BACKGROUND
the methanogens are a group of microorganisms within the kingdom euryarchaeota of the domain archaea  <cit> .
methanogenesis is a process confined exclusively to the methanogens and utilises substrates such as hydrogen, co <dig>  acetate, formate, methanol and methylamines for methane generation.
the methanogenic archaea are widely distributed in natural environments including the gastrointestinal tracts of ruminants, termites and humans  <cit> .
studies to date indicated that methanobrevibacter smithii is the predominant archaeal species present in the human large intestine  <cit> .
and so far only a second methanogenic species methanosphaera stadtmanae, also a member of the order methanobacteriales has been isolated from the human intestinal tract  <cit> .
the reasons underlying methanogen incidence and their seemingly low diversity in human populations are of considerable interest to gastrointestinal microbial ecologists.
an increasing interest in the methanogenic populations of the human gastrointestinal tract is also underpinned by their potential role as environmental factors in obesity  <cit>  and pneumatosis cystoides intestinalis  <cit>  and any possible association methanogens or methane production may have with gastrointestinal disease.
as yet, no pathogenic archaea have been characterised to date and the aetiological role, if any, methanogens play in disease is unknown  <cit> .
although no pathogenic archaeal species has been found  <cit> , they are routinely identified in samples from the sites of oral infections  <cit>  leading to speculation that they may be indirectly involved in disease processes by creating environmental conditions to facilitate the growth of microbes involved in pathogenesis.
in methane-producing humans methanogen numbers are approximately  <dig>  ×  <dig> to  <dig> × 109/g faeces  <cit> .
and in non-methanogenic humans  <dig> to  <dig> × 106/g faeces  <cit> .
traditionally, the characterization of methanogens from the human intestinal environment has largely focused on phenotypic, microscopic, biochemical and nutritional classification methodologies  <cit> .
and whilst these techniques are critical to gastrointestinal microbial studies they are time consuming and laborious  <cit> .
furthermore, molecular techniques for characterising microbial communities are considered more comprehensive and enable a more accurate overview of the diversity and functionality present in the human gastrointestinal tract  <cit> .
this study has undertaken the optimisation of appropriate culture independent methodologies with the aim of investigating and characterising methanogen incidence and diversity in human faecal samples.
a pcr and clone library methodology was employed using one set of previously published pcr primers  <cit>  specific to the α subunit of methyl-coenzyme m reductase , a functional gene that catalyzes the terminal step in the formation of methane by methanogens  <cit>  and a second set of novel primers which were designed to specifically target the mrta gene  of methanosphaera stadtmanae.
RESULTS
high molecular weight dna, free from pcr inhibitors is critical to pcr analysis  <cit>  and each dna sample in this study was tested in order to ensure that a negative mcra gene or mrta gene pcr result were not as a consequence of poor dna extraction and quality.
an initial pcr targeting the 16s rrna gene was used as a control measure for dna quality and all faecal dna extracts gave a positive signal for a 16s rrna gene pcr assay.
the mcra gene was chosen as a proxy for methanogen detection as this gene can be readily amplified from mbb.
smithii  which is the dominant methanogen in the human gut  <cit> .
furthermore, the detection limits of the primers for mcra gene amplification were calculated and a positive pcr result obtained using the mcra primer set from spiked samples at cell numbers ≥  <dig> cells per gram of faeces using mbb.
smithii pst as the test organism.
therefore, results recorded and reported in this study were obtained from faecal samples where methanogen cell numbers equalled or exceeded  <dig> cells per gram of faeces making its detection level equivalent to that of the methane breath test  <cit>  and real-time pcr assay using methanogen specific 16s rrna gene primers  <cit> .
interestingly, the results from the pcr survey of methanogen frequency in different groups of individuals found that in the control, colorectal cancer, polypectomised and irritable bowel syndrome groups, the percentage of individual's positive for methanogens were largely equivalent and fell within a range of  <dig> – 50% .
the percentage of individuals within the inflammatory bowel disease cohorts harboured methanogens at a lower rate of 30% for crohn's disease  and a significantly lower rate of 24% for ulcerative colitis compared to the control group  .
although these data can not be directly correlated with previous published studies, the same general trend was observed in a methane detection survey where methane excretion was detected in 54% of healthy controls, 53% of non gastrointestinal patients and 32% of gastrointestinal patients  <cit> .
