmosphere M. oralis as a periodontal pathogen 2 2 (13). Because methanogenic archaea such as Methanogenic archaea M. oralis satisfying 4 of M. oralis in periodontal pockets used the end 5 criteria proposed by Socransky could be products (acetate, methylamines and H2/CO2) therefore confirmed as a periodontal pathogen produced by anaerobic bacteria including the (33, 34). Indeed, the criterion, ‘association with sulphate-reducing bacteria (SRB), removal of disease’ states that the putative pathogen could these metabolic products could increase SRB have a significantly higher number or propor- activity, contributing to local tissue damage tion in diseased sites than at healthy sites (33). (28, 31). Moreover, M. oralis plays a role in reg- The pooled prevalence showed that 55% of ulating the local pH by fixation of hydrogen

8 © 2012 The Authors APMIS © 2012 APMIS ARCHAEA IN SUBGINGIVAL SITES protons released by growing anaerobic bacteria. pockets may contribute to the diagnosis, the Therefore, M. oralis and SRB may both be prognosis and the follow-up of periodontitis, involved in destructive periodontal diseases. including refractory ones (21).

Implication for diagnosis of periodontitis Implication for treatment of periodontitis Studies analysing periodontal pocket microbi- Tetracyclines used to be prescribed to treat ota found that methanogenic archaea were periodontal infections (43) and refractory peri- always associated with anaerobic bacteria such odontitis has been associated with a microflora as SRB (21, 31), acetogenic bacteria (21) and relatively resistant to this antibiotic (44). Meta- other anaerobes (14, 36). Because average bolic process and cell wall structure of archaea loads of both methanogenic archaea and SRB differ from those of bacteria (3), suggesting were significantly elevated in severe cases of that some antibiotics effective against bacteria periodontitis, the mcrA gene of M. oralis and are not effective against methanogenic archaea. the dsrAB gene of sulfate-reducing bacteria It is worth noting that M. oralis is resistant to (SBR) were proposed as potential biomarkers many antibiotics including tetracyclines with for diagnosis of periodontitis progression (21). minimum inhibitory concentration (MIC) In addition, an antagonistic interaction 100 mg/L, but it is susceptible to metronida- between acetogenic bacteria and methanogens- zole with MIC 1 mg/L (45). Interestingly, SBR was observed, indicating that these three the most common and successful antibiotic hydrogenotrophic microbial groups may func- regimen for periodontitis was a combination tion as alternative syntrophic partners of sec- of metronidazole and amoxicillin (46). Efficacy ondary fermenting periodontal pathogens (21). of this antibiotic combination may therefore Also, the relative abundance of treponemal rely in part on the elimination of M. oralis by 16S rRNA gene was significantly lower in sites metronidazole. with the archaeal 16S rRNA gene than in sites without the archaeal 16S rRNA gene (p < 0.05, t-test) (14). This study supported CONCLUSIONS the hypothesis that members of the Treponema genus including the well-known periodontal Methanogenic archaea including M. oralis are pathogen Treponema denticola were potential significantly associated with periodontitis. hydrogen competitors of M. oralis (14). M. oralis is an emerging periodontal pathogen. Accordingly, analyses of subgingival plaque Quantification of M. oralis in periodontal may not definitely identify pathogens that are pockets may help in the diagnosis of periodon- responsible for periodontitis and peri-implanti- titis severity and in the follow-up of this dis- tis in all clinical cases (37, 38), a microbiologi- ease under treatment. The sources, diversity cal test could contribute to diagnosis and and pathogenesis of methanogenic archaea therapeutics. In fact, culture is the only present in subgingival plaque need to be fur- method assessing the antibiotic susceptibility ther investigated. and probable detection unexpected periodontal pathogens whereas culture-independent molecular methods are useful to broadly identify the REFERENCES fastidious microorganisms (39). Studies using 16S rRNA gene pyrosequencing and high- 1. Woese CR, Fox GE. Phylogenetic structure of throughput sequencing of the bacterial 16S the prokaryotic domain: the primary kingdoms. rRNA gene established the subgingival micro- Proc Natl Acad Sci USA 1977;74:5088–90. biota (40–42). However, the archaeal 16S 2. Kandler O, Konig H. Cell wall polymers in Archaea (Archaebacteria). Cell Mol Life Sci rRNA gene was not detected in these studies, 1998;54:305–8. which completely ignored the archaeal popula- 3. Koga Y, Morii H. Biosynthesis of ether-type tion. As methanogenic archaea were associated polar lipids in archaea and evolutionary consid- with severe periodontitis, the detection and erations. Microbiol Mol Biol Rev 2007;71:97– quantification of M. oralis in periodontal 120.

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______Chapitre 2

Chapitre 2

Susceptibility of Archaea to antimicrobial agents: applications to clinical microbiology

Saber Khelaifia and Michel Drancourt1*

1 Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UMR CNRS

6236 IRD 3R198, Méditerranée Infection, Faculté de Médecine, Aix-marseille-Université,

Marseille, France

*Corresponding author: Prof. Michel Drancourt, Unité des Rickettsies, Faculté de Médecine,

27, Boulevard Jean Moulin-Cedex 5- France. Tel: 00 33 4 91 38 55 17. Fax: 00 33 4 91 38 77

72. Email: [email protected]

Key words: archaea, methanogenic archaea, microbiota, antimicrobial agent, susceptibility

testing.

Clinical Microbiology and Infection (2012)

______Chapitre 2

Chapitre 2: Préambule

L’implication des archaea méthanogènes dans les maladies du tube digestif et l'obésité n'a pas

été fermement établie, mais des preuves récentes les impliquent directement aux infections de parodontites. Leur comportement envers les agents antimicrobiens d’intérêt clinique reste encore controversé. Dans ce chapitre nous avons revue l'état des connaissances concernant la sensibilité in-vitro et in-vivo des archaea aux agents antimicrobiens, y compris des nouvelles molécules. En effet, certaines archaea associées au microbiote humain ont été impliquées dans des maladies telles que les parodontites. Les archaea sont caractérisées par leur large spectre de résistance aux agents antimicrobiens. En particulier, leur paroi cellulaire est exempte de peptidoglycane, ce qui les rend résistantes aux agents antimicrobiens interférant avec la biosynthèse du peptidoglycane. Les archaea sont toutefois sensibles à l'acide fusidique, un agent antimicrobien inhibant la synthèse des protéines et aux dérivés de l'imidazole. En outre, la squalamine, un agent antimicrobien agissant sur la paroi cellulaire, s'est avérée efficace contre les archaea méthanogènes humaines. Les données de sensibilité in-vitro pourraient être utilisées pour concevoir des protocoles pour la décontamination du microbiote complexe en vue et de l'isolement sélectif des archaea par culture anaérobie.

REVIEW 10.1111/j.1469-0691.2012.03913.x

Susceptibility of archaea to antimicrobial agents: applications to clinical microbiology

S. Khelaiﬁa and M. Drancourt Unite´ de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UMR CNRS 6236 IRD 3R198, Me´diterrane´e Infection, Faculte´ de Me´decine, Aix-marseille-Universite´, Marseille, France

Abstract

We herein review the state of knowledge regarding the in vitro and in vivo susceptibility of archaea to antimicrobial agents, including some new molecules. Indeed, some archaea colonizing the human microbiota have been implicated in diseases such as periodontopathy. Archaea are characterized by their broad-spectrum resistance to antimicrobial agents. In particular, their cell wall lacks peptidoglycan, making them resistant to antimicrobial agents interfering with peptidoglycan biosynthesis. Archaea are, however, susceptible to the protein synthesis inhibitor fusidic acid and imidazole derivatives. Also, squalamine, an antimicrobial agent acting on the cell wall, proved effective against human methanogenic archaea. In vitro susceptibility data could be used to design protocols for the decontamination of complex microbiota and the selective isolation of archaea in anaerobic culture.

Keywords: Antimicrobial agent, archaea, methanogenic archaea, microbiota, susceptibility testing

Clin Microbiol Infect

Corresponding author: M. Drancourt, Unite´ des Rickettsies, Fa- culte´ de Me´decine, 27, Boulevard Jean Moulin-Cedex 5, France E-mail: [email protected]

Methanogenic archaea (herein referred to as methano- Introduction gens) are the sole organisms producing methane from

H2 +CO2 [6]. They are widely distributed in nature in terms Archaea form a distinct kingdom of life, in addition to of their adaptation to different conditions of temperature, eukaryotes, bacteria, and large DNA viruses [1,2]. Archaea pH, and salinity, but remain conﬁned to strictly anaerobic comprise three phylogenetically distinct groups: the environments. The observation that human breathing Crenarchaeota mainly consist of hyperthermophilic sul- released methane led to the isolation of the ﬁrst human phur-dependent organisms, the Euryarchaeota contain metha- intestinal methanogen, Methanobrevibacter smithii [7]. Two nogens and extreme halophiles, and molecular evidence has other species, Methanosphaera stadtmanae [8] and Methano- indicated the presence of the Korarchaeota in hyperthermo- massiliicocus luminyensis [9], were then isolated from the philic environments similar to those inhabited by the Cre- human gut microbiota by the use of anaerobic culture narchaeota [3]. On the basis of their physiology, archaea can [10,11]. Methanobrevibacter oralis [12] was detected and iso- be organized into methanogens, extreme halophiles, and lated from periodontitis specimens, with the same proce- (hyper)thermophiles [4]. In addition to unifying features that dure. Methanogens are perfectly adapted to their distinguish archaea from bacteria, archaea exhibit other environment, and play a role in the oral and intestinal micro- unique structural or biochemical characteristics related to biota [13]. M. smithii is the dominant methanogen in the their particular habitats [4]. Antimicrobial susceptibility pat- human gut, being detected with a high prevalence of 95.7%, terns clearly distinguish archaea from the other organisms, whereas Methanosphaera stadtmanae and Methanomassiliicocus and antimicrobials active against most bacteria are ineffective luminyensis are detected in 29.4% and 4% of individuals, against archaea [5]. respectively [14].

ª2012 The Authors Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases 2 Clinical Microbiology and Infection CMI

The gut is usually sterile at birth [15]. The development The density of the archaeal inoculum is paramount, and must and establishment of the intestinal microflora is a complex be adjusted with a photometer. Inocula were transferred in process. It takes weeks or months to stabilize as a climax each Petri dish, resulting in a final inoculum of 105 cells/mL microflora, a process that is influenced by diet. Methanogens [24,27]. The reliability of tests is influenced by many parame- are detected until after weaning. It was generally reported ters that must be strictly controlled. The culture medium that this marked change in diet was concomitant with an that allows growth of archaea does not contain antimicrobial increase in the density and complexity of the microflora suf- inhibitors [28]. The concentration of calcium and magnesium ficient to produce the conditions that would allow further should be monitored, as concentrations above 10 mM may colonization by methanogens [15]. Studies of the genetic inhibit the activity of certain antimicrobials acting on mem- diversity of the human intestinal microbial community in rela- branes [29]. Likewise, the pH influences the activity of sev- tion to obesity, using culture-independent, molecular, phylo- eral antimicrobials [28]. The temperature and delay of genetic and ecological statistical methods, showed that obese incubation must be fixed [30]. Using a susceptible organism individuals have distinctly different intestinal communities as a positive control is mandatory [5]. than normal-weight individuals, confirming an association between methanogens and obesity [16,17]. Methanogens are Antimicrobials Acting on the Cell Wall also detected in the human vagina [18]. It was shown that diseased patients had a greater likelihood of being methanogen-positive, but no relationship was demonstrated between Cell-wall synthesis inhibitors patient condition and the presence of methanogens in the Bacterial cell walls contain peptidoglycan, with N-acetylmu- vagina [18]. The potential role of archaea in digestive tract ramic acid being the molecular signature for the presence of disease, obesity and vaginal infection has not been firmly peptidoglycan [31]. Archaea are considerably more diverse in established [17–19], whereas evidence has accumulated impli- the composition of the cell wall; they lack peptidoglycan in cating archaea in periodontitis [20,21]. any form, but instead, proteins, glycoproteins and polysac- Archaea are characterized by their broad-spectrum resis- charides cover the outside of the cell membrane [32]. In any tance to antimicrobial agents [5]. Knowledge about their case, the functions of the cell wall remain the same: contain- behaviour towards antimicrobials is needed in the perspec- ing the cytoplasm, shaping the organism, and adapting to and tive of their potential pathogenic role. Also, antimicrobial interacting with the environment [33]. susceptibility patterns can be used to design protocols b-Lactams, glycopeptides, lipoglycopeptide and fosfomycin for the decontamination of complex microbiota to select are the principal families of antimicrobials acting on the bac- archaea [22]. We herein review data regarding the antimi- terial cell wall or bacterial cell-wall synthesis (Fig. 1). The crobial susceptibility patterns of archaea, emphasizing the b-lactams include many bactericidal molecules. Their com- methanogens found in humans. mon features are a b-lactam nucleus and a similar mode of action by inhibiting the final step of peptidoglycan synthesis [34]. Glycopeptides are huge molecules that cannot pass Testing the Susceptibility of Archaea through the porins. Their spectrum of activity is limited to Gram-positive bacteria. Glycopeptides inhibit the synthesis In vitro susceptibility testing of peptidoglycan in its final phase. The three-dimensional In liquid medium, an archaeal inoculum of 10% (v/v) of a structure of these molecules covers the D-Ala-D-Ala of stock solution is distributed in a series of tubes containing the pentapeptide-disaccharide, ready to be incorporated in the antimicrobial (macrodilution method). The inoculum is the peptidoglycan, preventing the action of glycosyl transfer- determined by a 0.4 optical density at 580 nm corresponding ases and transpeptidases, and blocking the elongation of to (4.4212 ± 1.8411) cells/mL. After incubation, the MIC is peptidoglycan [35]. Fosfomycin acts at the earliest stage of indicated by the first tube exhibiting no visible growth [5]. In peptidoglycan synthesis, and must enter the cell to be solid medium, the antimicrobial is incorporated into agar active [36]. poured into Petri dishes. The archaeal inocula are then Fosfomycin and antimicrobials directed against peptido- spread over the surface of the agar. After incubation, the glycan biosynthesis have no growth-inhibitory effect against MIC is determined by the inhibition of growth on the med- archaea with MICs of >50–100 mg/L [5,37]. The activity of ium containing the lowest concentration of the antimicrobial these antimicrobials against M. smithii has been investigated [23]. Agar dilution, performed with a range of concentrations with the reference strain DSMZ 861 (http://www.dsmz.de). in a geometric progression, is the reference method [24–26]. The high level of resistance of this strain to b-lactams and

ª2012 The Authors Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases, CMI CMI Khelaiﬁa and Drancourt Susceptibility of archaea 3

FIG. 1. Mode of action of antimicrobial agents against archaea. , Anti-archaeal activity observed. , No anti-archaeal activity observed. PAB, p-aminobenzoic acid.

glycopeptides was demonstrated by isolation procedures be resistant to polymyxin B [41]. The susceptibility of [22]. The resistance pattern of the faecal isolates agrees with halophilic archaea to this family of antimicrobial agents the structural differences between bacteria and archaea [5], appears to be dependent on the strain tested, and may differ and this resistance is a natural attribute of these microorgan- between closely related species [41,42]. Human methanogens isms [22]. Lack of peptidoglycan is the only documented were found to be susceptible to bacitracin, with MICs of mechanism of resistance. Indeed, different mesophilic metha- <4 mg/L and <25 mg/L for M. oralis [5]. Such concentrations nogenic and extremely halophilic archaea containing pseu- are achieved by topical utilization of bacitracin in oral formu- domurein or glycoprotein cell walls were tested for b- lations [43], suggesting that oral bacitracin could be used for lactamase activity, with the chromogenic b-lactam nitrocefin the treatment of periodontitis where M. oralis has been as substrate. No b-lactamase activity was detected in any of implicated as a co-pathogen [20,21,44]. Susceptibility of the archaeal organisms [38]. This supports the view that b- human archaea to bacitracin has been exploited in the for- lactamases are absent in archaea and are restricted to bacte- mulation of a medium for the selective isolation of Streptococ- ria. Resistance to peptidoglycan inhibitors could be exploited cus mutans from human dental plaque [45]. for the selective isolation of archaea from complex microbiota. b-Lactams, glycopeptides and lipoglycopeptide are fre- Amphotericin B. In 2010, a study focused on halophilic archaea quently used to isolate methanogens from human specimens colonizing the human intestinal mucosa demonstrated the containing a mixed microbiota, with the use of selective resistance of these microorganisms to this antifungal agent. media to purify methanogen cultures [22]. Amphotericin B was therefore used in association with penicillin and erythromycin at 100 mg/L, to repress growth of Cell-wall-alterating antimicrobials salt-tolerant bacteria and fungi, with the aim of cultivating Polymyxin. Polymyxin B and polymyxin E (also known as colis- halophilic archaea from the human intestinal mucosa speci- tin) are the two antimicrobial polypeptides used in clinical men [46]. No data were provided for the other archaeal practice. They have a rapid bactericidal action by disrupting families, including methanogens. the lipidic components of membranes, including the lipopoly- saccharide and the phospholipids [39]. Antimicrobial suscep- Squalamine and its derivatives. Squalamine is a potent, broad- tibility testing to polymyxin E was performed on the spectrum antimicrobial molecule extracted from the livers of haloalkaliphilic archaeon Halalkalicoccus tibetensis [40]. This dogfish and other shark species [47]. It acts on Gram-nega- strain was reported to be resistant to polymyxin E and tive bacteria by a mechanism similar to that of colistin, several other antimicrobials, including penicillin, ampicillin, requiring interactions with the negatively charged phosphate streptomycin, tetracycline, bacitracin, neomycin, and sul- groups of the bacterial outer membrane as the first step in a phafurazole, and to be susceptible to rifampicin and novobio- sequence of different events leading to the disruption of the cin, but no MIC was determined in these studies. The membrane; squalamine exhibits a depolarizing effect on halophilic archaeon Natronococcus amylolyticus was found to Gram-positive bacteria, resulting in rapid cell death [29].

