Variation of platelet function in clinical phenotypes of acute venous thromboembolism - Results from the GMP-VTE project.

BACKGROUND: The role of platelets in the pathogenesis of venous thromboembolism (VTE) is receiving increasing attention; however, limited information is available on platelet function in the acute phase of the disease. OBJECTIVE: To characterize platelet function according to VTE phenotypes. PATIENTS/METHODS: In total, 154 subjects (isolated pulmonary embolism [iPE], n = 28; isolated deep vein thrombosis [iDVT], n = 35; DVT+PE, n = 91) were included. In this study platelet function analyzer (PFA)-200, light transmission aggregometry (LTA), thrombin generation (TG) in presence (PRP) and absence (PFP) of platelets and platelet flow cytometry were investigated. LASSO regression was used to select clinical and platelet biomarkers that distinguish between VTE phenotypes. RESULTS: PFA-200 results did not differ between VTE phenotypes. LTA from DVT+PE subjects showed lowest maximum aggregation after epinephrine and adenosine diphosphate compared to iPE and iDVT. Lower % of PAC-1-positive platelets after in-vitro trigger were present in DVT+PE and iPE compared to iDVT. TG in PRP had lower peak height and velocity in DVT+PE and iPE against iDVT. The results of LASSO regression for the distinction between DVT+PE vs iDVT identified 18 variables (AUC =0.93) of which 72% were platelet biomarkers. For distinction between iPE and iDVT, 10 variables were selected (AUC = 0.96) of which 50% were platelet-related. Obesity was the only variable weakly discriminating between DVT+PE vs iPE (AUC = 0.66). CONCLUSION: This explorative study suggests an important distinction between PE-related phenotypes and iDVT when considering clinical and platelet function data. Lower platelet-dependent TG along with reduced platelet reactivity suggest higher platelet degranulation in PE-dependent phenotypes compared to iDVT.

Is There Reduced Hemodynamic Brain Activation in Multiple Sclerosis Even with Undisturbed Cognition?

Cognitive impairments related to changes in deep gray matter and other brain regions occur in up to 70% of people with multiple sclerosis. But do such brain changes also occur in patients without significant cognitive impairment? Eighteen participants with relapsing-remitting multiple sclerosis (RRMS) and fifteen healthy controls participated in this study. Cognitive status, depression, and fatigue were assessed using the Multiple Sclerosis Inventory of Cognition (MUSIC), Beck's Depression Inventory (BDI-II), and the Fatigue Severity Scale (FSS). fMRI was recorded while a participant performed the modified attention network test (ANT). The effects of ANT executive attention network on hemodynamic activation of a priori defined regions of interest, including the hippocampus, anterior cingulate cortex (ACC), thalamus, caudate nucleus, pallidum, and putamen were studied. The individual lesion load was estimated. For fMRI data analysis a general linear model with randomization statistics including threshold-free cluster enhancement as implemented in the FSL software was used. Participants with RRMS showed reduced activation of the executive attention network in the hippocampus, pallidum, and ACC. The thalamus was involved in both group activations but did not differ between groups. In summary, functional changes in the brain can also be demonstrated in RRMS patients without cognitive deficits. The affected brain regions can best be assigned to the attention network for executive control. This association could likely serve as a biological indicator of susceptibility to imminent cognitive impairment in MS.

Characterization of Thrombin Generation Curve Shape in Presence of Platelets from Acute Venous Thromboembolism Patients.

Background. Anticoagulant therapy, the cornerstone treatment in acute venous thromboembolism (VTE), strongly impacts thrombin generation (TG). Until now, the appearance of the TG curve in platelet rich plasma (PRP) from patients with acute VTE has not been investigated. Methods. We analyzed the shape of TG curves measured in PARP of 180 acute VTE patients. Results. Normal shape of TG curves was observed in 110 patients, 50 patients showed no TG and 20 patients showed biphasic TG curve. The linear regression analysis, adjusted for age, sex, VTE clinical phenotypes and therapy showed that the appearance of biphasic curves is significantly associated with female sex, presence of cancer and therapy with Factor Xa inhibitors. Conclusions. This study demonstrated that despite taking anticoagulants, TG in presence of platelets is still present in the majority of acute VTE patients. Appearance of unusual TG curves is strongly related to the intake of anti-Factor Xa inhibitors. The clinical relevance of biphasic TG curve appearance requires further investigation.

Comprehensive platelet phenotyping supports the role of platelets in the pathogenesis of acute venous thromboembolism - results from clinical observation studies.