in that study the patients within the gastrointestinal disease group that had inflammatory bowel disease had significantly lower methane detection; 13% for crohn's disease and 15% for ulcerative colitis  <cit> .
furthermore, methane production in the predominantly diarrhoeal conditions of ulcerative colitis and crohn's disease is reportedly almost non existent  <cit> , a finding that may be due to loss of slow growing methanogens that are displaced during conditions of rapid gut transit.
collectively, these data for reduced methanogens in ibd may support the hypothesis that methane is not in fact biologically inert but may play a role in intestinal motility with consequences for irritable bowel syndrome associated constipation  <cit> , diverticulitis  <cit>  and encopresis  <cit> .
no data was available on the time of collection of samples and all samples analysed in this study were participants in larger trials.
the individuals selected for each trial was done so in a randomised fashion with only the disease status of the host being the primary prerequisite for inclusion.
a cdai: crohn's disease activity index.
individuals with a cdai greater than  <dig> are considered to be in relapse  <cit> .
b diagnosed by clinician.
c see  <cit> .
d significantly  different from control group using fischers' exact statistical test.
the results from the healthy group reported in this study are also similar to those obtained from a real time-pcr analysis of methanogen incidence and diversity in the human gastrointestinal tract using 16s rrna gene as a target  <cit> .
only  <dig> adults were analysed as part of this study and 5/ <dig> adults  gave a positive pcr result.
in the majority of samples analysed in the total study pcr amplicons generated were beyond the quantitative limits of the pcr assay.
furthermore, mbb.
smithii was the predominant sequence obtained from extensive sequence analysis with no msp.stadtmanae sequences recovered  <cit> .
with respect to the diversity of methanogens in the present study, restriction fragment length polymorphism  analysis of the mcra gene clones  revealed only  <dig> unique rflp types  the results of which are outlined in table  <dig> and illustrated in figures  <dig> and  <dig>  in fact, all clone libraries generated with the exception of ulcerative colitis , crohn's disease  and irritable bowel syndrome  exhibited a uniform rflp profile identical to that obtained from mbb.
smithii pst and mbb.
smithii   with all clones that were subsequently sequenced showing 100% amino acid identity to mbb.
smithii.
this finding reinforces the prevailing knowledge that mbb.
smithii is the dominant methanogen in the human gastrointestinal tract.
three additional rflp profiles were identified in the uc, cd and ibs clone libraries.
amino acid sequence analysis of these clones indicated that rflp type b from the ibs and crohn's disease clone library was most closely related to methanobrevibacter oralis, type c from ulcerative colitis  which, although exhibiting a unique rflp profile, is most closely related to mbb.
smithii  and a final rflp type d  which was identified in the ulcerative colitis library.
phylogenetic analysis indicated that uc-6's closet cultured relative was the alkaliphilic, halophilic and methylotrophic archaea methanosalsum zhilinae  <cit>  .
the uc- <dig> sequence obtained in this study was also 100% identical to a sequence  obtained from a human faecal sample from a unpublished study indicating that uncultured members of the methanogen outside the methanobacteriales have been identified in other laboratories as well.
the constructed phylogenetic tree further highlights these relationships amongst clone isolates from this study with cultured and uncultured methanogens .
several mismatches to the mrta gene sequence of msp.stadtmanae were evident with the mcra primer set and phylogenetic analysis and tree construction illustrates that although the mrta gene of msp.
stadtmanae encodes an enzyme of the same function and inhabits the same niche it is phylogenetically distinct from mcra gene sequences of mbb.
smithii and related clones .
a second set of primers were designed and optimised for mrta gene amplification serving as a marker for msp.
stadtmanae detection.
pcr and clone library analysis indicate that the primers were highly specific for and readily amplified dna from pure msp.
stadtmanae dna at concentrations less than  <dig>  ng ul- <dig>  however, no positive pcr results were obtained for mrta gene from faecal dna extracts in this study.
further analysis of available 16s rrna gene libraries from published literature  <cit>  and analysis in silico of metagenomic clones libraries generated from human faecal samples also support the idea that msp.
stadtmanae is not a common feature of the human gastrointestinal microbiota.
therefore, it is quite possible that if msp.
