Laboratory tools for detection of archaea in humans

Authors

  • B. Dridi

    1. Centre de recherche en Infectiologie du Centre de recherche du CHUL and Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada
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Corresponding author: Bédis Dridi, Centre de recherche en Infectiologie du Centre de recherche du CHUL and Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada G1V 4G2.
E-mail: bedis.dridi@crchul.ulaval.ca

Abstract

Clin Microbiol Infect 2012; 18: 825–833

Abstract

This work represents an update of knowledge regarding the detection methods for human microbiome-associated archaea. Despite the fact that, during the last three decades, only four methanoarchaeal species have been isolated from the human mucosa, including faeces, subgingival plaque, and vaginal mucosa (Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanobrevibacter oralis and, most recently, ‘Methanomassiliicoccus luminyensis’), molecular studies, including PCR and metagenomic analyses, have detected DNA sequences indicative of the presence of additional methanoarchaea, as well as non-methanogenic archaea, in the human intestinal tract. Opinion is divided on the roles (if any) of these organisms in human disease, and certainly the data are still unclear. Future research and recently reported data highlighting the antimicrobial susceptibility of the human methanoarchaea could help in the design of selective media to discover additional human mucosa-associated archaea and ascertain their role in human infections involving complex flora.

Introduction

Methanoarchaea represent a distinct group of anaerobic archaea that produce methane under anaerobic conditions [1]. In fact, in the anaerobic microniches where they have been discovered, the activity of the primary microbial populations using available complex organic compounds leads to the accumulation of H2 + CO2 and volatile fatty acids. Formate is rapidly converted via the formic hydrogenlyase to H2 and CO2, and may not be detectable, as it is rapidly metabolized. The ultimate formation of methane and CO2 marks the last step in a series of dissimilatory reactions in which organic compounds are completely degraded. Methane is the most reduced form of carbon, and CO2 the most oxidized form of carbon [1,2]. Indeed, methane is one of the most abundant greenhouse gases in the earth’s atmosphere, constituting 18% of the global total and having a greenhouse effect 25 times more efficient than CO2 [3]. Methane is mainly produced from ruminant livestock; therefore, several studies have been conducted with the aim of limiting the production of this gas by these animals [4,5].

Methanoarchaea have been also found to be part of the intestinal microbiota in animals and humans, and even in intracellular niches in some protists [6–16]. The most predominant methanoarchaeal species in human and animal intestinal tracts belong to the genus Methanobrevibacter [1,7,9,17]. A few other genera, such as Methanosphaera, Methanosarcina, Methanobacterium, Methanomicrobium, Methanogenium and, most recently, ‘Methanomassiliicoccus’, have been also isolated from animals and humans [6,8,10,11,18–20]. Methanoarchaea play a paramount role in the digestion process and in preventing the accumulation of H2 and other reaction end-products [21–24]. In fact, in anoxic conditions, most gastrointestinal methanoarchaea, except members of the genera Methanosphaera and ‘Methanomassiliicoccus’, obtain energy by reduction of CO2 to methane by using hydrogen as the terminal electron acceptor [23]. Indeed, Methanosphaera and ‘Methanomassiliicoccus’ species require both H2 and methanol (oxidation of H2 and reduction of methanol) to produce methane (Table 1) [8,20]. Besides H2, Methanosarcina spp. may also use acetate, methanol, monomethylamine, dimethylamine, trimethylamine, H2CO2, and CO [25–27].

Table 1.   Morphological, phenotypic and G + C content of methanoarchaea isolated in humans Thumbnail image of

Although the precise ecological niches and the routes of acquisition of archaea in humans remain largely unknown, the environment is a likely source of human methanoarchaea. In fact, Angel et al. [28] recently demonstrated that methanoarchaea are globally ubiquitous in aerated soils and become active under wet anoxic conditions. These findings support our hypothesis that the acquisition of some methanoarchaea could occur through environmental contamination, and once these organisms find favourable physicochemical conditions and available substrates in the gut, stable colonization is established [29].