ª2012 The Authors Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases, CMI 4 Clinical Microbiology and Infection CMI

(a) Antimicrobials interfering with DNA

DNA replication inhibition DNA replication and transcription are targets for antimicrobials, including quinolones and novobiocin [48] (Fig. 1). Qui- nolones are synthetic antibacterial agents that were initially active against Gram-negative bacilli [48]. Quinolones enter cells by simple diffusion, and selectively inhibit DNA replication in bacteria and some archaea, acting at the level of supercoiling, which causes a reduction in the space occupied by DNA [48]. DNA gyrase is a topoisomerase involved in DNA supercoiling [49–51]. Quinolones form an irreversible ternary complex with the DNA gyrase, preventing gyrase activity and blocking replication. Coumermycin, a quinolone derivative, was studied on several archaea at concentrations up to 200 mg/L. The results (b) showed the susceptibility of halobacterial archaea to this compound, which also inhibits the growth of Sulfolobus acidocaldarius and members of the Methanobacteriales, Methanococ- cales and Methanomicrobiales [51]. The coumermycin MIC depended on the strain. Halophilic archaea were more susceptible, with an MIC of 5 mg/L, whereas thermophilic archaea exhibited an MIC of >200 mg/L [51]. Novobiocin is a bacteriostatic antimicrobial that is mainly active on Gram- positive bacteria by inhibiting DNA replication through (c) preventing ATP binding to the DNA gyrase b-subunit [52]. Novobiocin was used to demonstrate the action of antimicrobial agents on the anaerobic digestion process [53]. The inhibitory action of novobiocin specifically affects the different populations involved in the final stage of anaerobic digestion. This hypothesis was confirmed by the lack of utilization of acetate and the partial degradation of propionate and butyrate FIG. 2. Electronmicrographs showing the morphological effects of [53]. squalamine on the Methanobrevibacter smithii cell wall. (a) M. smithii Ansamycins form a family of secondary metabolites that without squalamine. (b) M. smithii +1lg/mL squalamine. (c) M. smi- show antimicrobial activity against many Gram-positive and thii after 12 h of incubation in a culture medium containing 1 lg/mL some Gram-negative bacteria [54]. Moreover, ansamycins squalamine. demonstrated antiviral activity towards bacteriophages and poxviruses [55]. Ansamycins inhibit the chaperone-mediated folding of Hsp90 substrates by blocking their ATP-dependent Squalamine is also effective against human methanogens, dissociation from Hsp90 [56]. The proeukaryote Hsp90 with an MIC of 1 mg/L [5] (S. Khelaifia and M. Drancourt, homologue HtpG is present in most bacterial species, but unpublished data). Our electron microscopy observations not in archaea [57]. suggest that squalamine breaks the M. smithii cell wall, induc- Accordingly, the ansamycin rifampicin was shown to be ing cytoplasm leakage and cell death by a mechanism similar ineffective on human archaea, with an MIC of >100 mg/L to that observed for Gram-negative bacteria (Fig. 2). Unpub- [5]. H. tibetensis [40] is a haloalkaliphilic archaeon previ- lished data from our laboratory indicate that human metha- ously reported to be resistant to resistant to polymyxin E nogens are susceptible to squalamine and some of its and several other antimicrobials, including penicillin, ampi- derivatives, with MICs between 0.1 and 1 mg/L (S. Khelaifia cillin, streptomycin, tetracycline, bacitracin, neomycin, and and M. Drancourt, unpublished data). sulphafurazole. This strain was reported to be susceptible

ª2012 The Authors Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases, CMI CMI Khelaiﬁa and Drancourt Susceptibility of archaea 5

to rifampicin, but no MIC was determined in these nopyridines inhibit the synthesis of folic acid, a key cofactor studies. in the synthesis of purine and pyrimidine bases in prokaryotes [70,71], whereas eukaryotes directly assimilate folic DNA-altering antimicrobials: imidazole and derivatives acid from the diet. A detailed inhibition study of carbonic The spectrum of activity of imidazoles is limited to organisms anhydrases belonging to the b and c carbonic anhydrase whose metabolism is anaerobic or at least micro-aerophilic, families from archaea with sulphonamides was presented for such as Helicobacter pylori and Gardnerella vaginalis [58,59]. the first time in 2004 [72]. The two susceptibility carbonic Indeed, imidazoles are prodrugs requiring partial reduction anhydrases from Methanosarcina thermophila showed very of the NO2 group by anaerobic organisms [60,61]. Reduced different inhibitory properties than those from Methanobac- imidazole derivatives are biologically active products that terium thermoautotrophicum. The most potent inhibitors were bind to DNA regions rich in adenine and thymine and cause sulphamic acid and acetazolamide, with MICs in the range oxidative cleavage of DNA stretches. Such DNA lesions are 63–96 nM [72]. followed by the death of archaea and bacteria [59,61]. Met- ronidazole, an imidazole derivative, was initially shown to Protein Synthesis Inhibitors inhibit unidentified faecal methanogens with MICs between 0.5 and 64 mg/L [22]. It also showed in vitro activity against human methanogens, with an MIC of 1 mg/L [5]. It was, The susceptibility of archaea to protein synthesis inhibitors indeed, observed that treatment of the digestive tract with has been determined by several groups [73,74] (Fig. 1). It has metronidazole in bone marrow transplant recipients elimi- been known for some time that even closely related archaeal nated detectable methanogens in stools: patients receiving species are remarkably heterogeneous in their sensitivity to metronidazole were negative for methanogen culture within ribosome-targeted antimicrobials [75]. Many of the classical the first week of therapy, and recolonization occurred within inhibitors of eubacterial 70S and eukaryotic 80S ribosomes several weeks [62]. Gut decontamination with metronidazole do not inhibit the growth of these organisms even at high suppressed or eliminated the methanogens, just as it did the concentrations; inhibition is caused by only a few compounds anaerobic bacteria [63]. that affect eubacterial and eukaryotic cells [5]. However, it is Nitrofurans are synthetic molecules used for treating unclear whether this lack of susceptibility is caused by the intestinal tract infections (furazolidone and nifuroxazide) and impermeability of these organisms to most antimicrobials or urinary tract infections (nitrofurantoin and hydroxymethyl-ni- by the lack of a ribosomal binding site [73]. The susceptibility trofurantoin) [64]. These molecules preferentially inhibit the of hyperthermophilic archaeal ribosomes to the inhibitory synthesis of inducible enzymes by blocking the initiation of actions of all known classes of aminoglycoside antimicrobial translation. The action of nitrofuran has implications for the has been tested on the hyperthermophilic Aquifex pyrophilus. regulation of gene expression in general [65]. Nitrofurans A. pyrophilus ribosomes are susceptible to all tested aminogly- target DNA after reduction of the NO2 group by aerobic cosides, including 2-deoxystreptamines, monosubstituted bacterial nitroreductase [66]. Reduced derivatives break and 2-deoxystreptamines, and streptidine [76]. The effect of induce mutations in DNA. Their effect is bacteriostatic or selected aminoglycoside antimicrobials on the translational bactericidal, depending on the dose [67]. The anti-archaeal accuracy of poly(U) programmed ribosomes derived from activity of nitrofurantoin was confirmed against the halo- the thermophilic archaea Thermoptasma acidophilum, Sulfolo- philic, aerobic archaea Halobiforma haloterrestris and Halogeo- bus solfataricus, Thermococcus celer and Desulfurococcus mobilis metricum borinquense, without the determination of MICs showed that the four species investigated are markedly [68]. diverse in their response to the miscoding-inducing action of aminoglycoside antimicrobials [77]. A study of the suscepti- DNA synthesis inhibitors: sulphonamides and benzylpyrimi- bility of human methanogens showed high in vitro resistance dines to gentamicin and streptomycin, with MICs of >100 mg/L Sulphonamides are synthetic molecules that are often com- [5]. bined with diaminopyridines (benzylpyrimidines) to increase Tetracyclines bind to the 30S subunit of the bacterial ribo- their activity and to reduce the risk of resistance emer- some [78]. First-generation tetracyclines were obtained from gence. Sulphonamides are derivatives of p-aminobenzene- chemical derivatives, doxycycline and minocycline, which sulphonic acid; the presence of a free amine and free have better bioavailability and increased tissue distribution, sulphur radicals directly substituting benzene are essential and a longer half-life for once-daily application [79]. Pacta- for the antibacterial activity [69]. Sulphonamides and diami- mycin was isolated from Streptomyces pactum as a potential

ª2012 The Authors Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases, CMI 6 Clinical Microbiology and Infection CMI

TABLE 1. Classiﬁcation of antimicrobial agents according to their mode of action

Cell-wall synthesis inhibitors DNA-interfering antimicrobials Protein synthesis inhibitors Cell-wall-alterating antimicrobials

()) b-Lactams ()) Ansamycins ()) Tetracyclines ()) Polymyxins ()) Glycopeptide and lipoglycopeptide (+) Quinolones ()) Macrolides ()) Amphotericin B ()) Fosfomycin (+) Novobiocin ()) Lincosamides (+) Squalamine (+) Imidazole ()) Erythromycin (+) Nitrofurans ()) Phenicols (+) Sulphonamides (+) Aminoglycosides (+) Benzylpyrimidines (+) Fusidic acid

()), no anti-archeal activity observed; (+), anti-archeal activity observed.

new human antitumour drug, but is in fact a potent inhibitor nism of action of chloramphenicol remains unclear, but these of translation in eukaryotes, bacteria, and archaea [78]. agents probably inhibit peptide binding and block chain elon- Testing of the susceptibility of human archaea showed that gation [85]. all tested human methanogens were resistant to tetracycline Archaea are generally less sensitive to phenicols; the at concentrations >100 mg/L [5]. growth of Halobacterium halobium and Sulfolobus acidocaldarius Fusidic acid inhibits polypeptide chain elongation by bind- is inhibited at elevated concentrations of chloramphenicol, at ing to the ribosome elongation factor-G–GDP complex, MICs of ‡100 mg/L [86]. The in vitro susceptibility of human thereby preventing its dissociation [80]. The interactions of archaea to chloramphenicol is variable. M. smithii, M. oralis fusidic acid with archaeal elongation factors were assayed by and Methanomassiliicocus luminyensis are resistant, with an using poly(U) programmed cell-free systems under optimal MIC up to 25 mg/L, in contrast to Methanosphaera stadtm- culture conditions for polyphenylalanine synthesis. The anae, which exhibits an MIC of 4 mg/L [5]. The M. smithii, effects of fusidic acid on the polyphenylalanine-synthesizing M. oralis and Methanomassiliicocus luminyensis genomes encode capacities of cell-free systems derived from representative a chloramphenicol O-acetyltransferase, an enzyme that inacti- members of the families Methanobacteriaceae, Methanomicrobi- vates chloramphenicol, but the gene for this is absent in the aceae, and Methanococcaceae, the reference eubacterial Esc- Methanosphaera stadtmanae genome [5]. herichia coli and the eukaryotic Saccharomyces cerevisiae were investigated. The elongation factor-G equivalent factor (elon- Conclusions gation factor-2) of all of the methanogens surveyed was sys- tematically inhibited by fusidic acid within the same range of effective concentrations as that affecting the functionally This review of the data regarding the susceptibility of ar- homologous factors of E. coli and S. cerevisiae, at an MIC of chaea to antimicrobial agents indicates that these organisms 0.5 mg/L [81], supporting the hypothesis that archaea are are broadly resistant to the antibiotics routinely used for the susceptible to molecules that are also active against bacteria treatment of bacterial infections in humans (Table 1). How- and eukaryotes [5]. ever, archaea are members of microbial communities, and Macrolides are active against Gram-positive and some rely on bacterial metabolism for their own survival and mul- Gram-negative bacteria [82] by inhibiting the elongation of tiplication. Therefore, the elimination of bacteria, including the peptide chain after binding to the 50S subunit of bacterial anaerobes, in these communities could result in the indirect, ribosomes [82,83]. As b-lactams, macrolides are frequently unexpected elimination of antibiotic-resistant archaea. If the used in association with other antimicrobial mixtures for lab- role of archaea in human infection is further documented oratory decontamination to isolate methanogens from [19], then anti-archaeal compounds in addition to metronida- human specimens [22]. zole and fusidic acid will be useful. Also, further studies As previously described, erythromycin, an antimicrobial of should aim to test the effectiveness of oral compounds such the macrolide family, was used to cultivate halophilic archaea as bacitracin against archaea implicated in periodontitis from human faeces [46]. These microorganisms were resis- [20,21]. tant to concentrations of approximately 100 mg/L [46]. Transparency Declaration Phenicols Chloramphenicol, thiamphenicol and ﬂorphenicol bind preferentially to the A site at the 50S subunit [84]. The mecha- The authors have no conﬂict of interest regarding this paper.

ª2012 The Authors Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases, CMI CMI Khelaiﬁa and Drancourt Susceptibility of archaea 7

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ª2012 The Authors Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases, CMI

______Chapitre 3

Chapitre 3

A semi-automated protocol for Archaea DNA extraction from stools

Saber Khelaifia1, Pierre-Yves Ramonet1, Marielle Bedotto Buffet1 and Michel Drancourt1*

1 Aix Marseille Université, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095, 13005,

Marseille, France

*Corresponding author: Professeur Michel Drancourt, Unité des Rickettsies, Faculté de Médecine,

27, Boulevard Jean Moulin-Cedex 5- France. Tel: 00 33 4 91 38 55 17. Fax: 00 33 4 91 38 77 72.

Email: [email protected]

Key words: human-associated archaea, methanogenic archaea, microbiota, DNA extraction,

archaeal DNA.

BMC research Notes (2013)

______Chapitre 3

Chapitre 3: Préambule

La détection de l’ADN des archaea par PCR dans des échantillons humains repose sur une technique d'extraction d'ADN efficace. Nous avons déjà mis en place un tel protocole ne faisant intervenir que des étapes manuelles. Dans le but de réduire la charge de travail, nous avons évalué un protocole semi-automatisé comparé au protocole manuel de référence et à un protocole automatisé pour l'extraction d'ADN archaea à partir d'échantillons humains.

110 échantillons de selles humaines ont été testées en utilisant les trois protocoles précédemment cités, le protocole semi-automatisé a produit des résultats comparables a ceux obtenus en utilisant le protocole manuel de référence en terme de prévalence avec des quantités plus importantes d’ADN d’archaea extrait. L’étape de digestion enzymatique à la protéinase-K et la lyse mécanique à la poudre de verre augmentent considérablement le rendement d'extraction de l'ADN d’archaea à partir des selles humaines. Le protocole semi- automatique décrit ici peut être utilisé comme un protocole de première ligne pour l'extraction de l'ADN total des selles à la recherche de nouvelles espèces d’archaea colonisant le tube digestif humain.

Les deux co-auteurs (SK et MD) sont co-inventeurs d'un brevet N / Réf: H52 888 CAS

13 FR (MD / SB 12.05.00329) pour la technique d’extraction d’ADN rapporté dans ce travail.

BMC Research Notes

This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon.

A semi-automated protocol for Archaea DNA extraction from stools

BMC Research Notes 2013, 6:186 doi:10.1186/1756-0500-6-186

Saber Khelaifia ([email protected]) Pierre-Yves Ramonet ([email protected]) Marielle Bedotto Buffet ([email protected]) Michel Drancourt ([email protected])

ISSN 1756-0500

Article type Technical Note

Submission date 20 September 2012

Acceptance date 17 April 2013

Publication date 7 May 2013

Article URL http://www.biomedcentral.com/1756-0500/6/186

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© 2013 Khelaifia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A semi-automated protocol for Archaea DNA extraction from stools

Saber Khelaifia 1 Email: [email protected]

Pierre-Yves Ramonet 1 Email: [email protected]

Marielle Bedotto Buffet 1 Email: [email protected]

Michel Drancourt 1,2,* Email: [email protected]

1 Aix Marseille Université, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095, 13005 Marseille, France

2 Unité des Rickettsies, Faculté de Médecine, 27, Boulevard Jean Moulin, Cedex 5 Marseille, France

* Corresponding author. Unité des Rickettsies, Faculté de Médecine, 27, Boulevard Jean Moulin, Cedex 5 Marseille, France

Abstract

Background

The PCR-based detection of archaea DNA in human specimens relies on efficient DNA extraction. We previously designed one such protocol involving only manual steps. In an effort to reduce the workload involved, we compared this manual protocol to semi-automated and automated protocols for archaea DNA extraction from human specimens.

Findings

We tested 110 human stool specimens using each protocol. An automated protocol using the EZ1 Advanced XL extractor with the V 1.066069118 Qiagen DNA bacteria card and the EZ1 ® DNA Tissue Kit (Qiagen, Courtaboeuf, France) yielded 35/110 (32%) positives for the real-time PCR detection of the Methanobrevibacter smithii 16S rRNA gene, with average Ct values of 36.1. A semi-automated protocol combining glass-powder crushing, overnight proteinase K digestion and lysis in the buffer from the EZ1 kit yielded 90/110 (82%) positive specimens ( P = 0.001) with an average Ct value of 27.4 ( P = 0.001). The manual protocol yielded 100/110 (91%) positive specimens ( P = 0.001) with an average Ct value of 30.33 ( P = 0.001). However, neither the number of positive specimens nor the Ct values were significantly different between the manual protocol and the semi-automated protocol ( P > 0.1 and P > 0.1) Conclusion

Proteinase K digestion and glass powder crushing dramatically increase the extraction yield of archaea DNA from human stools. The semi-automated protocol described here was more rapid than the manual protocol and yielded significantly more archaeal DNA. It could be applied for extracting total stool DNA for further PCR amplification.

Keywords

Human-associated archaea, Methanogenic archaea, Microbiota, DNA extraction, Archaeal DNA Findings

Archaea are permanent inhabitants of the human gut. Three methanogenic species, Methanobrevibacter smithii [1], Methanosphaera stadtmanae [2] and Methanomassiliicoccus luminyensis [3,4], have been isolated from human stools, and Methanobrevibacter oralis has been isolated from the subgingival plaque [4,5]. In addition to their role in the local homeostasis of anaerobic communities [6], methanogenic archaea are suspected to be involved in digestive tract diseases and obesity [7-9] and have been implicated in periodontitis [10-12]. In addition to fastidious isolation and culture, PCR-based techniques have provided additional information about cultured archaea [6,13] and further revealed the presence of as-yet uncultured archaea [3,13,14]. Several different approaches have been used to extract DNA from human feces [13-15], and various methods have been described [16-18]. We previously showed that an appropriate extraction protocol increased the archaeal DNA yield from human stools [14]. However, this protocol involved only manual steps, making it too labor intensive for routine diagnostic use. We therefore aimed to reduce the number of manual steps and compared automated, semi-automated and reference manual DNA extraction protocols for the real-time PCR detection of M. smithii in human feces sample.

This study included 110 stool specimens prospectively collected in 110 individuals from Marseille, France, between July and August 2011 as a part of routine diagnostic activity in the Microbiology laboratory, Timone Hospital, Méditerranée Infection, Marseille, France. No written consent was needed for this work in accordance with the “LOI n° 2004-800 relative à la bioéthique” published in the “Journal Officiel de la République Française” the 6 August 2004 since no additional sample was taken for the study. According to this law, patients were informed that stool specimens could be used for anonymised study. This study was approved by the local Ethics Committee IFR48. Three different DNA extraction protocols were performed in parallel. The reference manual protocol using the NucleoSpin ® Tissue Mini Kit (Macherey Nagel, Hoerdt, France) was performed as previously described [15]. The automated protocol involved DNA extraction using the EZ1 Advanced XL extractor with the V 1.066069118 Qiagen DNA bacteria card and the EZ1 ® DNA Tissue Kit (Qiagen, Courtaboeuf, France) as described by the manufacturer. A semi-automated protocol was performed as follows: approximately 1 gram of stool specimen was suspended in 5 mL Tris- HCl 0.05 M, pH 7.5. A 250 µL aliquot of the suspension was transferred to a sterile screw- cap Eppendorf tube containing 0.3 g of acid-washed beads ( ≤106 mm; Sigma, Saint-Quentin- Fallavier, France) and shaken in a FastPrep BIO 101 apparatus (Qbiogene, Strasbourg, France) at level 6.5 (full speed) for 90 s to achieve mechanical lysis. The supernatant was collected and incubated overnight at 56°C with 180 µL of lysis buffer and 25 µL proteinase K (20 mg/mL) from the Qiagen EZ1 ® DNA Tissue Kit. After a second cycle of mechanical lysis, the supernatant was incubated for 10 min at 100°C, and total DNA was then extracted using the Qiagen EZ1 ® DNA Tissue Kit in the EZ1 Advanced XL extractor with the V 1.066069118 Qiagen DNA bacteria card. Negative controls consisting of sterile DNA-free water were introduced at all steps and underwent the same extraction process that was used for the stool specimens. The working time required for each protocol was measured on three separate occasions.

Extracted total DNA was used as a template for the real-time PCR detection of the M. smithii 16S rRNA gene using PCR primers Smit.16S-740F: 5 ′-CCGGGTATCTAATCCGGTTC-3′ and Smit.16S-862R: 5 ′-CTCCCAGGGTAGAGGTGAAA-3′ and the probe Smit.16S FAM: 5′-CCGTCAGAATCGTTCCAGTCAG-3′, adapted from a previously described protocol [15]. A quantification synthetic plasmid was used as an internal control to monitor PCR inhibition; total bacterial load was measured a previously described [15]. Real time-PCR products were sequenced using the primers Smit.16S-740F, Smit.16S-862R, the BigDye Terminator 1.1 Cycle Sequencing kit and the 3130 Genetic Analyzer (Applied Biosystems, Villebon sur Yvette, France). Negative controls were incorporated into each assay. Sequences were analyzed using the Seqscape program (Applied Biosystems), and sequence similarity values were determined using the online BLAST program at NCBI (www.ncbi.nlm.nih.gov/BLAST/).