stadtmanae was present in any of the faecal samples analysed in this study its concentration was below the detection levels of this novel primer set.
methanobrevibacter smithii and msp.stadtmanae are phylogenetically diverse methanogens and the metabolic capacity of both microbes may provide evidence for the predominance of mbb.smithii in the human gastrointestinal tract.
genome comparisons between both methanogens indicate that mbb.
smithii is more adapted to the gut environment in terms of persistence, metabolic versatility and capacity for genomic evolution  <cit> .
not only is the mbb.
smithii genome significantly enriched for genes involved in co <dig>  h <dig> and formate utilisation during methanogenesis, mbb.
smithii also possesses the capacity for non methanogenic removal of other bacterial fermentation end-products, namely methanol and ethanol.
although both genomes exhibit limited global synteny, msp.stadtmanae has the most restricted energy metabolism of any archaea studied to date  <cit>  and can only use hydrogen to reduce methanol to methane.
it is possible that this limited substrate range could be a factor in competition between msp.stadtmanae and other microorganisms such as mbb.
smithii and the sulphate reducing bacteria which also require hydrogen for growth  <cit> .
however, further insight into msp.stadtmanae incidence and interaction is now possible with this novel primer set which also have application to the rumen gut and other ecosystems where msp.stadtmanae like sequences have been reported  <cit>
the functionality of methanogens appears to be strongly associated and facilitated by the presence and activities of other microbial groups  <cit> .
if indeed the carriage of methanogens is dependent upon or supported by other microbial groups and their activities, these differences observed between the methanogen frequency in certain disease and control groups compared to inflammatory bowel disease is quite possibly linked to the reduced bacterial diversity that is routinely reported in this gastrointestinal disease conditions  <cit>  considering these points, it is reasonable to suggest the presence of methanogens in human gastrointestinal tract is part due to the presence of other microbial group or groups with fermentation capacities that generate sufficient h <dig> and substrates to support methanogenesis in a competitive environment.
the presence of such a bacterial population could be present due to genetic host factors, diet, intestinal transit time, stochastic and other environmental factors that would support such an overall symbiotic community.
as such the reduced frequency of methanogens evident within the ibd groups could possibly serve as a biomarker of altered microbial diversity and metabolic processes within the human gastrointestinal tract.
CONCLUSIONS
this exploration of a functionally significant microbial group provides a comprehensive survey of methanogen incidence and diversity in both health and disease of the human gastrointestinal tract using a culture independent approach.
the limited diversity of methanogens as evidenced by the mbb.
smithii dominance of clone libraries and failure to amplify msp.
stadtmanae from faecal samples raises interesting ecological questions on the nature of microbial competition for resources in the human gut.
furthermore, the identification of a mcra gene sequences only distantly related to cultured methanogens is of significant interest and highlights the necessity of a culture independent approach when surveying diversity of this fastidious group of microorganisms and also the requirement to better our cultivation techniques to gain access to novel and uncultured microbes.
in addition, the results of this study have highlighted some important points on the nature of methanogen carriage and their prospect as markers for altered gastrointestinal function.
the reduced incidence of hydrogen utilising microbes in ibd could also serve as a biomarker for altered biochemical activities in the intestinal tract of certain individuals and warrants further exploration.
it is now possible with the methods outlined in this study that functional analysis of mcra gene as a proxy for methane production using rna as a template could have relevance for the further study of clinical conditions related to methanogen carriage and methane production and also the competitive interactions between methanogens and other microbial groups.