Studies on methanoarchaea started with classic bacteriological culture isolation and quantification methods [7,9,30]. More recently, molecular approaches have been developed to provide an alternative means of investigating the gastrointestinal ecosystem without bacteriological culture methodology [31–40], mostly because of the difficulty in isolating fastidious anaerobes, and the long incubation times and strict anaerobic cultivation methods needed, adding to the limited knowledge of the nutritional requirements and antibiotic sensitivity patterns of many methanoarchaea [29,30,41,42]. It was generally believed that methanoarchaea were the only group of archaea associated with the human mucosa, but molecular approaches have demonstrated the presence of some other archaeal groups in the human gastrointestinal tract, such as Thermoplasma, Crenarchaeota, and halophilic archaea [35,43–46].

We herein attempt to update the knowledge of the methods used during the last two decades to detect archaea in association with the human mucosa, and the implication of these methods for investigating the potential impact of these organisms on human health and disease.

How to Detect Archaea in Humans

Direct microscopic examination

Auto-fluorescence is an interesting feature of methanoarchaea, because they carry factor 420, causing blue–green auto-fluorescence when they are exposed to UV light at a wavelength of 420 nm [47] (Fig. 1). Thus, methanoarchaeal cells or colonies can be quickly identified by epifluorescence microscopy. This attribute has been and still is used in methanogen growth monitoring [7,9,17,20,46].

Figure 1.

 An organigram showing the main methods used for the detection, identification and isolation of methanoarchaea from human samples. MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight.

Anaerobic cultivation

On the basis of the observation that people expired methane, Nottingham and Hungate [30] deduced that some methanoarchaea could be present in the gut. The team succeeded in isolating, for the first time, methanoarchaea from human faeces on non-selective medium in an anoxic atmosphere with 80% H2 and 20% CO2 [30]. Combining the preceding methods with the development of an anaerobic chamber, Edwards and McBride [46] have proposed a new method for the rapid growth and detection of methanoarchaea on Petri plates. Isolation of methanoarchaea from complex specimens such as stools, dental plaque and environmental samples was performed with the roll-tube technique according to Hungate [42] (Fig. 1). Miller et al. [9] further isolated Methanobrevibacter smithii from human stool specimens of four healthy adults by using anaerobic cultures enriched with an H2/CO2 atmosphere (80 : 20) pressurized to 2 atm. A second methanoarchaeon isolated from human faeces was Methanosphaera stadtmanae; Miller and Wolin [8] have established that this archaeon requires H2 and methanol for growth, and uses H2 to reduce methanol to methane. In a further study, Belay et al. [48] succeeded in isolating strains having antigenic similarity with M. smithii and Methanosphaera stadtmanae from samples related to patients with some degree of periodontal disease. Thereafter, anaerobic sampling and culture of 12 vaginal specimens collected from eight healthy women, three women with bacterial vaginosis and one women with erosive lichen planus yielded methanoarchaea in only two of the specimens from patients with bacterial vaginosis. Methanoarchaea were tentatively identified as M. smithii on the basis of morphological, culture and immunological characteristics [49]. Brusa et al. [17] also cultivated methanoarchaea from the dental plaque of nine healthy subjects who also harboured archaeal organisms in the faeces. The third methanoarchaeon isolated in humans was Methanobrevibacter oralis, which was isolated in 1994 from the subgingival plaque of healthy individuals [2]. Finally, we most recently isolated ‘Methanomassiliicoccus luminyensis’ from the faeces of a healthy 86-year-old man; this archaeon exhibited a similar type of metabolism to that of Methanosphaera stadtmanae, by oxidizing H2 and reducing methanol to methane, but requires tungsten to grow [20].

The major obstacle explaining the low number of methanoarchaeal strains isolated until now from human samples is most probably the limited knowledge of the nutritional requirements and antibiotic sensitivity patterns of many archaea [29,30]. In fact, the sensitivity of human-associated archaea to antibiotics has, until recently, been investigated in few studies, predominantly limited to methanogenic strains of the gastrointestinal tract. Dermoumi and Ansorg tested the sensitivity of 15 faecal isolates of M. smithii and the reference strain DSM 861 to nine antibiotics. They concluded that only metronidazole inhibited all strains at MICs between 0.5 and 64 mg/mL [50]. These results were further confirmed by one subsequent study in bone marrow transplant recipients, in which the use of metronidazole targeted to faecal anaerobic bacteria also suppresses faecal methanoarchaea [51]. Following this work, and using the macrodilution method in Hungate tubes with optical microscope observation combined with monitoring of methane production (Fig. 1), we determined the antibiotic resistance characteristics of the eight methanoarchaeal strains isolated in humans for drugs of clinical interest and for squalamine, which is a new potent antimicrobial agent reported to inhibit fungi and both Gram-positive and Gram-negative bacteria [52]. The antimicrobial susceptibility data that we reported demonstrated the susceptibility of human methanoarchaea to only molecules also effective against both bacteria and eukaryotes, such as azoles and squalamine, in agreement with their phylogenetic location as a unique domain of life [53]. These results could help in the design of selective media for the isolation of new archaea from the human-associated microbiota.