The negative controls remained negative in all experiments, and no archaeal DNA was extracted from the water used as negative control. The internal plasmid control was detected in all specimens, with median Ct value of 24.6 for automated protocol; of 23.9 for semi- automated protocol and of 23.6 for manual protocol. Likewise, all bacteria detection was positive in all specimens with respective Ct value of 32.1, 25.9 and 26.4. These data indicated the absence of PCR inhibition in any of the tested protocols. All real time-PCR positive product sequences had 99% sequence similarity to the M. smithii reference sequence (GenBank accession number CP000678). This test was used as a positive control for real- time PCR detection. We compared three microbial DNA extraction protocols to identify an optimized protocol to obtain archaea DNA from human fecal specimens (Table 1). The automated protocol based on the EZ1 Advanced XL extractor and EZ1 ® DNA Tissue Kit yielded 35/110 (32%) positive specimens with an average Ct value of 36.1 ± 6.23, while the semi-automated protocol combining the EZ1 ® DNA Tissue Kit with mechanical disruption and enzymatic and chemical digestion yielded 90/110 (87%) positive specimens with an average cycle threshold (Ct) Ct value of 27.4 ± 7.1. Compared with the automated protocol, the semi-automated protocol yielded significantly more positive specimens ( P < 0.001; Student’s t test) and significantly lower Ct values ( P < 0.001). The previously described manual protocol [15] yielded 100/110 (91%) positive specimens with an average Ct value of 30.33 ± 6.36. The manual protocol yielded significantly more positive specimens and significantly lower Ct values than the automated protocol ( P < 0.001 and P < 0.001, respectively); however, neither the number of positive specimens nor the Ct values were significantly different between the manual protocol and the semi-automated protocol ( P > 0.1 and P > 0.1). Table 1 Statistical analysis applied to 110 human specimens extracted using three DNA extraction methods Manual protocol Semi-automated Automated protocol Positive specimens (%) 100 (91%) 90 (87%) 35 (32%) Negative specimens 10 20 75 Average, Ct 30.33 27.4 36.1 Standard deviation, Ct 6.36 7.1 6.23 P value 0.1 0.001 0.001 Ct , cycle threshold value in real-time PCR.

After the first step (overnight proteinase K digestion and lysis buffer), the semi-automated protocol took from 15 to 30 min without the intervention of an operator, compared to 3 hours for the manual technique, depending on the instruction of an operator. The automated protocol is performed into two steps, proteinase K digestion at 70°C for 10 min and the automated step for 15 min.

These data indicated that combining mechanical agitation in the presence of glass beads with enzymatic and chemical lysis significantly increased the yield of PCR-amplifiable archaeal DNA. The exact mechanism of these procedures was not tested here, but our previous experience suggests that these procedures not only efficiently break the cell walls, thus liberating the archaeal DNA, but also decrease the effects of PCR inhibitors [15]. We found that it was possible to further combine this manual part of the procedure with automated DNA extraction, thus significantly decreasing the protocol turn-around time and rendering archaeal DNA extraction and detection amenable to a routine procedure.

This study revealed that the DNA extraction method used strongly affects the apparent gut diversity and microbial community structure, as observed by real-time PCR tests. Each DNA extraction method revealed a different prevalence of M. smithii . Currently, no available stool DNA extraction method [16,17] is optimized to effectively extract archaeal DNA, contrary to that reported for plants [18,19]. Before the publication of the protocol described by Dridi et al in 2011 [15], the prevalence of M. smithii, the dominant archaea in the human digestive tract, was reported to be 30%, but it was detected in 91%-95.7% of stool samples using this protocol. This protocol has thus significantly increased the ability to detect archaea in the human gut. It has also allowed the PCR-based detection of a fourth archaeal species in the human gut, M. luminyensis, and led to its isolation and description [3]. However, the diversity of archaea in the human gut remains poorly studied. The DNA extraction protocol provided here can improve the exploration of the intestinal microflora, specifically the archaeal community; the identification of new species will increase knowledge in this area and promote the investigation of the potential roles of archaeal species in human diseases [11,12,20] and their effects on the bacterial microflora that colonize the human gastrointestinal tract.

In conclusion, proteinase K digestion and glass powder crushing dramatically increase the yield of archaea DNA from human stool samples. A semi-automated protocol could be used for extracting total stool DNA for further PCR amplification. Competing interest

The two co-authors (SK and MD) are co-inventors of a patent N/Réf: H52 888 cas 13 FR (MD/SB 12.05.00329) for the DNA extraction protocol reported here. Authors’ contributions

SK, PR, and MBB designed and performed the analyses; SK and MD interpreted the data and wrote the manuscript. All authors read and approved the final manuscript. References

1. Miller TL, Wolin MJ, de Macario EC, Macario AJ: Isolation of Methanobrevibacter smithii from human feces. Appl Environ Microbiol 1982, 43 :227–232.

2. Miller TL, Wolin MJ: Methanosphaera stadtmaniae gen. nov., sp. nov.: a species that forms methane by reducing methanol with hydrogen. Arch Microbiol 1985, 141 :116–122.

3. Dridi B, Fardeau ML, Ollivier B, Raoult D, Drancourt M: Methanomassiliicocus luminyensis, gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces. Int J Syst Evol Microbiol 2012, 62 :1902–1907.

4. Gorlas A, Robert C, Gimenez G, Drancourt M, Raoult D: Complete genome sequence of Methanomassiliicoccus luminyensis , the largest genome of a human-associated Archaea species. J Bacteriol 2012, 194 :4745.

5. Ferrari A, Brusa T, Rutili A, Canzi E, Biavati B: Isolation and characterization Methanobrevibacter oralis sp. nov. Curr Microbiol 1994, 29 :7–12.

6. Dridi B, Raoult D, Drancourt M: Archaea as emerging organisms in complex human microbiomes. Anaerobe 2011, 17 :56–63.

7. de Macario Conway E, Macario AJL: Methanogenic archaea in health and disease: a novel paradigm of microbial pathogenesis. Int J Med Microbiol 2009, 299 :99–108.

8. DiBaise JK, Zhang H, Crowell MD, Krajmalnik-Brown R, Decker GA, Rittmann BE: Gut microbiota and its possible relationship with obesity. Mayo Clin Proc 2008, 83 :460–469.

9. Zhang H, DiBaise JK, Zuccolo A, Kudrna D, Braidotti M, Yu Y, et al : Human gut microbiota in obesity and after gastric bypass. P Natl Acad Sci 2009, 106 :2365–2370.

10. Hans-Peter Horz GC: Methanogenic Archaea and oral infections - ways to unravel the black box. J Oral Microbiol 2011, 3:5940.

11. Lepp PW, Brinig MM, Ouverney CC, Palm K, Armitage GC, Relman DA: Methanogenic Archaea and human periodontal disease. P Natl Acad Sci USA 2004, 101 :6176–6181. 12. Yamabe K, Maeda H, Kokeguchi S, Tanimoto I, Sonoi N, Asakawa S, et al : Distribution of Archaea in Japanese patients with periodontitis and humoral immune response to the components. FEMS Microbiol Lett 2008, 287 :69–75.

13. Schleper C, Jurgens G, Jonuscheit M: Genomic studies of uncultivated archaea. Nat Rev Microbiol 2005, 3:479–488.

14. van der Maarel MJEC, Sprenger W, Haanstra R, Forney LJ: Detection of methanogenic archaea in seawater particles and the digestive tract of a marine fish species. FEMS Microbiol Lett 1999, 173 :189–194.

15. Dridi B, Henry M, El Khéchine A, Raoult D, Drancourt M: High prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using an improved DNA detection protocol. PLoS One 2009, 4:e7063.

16. Deuter R, Pietsch S, Hertel S, Müller O: A method for preparation of fecal DNA suitable for PCR. Nucleic Acids Res 1995, 23 :3800–3801.

17. Mûller A, Stellermann K, Hartmann P, Schrappe M, Fâtkenheuer G, Salzberger B, et al : A powerful DNA extraction method and PCR for detection of microsporidia in clinical stool specimens. Clin Diagn Lab Immun 1999, 6:243–246.

18. Bashalkhanov S, Rajora OP: Protocol: a high-throughput DNA extraction system suitable for conifers. Plant Methods 2008, 4:20.

19. Xin Z, Chen J: A high throughput DNA extraction method with high yield and quality. Plant Methods 2012, 8:26.

20. Scanlan P, Shanahan F, Marchesi J: Human methanogen diversity and incidence in healthy and diseased colonic groups using mcrA gene analysis. BMC Microbiol 2008, 8:79.

______Chapitre 4

Chapitre 4

Real-time PCR quantification of Methanobrevibacter oralis in periodontitis

Amélie Bringuier 1.2#, Saber Khelaifia1#, Hervé Richet1, Gérard Aboudharam 1.2, Michel Drancourt1*

1 Aix Marseille Université, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095, 13005, Marseille,

France

2 UFR Odontologie, Aix-Marseille Université, Marseille France.

# These two authors equally contributed to this work.

*Corresponding author: Professeur Michel Drancourt, Unité des Rickettsies, Faculté de Médecine, 27,

Boulevard Jean Moulin-Cedex 5- France. Tel: 00 33 4 91 38 55 17. Fax: 00 33 4 91 38 77 72. Email:

[email protected]

Key words: Periodontitis, periodontal disease, human, methanogens, Methanobrevibacter oralis.

Journal of Clinical Microbiology (2013)

______Chapitre 4

Chapitre 4: Préambule

La parodontite est une infection polymicrobienne où l’archaea méthanogène

Methanobrevibacter oralis a été impliquée comme un organisme pathogène. Nous avons développé un test de PCR en temps réel ciblant le gène cnp60 de M. oralis et appliqué cette analyse sur 20 prélèvements de patients atteints d’infection parodontale et 10 prélèvements de sujets sains. Un score a été établi pour déterminer le degré de sévérité de la parodontite.

Chez les patients, la valeur Ct a été significativement corrélée avec la sévérité de la maladie parodontale. Ces données impliquent directement M. oralis dans la pathologie de parodontopathie. La surveillance de la charge de M. oralis peut être utilisée comme un biomarqueur de la parodontite.

JCM01962-12- revised version-2

1 Real-time PCR quantification of Methanobrevibacter oralis in periodontitis

2 Running title : Methanobrevibacter oralis periodontitis

3 Amélie Bringuier 1.2#, Saber Khelaifia1#, Hervé Richet1, Gérard Aboudharam 1.2, Michel Drancourt1*

5 1 Aix Marseille Université, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095, 13005, Marseille,

6 France

7 2 UFR Odontologie, Aix-Marseille Université, Marseille France.

9 # These two authors equally contributed to this work.

11 *Corresponding author: Professeur Michel Drancourt, Unité des Rickettsies, Faculté de Médecine, 27,

12 Boulevard Jean Moulin-Cedex 5- France. Tel: 00 33 4 91 38 55 17. Fax: 00 33 4 91 38 77 72. Email:

13 [email protected]

15 Abstract word count: 49

16 Main text word count: 956

17 Number of table: 1

18 Number of references: 18

19 Key words: Periodontitis, periodontal disease, human, methanogens, Methanobrevibacter oralis.

1 JCM01962-12- revised version-2

21 Abstract

22 A real-time PCR assay developed to quantify Methanobrevibacter oralis indicated that its

23 inoculum significantly correlated with periodontitis severity (p = 0.003), despite a non-significant

24 difference in prevalence between controls (3/10) and patients (12/22) (p = 0.2, Fisher test).

25 Monitoring M. oralis load can be used as a biomarker for periodontitis.

2 JCM01962-12- revised version-2

27 28 Periodontitis is an anaerobic infection possibly leading to loss of teeth (4, 5, 15). It likely

29 results from infection with microbial complexes comprising methanogens (16, 2) mostly

30 Methanobrevibacter oralis (1, 7, 12). In this study, we correlated M. oralis load as measured by real-

31 time PCR in subgingival plaque with the severity of periodontitis.

32 All patients diagnosed with periodontitis were included from October 2011 to June 2012. The

33 control individuals with generally healthy gingival were volunteers. All patients underwent interview

34 for medical and dental history, intraoral examination to determinate the bleeding on probing (BOP),

35 probing depth (PD), plaque indice (PI) and radiographs (11); periodontal status was also scored as

36 previously reported (3) (Table1). All individuals signed an informed consent and the ethics committee

37 of the IFR 48, University of Aix-Marseille approved the protocol.

38 Subgingival plaque specimens (50 µL) collected from 3 to 12 mm periodontal pockets in both

39 patients and controls, were suspended into 1mL Tris HCl 0.05M, pH 7.5 buffer. After homogenisation,

40 a 250 µL-aliquot was shaken with 0.3 g of acid-washed beads (≤106 mm; Sigma, Saint-Quentin-

41 Fallavier, France) in a FastPrep-24 instrument (MP Biomedical Europe, Illkirch, France) at speed 6.5

42 m/s (full speed) for 90 seconds. The supernatant was incubated overnight at 56°C with 180 mL of

43 lysis buffer and 25µL proteinase K (20 mg/mL) in the Qiagen EZ1® DNA Tissue Kit (Qiagen,

44 Courtaboeuf, France). After a second cycle of mechanical lysis, the supernatant was incubated for 10

45 min at 100°C and total DNA was then extracted using the same kit. Negative controls consisting of

46 sterile DNA-free water were introduced in all the manipulations. The specificity of M. oralis-cnp602P

47 probe (6FAM-5’AGCAGTGCACCTGCTGATATGGAAGG-3’) (Applied Biosystems, Courtaboeuf,

48 France) and primer pair M. oralis-cnp602F (5’-GCTGGTGTAATCGAACCTAAACG-3’) and M.

49 oralis-cnp602R (5’-CACCCATACCCGGATCCATA-3’) (Eurogentec, Seraing, Belgium) was

3 JCM01962-12- revised version-2

50 verified in-silico using the BLAST program at NCBI (http://www.ncbi. nlm.nih.gov/BLAST) and

51 further experimentally ensured by incorporating DNA extracted from Methanobrevibacter smithii

52 ATCC35061T, Methanosphaera stadtmanae ATCC43021T and Methanomassiliicoccus luminyensis

53 CSURP135T. Escherichia coli, Salmonella enterica, Staphylococcus aureus and Treponema denticola

54 ATCC35405T. Real-time PCR assays were performed with a CFX96 TM Touch Real-Time PCR

55 Detection System (Biorad, Marnes-la-Coquette, France) using the Mastermix PCR Kit (Eurogentec)

56 with 5 pmol of each primer and probe and 5 µL about 2 µg of DNA into a 20-µL final volume. M.

57 oralis DSMZ 7256T DNA used as a positive control and one negative extraction control were

58 included in each reaction plate. Results were expressed as the number of M. oralis cell/mL of

59 specimen (Table1). A quantification synthetic plasmid was used as an internal control to monitor PCR

60 inhibition and total bacterial load was measured as previously described (6). The PCR programme

61 was 95°C for 5 min, followed by 40 cycles of 95°C for 1 s, 60°C for 35 s and 45°C for 30 s. A

62 calibration curve was done by measuring the cycle threshold (Ct) value of a serial dilution of M.

63 oralis (10E+01-10E+09). As for clinical specimens tested in duplicate, the real-time PCR was

64 regarded as positive for any Ct value < 40.

65 Ten male and 12 female periodontitis patients aged between 38 and 86 years and 10 age-

66 matched controls were prospectively enrolled. The clinical score varied from 4 to 8 for controls

67 and from 16 to 30 for patients (p < 10-3 Student test). In all PCR-based experiments, negative

68 controls remained negative whereas the quantification plasmid and the M. oralis control yielded

69 positive amplifications in 100% specimens. Total bacterial DNA was positive in 8/10 (80%)

70 controls and in 22/22 (100%) periodontitis patients (Table1). M. oralis DNA was detected in 3/10

71 (30%) controls with an average cell number of 5.39E+02 ± 5.58E+01 and in 12/22 (54%) patients

72 with an average cell number of 7.06E+05 ± 1.74E+06 (p=0.2, Student test). M. oralis detection

4 JCM01962-12- revised version-2

73 was negative in 3/3 (100%) patients with a clinical score < 20 and positive in 12/19 (63%)

74 patients with a clinical score > 20. M. oralis cell number significantly correlated with the

75 periodontal clinical score with r = 0.527, n = 30, p = 0.003. The Sig, Anova test or the Krusskal-

76 Wallis test (2-tailed) correlation was 0.003.

77 PCR systems previously used to detect methanogens in the subgingival plaque (1, 12, 13, 19)

78 were not completely specific for M. oralis. Indeed, other methanogens including Methanobrevibacter

79 smithii (10), Methanosphaera stadtmanae (1), Methanobacterium curvum / Methanobacterium

80 congolense and Methanosarcina mazeii (14, 8) have been detected in this situation. Here, we

81 developed a real-time PCR assay in the perspective of its routine use in clinical microbiology

82 laboratories. In-silico analyses indicated that the cnp60-gene system reported here was M. oralis-

83 specific. Accordingly, no amplification was obtained by incorporating seven other archaea and

84 bacteria. Furthermore, amplification of the plasmid control demonstrated the lack of PCR inhibitors in

85 all specimens and this system was calibrated. Total bacterial control indicated that for a Ct value >

86 35.5, M. oralis detection was not interpretable. Based on these controls, the detection of M. oralis

87 DNA in healthy individuals (Ct ≤ 35.5) was possible in contrast to previous studies (13, 17-19) and a

88 significant correlation was observed between the M. oralis load and periodontitis severity.

89 Because its sensitivity and specificity, the use of this real-time PCR system simplifies the

90 molecular-based detection of M. oralis in clinical specimens, rendering this task compatible with

91 a routine diagnosis activity. Data herein reported indicate that it is possible to measure the load of

92 M. oralis in the subgingival plaque by using real-time PCR. This study further established the

93 proof-of-concept that M. oralis load in the periodontal pockets correlates to a standardized

94 severity score of periodontitis. The increased amount of M. oralis in healthy and diseased patients

95 may indicate its use as a biomarker of altered microbiota. Monitoring the M. oralis load by using

5 JCM01962-12- revised version-2

96 real-time PCR could be useful for the diagnosis and staging of patients with periodontitis and

97 treatment follow-up. It could complement clinical evaluation in detecting individuals at higher

98 risk of developing severe periodontitis; and in evaluation the efficacy of antibiotic treatment as M.

99 oralis is highly resistant to antibiotics, except for metronidazole (9).

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100 References :

101 1. Belay N, Johnson R, Rajagopal B S, Conway de Macario E, Daniels L. 1988.

102 Methanogenic bacteria from human dental plaque. Appl. Environ. Microbiol. 54:600-603.

103 2. Brusa T, Conca R, Ferrara A, Ferrari A, Pecchioni A. 1987. The presence of

104 methanobacteria in human subgingival plaque. J. Clin. Periodontol. 14:470-471.

105 3. Chandra RV. 2007. Evaluation of a novel periodontal risk assessment model in patients

106 presenting for dental care. Oral Health Prev. Dent. 5:39-48.

107 4. D'Aiuto F, Parkar M, Nibali L, Suvan J, Lessem J, Tonetti MS. 2006. Periodontal

108 infections cause changes in traditional and novel cardiovascular risk factors: results from

109 a randomized controlled clinical trial. Am. Heart J. 151:977-984.

110 5. Dortbudak O, Eberhardt R, Ulm M, Persson GR. 2005. Periodontitis, a marker of risk

111 in pregnancy for preterm birth. J. Clin. Periodontol. 32:45-52.

112 6. Dridi B, Henry M, El Khéchine A, Raoult D, Drancourt M. 2009. High prevalence of

113 Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut

114 using an improved DNA detection protocol. PLoS ONE. 4:e7063.

115 7. Ferrari A, Brusa T, Rutli A, Canzi E, Biavati B. 1994. Isolation and characterization of

116 Methanobrevibacter oralis sp. nov. Curr. Microbiol. 29:7-12.

117 8. Horz HP, Seyfarth I, Conrads G. 2012. McrA and 16S rRNA gene analysis suggests a

118 novel lineage of Archaea phylogenetically affiliated with Thermoplasmatales in human

119 subgingival plaque. Anaerobe 18:373-377.

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120 9. Khelaifia S, Drancourt M. 2012. Susceptibility of archaea to antimicrobial agents:

121 applications to clinical microbiology. Clin Microbiol Infect.18:841-848.