 the methanogens are a group of microorganisms within the kingdom euryarchaeota of the domain archaea. methanogenesis is a process confined exclusively to the methanogens and utilises substrates such as hydrogen, co acetate, formate, methanol and methylamines for methane generation. the methanogenic archaea are widely distributed in natural environments including the gastrointestinal tracts of ruminants, termites and humans. studies to date indicated that methanobrevibacter smithii is the predominant archaeal species present in the human large intestine. and so far only a second methanogenic species methanosphaera stadtmanae, also a member of the order methanobacteriales has been isolated from the human intestinal tract. the reasons underlying methanogen incidence and their seemingly low diversity in human populations are of considerable interest to gastrointestinal microbial ecologists. an increasing interest in the methanogenic populations of the human gastrointestinal tract is also underpinned by their potential role as environmental factors in obesity and pneumatosis cystoides intestinalis and any possible association methanogens or methane production may have with gastrointestinal disease. as yet, no pathogenic archaea have been characterised to date and the aetiological role, if any, methanogens play in disease is unknown. although no pathogenic archaeal species has been found, they are routinely identified in samples from the sites of oral infections leading to speculation that they may be indirectly involved in disease processes by creating environmental conditions to facilitate the growth of microbes involved in pathogenesis. in methane-producing humans methanogen numbers are approximately × to × 109/g faeces. and in non-methanogenic humans to × 106/g faeces. traditionally, the characterization of methanogens from the human intestinal environment has largely focused on phenotypic, microscopic, biochemical and nutritional classification methodologies. and whilst these techniques are critical to gastrointestinal microbial studies they are time consuming and laborious. furthermore, molecular techniques for characterising microbial communities are considered more comprehensive and enable a more accurate overview of the diversity and functionality present in the human gastrointestinal tract. this study has undertaken the optimisation of appropriate culture independent methodologies with the aim of investigating and characterising methanogen incidence and diversity in human faecal samples. a pcr and clone library methodology was employed using one set of previously published pcr primers specific to the α subunit of methyl-coenzyme m reductase, a functional gene that catalyzes the terminal step in the formation of methane by methanogens and a second set of novel primers which were designed to specifically target the mrta gene of methanosphaera stadtmanae.  high molecular weight dna, free from pcr inhibitors is critical to pcr analysis and each dna sample in this study was tested in order to ensure that a negative mcra gene or mrta gene pcr result were not as a consequence of poor dna extraction and quality. an initial pcr targeting the 16s rrna gene was used as a control measure for dna quality and all faecal dna extracts gave a positive signal for a 16s rrna gene pcr assay. the mcra gene was chosen as a proxy for methanogen detection as this gene can be readily amplified from mbb. smithii which is the dominant methanogen in the human gut. furthermore, the detection limits of the primers for mcra gene amplification were calculated and a positive pcr result obtained using the mcra primer set from spiked samples at cell numbers ≥ cells per gram of faeces using mbb. smithii pst as the test organism. therefore, results recorded and reported in this study were obtained from faecal samples where methanogen cell numbers equalled or exceeded cells per gram of faeces making its detection level equivalent to that of the methane breath test and real-time pcr assay using methanogen specific 16s rrna gene primers. interestingly, the results from the pcr survey of methanogen frequency in different groups of individuals found that in the control, colorectal cancer, polypectomised and irritable bowel syndrome groups, the percentage of individual's positive for methanogens were largely equivalent and fell within a range of – 50%. the percentage of individuals within the inflammatory bowel disease cohorts harboured methanogens at a lower rate of 30% for crohn's disease and a significantly lower rate of 24% for ulcerative colitis compared to the control group. although these data can not be directly correlated with previous published studies, the same general trend was observed in a methane detection survey where methane excretion was detected in 54% of healthy controls, 53% of non gastrointestinal patients and 32% of gastrointestinal patients. in that study the patients within the gastrointestinal disease group that had inflammatory bowel disease had significantly lower methane detection; 13% for crohn's disease and 15% for ulcerative colitis. furthermore, methane production in the predominantly diarrhoeal conditions of ulcerative colitis and crohn's disease is reportedly almost non existent, a finding that may be due to loss of slow growing methanogens that are displaced during conditions of rapid gut transit. collectively, these data for reduced methanogens in ibd may support the hypothesis that methane is not in fact biologically inert but may play a role in intestinal motility with consequences for irritable bowel syndrome associated constipation, diverticulitis and encopresis. no data was available on the time of collection of samples and all samples analysed in this study were participants in larger trials. the individuals selected for each trial was done so in a