Molecular approaches

Recently, molecular methods have emerged as efficient alternatives with which to investigate the prevalence of archaea in human specimens and their potential association with human disease [29]. On the basis of 16S rDNA sequencing, many studies confirmed that the human gastrointestinal tract and oral cavity were dominated by Methanobrevibacter species [19,31,33,35,54,55] (Fig. 2). They also established the presence of M. smithii and Methanosphaera stadtmanae in the human gut, with variable and low prevalence, and in most cases a failure in Methanosphaera stadtmanae detection, in addition to sequences corresponding to uncultured methanoarchaeal clones [29].

Figure 2.

 Dendrogram depicting the major taxonomic groups of the Archaea. The dendrogram is based on representative 16S rRNA gene sequences, and derived from sequence alignment with the neighbour-joining algorithm in Mega (http://www.megasoftware.net). Branches with triangles correspond to genera with species previously detected or/and isolated in humans (red, detected and isolated in humans; green, sporadic detection of 16S rRNA or mcrA genes in the human gut; blue, sporadic detection of 16S rRNA or mcrA genes in the human gut and oral cavity). Note that this tree is not a rigorous phylogenetic analysis, but an attempt to convey the sequence relationships among archaeal organisms.

Besides 16S rDNA, the mcrA gene coding for a subunit of methyl-coenzyme M reductase, a vital enzyme in methane production, was used for the investigation of methanoarchaea in the human gut and oral cavity, and allowed the detection of Methanosarcina plus Methanoculleus sequences, in addition to M. smithii, Methanosphaera stadtmanae and Methanobrevibacter oralis sequences, and sequences related to uncultured methanoarchaea [19,31,35,36,56] (Fig. 2).

Metagenomic studies have also focused on archaeal presence in the human gastrointestinal tract, vagina, and oral cavity, and have confirmed the domination of Methanobacteriales and, essentially, M. smithii [37–40,57–59] (Fig. 2). On the basis of these data, the detection of M. smithii has been proposed as an indicator of faecal contamination of potable water [60].

In addition to methanoarchaea, conventional PCR yielded 16S rRNA sequences suggesting the presence of crenarchaeota, halophilic archaea and Thermoplasma organisms in the human digestive ecosystem [31,35,38,43–45] (Fig. 2).

Gas chromatography

Gas chromatography is used to monitor methanoarchaeal growth by detecting methane production rates [20,52].

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)

MALDI-TOF MS has recently emerged as a rapid and cost-effective technique for the identification of bacteria, eukaryotes, and giant viruses [61–66]. In the literature, only one study has reported the usefulness of MALDI-TOF MS for the identification of environmentally extremophilic archaea, belonging to quite distant phyla; these data were analysed with respect to a limited range of spectra between 500 and 2000 Da [67]. Hence, we developed a specific protocol for MALDI-TOF MS identification of archaea, and applied it to seven environmental human-associated strains of M. smithii, M. oralis, Methanosphaera stadtmanae, and the recently described ‘Methanomassiliicoccus luminyensis’. After mechanical lysis, we observed a unique protein profile for each organism, comprising 7–24 peaks ranging from 3015 to 10 632 Da, with a high quality score of 7.38 ± 1.26. Profiles were reproducible over successive experiments performed at 1-week, 2-week and 3-week growth durations, and unambiguously distinguished archaea from all of the three 995 bacterial spectra in the Brüker database. After the incorporation of the determined profiles into a local database, archaeal isolates were blindly identified within 10 min, with an identification score of 1.9–2.3. The MALDI-TOF MS-based clustering of these archaeal organisms was consistent with their 16S rDNA sequence-based phylogeny. The obtained data proved that MALDI-TOF MS profiling could be used as a first-line technique for the identification of human archaea [68] (Fig. 1).