122 10. Kulik EM, Sandmeier H, Hinni K, Meyer J. 2001. Identification of archaeal rDNA

123 from subgingival dental plaque by PCR amplification and sequence analysis. FEMS

124 Microbiol. Lett. 196:129-133.

125 11. Lang NP, Tonetti MS. 2003. Periodontal risk assessment (PRA) for patients in

126 supportive periodontal therapy (SPT). Oral Health Prev. Dent. 1:7-16.

127 12. Lepp PW, Brinig MM, Ouverney CC, Palm K, Armitage GC, Relman DA. 2004.

128 Methanogenic Archaea and human periodontal disease. Proc. Natl. Acad. Sci U S A

129 101:6176-6181.

130 13. Li CL, Liu DL, Jiang YT, Zhou YB, Zhang MZ, Jiang W, Liu B, Liang JP. 2009.

131 Prevalence and molecular diversity of Archaea in subgingival pockets of periodontitis

132 patients. Oral Microbiol. Immunol. 24:343-346.

133 14. Matarazzo F, Ribeiro AC, Feres M, Faveri M, Mayer MP. 2011. Diversity and

134 quantitative analysis of Archaea in aggressive periodontitis and periodontally healthy

135 subjects. J. Clin. Periodontol. 38:621-627.

136 15. Pihlstrom BL, Michalowicz BS, Johnson NW. 2005. Periodontal diseases. Lancet

137 366:1809-1820.

138 16. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. 1998. Microbial

139 complexes in subgingival plaque. J. Clin. Periodontol. 25:134-144.

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140 17. Vartoukian SR, Palmer RM, Wade WG. 2007. The division "Synergistes". Anaerobe

141 13:99-106.

142 18. Vianna ME, Holtgraewe S, Seyfarth I, Conrads G, Horz HP. 2008. Quantitative

143 analysis of three hydrogenotrophic microbial groups, methanogenic archaea, sulfate-

144 reducing bacteria, and acetogenic bacteria, within plaque biofilms associated with human

145 periodontal disease. J. Bacteriol. 190:3779-3785.

146 19. Yamabe K, Maeda H, Kokeguchi S, Tanimoto I, Sonoi N, Asakawa S, Takashiba S.

147 2008. Distribution of Archaea in Japanese patients with periodontitis and humoral

148 immune response to the components. FEMS Microbiol. Lett. 287:69-75.

149

150

9 JCM01962-12- revised version-2

151 Table legend:

152 M. oralis cell number and Ct value (cycle threshold) of two internal controls. The total bacterial

153 DNA was tested by real-time PCR and a quantification plasmid (Stdint) was introduced as an

154 internal control to monitor the absence of PCR inhibition. P: patient; C: controls; BOP: Bleeding

155 On Probing; PD: Probing Depth; PI: Plaque Indice. Clinical score was determined as previously

156 reorted (3).

157

10 M. oralis All Clinical Sample Stdint/Ct cells BOP PD (mm) PI (%) bacteria/Ct score number P1 21.9 33.3 / localized 6 32 19 P2 22.6 23.1 3.125E+04 generalized 8 63 28 P3 22.4 22.6 7.962E+05 localized 8 34 30 P4 21.2 21.4 8.965E+05 generalized 6 60 27 P5 24.0 27.9 6.325E+03 generalized 5 65 27 P6 20.9 35.1 / localized 5 28 16 P7 22.8 37.8 / generalized 6 56 18 P8 23.4 31.5 / generalized 7 34 23 P9 24.5 21.6 6.125E+06 generalized 6 38 27 P10 23.3 22.2 3.896E+05 localized 6 54 21 P11 21.8 37.6 / generalized 9 40 29 P12 22.2 27.2 2.005E+04 generalized 7 70 29 P13 24.3 28.5 2.654E+03 generalized 6 55 26 P14 241 26.3 9.652E+02 localized 6 59 27 P15 23.3 35.2 / generalized 7 59 24 P16 22.2 37.2 / generalized 6 62 28 P17 23.4 24.2 5.324E+03 localized 5 29 27 P18 23.9 35.6 / localized 10 85 30 P19 22.2 34.3 / localized 7 75 26 P20 23.6 23.9 1.956E+05 localized 7 12 28 P21 24.2 26.8 5.785E+03 generalized 7 33 28 P22 21.9 32.7 / generalized 7 22 27 C23 24.0 35.4 / / 4 C24 23.6 35.2 4.862E+02 / 8 C25 22.3 37.2 / / 5 C26 23.5 / / / 7 C27 23.2 37.3 / / 5 C28 23.5 35.2 / / 7 C29 22.7 32.3 5.329E+02 / 4 C30 24.1 33.5 5.974E+02 / 6 C31 23.3 / / / 5 C32 24.2 36.2 / / 4 T- / / / T- / / / T+ 22.2 18.3 10E+09 T+ 23.4 17.5 10E+09

______Chapitre 5

Chapitre 5

Tungsten-enhanced growth of Methanosphaera stadtmanae

Bédis Dridi1, Saber Khelaifia1, Marie-Laure Fardeau2, Bernard Ollivier2 and

Michel Drancourt1*

1 Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes UMR CNRS

6236 IDR 198, IFR48 IHU POLMIT, Université de la Méditerranée, Marseille, France.

2 Laboratoire de Microbiologie IRD, UMR D180, Microbiologie et Biotechnologie des

Environnements Chauds, Universités de Provence et de la Méditerranée, ESIL, Marseille,

France.

*Corresponding author: Professeur Michel Drancourt, Unité des Rickettsies, Faculté de Médecine,

27, Boulevard Jean Moulin-Cedex 5- France. Tel: 00 33 4 91 38 55 17. Fax: 00 33 4 91 38 77 72.

Email: [email protected]

Keywords: Methanogenic Archaea, Methanosphaera stadtmanae, Methanomassiliicoccus luminyensis, Tungsten, Selenium

BMC Research Notes (2012)

______Chapitre 5

Chapitre 5: Préambule

L’archaea méthanogène Methanosphaera stadtmanae a été détectée dans le microbiote intestinal humain par la méthode de culture anaérobie mise au point par Hungate et qui reste la technique de référence pour l’isolement de tels microorganismes fastidieux. La croissance de M. stadtmanae atteint une phase exponentielle après 5 à 7 jours de culture dans le milieu

DSM 322 (10% vol). L’isolement récent de Methanomassiliicoccus luminyensis, une archaea méthanogène qui nécessite une solution de tungstate/sélénite pour sa croissance et qui possède des caractéristiques métaboliques similaire à celles de M. stadtmanae nous a incité à étudier les effets du tungstène et du sélénium sur la croissance de M. stadtmanae. L’addition de 0,2 mg/L tungstate de sodium au milieu DSM 322 avait un effet fortement stimulant permettant d’accélérer vitesse de croissance de M. stadtmanae par un facteur 3. Ces données fournissent de nouvelles informations concernant les exigences nutritionnelles mal connues des archaea méthanogènes associées à l’Homme.

Dridi et al. BMC Research Notes 2012, 5:238 http://www.biomedcentral.com/1756-0500/5/238

SHORT REPORT Open Access Tungsten-enhanced growth of Methanosphaera stadtmanae Bédis Dridi1, Saber Khelaifia1, Marie-Laure Fardeau2, Bernard Ollivier2 and Michel Drancourt1*

Abstract Background: The methanogenic Archaea Methanosphaera stadtmanae has been detected in the human gut microbiota by both culture and culture-independent methods. Its growth reaches an exponential phase after 5 to 7-day culture in medium 322 (10% vol). Our recent successful isolation of Methanomassiliicoccus luminyensis,a tungstate-selenite-requiring Archaea sharing similar metabolism characteristics with M. stadtmanae prompted us to study the effects of tungsten and selenium on M. stadtmanae growth. Findings: Addition of 0.2 mg/L sodium tungstate to medium 322 yielded, 48 hours after inoculation, a growth rate equivalent to that obtained after 6 days with control culture as measured by methane monitoring and optical density measurement. Addition of 50 μg/mL sodium selenate had no effect on M. stadtmanae growth. Quantitative real-time PCRs targeting the M. stadtmanae 16S rRNA confirmed these data. Conclusions: These data provide new information regarding the poorly known nutritional requirements of the human gut colonizing organisms M. stadtmanae. Adding sodium tungstate to basal medium may facilitate phenotypic characterization of this organism and additionally aid the isolation of new Archaea from complex host microbiota. Keywords: Methanogenic Archaea, Methanosphaera stadtmanae, Methanomassiliicoccus luminyensis, Tungsten, Selenium

Findings [5,6]) within the order Methanobacteriales. We recently Methanosphaera stadtmanae is a spherical-shaped, non- isolated Methanomassiliicoccus luminyensis,thefirstcul- motile archaeon initially isolated from human feces [1]. tured representative of new order of methanoarchaea [7]. M. stadtmanae was the first human Archaea to be This archaeon exhibits a metabolic trait similar to that of genome sequenced and analysis of the genome confirmed M. stadtmanae by using hydrogen as electron donor and that M. stadtmanae belonged to Methanobacteriales [2]. methanol as electron acceptor [7]. Unexpectedly, we PCR-based analyses further indicated that M. stadtmanae- observed that addition of tungstate-selenite to culture specific sequences could be detected in stool specimen in medium had been a key factor for successful isolation of up to 30% of individuals [3]. However, M. stadtmanae is M. luminyensis and that this archaeon indeed required a fastidious organism, with only one M. stadtmanae iso- tungstate-selenite as an essential element for growth. We late reported and accordingly only one M. stadtmanae therefore tested the hypothesis that the addition of so- strain available in public collections. M. stadtmanae oxi- dium tungstate or sodium selenate or both to basal cul- dizes hydrogen to reduce methanol into methane [1,2]. ture medium would also enhance the growth of M. This metabolic trait has been already reported for M. stadtmanae. stadtmanae [4], and more recently for members of the M. stadtmanae DSMZ 3091 T (ATCC 43021T) pur- genus Methanobacterium (e.g. M. veterum and M. lacus; chased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) was * Correspondence: [email protected] grown on medium 322 (http://www.dsmz.de) incubated 1 Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes at 37°C in Hungate tubes (Dutscher, Issy-les-Moulineaux, UMR CNRS 6236 IDR 198, IFR48, Institut Méditerranée Infection, – Aix-Marseille-Université, Marseille, France France) under 2-bar pressure of a H2/CO2 (80 20) at- Full list of author information is available at the end of the article mosphere. The inoculated medium (10% vol) was

© 2012 Dridi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dridi et al. BMC Research Notes 2012, 5:238 Page 2 of 4 http://www.biomedcentral.com/1756-0500/5/238

incubated at 37°C with shaking. On the exponential phase of this first culture, a second inoculation was performed by 10% vol. in the same basal medium modified or not by the addition of Na2O4W (0.2 mg/L) and/or Na2O4Se (50 μg/L) (Sigma, Saint-Quentin Fallavier, France). Non-inoculated media were used as negative controls and each experiment was repeated ten times. Growth was assessed by optical microscope observation, parallel methane production measurement and measurement of the optical density of the medium. Methane production measurement used a GC-8A gas chromatograph (Shimadzu, Champs-sur-Marne, France) equipped with a thermal conductivity detector and a Chromosorb WAW 80/100 mesh SP100 column (Alltech, Carquefou, France). N2 at a pressure of 100 kPa Figure 1 Visualizations of H2 used (in mM; left Y axis) and CH4 was used as the carrier gas. The detector and the injector (in mM; right Y axis) produced by M. stadtmanae with and without addition of sodium tungstate solution (Na O W) (over temperatures were 200°C, and the column temperature 2 4 140 hours (X axis). ♦ H used with sodium tungstate (Na O W), was 150°C. H consumption and CH production were 2 2 4 2 4 ■ CH4 production with sodium tungstate (Na2O4W), ▲ H2 used measured every 6 hours for 24 hours and then every without sodium tungstate (Na2O4W), and ● CH4 production 12 hours for 6 days. The optical density at 580 nm without sodium tungstate (Na2O4W). was measured by inserting Hungate tubes into the spectrophotometer (Varian Cary50; Agilent Technologies, Massy, France). Experiment was done in triplicate and The addition of sodium selenate alone has no effect on average optical density value for the three replicates the growth curve of M. stadtmanae. However, the was calculated. addition of sodium tungstate alone or in combination M. stadtmanae DNA extraction, quantification and se- with sodium selenate shortened the lag period to 2 days quencing were performed as previously described based post-inoculation with an equivalent 16S rRNA and rpoB on specific quantitative real-time PCR targeting 16S genes copy number and with equivalent rates of me- rRNA gene [3]. thane production (Figure 1). In the absence of tungstate, Negative controls (with and without tungstate and sel- M. stadtmanae exhibited a 30-hour log phase. Adding enium) remained negative with no growth occurring tungsate to the culture medium reduced the delay of this after one-week incubation indicating that results herein log-phase so that it took 47 hours instead of 72 hours to reported did not merely result from carry-over of organ- achieve a 0.35 optical density of the culture (Figure 2). isms. The exponential phase of M. stadtmanae growth These results correlated with the fact that M. stadtma- cultured in medium 322 was reached at 6-day incuba- nae genome encodes a formylmethanofuran dehydro- tion. At this point microscopic observation disclosed genase comprising of five sub-units (Genes IDs: organisms with morphology compatible with M. stadt- 3855499-3855500-3855501-3855502-3855503), an en- manae and no contaminant. Also, qPCR detected an zyme found in methanogenic Archaea. In strict anaer- equivalent of 3.22E + 12 ± 1.53E + 11 copies of 16S rRNA obic micro-organisms, this enzyme catalyzes the gene/mL (Table 1). Sequencing of 16S rRNA gene PCR reversible dehydrogenation of formylmethanofuran into products from all specimens yielded a sequence similar- CO2 and methanofuran. The formylmethanofuran dehy- ity of 99-100% with the reference M. stadtmanae DSM drogenases are either molybdenum- or tungsten-iron- 3091 sequence. sulfur proteins. The tungsten is likely bound to the same

Table 1 M. stadtmanae 16S rDNA gene copy number after 48-hour culture with Na2O4W+Na2O4Se or only Na2O4W and a 6-day culture with no Na2O4W+Na2O4Se or only with Na2O4Se (Mean and standard deviation were calculated for 10 independent culture tests for each condition) 48-hour culture 6-day culture without with with with without

Na2O4W+Na2O4Se Na2O4W+Na2O4Se Na2O4WNa2O4Se Na2O4W+Na2O4Se Means 2.13E + 10 4.42E + 12 3.93E + 12 4.02E + 12 3.22E + 12 Standard deviation 5.56E + 09 1.84E + 11 3.67E + 11 2.23E + 11 1.53E + 11 Dridi et al. BMC Research Notes 2012, 5:238 Page 3 of 4 http://www.biomedcentral.com/1756-0500/5/238

Figure 2 The effect of addition of selenite/tungstate solution on growth of M. stadtmanae. ♦ Growth of M. stadtmanae with tungstate

(Na2O4W). ■ Growth of M. stadtmanae without tungstate (Na2O4W). skeleton as the molybdenum in the so-called molybdop- dehydrogenase [18]. Selenium was also reported as a terin dinucleotide cofactor [8-10]. component of both a hydrogenase [19] and tRNA [20]. Previous reports described the requirement of tungsten In the absence of tungstate, M. stadtmanae exhibited for growing numerous methanogens including Metha- a growth delay of 5–7 days which is long for testing nothermobacter wolfei which has an obligate requirement in vitro susceptibility to antibiotics [22]. As we now for tungsten to maintain autotrophic growth, Methano- observed that tungsten enhances the growth of two coccus vannielii requiring tungsten as a cofactor for the taxonomically unrelated methanogens, M. stadtmanae enzyme formate dehydrogenase [11], Methanogenium and M. luminyensis, we suggest that tungsten-containing tatii [12] and Methanocorpusculum parvum [13] also re- media could be incorporated into the panel of media quiring tungsten for growth (Table 2). Selenium has also used for the isolation and culture of new methanogens been reported as stimulatory and may be required for from clinical and environmental specimens, and for test- many methanogens, especially members of the genus ing their in-vitro susceptibility to antibiotics. Methanococcus as Methanococcus vannielii [11], Metha- Methanogenic Archaea recently emerged as normal nococcus jannaschii [14], Methanococcus maripaludis components of the human gastrointestinal and oral mi- [15], Methanococcus voltae [16] and Methanococcus ther- crobial ecosystems, where they could play important molithotrophicus [17] (Table 1). Requirement for selenium roles in health and diseases [23]. However, the isolation could have enzymatic basis, since it was reported that of such organisms requires long incubation times and M. vannielii possesses a selenium-dependant formate strict anoxic atmosphere and is hampered by the incom- plete knowledge of their nutritional requirements [23]. In fact, the result obtained in the present study may Table 2 Requirement of tungsten or/and selenium for growth of methanogens as reported in bibliography prompt further phenotypic characterization including extended antibiotic susceptibility testing [22] and even Species Tungsten Selenium References allowing isolation of new Archaea in order to assess Methanothermobacter wolfei YES NA [21] understanding their contribution in the physiology of Methanococcus vannielii YES YES [11,18] complex human microbiomes and their potential role in Methanogenium tatii YES NA [12] the course of infections. Methanocorpusculum parvum YES NA [13] Methanococcus jannaschii NA YES [14] Competing interests The authors declare that they have no competing interests. Methanococcus maripaludis NA YES [15] Methanococcus voltae NA YES [16] Author’s contributions Methanococcus thermolithotrophicus NA YES [17] BD, SK, MLF designed and performed analyses, BO, MD interpreted data and wrote the draft. All authors read and approved the final manuscript. Dridi et al. BMC Research Notes 2012, 5:238 Page 4 of 4 http://www.biomedcentral.com/1756-0500/5/238