randomised fashion with only the disease status of the host being the primary prerequisite for inclusion. a cdai: crohn's disease activity index. individuals with a cdai greater than are considered to be in relapse. b diagnosed by clinician. c see. d significantly different from control group using fischers' exact statistical test. the results from the healthy group reported in this study are also similar to those obtained from a real time-pcr analysis of methanogen incidence and diversity in the human gastrointestinal tract using 16s rrna gene as a target. only adults were analysed as part of this study and 5/ adults gave a positive pcr result. in the majority of samples analysed in the total study pcr amplicons generated were beyond the quantitative limits of the pcr assay. furthermore, mbb. smithii was the predominant sequence obtained from extensive sequence analysis with no msp.stadtmanae sequences recovered. with respect to the diversity of methanogens in the present study, restriction fragment length polymorphism analysis of the mcra gene clones revealed only unique rflp types the results of which are outlined in table and illustrated in figures and in fact, all clone libraries generated with the exception of ulcerative colitis, crohn's disease and irritable bowel syndrome exhibited a uniform rflp profile identical to that obtained from mbb. smithii pst and mbb. smithii with all clones that were subsequently sequenced showing 100% amino acid identity to mbb. smithii. this finding reinforces the prevailing knowledge that mbb. smithii is the dominant methanogen in the human gastrointestinal tract. three additional rflp profiles were identified in the uc, cd and ibs clone libraries. amino acid sequence analysis of these clones indicated that rflp type b from the ibs and crohn's disease clone library was most closely related to methanobrevibacter oralis, type c from ulcerative colitis which, although exhibiting a unique rflp profile, is most closely related to mbb. smithii and a final rflp type d which was identified in the ulcerative colitis library. phylogenetic analysis indicated that uc-6's closet cultured relative was the alkaliphilic, halophilic and methylotrophic archaea methanosalsum zhilinae. the uc- sequence obtained in this study was also 100% identical to a sequence obtained from a human faecal sample from a unpublished study indicating that uncultured members of the methanogen outside the methanobacteriales have been identified in other laboratories as well. the constructed phylogenetic tree further highlights these relationships amongst clone isolates from this study with cultured and uncultured methanogens. several mismatches to the mrta gene sequence of msp.stadtmanae were evident with the mcra primer set and phylogenetic analysis and tree construction illustrates that although the mrta gene of msp. stadtmanae encodes an enzyme of the same function and inhabits the same niche it is phylogenetically distinct from mcra gene sequences of mbb. smithii and related clones. a second set of primers were designed and optimised for mrta gene amplification serving as a marker for msp. stadtmanae detection. pcr and clone library analysis indicate that the primers were highly specific for and readily amplified dna from pure msp. stadtmanae dna at concentrations less than ng ul- however, no positive pcr results were obtained for mrta gene from faecal dna extracts in this study. further analysis of available 16s rrna gene libraries from published literature and analysis in silico of metagenomic clones libraries generated from human faecal samples also support the idea that msp. stadtmanae is not a common feature of the human gastrointestinal microbiota. therefore, it is quite possible that if msp. stadtmanae was present in any of the faecal samples analysed in this study its concentration was below the detection levels of this novel primer set. methanobrevibacter smithii and msp.stadtmanae are phylogenetically diverse methanogens and the metabolic capacity of both microbes may provide evidence for the predominance of mbb.smithii in the human gastrointestinal tract. genome comparisons between both methanogens indicate that mbb. smithii is more adapted to the gut environment in terms of persistence, metabolic versatility and capacity for genomic evolution. not only is the mbb. smithii genome significantly enriched for genes involved in co h and formate utilisation during methanogenesis, mbb. smithii also possesses the capacity for non methanogenic removal of other bacterial fermentation end-products, namely methanol and ethanol. although both genomes exhibit limited global synteny, msp.stadtmanae has the most restricted energy metabolism of any archaea studied to date and can only use hydrogen to reduce methanol to methane. it is possible that this limited substrate range could be a factor in competition between msp.stadtmanae and other microorganisms such as mbb. smithii and the sulphate reducing bacteria which also require hydrogen for growth. however, further insight into msp.stadtmanae incidence and interaction is now possible with this novel primer set which also have application to the rumen gut and other ecosystems where msp.stadtmanae like sequences have been reported the functionality of methanogens appears to be strongly associated and facilitated by the presence and activities of other microbial groups. if indeed the carriage of methanogens is dependent upon or supported by other microbial groups and their activities, these differences observed between the methanogen frequency in certain disease and control groups compared to inflammatory bowel disease is quite possibly linked to the reduced bacterial diversity that is routinely reported in this gastrointestinal disease conditions considering these points, it is reasonable to suggest the presence of methanogens in human gastrointestinal tract is part due to the presence of other microbial group or groups with fermentation capacities that generate sufficient h and substrates to support methanogenesis in a competitive environment. the presence of such a bacterial population could be present due to genetic host factors, diet, intestinal transit time, stochastic and other environmental factors that would support such an overall symbiotic community. as such the reduced frequency of methanogens evident within the ibd groups could possibly serve as a biomarker of altered microbial diversity and metabolic processes within the human gastrointestinal tract.  