Contribution of Archaeal Detection in Clinical Microbiology

Human gut

For determination of the contribution of active methanoarchaea to disease, most studies were based on measurement of breath methane by gas chromatography. The relationship between methanoarchaea in the colon and colonic vascular circulation diseases has been investigated in patients with aortoiliac and femoropopliteal diseases [69]. The proportions of methane excretors in the control group and in those with femoropopliteal disease were 43% and 30%, respectively; in patients with aortoiliac pathology, the proportion of methane excretors was significantly higher (83%). As compared with healthy patients, elevated rates of breath methane have been detected in patients with ulcerative colitis, colonic polyposis, and colon cancer [70]. Most recently, Basseri et al. [71] have demonstrated that a higher concentration of methane detected by breath testing is a predictor of significantly greater obesity in overweight subjects. This method is also used for methanoarchaeal growth monitoring by detecting methane production rates [20,52].

Scanlan et al. compared the incidence of methanoarchaeal diversity in healthy and diseased colon groups through mcrA gene analysis. They established a reduced methanoarchaeal incidence in the inflammatory bowel disease groups: 24% for ulcerative colitis, and 30% for Crohn’s disease [36]. On the other hand, Samuel and Gordon demonstrated that methanoarchaea could enhance the activity and growth of polysaccharide consumers such as members of the Bacteroidetes and Firmicutes by removing H2 [72]. Thus, methanoarchaea indirectly promote calorie intake by the body and, consequently, accumulation of fat deposits, leading to obesity in individuals on a high-fibre diet. These results were further confirmed by comparing lean controls with obese patients before and after gastric bypass; the authors observed an increase in Methanobrevibacter load in obese patients [39].

By applying an improved DNA detection protocol based on quantitative real-time PCR targeting 16S rRNA and rpoB genes in a large number of specimens (650 individuals), we recently established a high prevalence of the methanoarchaea M. smithii (95.5%) and Methanosphaera stadtmanae (29.4%) in the human gut, with the former being an almost ubiquitous inhabitant of the intestinal microbiome [33].

Most recently, using the molecular system designed in the last study [33], Armougom et al. obtained results in agreement with those of Samuel and Gordon, reporting a 1.72-fold increase in the M. smithii load in the obese group as compared with the lean one; surprisingly, they observed a significantly higher M. smithii load in the anorexic population than in lean patients (p 0.0171), and gave two possible reasons for this—adaptive use of the very low calorie diet in anorexic cases, or constipation, a common phenomenon in anorexia nervosa patients [34].

Oral cavity

Several studies have principally focused on the role of oral archaea in periodontal disease and endodontic infections. Using PCR amplification of archaeal 16S rDNA on pooled subgingival plaque samples, Kulik et al. [73] found Methanobrevibacter species in 37 of 48 cases of periodontal disease. Quantitative PCR targeting archaeal 16S rDNA sequences allowed Lepp et al. [74] to detect methanoarchaea, mostly related to the genus Methanobrevibacter, in the lesion sites of 36% of tested patients, and to demonstrate the direct correlation between the amount of methanoarchaea present in the subgingival crevice and the severity of periodontitis. The authors hypothesized that,. in periodontal pockets, methanoarchaea act syntrophically by serving as an H2 sink, and thus favour the proliferation of pathogens. Through quantitative real-time PCR based on the functional gene mcrA and on archaeal 16S rRNA genes, Vianna et al. detected a methanogenic pool dominated by M. oralis-like phylotypes in five of 20 necrotic uniradicular teeth with no previous endodontic treatment. They estimated the size of archaeal population as approximately 2.5% of the total prokaryotic community [54]. In a subsequent study, the same authors used an identical molecular approach to perform a quantitative analysis of methanoarchaea within plaque biofilms associated with human periodontal disease; they consistently detected hydrogenotrophic groups in periodontal pockets with significantly elevated loads of methanoarchaea and sulphate reducers, and confirmed the impact of antagonistic interactions between H2 consumers and producers on the severity of periodontitis [55]. Moreover, the authors established the presence of an M. oralis-related phylotype in 44 periodontitis patients [55]. These results were confirmed by subsequent studies using similar molecular approaches; 16S rDNA sequences related to an M. oralis-like species were detected in the root canals of two patients, and it was established that archaea were always found in combination with bacteria [75]. These results reinforce the antagonism hypothesis. Most recently, Vianna et al. performed a terminal restriction fragment length polymorphism-based mcrA gene analysis of methanoarchaea associated with oral infections. They established a positive association between methanoarchaea and Synergistes species, and detected DNA of a novel Methanobrevibacter phylotype in five periodontal samples and in one endodontic sample, in addition to M. oralis [56].