Author details 19. Yamazaki S: A selenium-containing hydrogenase from Methanococcus 1Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes vannielii, Identification of the selenium moiety as a selenocysteine UMR CNRS 6236 IDR 198, IFR48, Institut Méditerranée Infection, residue. J Biol Chem 1982, 257:7926–7929. Aix-Marseille-Université, Marseille, France. 2Laboratoire de Microbiologie IRD, 20. Ching WM, Wittwer AJ, Tsai L, Stadtman TC: Distribution of two UMR D180, Microbiologie et Biotechnologie des Environnements Chauds, selenonucleosides among the selenium-containing tRNAs from Aix-Marseille-Université, ESIL, Marseille, France. Methanococcus vannielii. Proc Natl Acad Sci U S A 1984, 81:57–60. 21. König H, Semmler R, Lerp C, Winter J: Evidence for the occurrence of Received: 16 November 2011 Accepted: 25 April 2012 autolytic enzymes in Methanobacterium wolfei. Arch Microbiol 1985, Published: 15 May 2012 141:177–180. 22. Dridi B, Fardeau ML, Ollivier B, Raoult D, Drancourt M: The antimicrobial resistance pattern of cultured human methanogens reflects the unique References phylogenetic position of archaea. J Antimicrob Chemother 2011, 1. Miller TL, Wolin MJ: Methanosphaera stadtmaniae gen. nov., sp. nov.: a 66:2038–2044. species that forms methane by reducing methanol with hydrogen. Arch 23. Dridi B, Raoult D, Drancourt M: Archaea as emerging organisms in – Microbiol 1985, 141:116 122. complex human microbiomes. Anaerobe 2011, 17:56–63. 2. Fricke WF, Seedorf H, Henne A, Kruer M, Liesegang H, Hedderich R, et al: The genome sequence of Methanosphaera stadtmanae reveals why this doi:10.1186/1756-0500-5-238 human intestinal archaeon is restricted to methanol and H2 for methane – Cite this article as: Dridi et al.: Tungsten-enhanced growth of formation and ATP synthesis. J Bacteriol 2006, 188:642 658. Methanosphaera stadtmanae. BMC Research Notes 2012 5:238. 3. Dridi B, Henry M, El Khéchine A, Raoult D, Drancourt M: High prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using an improved DNA detection protocol. PLoS One 2009, 4:e7063. 4. Kendall M, Boone D: The Order Methanosarcinales.InThe Prokaryotes. Edited by Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E. New York: Spriger; 2006:244–256. 5. Krivushin KV, Shcherbakova VA, Petrovskaya LE, Rivkina EM: Methanobacterium veterum sp. nov., from ancient Siberian permafrost. Int J Syst Evol Microbiol 2010, 60:455–459. 6. Borrel G, Joblin K, Guedon A, Colombet J, Tardy V, Lehours AC, Fonty G: Methanobacterium lacus sp. nov., a novel hydrogenotrophic methanogen from the deep cold sediment of a meromictic lake. Int J Syst Evol Microbiol 2011, Sep 2. [Epub ahead of print] PubMed PMID: 21890730. 7. Dridi B, Fardeau ML, Ollivier B, Raoult D, Drancourt M: Methanomassiliicoccus luminyensis, gen. nov., sp. nov., a novel methanogenic Archaea isolated from human feces. Int J Syst Evol Microbiol 2012, 62:1902–1907. in press. 8. Karrasch M, Borner G, Enssle M, Thauer RK: The molybdoenzyme formylmethanofuran dehydrogenase from Methanosarcina barkeri contains a pterin cofactor. Eur J Biochem 1990, 194:367–372. 9. Karrasch M, Borner G, Enssle M, Thauer RK: Formylmethanofuran dehydrogenase from methanogenic bacteria, a molybdoenzyme. FEBS Lett 1989, 253:226–230. 10. Borner G, Karrasch M, Thauer RK: Molybdopterin adenine dinucleotide and molybdopterin hypoxanthine dinucleotide in formylmethanofuran dehydrogenase from Methanobacterium thermoautotrophicum (Marburg). FEBS Lett 1991, 290:31–34. 11. Jones JB, Stadtman TC: Methanococcus vannielii: culture and effects of selenium and tungsten on growth. J Bacteriol 1977, 130:1404–1406. 12. Zabel HP, König H, Winter J: Isolation and characterization of a new coccoid methanogen, Methanogenium tatii spec. nov. from a solfataric field on Mount Tatio. Arch Microbiol 1984, 137:308–315. 13. Zellner G, Alten C, Stackebrandt E, Conway De Macario E, Winter J: Isolation and characterization of Methanocorpusculum parvum gen. nov., spec. nov., a new tungsten requiring, coccoid methanogen. Arch Microbiol 1987, 147:13–20. 14. Jones WJ, Leigh JA, Mayer F, Woese CR, Wolfe RS: Methanococcus jannaschii sp. nov., an extremely thermophilic methanogen from a submarine hydrothermal vent. Arch Microbiol 1983, 136:254–261. Submit your next manuscript to BioMed Central 15. Jones WJ, Paynter MJB, Gupta R: Characterization of Methanococcus and take full advantage of: maripaludis sp. nov., a new methanogen isolated from salt marsh sediment. Arch Microbiol 1983, 135:91–97. • Convenient online submission 16. Berghöfer Y, Agha-Amiri K, Klein A: Selenium is involved in the negative regulation of the expression of selenium-free [NiFe] hydrogenases in • Thorough peer review Methanococcus voltae. Mol Gen Genet 1994, 242:369–373. • No space constraints or color ﬁgure charges 17. Belay N, Sparling R, Daniels L: Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus. Appl Environ • Immediate publication on acceptance Microbiol 1986, 52:1080–1085. • Inclusion in PubMed, CAS, Scopus and Google Scholar 18. Jones JB, Stadtman TC: Selenium-dependent and selenium-independent • Research which is freely available for redistribution formate dehydrogenases of Methanococcus vannielii. Separation of the two forms and characterization of the purified selenium-independent form. J Biol Chem 1981, 256:656–663. Submit your manuscript at www.biomedcentral.com/submit

______Chapitre 6

Chapitre 6

A versatile medium for cultivating methanogenic archaea

Saber Khelaifia, Didier Raoult, and Michel Drancourt#

1Aix Marseille Université, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095, 13005,

Marseille, France

#Corresponding author: Professeur. Michel DRANCOURT

Unité des Rickettsies, Faculté de Médecine, 27, Boulevard Jean Moulin-Cedex 5- France.

Tel: 00 33 4 91 38 55 17, Fax: 00 33 4 91 38 77 72, Email: [email protected]

Keywords: anaerobic culture, culture medium, nutrient requirements, human methanogenic

archaea

PLoS ONE (2013)

______Chapitre 6

Chapitre 6: Préambule

Peu d’archaea méthanogènes ont été cultivées à partir du microbiote digestif ou de la cavité buccale de l’Homme. Parmi ces méthanogènes on compte Methanobrevibacter smithii,

Methanosphaera stadtmanae, Methanomassilicoccus luminyensis, Methanobrevibacter arboriphilicus et Methanobrevibacter oralis. Elles sont toutes anaérobie strict caractérisées par leur croissance lente exigeant un milieu spécifique pour une croissance optimale de chaque espèce. Ces spécificités limitent l'isolement et la culture des archaea méthanogènes à partir d'échantillons cliniques. Un nouveau milieu de culture ici désigné comme milieu de culture SAB a été optimisé et testé pour la culture des archaea méthanogènes associées au microbiote humain, ainsi que deux archaea méthanogènes mésophile, Methanobacterium beijingense et Methanosaeta concilii et pour l'isolement des archaea méthanogène à partir de

20 échantillons de selles humaines, dont 10 échantillons testés positifs pour M . smithii par

PCR et 10 négatifs. Le milieu de culture SAB favorise la croissance rapide des archaea méthanogènes humaine ainsi que les deux méthanogènes mésophiles. C’est un support polyvalent qui devrait simplifier la détection des archaea méthanogènes par la culture dans les

échantillons cliniques et environnementaux.

Les trois co-auteurs sont co-inventeurs d'un brevet N / Réf: H52 888 CAS 13 FR (MD

/ SB 12.05.00329) pour le milieu de culture rapporté ici.

A Versatile Medium for Cultivating Methanogenic Archaea

Saber Khelaifia, Didier Raoult, Michel Drancourt* Aix Marseille Universite´, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095, 13005, Marseille, France

Abstract

Background: Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, Methanomassilicoccus luminyensis and Methanobrevibacter arboriphilicus have been cultured from human digestive microbiota. Each one of these fastidious methanogenic archaea requires a specific medium for its growth, hampering their routine isolation and the culture.

Methodology/Principal Findings: A new culture medium here referred as SAB medium was optimized and tested to cultivate methanogens associated with human microbiota, as well as two mesophile methanogens Methanobacterium beijingense and Methanosaeta concilii. It was further tested for the isolation of archaea from 20 human stool specimens including 10 specimens testing positive for PCR detection of M. smithii. After inoculating 105 colony-forming-unit archaea/ mL or 1 g stool specimen in parallel in SAB medium and reference DSMZ medium in the presence of negative controls, growth of archaea was determined by optical microscopy and the measurement of methane production by gas chromatography. While the negative controls remained sterile, all tested archaea grew significantly more rapidly in SAB medium than in reference medium in 1–3 days (P,0.05, Student test). Among PCR-positive stool specimens, 10/10 grew in the SAB medium, 6/10 in DSMZ 119 medium, 5/10 in DSMZ 322 medium and 3/10 in DSMZ 334 c medium. Four out of ten PCR-negative stool specimens grew after a 3-week incubation in the SAB-medium whereas no growth was detected in any of the reference media. 16S rRNA gene sequencing yielded 99–100% sequence similarity with reference M. smithii except for one specimen that yielded 99–100% sequence similarity with reference Methanobrevibacter millerae.

Conclusions/Significance: SAB medium allows for the versatile isolation and growth of methanogenic archaea associated with human gut microbiota including the archaea missed by inoculation of reference media. Implementation of the SAB medium in veterinary and medical microbiology laboratories will ease the routine culture-based detection of methanogenic archaea in clinical and environmental specimens.

Citation: Khelaifia S, Raoult D, Drancourt M (2013) A Versatile Medium for Cultivating Methanogenic Archaea. PLoS ONE 8(4): e61563. doi:10.1371/ journal.pone.0061563 Editor: Arnold Driessen, University of Groningen, The Netherlands Received January 25, 2013; Accepted March 11, 2013; Published April 17, 2013 Copyright: ß 2013 Khelaifia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction the most restricted energy metabolism of all known human- associated methanogenic archaea, generating methane through So far, new laboratory techniques for cultivating strict the reduction of methanol with H2 and being strictly dependent on anaerobes, including the technique of Hungate [1,2] have allowed acetate as a carbon source [13]. Recently, we showed that a isolation and further characterization of several new species of the selenite/tungstate solution boosted the growth of this archaea [14] methanogenic archaea associated with human microbiota [3–8]. and was required for culturing a new methanogen, the However, isolating methanogenic archaea remains a fastidious Methanomassilicoccus luminyensis from human stool specimens [3]. process because of the slow growth of these archaea and because of The growth of other archaeal species requires aromatic amino- their extreme intolerance to oxygen [9]. Accordingly, an acid tryptophan, thiamine, pyridoxine, p-aminobenzoic acid atmosphere consisting of 80% H2 and 20% CO2 is one of the (PABA), branched-chain fatty acids, acetate and other unknown specific requirements for the optimal growth of these archaea [10]. factors [15,16]. Methanogens further require metal ions such as Mastering the techniques of anaerobic culture is not sufficient to nickel, which is present in F430, hydrogenase and carbon isolate and cultivate methanogens and additional knowledge in monoxide dehydrogenase [17,18]. nutrient requirements has proved useful to design methanogenic Because of these specific requirements, laboratories are archaea culture media [11]. currently preparing one specific culture medium for each one of Some methanogenic archaea, such as Methanobrevibacter smithii the various methanogenic archaea species. As a consequence, [7], Methanobrevibacter oralis [4] and Methanobrevibacter arboriphilicus methanogens are excluded from the routine culture of human gut [12] that we recently isolated from two human stool specimens (S. microbiota, despite the fact that they have been associated with Khelaifia and M. Drancourt, unpublished data) were reported to some pathologies including digestive tract diseases, obesity and require ruminal fluid for growth. Methanosphaera stadtmanae [8] has

PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e61563 Improved Culture of Methanogens vaginal infection [19–21] and more convincingly periodontitis were PCR-negative. No written consent was needed for this work [22–24]. In this study, we aimed to design a new, versatile culture in accordance with the Law regarding bioethics ‘‘nu 2004–800 medium that would allow for the culture of all methanogenic relative a` la bioe´thique’’ published in the ‘‘Journal Officiel de la archaea. Re´publique Franc¸aise’’ the 6 August 2004 since no additional sample was taken for the study. This study was approved by the Materials and Methods local ethic committee of the Institut Fe´de´ratif de Recherche 48, Faculty of Medicine, Marseille, France of 17.02-07 which Archaea Organisms exempted this study from requiring written informed consent. M. smithii ATCC 35061T DSMZ 861, M. smithii DSMZ 2374, The samples were analysed anonymously. M. smithii DSMZ 2375, M. smithii DSMZ 11975, M. oralis DSMZ Approximately, 1 g of each stool specimen was inoculated in a 7256 T, M. stadtmanae ATCC 43021T DSMZ 3091, M. beijingense bottle containing 50 mL of each medium. One decimal dilution DSMZ 15999 and M. concilii DSMZ 2139 purchased from the was made on the supernatant, in Hungate tubes, to eliminate a German Collection of Microorganisms and Cell Cultures (DSMZ, maximum of stools and to promote growth only on the culture Braunschweig, Germany). The M. arboriphilicus strain tested in this medium. Vancomycin 100 mg/L and imipenem 100 mg/L study was recently isolated in our laboratory from one human stool (Mylan SAS, Saint Priest, France) and amphotericin B 50 mg/L specimen. The M. smithii strains, M. arboriphilicus and M. beijingense (Bristol-Myers-Squibb, Rueil-Malmaison, France) were added to [6] were grown in liquid medium 119 (http://www.dsmz.de). To the culture medium to eliminate contaminants from intestinal cultivate M. oralis, medium 119 was modified by the addition of microflora. Growth delay for clinical specimens incubated in the 1 g/L of yeast extract and 1 g/L of peptone, and a 2.5 bar H2/ SAB-medium was compared to growth delay in three standard CO2 (80%-20%) atmosphere was used. Medium 322 (http:// DSMZ culture media 119, 322 and 334c (http://www.dsmz.de). www.dsmz.de) was used to cultivate M. stadtmanae. Medium 334c was used (http://www.dsmz.de) to cultivate M. concilii at 37uCin SAB Medium Hungate tubes (Dutscher, Issy-les-Moulineaux, France) under a 2- To optimize the SAB-medium, we used a basal medium bar H2/CO2 (80%–20%) atmosphere with agitation. M. luminyensis consisting of components shared by all studied DSMZ media and T CSUR P135 was cultivated using the Methanobrevibacter medium other media used to isolate and cultivate some methanogenic (medium 119: http://www.dsmz.de) modified by the addition of archaea [3,5–8,14,25]. Thereafter, we added to this medium some methanol and selenite/tungstate solution under 2-bar of H2/CO2 compounds known to enhanced growth of methanogenic archaea (80%-20%) atmosphere with agitation [3]. [11,14,16–18,26–31] and we monitored the effect of each compound on the methanogens’ growth. The definite SAB Clinical Specimens medium contains the following: NiCl2 . 6H2O, 1.5 mg/L; FeSO4 This study included 20 stool specimens prospectively collected . H2O, 0.5 mg/L; MgSO4 . 7H2O, 0.8 g/L; KH2PO4, 0.5 g/L; in 20 individuals, in Marseille, France, between July and August K2HPO4, 0.5 g/L; KCl, 0.05 g/L; CaCl2 . 7H2O, 0.05 g/L; 2011. Ten specimens were PCR-positive for M. smithii, and ten NaCl, 1.5 g/L; NH4Cl, 1 g/L; MnSO4 . 7H2O, 0.6 mg/L;

Figure 1. Growth time of ten methanogenic archaea strains growing in culture medium SAB-medium or the standard DSMZ media. (Triplicate experiment). doi:10.1371/journal.pone.0061563.g001

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Figure 2. Growth monitoring of the five human-associated archaea strains by monitoring the CH4 production and H2 used by methanogens growing in culture medium SAB-medium or the standard DSMZ media. A: Methanobrevibacter smithii; B: Methanobrevibacter oralis;C:Methanosphaera stadtmanae;D:Methanomassilicoccus luminyensis; E: Methanobrevibacter arboriphilicus (Triplicate experiment). doi:10.1371/journal.pone.0061563.g002

ZnSO4 . 7H2O, 0.1 mg/L; CuSO4 . 5H2O, 0.02 mg/L; Growth Detection 5 KAl(SO4)2 . 12H2O, 0.2 mg/L; H3BO3,7mg/L; CoSO4 . After inoculation of the culture media with 10 colony-forming 7H2O, 4 mg/L; Na2MoO4 . 2H2O, 0.5 mg/L; Na2SeO3 . units (CFU)/mL of archaea or 1 g stool specimen, growth of the 5H2O, 3 mg/L; Na2WO4 6 2H2O, 4 mg/L; Nitrilotriacetic acid, methanogens was verified (i) by monitoring the presence of 0.15 mg/L; sodium acetate, 1 g/L; trypticase, 2 g/L; yeast organisms by optical microscopy observations (ii) by measuring extract, 2 g/L; L-cysteine hydrochloride monohydrate, 0.5 g/L; methane production using a GC-8A gas chromatograph (Shi- valeric acid, 5 mM; isovaleric acid, 5 mM; 2-methylbutyric acid, madzu, Champs-sur-Marne, France) equipped with a thermal 5 mM; isobutyric acid, 6 mM; 2-methyl valeric acid, 5 mM; conductivity detector and a Chromosorb WAW 80/100 mesh resazurin, 1 mg/L. The medium was boiled under a nitrogen flux. SP100 column (Alltech, Carquefou, France). N2 at a pressure of Bottles were then closed using a lid of aluminum foil and then 100 kPa was used as the carrier gas. The detector and injector cooled off to room temperature under N2 or 80% N2/20%CO2 temperatures were 200uC and the column temperature was 150uC flushing until the medium became transparent. The following (iii) by measuring optical density by spectrophotometry (Cary compounds were prepared and autoclaved anaerobically under N2 50 Scan UV-visible spectrophotometer, Agilent Technologies, and aseptically added to the medium to a final concentration of Paris, France) (iv) by PCR and sequencing of any growing 2% (v/v): NaHCO3, 10%; Na2S, 2%; methanol, 4 M; sodium organism (see below). The DSMZ culture medium inoculated with format, 8 M; and vitamin solution [32]. All of the solutions were 105 CFU/mL of the corresponding strain was used as positive prepared in anaerobic water, with N2/CO2 flushing to replace the control culture and non-inoculated culture media were used as oxygen [9,33]. pH was adjusted to 7.5 with 10 M KOH. The negative controls. The growth was monitored and compared for culture was incubated using a gas mixture of 80% H2+20% CO2 each strain in the corresponding standard DSMZ medium and in at 2.5-bar pressure required for the growth of methanogenic the SAB-medium inoculated with the same cell concentration. The archaea [10,14]. For all the methanogenic archaea strains, the experiments were performed in triplicate. culture was performed in Hungate tubes incubated at 37uC with agitation. PCR-sequencing Identification Identification of detected archaea was performed by PCR amplification and sequencing of the 16S RDNA gene after total

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DNA extraction as previously described [22]. Archaea 16S rDNA similarity of 99–100% with the studied archaea strain. Reproduc- was PCR amplified using archaeal primers SDArch0333aS15-59- ible results were obtained for the triplicate experiments. TCCAGGCCCTACGGG-39 and SDArch0958aA19- 59YCCGGCGTTGAMTCCAATT-39 [23]. Each 50 ml PCR Growth of Clinical Specimens consisted 16 buffer (Qiagen, Courtaboeuf, France), 200 mM each All PCR-positive stool specimens (10/10) grew in SAB medium dNTP, 0.2 mM each primer, 2.5 U hotstart Taq DNA polymerase after an incubation of one day (six specimens), 3 days (one (Qiagen) and 5 ml DNA. Archaeal 16S rDNA genes were specimen), 7 days (two specimens) and 10 days (one specimen). amplified using the following cycle conditions: 15-min at 95u,40 After incubation in the 119 DSMZ culture medium, 6/10 cycles of 95uC (30 s), 58uC (45 s) and 72uC (90 s) followed by a 5- specimens yielded detectable growth after incubation of 7 days min extension step at 72uC. Negative controls consisted in PCR (five specimens) and 10 days (one specimen). After incubation in buffer with no DNA. PCR products were purified and sequenced the 322 DSMZ culture medium, 5/10 specimens yielded detect- using the BigDye Terminator 1.1 Cycle Sequencing kit and the able growth after incubation of 7 days (three specimens) and 3130 genetic analyzer (Applied Biosystems, Villebon-sur-Yvette, 10 days (two specimens). In 334c DSMZ culture medium, growth France). Sequences were analyzed using the Seqscape program was detected for three specimens after an incubation of 7 days (one (Applied Biosystems) and similarity values were determined using specimen), 10 days (one specimen) and 15 days (one specimen) the online BLAST program at NCBI (www.ncbi.nlm.nih.gov/ (P,0.05). As for PCR-negative specimens, 4/10 specimens grew BLAST/). after a 3-week incubation in the SAB-medium whereas they were all sterile in all tested DSMZ culture media (P,0.05). In all cases, Statistical Analysis microscopic observation disclosed organisms with a morphology P values were calculated using the non-parametric Kruskal- compatible with M. smithii and no contaminant. Sequencing of 16S Wallis test and were used to assess statistical significance for the rRNA gene PCR products from all growing specimens yielded a delay in growth in both culture media. A P value ,0.05 was sequence similarity of 99–100% with the reference M. smithii indicative of a significant result. ATCC 35061 except for one PCR-negative specimen which yielded a sequence similarity of 99–100% with the reference Results Methanobrevibacter millerae DSMZ 16643T.