BACKGROUND: The pathogenesis of arterial and venous thrombosis is in large part interlaced. How much platelet phenotype relates to acute venous thromboembolism (VTE) independent of the underlying cardiovascular profile is presently poorly investigated. METHODS: Platelet count and mean platelet volume (MPV), platelet aggregation in whole blood and platelet rich plasma (PRP), platelet-dependent thrombin generation (TG) and platelet surface activation markers were measured under standardized conditions. Machine learning was applied to identify the most relevant characteristics associated with VTE from a large array (N = 58) of clinical and platelet-related variables. FINDINGS: VTE cases (N = 159) presented with lower platelet count and MPV vs controls (N = 140). Whole blood aggregation showed shorter collagen/Epinephrine closure times in cases, particularly within acetylsalicylic acid (ASA) users. Within ASA users, higher PRP aggregation after adenosine diphosphate (ADP), epinephrine, collagen and arachidonic acid was observed in cases vs controls. Within non-ASA and/or subjects on anticoagulants, cases presented with lower aggregation after ADP and collagen vs controls. Lower platelet-dependent TG, higher CD63 on resting and lower PAC-1 expression after collagen/ADP in-vitro stimulated platelets further characterized VTE cases vs controls, independent of therapy. Lasso regression analysis identified 26 variables associated with VTE of which 69% were platelet-related. INTERPRETATION: Comprehensive phenotyping of platelet function identified a large proportion of low responders to ASA in VTE cases. Lower platelet-dependent TG and lower platelet reactivity after ex-vivo stimulation characterized the "platelet exhausted syndrome" in cases. Finally, from a large array of covariates including clinical risk factors, platelet biomarkers comprised 69% of all selected variables differentiating VTE cases vs controls. FUNDING: German Federal Ministry of Education and Research, CTH-Mainz and Bayer AG.

Relation between platelet coagulant and vascular function, sex-specific analysis in adult survivors of childhood cancer compared to a population-based sample.

Female sex is a risk factor for long-term adverse outcome in cancer survivors, however very little is known for the underlying pathophysiological mechanisms rendering the increased risk. This study investigated sex-specifically the relation between thrombin generation (TG) with and without presence of platelets and vascular function in 200 adult survivors of a childhood cancer compared to 335 population-based control individuals. TG lag time, peak height and endogenous thrombin potential (ETP) measured in presence and absence of platelets were correlated to reflection index (RI) and stiffness index (SI). A sex-specific correlation analysis showed a negative relation in female survivors for platelet-dependent peak height and/or ETP and RI only. An age adjusted linear regression model confirmed the negative association between RI and platelet-dependent ETP (beta estimate: -6.85, 95% confidence interval: -12.19,-1.51) in females. Adjustment for cardiovascular risk factors resulted in loss of the association, whereby arterial hypertension and obesity showed the largest effects on the observed association. No other relevant associations were found in male and female cancer survivors and all population-based controls. This study demonstrates a link between platelet coagulant and vascular function of resistance vessels, found in female cancer survivors, potentially mediated by the presence of arterial hypertension and obesity.

Association of bruxism and anxiety symptoms among military firefighters with frequent episodic tension type headache and temporomandibular disorders.

OBJECTIVE: To assess the presence of bruxism and anxiety among military firefighters with frequent episodic tension-type headache and painful temporomandibular disorders (TMDs). METHODS: The sample consisted of 162 individuals aged 18 to 55 years divided into four groups. Headache was diagnosed in accordance with the International Classification of Headache Disorders-III. The Research Diagnostic Criteria for Temporomandibular Disorders questionnaire was used to classify TMDs and awake bruxism; sleep bruxism was diagnosed in accordance with the International Classification of Sleep Disorders-3; and anxiety was classified using the Beck Anxiety Inventory. In statistical models, a significance level of 95% was used. The chi-square test was used to assess anxiety. RESULTS: Associations were found among frequent episodic tension-type headache, painful TMDs, awake bruxism and anxiety (p < 0.0005). Sleep bruxism was not a risk factor (p = 0.119) except when associated with awake bruxism (p = 0.011). CONCLUSION: Anxiety and awake bruxism were independent risk factors for developing frequent episodic tension-type headache associated with painful TMDs; only awake bruxism was a risk factor for frequent episodic tension-type headache with non-painful TMDs.