this exploration of a functionally significant microbial group provides a comprehensive survey of methanogen incidence and diversity in both health and disease of the human gastrointestinal tract using a culture independent approach. the limited diversity of methanogens as evidenced by the mbb. smithii dominance of clone libraries and failure to amplify msp. stadtmanae from faecal samples raises interesting ecological questions on the nature of microbial competition for resources in the human gut. furthermore, the identification of a mcra gene sequences only distantly related to cultured methanogens is of significant interest and highlights the necessity of a culture independent approach when surveying diversity of this fastidious group of microorganisms and also the requirement to better our cultivation techniques to gain access to novel and uncultured microbes. in addition, the results of this study have highlighted some important points on the nature of methanogen carriage and their prospect as markers for altered gastrointestinal function. the reduced incidence of hydrogen utilising microbes in ibd could also serve as a biomarker for altered biochemical activities in the intestinal tract of certain individuals and warrants further exploration. it is now possible with the methods outlined in this study that functional analysis of mcra gene as a proxy for methane production using rna as a template could have relevance for the further study of clinical conditions related to methanogen carriage and methane production and also the competitive interactions between methanogens and other microbial groups. 
in addition, mcra gene sequences most closely related to methanobrevibacter oralis and members of the order methanosarcinales were also recovered. colorectal cancer, polypectomised, irritable bowel syndrome and the control group had largely equivalent numbers of individuals positive for methanogens. four unique mcra gene restriction fragment length polymorphism profiles were identified and bioinformatic analyses revealed that the majority of all sequences retrieved from libraries were 100% identical to methanobrevibacter smithii mcra gene. although methanobrevibacter smithii is the dominant methanogen in both the distal colon of individuals in health and disease, the diversity of methanogens is greater than previously reported. a pcr and clone library methodology targeting the mcra gene was adopted to facilitate the two-fold aim of surveying the relative incidence of methanogens in health and disease groups and also to provide an overview of methanogen diversity in the human gastrointestinal tract. methanogen incidence in the inflammatory bowel disease groups was reduced, 24% for ulcerative colitis and 30% for crohn's disease. the mcra gene serves as a useful biomarker for methanogen detection in the human gut and the varying trends of methanogen incidence in the human gut could serve as important indicators of intestinal function. in conclusion, the low incidence of methanogens in inflammatory bowel disease, the functionality of the methanogens and impact of methane production in addition to competitive interactions between methanogens and other microbial groups in the human gastrointestinal tract warrants further investigation.
 the incidence and diversity of human methanogens are insufficiently characterised in the gastrointestinal tract of both health and disease. a pcr and clone library methodology targeting the mcra gene was adopted to facilitate the two-fold aim of surveying the relative incidence of methanogens in health and disease groups and also to provide an overview of methanogen diversity in the human gastrointestinal tract.  dna faecal extracts from a group of healthy controls and five gastrointestinal disease groups were investigated. colorectal cancer, polypectomised, irritable bowel syndrome and the control group had largely equivalent numbers of individuals positive for methanogens. methanogen incidence in the inflammatory bowel disease groups was reduced, 24% for ulcerative colitis and 30% for crohn's disease. four unique mcra gene restriction fragment length polymorphism profiles were identified and bioinformatic analyses revealed that the majority of all sequences retrieved from libraries were 100% identical to methanobrevibacter smithii mcra gene. in addition, mcra gene sequences most closely related to methanobrevibacter oralis and members of the order methanosarcinales were also recovered.  the mcra gene serves as a useful biomarker for methanogen detection in the human gut and the varying trends of methanogen incidence in the human gut could serve as important indicators of intestinal function. although methanobrevibacter smithii is the dominant methanogen in both the distal colon of individuals in health and disease, the diversity of methanogens is greater than previously reported. in conclusion, the low incidence of methanogens in inflammatory bowel disease, the functionality of the methanogens and impact of methane production in addition to competitive interactions between methanogens and other microbial groups in the human gastrointestinal tract warrants further investigation. 