Growth Delay Monitoring Discussion While negative controls remained sterile in all the experiments, all the methanogenic archaea grew as expected, in the recom- The data reported here demonstrate that, under the appropriate mended standard DSMZ culture medium. All of the methano- temperature and atmosphere conditions, the SAB medium genic archaea also grew in the SAB medium. Microscopic promotes the growth of several methanogenic archaea. This fact observation disclosed organisms with a morphology compatible has been confirmed since all the negative controls have remained with the studied archaea organism in each essay and no negative in both culture-based and PCR-based experiments; and contaminant. Significant differences were observed in the time since all growing organisms have been identified by 16S rRNA required for detecting growth of each methanogenic archaea gene sequencing. In particular, the medium equally supported the (Figure 1). M. smithii DSMZ 2374, M. smithii DSMZ 2375 and M. growth of the five methanogens otherwise cultured from human smithii ATCC 35061T grew after 24-hour incubation with a 3-hour feces specimens in our laboratory. This is a contributive doubling time in SAB medium versus 72-hour incubation and a 9- observation as culturing each one of these methanogenic archaea hour doubling time in standard 119 DSMZ medium (P,0.05, currently requires a species-specific culture medium, thus increas- Student test). M. smithii DSMZ 11975 grew after 5-day incubation ing the time required for the preparation of the culture medium with a 11-hour doubling time in SAB medium versus 10-day beyond the routine capacity of most laboratories. Moreover, we incubation and a 21-hour doubling time in standard 119 DSMZ observed that growth of methanogens was significantly more rapid medium (P,0.05). M. oralis DSMZ 7256 grew after 3-day in the SAB medium than in the reference DSMZ medium. incubation with a 18-hour doubling time in SAB medium versus The design of the SAB medium benefited from careful a 7-day incubation and a 21-hour doubling time in standard examination of the media currently used to culture methanogenic 119 DSMZ medium modified by the addition of 1 g of yeast archaea and from the knowledge we acquired after the isolation extract and 2.5 bar of H2/CO2 (80%-20%) atmosphere (P,0.05). and culture of M. luminyensis [3]. In particular, several elements M. stadtmanae ATCC 43021T grew after 24-hour incubation with a previously reported to be required for growing methanogenic 3-hour doubling time in SAB medium versus 72-hour and a 9- archaea, such as trace metals [27–29] were added to SAB-medium hour doubling time in standard 322 DSMZ medium (P,0.05). M. at a defined concentration. Indeed, it was reported that the rumen luminyensis CSUR P135T grew after 3-day incubation in SAB fluid was supplying acetate, branched-chain fatty acids [26] and medium with a 18-hour doubling time versus 10-day incubation coenzyme M (2-mercaptoethanesulfonic acid) [34]. The anaerobic with a 21-hour doubling time in standard 119 DSMZ medium conversion of organic matter leads to the intermediate formation (P,0.05). M. beijingense DSMZ 15999 grew after 24-hour incuba- of volatile fatty acids, primarily butyrate, propionate and acetate tion with a 3-hour doubling time in SAB medium versus 72-hour [35]; fecal extract is required for the growth of some methanogens incubation and a 9–11-hour doubling time in standard and a volatile fatty acid mixture is highly stimulatory [4]. Some 119 DSMZ medium (P,0.05). M. concilii DSMZ 2139 grew after species of the genus Methanosarcina, isolated from an anaerobic 3-day incubation with a 9–11-hour doubling time in SAB medium sludge digester, were originally reported to require an anaerobic versus 7-day incubation and a 18-hour doubling time in standard sludge supernatant for growth. However, it was found that the 334c DSMZ medium (P,0.05). M. arboriphilicus grew after 3-day sludge supernatant could be replaced with 1 g/L yeast extract, incubation with a 18-hour doubling time in SAB medium versus 5- 6 mM bicarbonate-CO2 and trace metals [11]. The cysteine used day incubation and a 18-hour doubling time in standard to reduce the medium could also serve as a nitrogen source [11]. 119 DSMZ medium (P,0.05) (Figure 2). Sequencing the 16S Methanomicrobium mobile requires the aromatic amino-acid trypto- rRNA gene PCR products from all specimens yielded a sequence phan and the vitamins thiamine, pyridoxine and PABA [36].

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Whereas the composition of yeast extract is variable, the analysis lished for the in vitro susceptibility testing of these methanogens of a typical batch showed that 1 g/L would provide 0.15 mM [37] and the SAB medium could be implemented for such testing. PABA; accordingly, the vitamin solution we used provided 0.3 mM We developed a unique medium for the rapid growth of PABA [32]. These levels of PABA are adequate for the optimal methanogens. This medium is now routinely used for the culture- growth of and methanogenesis by archaea of the genera based detection of methanogens from appropriate clinical Methanosarcina and Methanomicrobium. Without the sludge superna- specimens in our medical microbiology laboratory. Laboratories tant as a PABA source, it was necessary to increase the looking for methanogens of veterinary interest may also want to concentration of yeast extract and add 1 g/L of peptone to the implement this medium in their routine practices. SAB medium, in addition to a mixture of volatile fatty acids and vitamin solution. This change led to a considerable reduction in Transparency Declaration the growth time for all of the tested strains (Figure 1). The three co-authors are co-inventors of a patent N/Re´f: H52 We propose that the SAB medium provides an opportunity for 888 cas 13 FR (MD/SB 12.05.00329) for the SAB medium here laboratories to implement the routine culture-based detection of reported. methanogenic archaea for culturing specimens known to contain such methanogens such as periodontal pockets, feces and vaginal Author Contributions discharge [19–24]. Using such a medium renders the otherwise Conceived and designed the experiments: DR MD. Performed the fastidious methanogens more amenable to routine practice in experiments: SK. Analyzed the data: SK DR MD. Contributed microbiology laboratories. Moreover, protocols have been pub- reagents/materials/analysis tools: DR. Wrote the paper: SK DR MD.

References 1. Hungate RE (1969) Roll-tube method for the cultivation of strict anaerobes. 20. Conway de Macario E, Macario AJL (2009) Methanogenic archaea in health Method Microbiol 3B: 117–32. and disease: A novel paradigm of microbial pathogenesis. Int J Med Microbiol 2. Miller TL, Wolin MJ (1974) A serum bottle modification of Hungate technique 299: 99–108. for cultiving obligate anaerobes. Appl Microbiol 27: 985–7. 21. Zhang H, DiBaise JK, Zuccolo A, Kudrna D, Braidotti M, et al. (2009) Human 3. Dridi B, Fardeau ML, Ollivier B, Raoult D, Drancourt M (2012) Methanomassi- gut microbiota in obesity and after gastric bypass. P Natl Acad Sci USA 106: liicocus luminyensis, gen. nov., sp. nov., isolated from the human gut microbiota. 2365–70. Int J Syst Evol Micr 62: 1902–7. 22. Bringuier A, Khelaifia S, Richet H, Aboudharam G, Drancourt M (2012) Real- 4. Ferrari A, Brusa T, Rutili A, Canzi E, Biavati B (1994) Isolation and time PCR quantification of Methanobrevibacter oralis in periodontitis. J Clin characterization Methanobrevibacter oralis sp. nov. Curr Microbiol 29: 7–12. Microbiol in-press. 5. Patel GB, Sprott GD (1990) Methanosaeta concilii gen. nov., sp. nov. (Methanothrix 23. Lepp PW, Brinig MM, Ouverney CC, Palm K, Armitage GC, et al. (2004) concilii)andMethanosaeta thermoacetophila nom. rev., comb. nov. Int J Syst Bacteriol Methanogenic archaea and human periodontal disease. P Natl Acad Sci USA 40: 79–82. 101: 6176–81. 6. Ma K, Liu X, Dong X (2005) Methanobacterium beijingense sp. nov., a novel 24. Yamabe K, Maeda H, Kokeguchi S, Tanimoto I, Sonoi N, et al. (2008) methanogen isolated from anaerobic digesters. Int J Syst Evol Micr 55: 325–9. Distribution of archaea in Japanese patients with periodontitis and humoral 7. Miller TL, Wolin MJ, de Macario EC, Macario AJ (1982) Isolation of immune response to the components. FEMS Microbiol Lett 287: 69–75. Methanobrevibacter smithii from human feces. Appl Environ Microbiol 43: 227–32. 25. Rother M, Metcalf WW (2004) Anaerobic growth of Methanosarcina acetivorans 8. Miller TL, Wolin MJ (1985) Methanosphaera stadtmaniae gen. nov., sp. nov.: a C2A on carbon monoxide: An unusual way of life for a methanogenic archaeon. species that forms methane by reducing methanol with hydrogen. Arch P Natl Acad Sci USA 101: 16929–34. Microbiol 141: 116–22. 26. Bryant MP, Tzeng SF, Robinson IM, Joyner AE (1971) Nutrient requirements 9. Wolfe RS, Metcalf WW (2010) A vacuum-vortex technique for preparation of of methanogenic bacteria. In: Anaerobic Biological Treatment Processes. anoxic solutions or liquid culture media in small volumes for cultivating American Chemical Society. 23–40. methanogens or other strict anaerobes. Anaerobe 16: 216–9. 27. Hartzell PL, Wolfe RS (1986) Requirement of the nickel tetrapyrrole F430 for 10. Dridi B, Raoult D, Drancourt M (2011) Archaea as emerging organisms in in vitro methanogenesis: reconstitution of methylreductase component C from complex human microbiomes. Anaerobe 17: 56–63. its dissociated subunits. P Natl Acad Sci USA 83: 6726–30. 11. Murray PA, Zinder SH (1985) Nutritional requirements of Methanosarcina sp. 28. Hensgens CMH, Nienhuis-Kuiper ME, Hansen TA (1994) Effects of tungstate strain TM-1. Appl Environ Microbiol 50: 49–55. on the growth of Desulfovibrio gigas NCIMB 9332 and other sulfate-reducing 12. Asakawa S, Morii H, Akagawa-Matsushita M, Koga Y, Hayano K. (1993) bacteria with ethanol as a substrate. Arch Microbiol 162: 143–7. Characterization of Methanobrevibacter arboriphilicus SA isolated from a paddy field 29. Jarrell KF, Kalmokoff ML (1988) Nutritional requirements of the methanogenic soil and DNA-DNA hybridization among M. arboriphilicus strains. Int J Syst archaebacteria. Can J Microbiol 34: 557–76. Bactriol 43: 683–6. 30. Moore JM, Salama NR (2005) Mutational analysis of metronidazole resistance 13. Fricke WF, Seedorf H, Henne A, Kru¨er M, Liesegang H, et al. (2006) The in Helicobacter pylori. Antimicrob Agents Ch 49: 1236–7. genome sequence of Methanosphaera stadtmanae reveals why this human intestinal 31. Patel GB, Baudet C, Agnew BJ (1988) Nutritional requirements for growth of archaeon is restricted to methanol and H2 for methane formation and ATP Methanothrix concilii. Can J Microbiol 34: 73–7. synthesis. J Bacteriol 188: 642–58. 32. Balch WE, Fox GE, Magrum LJ, Woese CR, Wolfe RS (1979) Methanogens: 14. Dridi B, Khelaifia S, Fardeau ML, Ollivier B, Drancourt M (2012) Tungsten- reevaluation of a unique biological group. Microbiol Rev 43: 260–96. enhanced growth of Methanosphaera stadtmanae. BMC Res Notes 5: 238. 33. Bryant MP (1972) Commentary on the Hungate technique for culture of 15. Scherer P, Sahm H (1981) Effect of trace elements and vitamins on the growth of anaerobic bacteria. Am J Clin Nutr 25: 1324–8. Methanosarcina barkeri. Acta Biotechnol 1: 57–65. 34. Taylor CD, McBride BC, Wolfe RS, Bryant MP (1974) Coenzyme M, Essential 16. Tanner RS, Wolfe RS (1988) Nutritional requirements of Methanomicrobium for Growth of a Rumen Strain of Methanobacterium ruminantium. J Bacteriol 120: mobile. Appl Environ Microbiol 54: 625–8. 974–5. 17. Diekert G, Konheiser U, Piechulla K, Thauer RK (1981) Nickel requirement 35. Wang Q, Kuninobu M, Ogawa HI, Kato Y (1999) Degradation of volatile fatty and factor F430 content of methanogenic bacteria. J Bacteriol 148: 459–64. acids in highly efficient anaerobic digestion. Biomass Bioenerg 16: 407–416. 18. Hammel KE, Cornwell KL, Diekert GB, Thauer RK (1984) Evidence for a 36. Tanner RS, Wolfe RS (1988) Nutritional requirements of Methanomicrobium nickel-containing carbon monoxide dehydrogenase in Methanobrevibacter arbor- mobile. Appl Environ Microbiol 54: 625–8. iphilicus. J Bacteriol 157: 975–8. 37. Dridi B, Fardeau ML, Ollivier B, Raoult D, Drancourt M (2011) The 19. Belay N, Mukhopadhyay B, Conway de Macario E, Galask R, Daniels L (1990) antimicrobial resistance pattern of cultured human methanogens reflects the Methanogenic bacteria in human vaginal samples. J Clin Microbiol 28: 1666–8. unique phylogenetic position of archaea. J Antimicrob Chemoth 66: 2038–44.

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______Chapitre 7

Chapitre 7

Culturomics reveals hidden intestinal archaea

Saber Khelaifia and Michel Drancourt*

Aix Marseille Université, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095, 13005,

Marseille, France

*Corresponding author: Professeur. Michel DRANCOURT

Unité des Rickettsies, Faculté de Médecine, 27, Boulevard Jean Moulin-Cedex 5- France.

Tel: 00 33 4 91 38 55 17, Fax: 00 33 4 91 38 77 72, E-mail: [email protected]

Keywords: Methanogenic archaea, human microbiota

Submitted to the ISME journal

______Chapitre 7

Chapitre 7: Préambule

Les études de métagénomique ont montré une grande diversité d’archaea halophiles et méthanogènes associées au microbiote intestinal humain, mais seulement trois archaea méthanogènes ont été isolées à partir de son tube digestif. Dans ce travail, une approche fondée sur la culture utilisant un milieu de culture polyvalent a été utilisée pour cultiver des archaea méthanogènes à partir d’échantillons de selles testés négatifs par PCR pour l’archaea méthanogène dominante Methanobrevibacter smithii. Parmi les 12 échantillons de selles inoculés, six ont montré une production de méthane témoignant d’une croissance d’archaea méthanogènes. L’analyser des séquences d'ADNr 16S a donné une similarité de séquence de

99% avec Methanobrevibacter arboriphilicus pour un isolat, Methanobrevibacter oralis pour le second, Methanobrevibacter millerae pour le troisième et M. smithii pour trois autres.

Néanmoins, Cette étude limitée a permis d’isoler trois archaea méthanogènes supplémentaires

à partir de l'intestin humain et doublant ainsi le nombre de ces derniers à six espèces cultivées.

Cette étude montre qu’en plus de la détection moléculaire, les approches basée sur la culture peuvent fournir des données supplémentaires concernant la diversité des archaea dans microbiote humain.

ISMEJ-13-00137C-revised

1 Culturomics reveals hidden intestinal archaea

2 Running title: human-associated archaea

3 Saber Khelaifia and Michel Drancourt*

5 Aix Marseille Université, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095, 13005,

6 Marseille, France

8 *Corresponding author: Prof. Michel DRANCOURT

9 Unité des Rickettsies, Faculté de Médecine, 27, Boulevard Jean Moulin-Cedex 5- France. Tel:

10 00 33 4 91 38 55 17, Fax: 00 33 4 91 38 77 72, E-mail: [email protected]

13 Text word count: 942

14 Abstract word count: 147

15 Number of figures: 2

17 Keywords: Methanogenic archaea, human gut microbiota, Methanobrevibacter smithii ,

18 Methanobrevibacter oralis , Methanosphaera stadtmanae , Methanobrevibacter arboriphilicus,

19 Methanobrevibacter millerae

ISMEJ-13-00137C-revised

21 Abstract

22 Metagenomic studies showed a diversity of human-associated halophilic and methanogenic

23 archaea, but only three methanogenic archaea have been isolated from the human intestinal

24 microbiota. Here, a culture-based approach incorporating a versatile culture medium was used

25 to cultivate methanogenic archaea from stool specimens tested negative by PCR for the

26 dominant methanogenic archaea Methanobrevibacter smithii . Among 12 inoculated stool

27 specimens, six yielded evidence for methanogenic archaea growth. Analyzing the 16S rDNA

28 sequences yielded 99% sequence similarity with the reference Methanobrevibacter

29 arboriphilicus for one isolate, Methanobrevibacter oralis for a second isolate,

30 Methanobrevibacter millerae for a third isolate and M. smithii for three additional isolates.

31 This limited study nevertheless yielded three additional methanogenic archaea from the

32 human gut, doubling the number of human-associated archaea to six cultivated species. This

33 study illustrates that, in addition to molecular-based detection, culture-based approaches can

34 provide additional data regarding the diversity of archaea in human microbiota.

ISMEJ-13-00137C-revised

36 Molecular analyses and metagenomics of the human digestive tract microbiota

37 disclosed a diversity of archaea chiefly methanogenic archaea (Oxley et al, 2010). This was

38 confirmed by a recent analysis of human colon biopsy (Nava et al, 2012). However, only three

39 methanogenic archaea including Methanobrevibacter smithii (Miller et al, 1982),

40 Methanosphaera stadtmanae (Miller & Wolin, 1985) and Methanomassiliicoccus luminyensis

41 (Dridi et al, 2012) have been isolated in pure culture from human stool specimens.

42 In order to further describe the repertoire of methanogenic archaea associated with the human

43 gut microbiota, we here used an alternative, culturomics approach to cultivate methanogenic

44 archaea in colon-associated microbiota.

45 Among specimens anonymously collected between July 2011 and August 2012, 12 specimens

46 which tested negative by PCR for M. smithii (Dridi et al, 2009) were analysed by culture for

47 methanogenic archaea. This study was approved by the local Ethic Committee IFR48.

48 Approximately 1g of each stool specimen was inoculated into one Hungate tube containing 5

49 mL of a home-made versatile culture medium incorporating vancomycin 100 mg/L, imipenem

50 100 mg/L (Mylan S.A.S, Saint Priest, France) and amphotericin B 50 mg/L (Bristol-Myers

51 Squibb, Reuil-Malmaison, France). Tubes were incubated under an atmosphere comprising of

52 80% H 2 + 20% CO 2 at two atmospheres of pressure at 37°C with agitation. Non-inoculated

53 negative controls, were introduced in all manipulations. Growth of methanogens was assessed

54 by methane production measurement using a GC-8A gas chromatograph (Shimad zu, Champs-

55 sur-Marne, France) and fluorescent microscopy observation o f methane-positive tubes. For

56 identification, 16S rDNA extracted as previously described (Bringuier et al, 2012) was PCR-

57 amplified using primers SDArch0333aS15-5′-TCCAGGCCCTACGGG-3′ and

58 SDArch0958aA19-5′YCCGGCGTTGAMTCCAATT-3′ (Lepp et al, 2004) . PCR was done as

59 previously described (Lepp et al, 2004) , incorporating 1× buffer (Qiagen, Courtaboeuf,

60 France), 200 µM each dNTP, 0.2 µM each primer, 2.5 U hotstart Taq DNA polymerase

ISMEJ-13-00137C-revised

61 (Qiagen) and 5 µl DNA. PCR purified products were sequenced using the BigDye Terminator

62 1.1 Cycle Sequencing kit and the 3130 genetic analyzer (Applied Biosystems, Villebon-sur-

63 Yvette, France). One negative control consisting in sterile DNA-free water was introduced in

64 each assay. Sequences were analyzed using the Seqscape program (Applied Biosystems) and

65 similarity values were determined using the online BLAST program at NCBI

66 (www.ncbi.nlm.nih.gov/ BLAST/).