Association of bruxism and anxiety symptoms among military firefighters with frequent episodic tension type headache and temporomandibular disorders / Associação de bruxismo e sintomas de ansiedade em bombeiros militares com cefaleia do tipo tensional episódica frequente e desordens temporomandibulares

ABSTRACT Objective To assess the presence of bruxism and anxiety among military firefighters with frequent episodic tension-type headache and painful temporomandibular disorders (TMDs). Methods The sample consisted of 162 individuals aged 18 to 55 years divided into four groups. Headache was diagnosed in accordance with the International Classification of Headache Disorders-III. The Research Diagnostic Criteria for Temporomandibular Disorders questionnaire was used to classify TMDs and awake bruxism; sleep bruxism was diagnosed in accordance with the International Classification of Sleep Disorders-3; and anxiety was classified using the Beck Anxiety Inventory. In statistical models, a significance level of 95% was used. The chi-square test was used to assess anxiety. Results Associations were found among frequent episodic tension-type headache, painful TMDs, awake bruxism and anxiety (p < 0.0005). Sleep bruxism was not a risk factor (p = 0.119) except when associated with awake bruxism (p = 0.011). Conclusion Anxiety and awake bruxism were independent risk factors for developing frequent episodic tension-type headache associated with painful TMDs; only awake bruxism was a risk factor for frequent episodic tension-type headache with non-painful TMDs.

RESUMO Objetivo Avaliar a presença de bruxismo e sintomas de ansiedade entre bombeiros militares com cefaleia do tipo tensional episódica frequente (CTTEF) e desordens temporomandibulares Dolorosas (DTMs). Métodos A amostra foi composta por 162 indivíduos com idade entre 18 e 55 anos divididos em quatro grupos. A CTTEF foi diagnosticada de acordo com o ICHD-III. O RDC / TMD foi usado para classificar as DTMs e o bruxismo acordado; o bruxismo do sono foi diagnosticado de acordo com o ICSD-3; e a ansiedade foi classificada usando o Inventário de Ansiedade de Beck. Nos modelos estatísticos, utilizou-se um nível de significância de 95%. O teste Qui-quadrado avaliou a ansiedade. Resultados Associações foram encontradas entre CTTEF, DTMs dolorosas, bruxismo diurno e ansiedade (p < 0,0005). O bruxismo do sono não foi fator de risco (p = 0,119), mas quando associado à atividade diurna (p = 0,011). Conclusão Ansiedade e bruxismo diurno foram fatores de risco independentes para o desenvolvimento de CTTEF associado a DTMs dolorosas. Apenas o bruxismo diurno foi fator de risco para CTTEF com DTMs não dolorosas.

Relevance of Polypharmacy for Clinical Outcome in Patients Receiving Vitamin K Antagonists.

BACKGROUND: Although polypharmacy is associated with a negative clinical outcome in various settings and commonly observed in patients receiving oral anticoagulation therapy, evidence on the relevance for the clinical outcome of anticoagulated patients is currently limited. The aim of the study was to investigate the effect of polypharmacy on the clinical outcomes among patients taking phenprocoumon. DESIGN: Prospective cohort study. SETTING: Regular medical care. PARTICIPANTS: Information on 2011 individuals receiving vitamin K antagonists was available for analysis from the prospective multicenter thrombEVAL study. MEASUREMENTS: Data were obtained from clinical visits, computer-assisted interviews, and laboratory measurements. Information on clinical outcome was obtained during a 3-year follow-up period and subsequently validated via medical records. RESULTS: The prevalence of polypharmacy (five drugs or more) was 84.1% (n = 1691). Quality of anticoagulation therapy assessed by time in therapeutic range was lower in individuals on five to eight drugs and nine drugs or more (70.7% and 64.7%, respectively) compared with subjects without polypharmacy (73.4%). In addition, a significantly higher variability of international normalized ratio measurements was found in the presence of polypharmacy. The cumulative incidence of bleeding, hospitalization, and all-cause mortality, but not for thromboembolic events, increased across groups of medication. In adjusted Cox regression analysis, polypharmacy is an independent risk factor for bleeding (hazard ratio [HR]≥ 9 drugs vs 1-4 drugs = 1.62; 95% confidence interval [CI] = 1.04-2.52; p = .033); hospitalization (HR≥ 9 drugs vs 1-4 drugs = 1.60; 95% CI = 1.26-2.03; p < .001; and all-cause mortality (HR≥ 9 drugs vs 1-4 drugs = 2.16; 95% CI = 1.43-3.27; p < .001) in a dose-dependent relationship. Per additional drug, bleeding risk was increased by 4%. CONCLUSIONS: Polypharmacy influences the quality of anticoagulation therapy and translates into an elevated risk of adverse events in anticoagulated patients. This suggests that additional medication intake in such patients should be critically reviewed by physicians, and it highlights the importance of initiating investigations aimed at reducing multiple medication intake. J Am Geriatr Soc 67:463-470, 2019.