By means of RT-PCR, based on universal archaeal 16S rDNA sequences, Jiang et al. investigated the presence and associations of archaea in primary and secondary root canal infections. In primary infections, archaea were detected in 16 of 42 (38%) specimens, but in only six of 35 (17%) cases with secondary root-infected canals [76].

Latterly, by combining archaeal 16S rRNA gene library sequencing and quantitative PCR, Matarazzo et al. demonstrated the presence of high levels of archaea and high archaeal/total prokaryote ratios in individuals with generalized aggressive periodontitis as compared with periodontally healthy subjects, indicating a possible role of some of these microorganisms as environmental modifiers in generalized aggressive periodontitis [77]. Similarly, by sequencing archaeal 16S rRNA gene libraries, Faveri et al. [78] demonstrated an increased prevalence of archaea in peri-implantitis sites, and suggested that the potential role of archaea in pathogenesis should be further investigated.

Perspectives

It is well established that archaea and, especially, methanoarchaea inhabit humans, and high numbers have been found in the colon, mouth, and vagina. M. smithii, Methanosphaera stadtmanae, M. oralis and ‘Methanomassiliicoccus luminyensis’ have been, until now, the only four archaeal organisms isolated in humans by culture approaches [2,7–9,17,48,49] (B. Dridi et al., unpublished data). The prevalence and quantity of human gut methanoarchaea have been underestimated for a long time, undoubtedly because of the complicated nature of their cultivation and the fact that standard protocols for DNA extraction have not been optimized for such fastidious microorganisms.

Although, until now, no archaea have been described as being directly pathogenic, the occurrence in and coincidence of these organisms, and especially methanoarchaea, with diverse diseases and pathological conditions have raised many unanswered questions concerning the role that they might have in these pathologies. Many hypotheses have been formulated to explain the possible contribution of methanoarchaea to diseases, and the most plausible is based on syntrophic interactions with other microorganisms [79]. The fact that methanoarchaea indirectly promote obesity indicates potential stratagems to control either obesity, by inhibition of methanoarchaea to decrease caloric intake, or starvation-related weakness, by adding methanoarchaea to the intestinal flora.

Besides methanoarchaea, some other archaeal groups, such as Thermoplasma, the Crenarchaeota, and halophilic archaea, have been transiently detected in human faeces through the use of molecular tools such as PCR and metagenomics [35,38,43–45]. Given the relative difficulty of their isolation from stool samples, owing to their low proportions, the role that these non-methanogenic archaea may play in the human digestive tract is still unknown. Their acquisition could also be accidental: for halophilic archaea-related DNA, the authors suggested the possibility that the sequences obtained originated from pre-colonoscopy saline lavage solutions [44]; and members of the Crenarchaeota have been found in fermented seafood [80]. In fact, the most plausible explanation for the fact that most archaea cannot colonize the human mucosa could be the exceptionality of their biochemistry; they use a variety of ‘unusual’ cofactors that bacteria and eukaryotes neither require nor produce [81]. Thus, from the standpoint of nutritional requirements, the human mucosa could not constitute a favourable environment for non-methanogenic archaea, because they are, as ‘biochemical outsiders’, inferior regarding competition with bacteria.

In conclusion, the involvement of archaea in human disease and health warrants further investigation: first, by completing the antimicrobial susceptibility patterns, by testing a wide spectrum of antimicrobial agents, to improve isolation conditions for such fastidious organisms and to develop new families of molecules for the specific inhibition of archaea, particularly with regard to the predicted potential role of these organisms in human infections; and second, through using adapted and specific approaches such as metagenomics with high-throughput sequencing methods, PCR-based methods, microscopy, fluorescence in situ hybridization, DNA microarrays, and mass spectrometry, which are rapidly emerging as powerful tools for the phenotypic detection of many human pathogens [61,64,66,68,82–88].

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