67 All the negative controls introduced in both culture-based and PCR-based experiments

68 remained negative, supporting the interpretation that data herein reported are verified. Six of

69 12 inoculated stool specimens yielded evidence for methanogenic archaea growth after three-

70 week incubation. One specimen yielded detectable CH4 Methanobrevibacter -like organism

71 (strain ANOR1, Collection des Souches de l’Unité des Rickettsies (CSUR) n° P1715,

72 GenBank accession number KC616344) which yielded 99% 16S rDNA sequence similarity

73 with reference Methanobrevibacter arboriphilicus DSM 1125 (GenBank accession number

74 AY196665). One further specimen yielded detectable CH 4 after four-week incubation,

75 Methanobrevibacter -like organism (strain GMR01, CSUR n° P1714, GenBank accession

76 number KC616346) with 99% 16S rDNA sequence similarity with reference

77 Methanobrevibacter oralis DSM 7256 (GenBank accession number HE654003). One

78 specimen yielded detectable CH 4 after three-week incubation Methanobrevibacter -like

79 organism (strain CMR-6, CSUR n° P1712, GenBank accession number KC616345) with 99%

80 16S rDNA sequence similarity with reference Methanobrevibacter millerae DSMZ 16643

81 (GenBank accession number AY196673). After three-week incubation three additional

82 specimens grew Methanobrevibacter -like organisms (including JMR02, CSUR n° P1713,

83 GenBank accession number KC616347), identified as M. smithii on the basis of sequence

84 similarity of 99% with the reference M. smithii DSM 861 (GenBank accession number

85 U55233) (Figure 1).

ISMEJ-13-00137C-revised

87 Figure 1: Phylogenetic tree showing the positions of the 16S ribosomal RNA (rRNA) gene

88 sequences of cultured and uncultured human-associated archaea. Sequences of human-

89 associated archaea isolated in this study are shaded (GenBank accession of ANOR1, CMR-6,

90 GMR01 and JMR02 are respectively KC616344, KC616345, KC616346, KC616347).

91 ☼: Uncultured archaeon; the scale bar corresponds to 0.1 substitutions per nucleotide.

ISMEJ-13-00137C-revised

94 Here, M. smithii cultured from three human stool specimens initially tested negative by M.

95 smithii -specific PCR (Dridi et al, 2009). This observation illustrates that culture is more

96 sensitive than PCR for the detection of this methanogenic archaea in human gut microbiota.

97 Therefor, a 97.5% M. smithii prevalence that we previously derived from PCR-based analysis

98 (Dridi et al, 2009) probably underscores its actual prevalence. Nevertheless, we here observed

99 nine individuals with both PCR-based and culture-based negative detection of this

100 methanogenic archaea. In three of these individuals, we cultured three different methanogenic

101 archaea previously not isolated from the human gut microbiota (Figure 2). M. millerae has

102 been previously detected by PCR and cultured in cows, lambs and alpaca (Popova et al,

103 2012). The significance of M. millerae in one human individual is completely unknown as

104 present study was anonymous and did not allow to access to individual characteristics.

105 Likewise, M. arboriphilicus was initially isolated from wet wood of living trees as

106 Methanobacterium arbophilicum sp. nov (Balch et al, 1979) . It was further designed as

107 Methanobrevibacter arboriphilus before the correct form of the epithet ( arboriphilicus ) was

108 reestablished after the isolation of an additional strain from a paddy field soil (Asakawa et al,

109 1993). Interestingly, M. arboriphilicus -specific sequences have been previously detected in

110 human colonic mucosal biopsies (Oxley et al, 2010) and we report first isolation from the

111 human gut. This observation suggests that, as for methanogenic archaea, stool microbiota is

112 representative of colon biopsy microbiota. At last, M. oralis is a known inhabitant of the oral

113 cavity in human periodontal disease (Bringuier et al, 2012). We cultured one M. oralis isolate

114 from the human gut, indicating that this methanogenic archaea is able to colonize several

115 mucosal niches in humans. Interestingly, two of these methanogenic species had never been

116 previously detected at all in the human digestive tract including metagenomic studies.

ISMEJ-13-00137C-revised

117

118 Figure 2: Diversity of cultured methanogenic archaea isolated from the human intestinal tract.

119 Strains isolated in this study are shaded.

120

ISMEJ-13-00137C-revised

121 At the opposite, molecular studies found Methanosaeta concilii in colonic mucosal biopsies in

122 association with some halophilic archaea (Oxley et al, 2010); these archaea remain to be

123 isolated and cultivated in humans.

124 Altogether, this limited work conduced on only 12 human specimens in only one laboratory,

125 doubles the number of methanogenic archaea cultured in this situation; four of six of these

126 organisms have been isolated in our laboratory (Dridi et al, 2012). Culturomics (Lagier et al,

127 2012) contributes revealing the archaeal diversity which cannot be deduced from molecular

128 studies only.

129

130 Conflicts of interest

131 The medium used to cultivate methanogenic archaea in this work have been patented by the

132 co-authors are as co-inventors N/Réf: H52 888 cas 13 FR (MD/SB 12.05.00329) for the

133 medium here reported.

134

135

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136 References

137 Asakawa S, Morii H, Akagawa-Matsushita M, Koga Y, Hayano K. (1993). Characterization of

138 Methanobrevibacter arboriphilicus SA isolated from a paddy field soil and DNA-

139 DNA hybridization among M. arboriphilicus strains. Int J Syst Bacteriol 43 : 683-686.

140 Balch WE, Fox GE, Magrum LJ, Woese CR, Wolfe RS. (1979). Methanogens: reevaluation

141 of a unique biological group. Microbiol Rev 43 : 260-296.

142 Bringuier A, Khelaifia S, Richet H, Aboudharam G, Drancourt M. Real-time PCR

143 quantification of Methanobrevibacter oralis in periodontitis. J Clin Microbiol 2012; e-

144 pub ahead of print 19 December 2012, doi:10.1128/JCM.02863-12 .

145 Conway de Macario E, Macario AJ. (2009). Methanogenic archaea in health and disease: A

146 novel paradigm of microbial pathogenesis. Int J Med Microbiol 299 : 99-108.

147 Dridi B, Henry M, El Khéchine A, Raoult D, Drancourt M. (2009). High prevalence of

148 Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human

149 gut using an improved DNA detection protocol. PLoS ONE 4: e7063.

150 Dridi B, Fardeau ML, Ollivier B, Raoult D, Drancourt M. (2012) Methanomassiliicoccus

151 luminyensis gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces.

152 Int J Syst Evol Microbiol 62: 1902-1907.

153 Dridi B, Raoult D, Drancourt M. (2011). Archaea as emerging organisms in complex human

154 microbiomes. Anaerobe 17: 56-63.

155 Ferrari A, Brusa T, Rutili A, Canzi E, Biavati B. (1994). Isolation and characterization

156 Methanobrevibacter oralis sp. nov. Curr Microbiol 29 : 7-12

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157 Lagier JC, Armougom F, Million M, Hugon P, Pagnier I, Robert C et al . (2012) Microbial

158 culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect

159 18:1185-1193.

160 Lepp PW, Brinig MM., Ouverney, CC, Palm K, Armitage GC, Relman DA. (2004).

161 Methanogenic Archaea and human periodontal disease. P Natl Acad Sci USA 101 :

162 6176-6181

163 Miller TL, Wolin MJ, de Macario EC, Macario AJ. (1982). Isolation of Methanobrevibacter

164 smithii from human feces. Appl Environ Microb 43: 227-232.

165 Miller TL, Wolin MJ. (1985). Methanosphaera stadtmaniae gen. nov., sp. nov.: a species that

166 forms methane by reducing methanol with hydrogen. Arch Microbio 141 : 116-122.

167 Nava GM, Carbonero F, Croix JA, Greenberg E, Gaskins HR. (2013). Abundance and

168 diversity of mucosa-associated hydrogenotrophic microbes in the healthy human

169 colon. ISME J 6: 57-70.

170 Oxley AP, Lanfranconi MP, Würdemann D, Ott S, Schreiber S, McGenity TJ et al . (2010).

171 Halophilic archaea in the human intestinal mucosa. Environ Microbiol 12: 2398-2410.

172 Popova M, Morgavi DP, Martin C. Methanogens and methanogenesis in the rumen and cecum

173 of lambs fed two different high-concentrate diets. Appl Environ Microbiol 2012 e-pub

174 ahead of print 14 December 2012, doi: 10.1128/AEM.03115-12 .

175 Yamabe K, Maeda H, Kokeguchi S, Tanimoto I, Sonoi N, Asakawa S, Takashiba S. (2008).

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178 Zhang H, DiBaise JK, Zuccolo A, Kudrna D, Braidotti M, Yu Y, Parameswaran P, Crowell

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181

102

______Chapitre 8

Chapitre 8

Hydrophobicity of imidazole derivatives correlates with improved activity against

human methanogenic archaea

Saber Khelaifia1, Jean Michel Brunel2, Didier Raoult1 and Michel Drancourt1*

1. Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UMR CNRS

6236 IRD 3R198, FR IDMM, Méditerranée Infection, Aix-Marseille-Université, Marseille,

France.

2. Centre de Recherche en Cancérologie de Marseille (CRCM), CNRS, UMR7258; Institut

Paoli Calmettes; Aix-Marseille Université, UM 105; Inserm, U1068, F-13009, Marseille,

France.

*Corresponding author: Professor Michel Drancourt, Unité des Rickettsies, Faculté de

Médecine, 27, Boulevard Jean Moulin-Cedex 5- France. Tel: 00 33 4 91 38 55 17. Fax: 00 33

4 91 38 77 72. Email: [email protected]

Key words: Human methanogenic archaea, microbiota, anti-archaea agents, susceptibility testing, in-vitro activity, metronidazole derivative

International Journal of Antimicrobial Agents (2013)

______Chapitre 8

Chapitre 8: Préambule

Les archaea méthanogènes sont des microorganismes caractérisés par leur large spectre de résistance aux agents antimicrobiens à l'exception des dérivés de l’imidazole. Dans cette

étude, nous avons synthétisé 10 dérivés de l'imidazole et testé leur cytotoxicité in-vitro ainsi que leur activité anti-archaea contre six archaea méthanogènes y compris Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, Methanobacterium beijingense, Methanosaeta concilii et Methanomassiliicoccus luminyensis. Les résultats obtenus indiquent un index thérapeutique de 20-400 pour ces composés par rapport au métronidazole. Ces composés ont manifesté une activité anti-archaea accrue contre les archaea méthanogènes cultivées à partir du microbiote humain. Ces composés sont donc des molécules prometteuses pour le traitement des infections liées aux archaea méthanogènes.

Les trois co-auteurs (JMB, DR, MD) sont co-inventeurs d'un brevet (N / Réf: 1h53 316 CAS 1

BN FR) sur les composés rapportés dans ce travail.

______Chapitre 9

Chapitre 9

In-vitro archaeacidal activity of biocides against human-associated archaea

Saber Khelaifia1, Jean Michel Brunel2 and Michel Drancourt1*

1. Aix Marseille Université, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095, 13005,

Marseille, France

2. Aix-Marseille Université, Centre de Recherche en Cancérologie de Marseille, Laboratory

of Integrative Structural & Chemical Biology (iSCB), UMR CNRS 7258, Inserm-U1068, Faculté de Pharmacie, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, France.

* Corresponding author: Professeur. Michel DRANCOURT

Unité des Rickettsies, Faculté de Médecine, 27, Boulevard Jean Moulin-Cedex 5- France.

Tel: 00 33 4 91 38 55 17, Fax: 00 33 4 91 38 77 72, E-mail: [email protected]

Keywords: archaea, biocides, peracetic acid, chlorexhidine, squalamine.

PLoS ONE (2013)

______Chapitre 9

Chapitre 9: préambule

Des archaea méthanogènes ont été détectées dans le microbiote intestinal humain. Ces archaea intestinales peuvent contaminer les dispositifs médicaux tels que les coloscopes.

Cependant, aucune activité biocide n’a été rapportée pour de tels microorganismes associés au microbiote intestinal humain. La concentration minimale archaeacide (MAC) de l'acide peracétique, la chlorhexidine, la squalamine ainsi que ses dix dérivés synthétiques rapportés dans cette étude a été déterminée contre cinq archaea méthanogènes associées à l’Homme y compris Methanobrevibacter smithii, Methanobrevibacter oralis, Methanobrevibacter arboriphilicus, Methanosphaera stadtmanae, Methanomassiliicoccus luminyensis et deux archaea méthanogènes environnementales Methanobacterium beijingense et Methanosaeta concilii en utilisant la technique de dilution en série dans les tubes Hungates. Le dérivé squalamine S1 a montré une plus grande activité archaeacide (10-200) que le reste des diocides. Les études précédentes ont indiqué que les dérivés de squalamine sont actifs à la fois contre les bactéries gram-positif et gram-négatif, donc, le dérivé S1 est un composé prometteur pour la décontamination des dispositifs médicaux contaminés par les archaea méthanogènes associées à l’homme.

In-Vitro Archaeacidal Activity of Biocides against Human- Associated Archaea

Saber Khelaifia1, Jean Brunel Michel2, Michel Drancourt1* 1 Aix Marseille Universite´, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095, Marseille, France, 2 Centre de Recherche en Cance´rologie de Marseille (CRCM), CNRS, UMR7258; Institut Paoli Calmettes; Aix-Marseille Universite´, UM 105; Inserm, U1068, Marseille, France

Abstract

Background: Several methanogenic archaea have been detected in the human intestinal microbiota. These intestinal archaea may contaminate medical devices such as colonoscopes. However, no biocide activity has been reported among these human-associated archaea.

Methodology: The minimal archaeacidal concentration (MAC) of peracetic acid, chlorhexidine, squalamine and twelve parent synthetic derivatives reported in this study was determined against five human-associated methanogenic archaea including Methanobrevibacter smithii, Methanobrevibacter oralis, Methanobrevibacter arboriphilicus, Methanosphaera stadtmanae, Methanomassiliicoccus luminyensis and two environmental methanogens Methanobacterium beijingense and Methanosaeta concilii by using a serial dilution technique in Hungates tubes.

Principal Findings: MAC of squalamine derivative S1 was 0.05 mg/L against M. smithii strains, M. oralis, M. arboriphilicus, M. concilii and M. beijingense whereas MAC of squalamine and derivatives S2–S12 varied from 0.5 to 5 mg/L. For M. stadtmanae and M. luminyensis, MAC of derivative S1 was 0.1 mg/L and varied from 1 to $10 mg/L for squalamine and its parent derivatives S2–S12. Under the same experimental conditions, chlorhexidine and peracetic acid lead to a MAC of 0.2 and 1.5 mg/L, respectively against all tested archaea.

Conclusions/Significance: Squalamine derivative S1 exhibited a 10–200 higher archaeacidal activity than other tested squalamine derivatives, on the majority of human-associated archaea. As previously reported and due to their week corrosivity and their wide spectrum of antibacterial and antifungal properties, squalamine and more precisely derivative S1 appear as promising compounds to be further tested for the decontamination of medical devices contaminated by human- associated archaea.

Citation: Khelaifia S, Michel JB, Drancourt M (2013) In-Vitro Archaeacidal Activity of Biocides against Human-Associated Archaea. PLoS ONE 8(5): e62738. doi:10.1371/journal.pone.0062738 Editor: Arnold Driessen, University of Groningen, Netherlands Received January 24, 2013; Accepted March 25, 2013; Published May 3, 2013 Copyright: ß 2013 Khelaifia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction received a purge [13]. Therefore, these human-associated archaea may contaminate any medical device soiled by feces such as An increasing number of methanogenic archaea are being colonoscopes. This contamination could be problematic because found in the human microbiota [1]. Since Miller and collaborators archaea significantly differ from bacteria so that the archaeacidal reported the isolation of methanogenic archaea Methanobrevibacter activity of biocides cannot be simply deduced from their smithii [2] from human feces, new strains were recently identified. bactericidal activity. Moreover, human-associated archaea have Thus, Methanosphaera stadtmanae [3] and Methanomassiliicoccus been found to be highly resistant to most commonly used luminyensis were isolated from human feces [4] and Methanobrevi- antibiotics [14,15]. In the perspective of broadening the spectrum bacter oralis was identified from the human subgingival plaque [5– of new active molecules, and more precisely against archaea 7]. Recently, we isolated Methanobrevibacter arboriphilicus [8] and organisms colonizing the human gut, squalamine and its Methanobrevibacter millerae [9] from human feces specimens (S. derivatives appear to be among the few antimicrobial agents able Khelaifia, M. Drancourt, unpublished data). Whereas M. smithii is to demonstrate an efficient anti-archaea activity [14,15]. Squala- an almost constant inhabitant of the human gut [10], M. stadtmanae mine is a natural aminosteol compound which is extracted from was only found in about one-third of individuals [10] and M. the spiny dogfish shark live [16]. Among various properties, it was luminyensis in an average of 4% individuals with an age-dependent found to be a potent antimicrobial compound active against both prevalence [11]. It has been shown that purge used prior to Gram-positive and Gram-negative bacteria and fungi [17]. We colonoscopy, may not eliminate these particular methanogenic previously observed its in-vitro activity against four methanogenic archaea: for instance, halophilic and methanogenic archaea M. archaea [15]. Here, we investigated the in-vitro archaeacidal smithii, M. stadtmanae, M. arboriphilicus and Methanosaeta concilii [12] were detected in colonic mucosal biopsies from patients who had

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Figure 1. Structure of squalamine, chlorhexidine and peracetic acid. doi:10.1371/journal.pone.0062738.g001 activity of 15 biocides including original squalamine derivatives quenched by adding water (1 mL) and stirring was maintained at against seven archaea including five human-associated archaea. room temperature for 20 minutes. The resulting inorganic precipitate was filtered off over a pad of Celite and washed with Materials and Methods Et2O and ethylacetate. The combined organic extracts were dried over Na2SO4, filtered and concentrated in vacuo. The crude Archaea product was placed in 10 mL of a MeOH/CHCl3 (1/1) solution, M. smithii ATCC 35061T DSMZ 861, M. smithii DSMZ 2374, 128 mg of K2CO3 (0.93 mmol) were added and the mixture was M. smithii DSMZ 2375, M. smithii DSMZ 11975, M. oralis DSMZ placed under stirring for 24 h. The solvents were evaporated and 7256 T, M. stadtmanae ATCC 43021T DSMZ 3091, Methanobacter- the mixture was extracted with water and ethylacetate. The ium beijingense [18] DSMZ 15999 and M. concilii DSMZ 2139, combined organic extracts were dried over Na2SO4, filtered and purchased from the German Collection of Microorganisms and concentrated in vacuo. Subsequent purification by flash chroma- Cell Cultures (DSMZ, Braunschweig, Germany). M. arboriphilicus tography on silicagel (eluent: CH2Cl2/MeOH/NH4OH(32%), strain tested in this study was recently isolated in our laboratory 7:3:1) led to a pale yellow solid. All squalamine derivatives from human feces (S. Khelaifia, M. Drancourt, unpublished data). reported here were prepared by using procedures similar to those M. smithii strains, M. arboriphilicus and M. beijingense were grown on described above and all the NMR and MS analyses were in liquid media 119 (http://www.dsmz.de). The media 119 modified accordance with the expected data. by addition of 1 g of Yeast extract and 2.5-bar of H2/CO2 (80/20) atmosphere was used to cultivate M. oralis. The media 322 (http:// Testing Archaeacidal Activity www.dsmz.de) was used to cultivate M. stadtmanae and the media A filtered aqueous solution of each one of the 12 biocides 334c (http://www.dsmz.de) to cultivate M. concilii,at37uCin (Figures 1, 2) was anaerobically added at a final 5 mg/L Hungate tubes (Dutscher, Issy-les-Moulineaux, France) under a 2- concentration into Hungate tubes [19] containing distilled water; bar H2/CO2 (80/20) atmosphere under stirring. M. luminyensis tubes were previously sterilized by autoclaving at 120uC for CSUR P135T was cultivated using Methanobrevibacter medium 30 min under an H2/CO2 (80/20) atmosphere. The in-vitro (medium 119: http://www.dsmz.de) modified by the addition of archaeacidal activity of the biocides was determined by transfer- methanol and selenite/tungstate solution under 2-bar of H2/CO2 ring 10E+05 archaea cells/mL of an exponentially growing culture (80/20) atmosphere under stirring [4]. into 4.5 mL of fresh medium containing 0.01, 0.05, 0.1, 0.2, 0.4, 0.8, 1, 5 or 10 mg/L of biocide. Tubes were incubated at 37uC Biocides and Squalamine Derivatives under stirring and archaea growth was observed after a 5-day Chlorhexidine (MP Biomedicals, Illkirch, France) and peracetic incubation. Cultures were centrifuged at 11,000 g for three acid (ANIOS, Lille-Hellemmes, Fance) were tested in this study. minutes at room temperature, washed with fresh medium to Concerning the synthesis of squalamine derivatives, all the solvents remove traces of biocide and reinoculated into a new culture were purified according to reported procedures and the reagents medium. Control cultures without biocide were incubated in used were commercially available. Methanol, ethyl acetate, parallel. Growth of archaea was assessed by optical microscopy dichloromethane, ammonia and petroleum ether (35–60uC) were observation and parallel measurement of methane production purchased from Solvants Documentation Synthe`ses (Peypin, using a GC-8A gas chromatograph (Shimadzu, Champs-sur- France) and used without further purification. Column chroma- Marne, France) equipped with a thermal conductivity detector and tography was performed on silica gel (70–230 mesh). 1H NMR a Chromosorb WAW 80/100 mesh SP100 column (Alltech, 13 and C NMR spectra were recorded in CDCl3 on a Bruker AC Carquefou, France). N2 at a pressure of 100 kPa was used as the 300 spectrometer working at 300 MHz and 75 MHz, respectively carrier gas. The detector and the injector temperature was 200uC (the usual abbreviations are used: s: singlet, d: doublet, t: triplet, q: and the column temperature was 150uC. The in-vitro activity of quadruplet, m: multiplet). Tetramethylsilane was used as internal biocide under these culture conditions was verified as follows. standard. All chemical shifts are given in ppm. A mixture of the Culture media 119, 322, 334 (http://www.dsmz.de) and M. lumi- desired ketosterol 1 or 2 (0.78 mmol), titanium (IV) isopropoxide nyensis medium were supplemented with a final concentration of (302 mL, 1.03 mmol) and the desired amine (2.34 mmol) in 0.01, 0.05, 0.1, 0.2, 0.4, 0.8, 1, 5 or 10 mg/L of biocide and were absolute methanol (5 mL) was stirred under argon at room incubated at 37uCinaH2/CO2 (80/20) atmosphere for 10 days. temperature for 12 hours. Sodium borohydride (29 mg, The activity of each biocide was controlled using clinical isolates of 0.78 mmol) was then added at 278uC and the resulting mixture Escherichia coli and Staphylococcus aureus [17,20] in the same culture was stirred for an additional 2 hours. The reaction was then conditions as the tested archaea. Growth controls with appropriate