Painful temporomandibular disorder, sleep bruxism, anxiety symptoms and subjective sleep quality among military firefighters with frequent episodic tension-type headache. A controlled study.

OBJECTIVE: To investigate associations of temporomandibular disorders (TMDs), bruxism, anxiety and sleep quality among military firefighters with frequent episodic tension-type headache (FETTH). METHODS: The sample comprised two groups (80 individuals): controls (mean age 35.2 years) and study group (mean age 38.5 years). Headache was diagnosed in accordance with the ICHD-III. The Research Diagnostic Criteria for TMDs were used to classify the TMDs; bruxism was diagnosed in accordance with the International Classification of Sleep Disorders; anxiety was classified using the Beck Anxiety Inventory; and sleep quality was assessed using the Pittsburgh Sleep Quality Index. In the statistical models, we used a significance level of 95%. RESULTS: Associations were found between participants with FETTH and TMDs (p < 0.001) and anxiety (p = 0.002).Poor quality of sleep (p = 0.687) and bruxism (p = 0.670) were not risk factors. CONCLUSION: The study found that TMDs and anxiety among firefighters were associated with FETTH.

Painful temporomandibular disorder, sleep bruxism, anxiety symptoms and subjective sleep quality among military firefighters with frequent episodic tension-type headache. A controlled study / Desordens temporomandibulares dolorosas, bruxismo do sono, sintomas de ansiedade e qualidade subjetiva do sono em bombeiros militares com cefaleia do tipo tensional episódica frequente. Estudo controlado

ABSTRACT Objective To investigate associations of temporomandibular disorders (TMDs), bruxism, anxiety and sleep quality among military firefighters with frequent episodic tension-type headache (FETTH). Methods The sample comprised two groups (80 individuals): controls (mean age 35.2 years) and study group (mean age 38.5 years). Headache was diagnosed in accordance with the ICHD-III. The Research Diagnostic Criteria for TMDs were used to classify the TMDs; bruxism was diagnosed in accordance with the International Classification of Sleep Disorders; anxiety was classified using the Beck Anxiety Inventory; and sleep quality was assessed using the Pittsburgh Sleep Quality Index. In the statistical models, we used a significance level of 95%. Results Associations were found between participants with FETTH and TMDs (p < 0.001) and anxiety (p = 0.002).Poor quality of sleep (p = 0.687) and bruxism (p = 0.670) were not risk factors. Conclusion The study found that TMDs and anxiety among firefighters were associated with FETTH.

RESUMO Objetivo Investigar associações de distúrbios temporomandibulares (DTM), bruxismo, ansiedade e qualidade subjetiva do sono entre bombeiros militares com cefaleia do tipo tensional episódica frequente (CTTEF). Método A amostra consistiu em dois grupos com 80 indivíduos cada: controles (idade média 35,2 anos) e grupo de estudo (idade média 38,5 anos). A dor de cabeça foi diagnosticada de acordo com a Classificação Internacional de Cefaleias, 3a edição. Os critérios de diagnóstico de pesquisa para DTM (RDC / TMD) foram utilizados para classificar DTM; o bruxismo foi diagnosticado de acordo com a Classificação Internacional de Distúrbios do Sono; a ansiedade foi classificada usando o Inventário de Ansiedade Beck; e a qualidade do sono foi avaliada usando o Índice de Qualidade do Sono de Pittsburgh (PSQI). Nos modelos estatísticos utilizamos um nível de significância de 95%. Resultados Foram encontradas associações entre indivíduos com CTTEF e presença de DTM (p <0,001) e ansiedade (p = 0,002). A baixa qualidade do sono (p = 0,687) e o bruxismo (p = 0,670) não foram fatores de risco para CTTEF. Conclusão O estudo verificou que DTM e ansiedade entre os bombeiros estavam associados a CTTEF, mas a qualidade subjetiva do sono e o bruxismo não foram fatores de risco.

Stable isotope probing of rRNA and DNA reveals a dynamic methylotroph community and trophic interactions with fungi and protozoa in oxic rice field soil.