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Figure 2. Overview of the pathway for the synthesis of squalamine derivatives S1–S12. doi:10.1371/journal.pone.0062738.g002 media instead of derivative dilutions were introduced in all incubation. All the positive control cultures of M. smithii, M. oralis, experiments. The minimal archaeacidal concentration (MAC) was M. arboriphilicus, M. stadtmanae, M. luminyensis, M. beijingense and defined as the lowest biocide concentration killing archaea M. concilii incubated without biocide grew as expected with a organisms. This was measured by observing the inhibition of methane production starting at day 3. As for four M. smithii strains, methane production and the absence of microscopically visible M. oralis, M. arboriphilicus, M. concilii and M. beijingensis, MAC was of growth of this archaea. 0.05 mg/L for squalamine derivative S1; 0.5 mg/L for squalamine and derivatives S2–S6; and 2 to 5 mg/L for derivatives S7– Results S12. As for M. stadtmanae and M. luminyensis, MAC was of 0.1 mg/ L for S1; 1 mg/L for squalamine and derivatives S2–S6; and The activity of the tested biocides incubated at 37uC under an 5 mg/L to $10 mg/L for derivatives S7–S12. Chlorhexidine and H2/CO2 (80/20) atmosphere was confirmed by observing the peracetic acid lead to a MAC of 0.2 and 1.5 mg/L, respectively killing of E. coli and S. aureus strains used as controls, after a 5-day against all tested archaea (Table 1).

Table 1. Minimal archaeacidal concentration (MAC, mg/mL) of 15 biocides against archaea strains.

Peracetic S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 Squalamine Chlorhexidine acid

M. smithii 0.05 0.5 0.5 0.5 0.5 0.5 5 5 5 5 5 2 0.5 0.2 1.5 ATCC35061T M. smithii 0.05 0.5 0.5 0.5 0.5 0.5 5 5 5 5 5 2 0.5 0.2 1.5 DSMZ 2374 M. smithii 0.05 0.5 0.5 0.5 0.5 0.5 5 5 5 5 5 2 0.5 0.2 1.5 DSMZ 2375 M. smithii 0.05 0.5 0.5 0.5 0.5 0.5 5 5 5 5 5 2 0.5 0.2 1.5 DSMZ 11975 M. oralis 0.05 0.5 0.5 0.5 0.5 0.5 5 5 5 5 5 2 0.5 0.2 1.5 DSMZ 7256 M. arboriphilicus 0.05 0.5 0.5 0.5 0.5 0.5 5 5 5 5 5 2 0.5 0.2 1.5 M. beijingense 0.05 0.5 0.5 0.5 0.5 0.5 5 5 5 5 5 2 0.5 0.2 1.5 DSMZ 15999 M. consilii 0.05 0.5 0.5 0.5 0.5 0.5 5 5 5 5 5 2 0.5 0.2 1.5 DSMZ 2139 M. stadtmanae 0,1111 1 1 10101010105 1 0.2 1.5 ATCC 43021T M. luminyensis 0.1111 1 1 10101010105 1 0.2 1.5 CSUR P13T

doi:10.1371/journal.pone.0062738.t001

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Figure 3. Images by electron microscopy demonstrating the morphological effects of squalamine on M. smithii cell wall. (A) M. smithii without squalamine. (B) M. smithii +1 mg/L squalamine. White arrows show holes caused by squalamine on M. smithii cell wall. The scale bar corresponds to 500 nm. doi:10.1371/journal.pone.0062738.g003

Discussion phospholipid molecules are fused into a single molecule with two polar heads [23]. This fusion may render membranes more rigid Although several reports have documented the presence of and stable in harsh environments [24]. Archaea lipids are based archaea in the human gut microbiota, they remain a neglected upon long isoprene side chain and often cyclopropane or field in medical microbiology. This is illustrated by the complete cyclohexane rings. These branched chains may keep archaeal lack of study addressing the archaeacidal activity of biocides. This membranes from leaking at high temperatures or help them resist is surprising considering intestinal archaea can potentially to disrupting membrane agents [25,26]. Another broad-spectrum contaminate medical devices such as colonoscopes. Moreover, it mechanism relies on efflux of molecules. Whereas the M. smithii has been shown that a purge prior to colonoscopy does not ATCC 35061 complete genome (GenBank accession number eliminate archaea from the gut [13]. Indeed, non-methanogenic CP000678) encodes for six efflux pumps representing 3/1000 of halophilic archaea have been detected in one purge preparation the genome size, M. stadtmanae DSM 3091 complete genome [13]. Accordingly, transmission of methanogenic archaea between sequence (GenBank accession number NC 007681) encodes for patients via reusable medical equipments such as colonoscopes four efflux pumps representing 4/1000 of the genome size. may alter the intestinal microbiota, causing pathologies such as Two commonly used biocides chlorhexidine and peracetic acid, digestive tract diseases and obesity [21,22]. Studying the exhibited an archaeacidal activity under current decontamination archaeacidal activity of some biocides is even more urgent, as protocols and by using concentrations of 0.2 and 1.5 g/L, archaea exhibit a unique cell wall structure and composition which respectively. However, squalamine and its derivatives exhibited a keep the results from being simply extrapolated from what was higher activity than these two usual biocides on the majority of already known for bacteria. Accordingly, in general, archaea are here tested archaea, not on all archaea. Interestingly, these more resistant to antibiotics than bacteria [14,15]. compounds are equally active against Gram-negative and Gram- Therefore, there was a need to assess the archaeacidal activity of positive bacteria, including bacteria from the human intestinal biocides used in routine. Here, since the controls introduced in all microbiota [17,20]. They act directly on the cell membrane of the experiments produced the expected results, the reported data have bacteria by creating holes (Figure 3), emptying the cell cytoplasms been interpreted as authentic. In particular, we controlled the in- which lead to the death of the bacteria [17]. Data herein reported vitro activity of molecules herein tested under the unusual indicate that some squalamine derivatives exhibited an increased atmosphere comprising of 80% H2 and 20% CO2 required for in-vitro activity against methanogenic archaea, particularly for growing methanogenic archaea. In addition, we tested squalamine derivative S1. Indeed, the structure of the different squalamine as a positive control molecule and observed a MAC value in the derivatives greatly influences their archaeacidal activity: aminos- range of the previously reported value [17,20]. In the present terol derivatives from dehydroepiandrosterone (DHEA) demon- study, we extended data on squalamine to M. arboriphilicus, M. strate a lower activity (around 5 mg/mL) compared to pregnen- beijingense and M. concilii which have not been previously tested for olone derivatives (0.05 to 0.5 mg/mL) while they differ only by the their susceptibility to squalamine. length of the side chain in position 17 suggesting a required We observed that the susceptibility of archaea to biocides varies specific conformation by targeting archaea. On the other hand, depending on both the archaea species and the nature of the tested even in the same series the activity is conserved whatever the M. stadmanae biocide. In particular, , an archaea found in almost nature of the amino side chain introduced except for derivative S1 one-third of individuals [10], was twice more resistant to biocides which is ten times more active and which differs only by the than the other tested archaea. This observation is in line with our presence of three positive charges instead of two in all the other previous observation that M. stadmanae is more resistant to products suggesting a potent interaction of the positive charge of antibiotics than M. smithii [14,15]. The mechanisms underlying the compound with the negative charge of the archaeal membrane such differences are unknown but they should be broad-spectrum, (Figure 1). All these features constitute a basis for the development relatively poorly specific mechanisms such as differences in the cell of a new class of biocides devoted to the decontamination of wall composition. Archaea possess membranes made of chemically archaea-contaminated medical devices. stable glycerol-ether lipid bonds. In some archaea the lipid bilayer is replaced by a monolayer, in which the tails of two independent

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Conclusion rationale and improve their potent routinely use for disinfection of The data reported here indicate that peracetic acid, which is medical devices including colonoscopes. routinely used for the desinfection of medical devices including colonoscopes, is effective against human-associated archaea. Author Contributions Nevertheless, less corrosive agents such as squalamine and its Conceived and designed the experiments: MD. Performed the experi- parent derivatives appear as better promising biocides against the ments: SK JMB. Analyzed the data: SK JMB MD. Contributed reagents/ majority of human-associated archaea. Studies are now under materials/analysis tools: MD. Wrote the paper: SK JMB MD. current investigation to understand their involved mechanistic

References 1. Dridi B, Raoult D, Drancourt M (2011) Archaea as emerging organisms in 14. Khelaifia S, Drancourt M (2012) Susceptibility of archaea to antimicrobial complex human microbiomes. Anaerobe 17: 56–63. agents: applications to clinical microbiology. Clin Microbiol Infect 18: 841–8. 2. Miller TL, Wolin MJ, Conway de Macario E, Macario AJL (1982) Isolation of 15. Dridi B, Fardeau ML, Ollivier B, Raoult D, Drancourt M (2011) The Methanobrevibacter smithii from human feces. Appl Environ Microbiol 43: 227–32. antimicrobial resistance pattern of cultured human methanogens reflects the 3. Miller TL, Wolin MJ (1985) Methanosphaera stadtmaniae gen. nov., sp. nov.: a unique phylogenetic position of archaea. J Antimicrob Chemother 66: 2038–44. species that forms methane by reducing methanol with hydrogen. Arch 16. Moore KS, Wehrli S, Roder H, Rogers M, Forrest JN Jr, et al. (1993) Microbiol 141: 116–22. Squalamine: an aminosterol antibiotic from the shark. Proc Natl Acad Sci U S A 4. Dridi B, Fardeau ML, Ollivier B, Raoult D, Drancourt M (2011) Methanomassi- 90: 1354–8. liicocus luminyensis, gen. nov., sp. nov., isolated from the human gut microbiota. 17. Alhanout K, Malesinki S, Vidal N, Peyrot V, Rolain JM, et al (2010) New Int J Syst Evol Microbiol 62: 1902–7. insights into the antibacterial mechanism of action of squalamine. J Antimicrob 5. Belay N, Johnson R, Rajagopal BS, Conway de Macario E, Daniels L (1988) Chemother 65: 1688–93. Methanogenic bacteria from human dental plaque. Appl Environ Microbiol 54: 18. Ma K, Liu X, Dong X (2005) Methanobacterium beijingense sp. nov., a novel 600–3. methanogen isolated from anaerobic digesters. Int J Syst Evol Microbiol 55: 6. Ferrari A, Brusa T, Rutili A, Canzi E, Biavati B (1994) Isolation and 325–9. characterization Methanobrevibacter oralis sp. nov. Curr Microbiol 29: 7–12. 19. Miller TL, Wolin MJ (1974) A serum bottle modification of Hungate technique 7. Bringuier A, Khelaifia S, Richet H, Aboudharam G, Drancourt M (2013) Real- for cultiving obligate anaerobes. Appl Microbiol 27: 985–7. time PCR quantification of Methanobrevibacter oralis in periodontitis. J Clin Microbiol 51: 993–4. 20. Salmi C, Loncle C, Vidal N, Letourneux Y, Fantini J, et al (2008) Squalamine: 8. Asakawa S, Morii H, Akagawa-Matusushita M, Koga Y, Hayano K (1993) An appropriate strategy against the emergence of multidrug resistant gram- Characterization of Methanobrevibacter arboriphilicus SA isolated from a paddy field negative bacteria? PLoS ONE 3: e2765. soil and DNA-DNA hybridization among M. arboriphilicus strains. Int J Syst 21. Conway de Macario E, Macario AJL (2009) Methanogenic archaea in health Bacteriol 43: 683–6. and disease: A novel paradigm of microbial pathogenesis. Int J Med Microbiol 9. Rea S, Bowman JP, Popovski S, Pimm C, Wright AD (2007) Methanobrevibacter 299: 99–108. millerae sp. nov. and Methanobrevibacter olleyae sp. nov., methanogens from the ovine 22. DiBaise JK, Zhang H, Crowell MD, Krajmalnik-Brown R, Decker GA, et al. and bovine rumen that can utilize formate for growth. Int J Syst Evol Microbiol (2008) Gut microbiota and its possible relationship with obesity. Mayo Clin Proc 57: 450–6. 83: 460–9. 10. Dridi B, Henry M, El Khe´chine A, Raoult D, Drancourt M (2009) High 23. Kandler O (1995) Cell wall biochemistry in Archaea and its phylogenetic prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in implications. J Biol Phys 20: 165–9. the human gut using an improved DNA detection protocol. PLoS ONE 4: 24. Woese CR, Kandler O, Wheelis ML (1990) Towards a natural system of e7063. organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl 11. Dridi B, Henry M, Richet H, Raoult D, Drancourt M (2012) Age-related Acad Sci USA 87: 4576–9. prevalence of Methanomassiliicoccus luminyensis in the human gut microbiome. 25. Dopson M, Baker-Austin C, Hind A, Bowman JP, Bond PL (2004) APMIS 120: 773–7. Characterization of Ferroplasma isolates and Ferroplasma acidarmanus sp. nov., 12. Patel GB, Sprott GD (1990) Methanosaeta concilii gen. nov., sp. nov. (Methanothrix extreme acidophiles from acid mine drainage and industrial bioleaching concilii)andMethanosaeta thermoacetophila nom. rev., comb. nov. Int J Syst Bacteriol environments. Appl Environ Microbiol 70: 2079–88. 40: 79–82. 26. Koga Y (2012) Thermal adaptation of the archaeal and bacterial lipid 13. Oxley AP, Lanfranconi MP, Wu¨rdemann D, Ott S, Schreiber S, et al (2010) membranes. Archaea 2012: 789652, doi: 10.1155/2012/789652. Halophilic archaea in the human intestinal mucosa. Environ Microbiol 12: 2398–410.

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______Conclusions et perspectives

Conclusions et perspectives

De nombreuses investigations récentes ont montré l’adaptation des archaea méthanogènes aux muqueuses intestinale et buccale humaines. Avant de débuter notre travail de thèse, la diversité des archaea dans la muqueuse intestinale était limitée à quatre espèces cultivées, et celle de la muqueuse bucco-dentaire à une espèce cultivée. Ces données n’étaient pas en concordance avec la grande diversité de ce domaine tellement vaste. De plus, les analyses moléculaires des flores associées aux muqueuses intestinale et bucco-dentaire humaines ont fourni plusieurs évidences de la présence d'autres groupes d'archaea, y compris

Methanosarcina, Thermoplasma, Crenarchaeota, ainsi que des archaea halophiles.

Le premier objectif de cette thèse était de mettre au point des techniques d’identification moléculaire des archaea dans des échantillons cliniques pour mieux adapter par la suite les conditions de culture. La détection moléculaire de l’ADN des archaea par PCR dans des

échantillons humains repose sur une technique d'extraction d'ADN efficace. Nous avons déjà mis en place un tel protocole ne faisant intervenir que des étapes manuelles. Dans le but de réduire la charge de travail, nous avons mis au point un protocole semi-automatique qui peut

être utilisé pour l'extraction de l'ADN totale des selles à la recherche de nouvelles espèces d’archaea colonisant le tube digestif humain. Nous nous sommes par la suite intéressés à l’implication de l’archaea méthanogène Methanobrevibacter oralis dans la parodontite. Le test de PCR en temps réel que nous avons développé nous a permis de quantifier la charge de

M. oralis et d’établir un score pour déterminer le degré de sévérité de la parodontite. Les données obtenues ont impliqué directement M. oralis dans la parodontopathie. La surveillance de la charge de M. oralis peut être utilisée comme un biomarqueur de la parodontite.

Après l’isolement de Methanomassiliicoccus luminyensis dans notre laboratoire, nous avons constaté que l’addition d’une solution de tungstate /sélénate était essentielle pour sa croissance et que l’addition de cette solution dans une culture de M. stadtmanae permet de

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______Conclusions et perspectives stimuler et d’accélérer la croissance de cette dernière par un facteur 3, ceci a donc permis d’étendre les connaissances sur les besoins nutritifs de ces organismes aussi fastidieux et difficiles à isoler. Nous avons par la suite développé et optimisé un nouveau milieu de culture que nous avons testé pour la culture de différentes espèces d’archaea méthanogènes et pour l'isolement des archaea méthanogènes à partir d’échantillons de selles humaines. Le nouveau milieu de culture a favorisé la croissance rapide de toutes les archaea méthanogènes testées et il nous a permis aussi d’isoler trois archaea méthanogènes supplémentaires à partir de l'intestin humain doublant ainsi le nombre de ces derniers à six espèces cultivées. C’est un support polyvalent qui devrait simplifier la détection des archaea méthanogènes par la culture dans les échantillons cliniques et environnementaux.

Les archaea méthanogènes sont des microorganismes caractérisés par leur large spectre de résistance aux agents antimicrobiens à l'exception des dérivés de l’imidazole et de la squalamine. Au cours des études que nous avons mené, nous avons synthétisé 8 dérivés de l'imidazole et 10 dérivés de la squalamine afin de tester in-vitro leur activité anti-archaea contre les archaea méthanogènes qui colonisent les muqueuses intestinale et buccale humaine.

Les dérivés de l'imidazole ont manifesté une activité anti-archaea accrue contre toutes les archaea méthanogènes tetsées. Ces composés sont donc des molécules prometteuses pour le traitement des infections liées aux archaea méthanogènes. Les dérivés de la squalamine ont montré une plus grande activité archaeacide que le reste des biocides testés. Des études précédentes ont indiqué que les dérivés de squalamine sont actifs à la fois contre les bactéries gram-positif et gram-négatif, donc, ces dérivés sont des composés prometteurs pour la décontamination des dispositifs médicaux contaminés par les archaea méthanogènes associées

à l’Homme.

Le développement de nouvelles méthodes d'identification et de culture des archaea à partir d'échantillons cliniques permettra d’isoler de nouvelles espèces d’archaea pour les

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______Conclusions et perspectives caractériser phénotypiquement, d'explorer leur génome par séquençage et d’étudier la dynamique des populations notamment au cours des pathologies pour préciser leur rôle exact au sein des flores complexes associées aux muqueuses de l'Homme.

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