Stable isotope probing (SIP) is a novel technique to characterize structure and in situ function of active microbial populations, which is based on the incorporation of 13C-labelled substrates into nucleic acids. Here, we have traced methylotrophic members of a rice field soil microbial community, which became active upon continuous addition of 13C-methanol (< 22 mM) as studied in microcosms. By combining rRNA- and DNA-based SIP, as well as domain-specific real-time PCR detection of templates in fractions of centrifugation gradients, we were able to detect 13C-labelled bacterial rRNA after 6 days of incubation. Fingerprinting and comparative sequence analysis of 'heavy' bacterial rRNA showed that mostly members of the Methylobacteriaceae and a novel clade within the Methylophilaceae formed part of the indigenous methylotrophic community. Over time, however, the Methylophilaceae were enriched. Unexpectedly, nucleic acids of eukaryotic origin were detected, mostly in intermediately 13C-labelled gradient fractions. These eukaryotes were identified as fungi mostly related to Fusarium and Aspergillus spp., and also Cercozoa, known as predatory soil flagellates. The detection of fungi and protozoa in 13C-enriched nucleic acid fractions suggests a possible involvement in either direct assimilation of label by the fungi, or a food web, i.e. that primary 13C-methanol consuming methylotrophs were decomposed by fungi and grazed by protozoa.

Molecular profiling of 16S rRNA genes reveals diet-related differences of microbial communities in soil, gut, and casts of Lumbricus terrestris L. (Oligochaeta: Lumbricidae).

Earthworms are important members of the soil macrofauna. They modify soil physical properties, soil organic matter decomposition, and thus regulate carbon and nitrogen cycling in soil. However, their interactions with soil microorganisms are still poorly understood, in particular the effect of gut passage on the community structure of ingested microorganisms. Moreover, it is still unsolved, if earthworms, like many other soil-feeding invertebrates, possess an indigenous gut microbial community. Therefore, we investigated the bacterial and archaeal community structure in soil (with and without additional beech litter), gut, and fresh casts of Lumbricus terrestris, an anecic litter-feeding earthworm, by means of terminal-restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene fragments. Ecological indices of community diversity and similarity, calculated from the T-RFLP profiles, revealed only small differences between the bacterial and archaeal communities in soil, gut, and fresh casts under both feeding conditions, especially in comparison to other soil-feeding invertebrates. However, multivariate statistical analysis combining multidimensional scaling and discriminant function analysis proved that these differences were highly significant, in particular when the earthworms were fed beech litter in addition. Because there were no dominant gut-specific OTUs detectable, the existence of an abundant indigenous earthworm microbial community appears unlikely, at least in the midgut region of L. terrestris.

Microbial community structure in midgut and hindgut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae).

The guts of soil-feeding macroinvertebrates contain a complex microbial community that is involved in the transformation of ingested soil organic matter. In a companion paper (T. Lemke, U. Stingl, M. Egert, M. W. Friedrich, and A. Brune, Appl. Environ. Microbiol. 69:6650-6658, 2003), we show that the gut of our model organism, the humivorous larva of the cetoniid beetle Pachnoda ephippiata, is characterized by strong midgut alkalinity, high concentrations of microbial fermentation products, and the presence of a diverse, yet unstudied microbial community. Here, we report on the community structure of bacteria and archaea in the midgut, hindgut, and food soil of P. ephippiata larvae, determined with cultivation-independent techniques. Clone libraries and terminal restriction fragment length polymorphism analysis of 16S rRNA genes revealed that the intestines of P. ephippiata larvae contain a complex gut microbiota that differs markedly between midgut and hindgut and that is clearly distinct from the microbiota in the food soil. The bacterial community is dominated by phylogenetic groups with a fermentative metabolism (Lactobacillales, Clostridiales, Bacillales, and Cytophaga-Flavobacterium-Bacteroides [CFB] phylum), which is corroborated by high lactate and acetate concentrations in the midgut and hindgut and by the large numbers of lactogenic and acetogenic bacteria in both gut compartments reported in the companion paper. Based on 16S rRNA gene frequencies, Actinobacteria dominate the alkaline midgut, while the hindgut is dominated by members of the CFB phylum. The archaeal community, however, is less diverse. 16S rRNA genes affiliated with mesophilic Crenarchaeota, probably stemming from the ingested soil, were most frequent in the midgut, whereas Methanobacteriaceae-related 16S rRNA genes were most frequent in the hindgut. These findings agree with the reported restriction of methanogenesis to the hindgut of Pachnoda larvae.

Phylogenetic diversity, abundance, and axial distribution of bacteria in the intestinal tract of two soil-feeding termites (Cubitermes spp.).

The hindgut of soil-feeding termites is highly compartmentalized and characterized by pronounced axial dynamics of the intestinal pH and microbial processes such as hydrogen production, methanogenesis, and reductive acetogenesis. Nothing is known about the bacterial diversity and the abundance or axial distribution of the major phylogenetic groups in the different gut compartments. In this study, we showed that the variety of physicochemical conditions is reflected in the diversity of the microbial communities in the different gut compartments of two Cubitermes species (TERMITIDAE: Termitinae). 16S rRNA gene clones from the highly alkaline first proctodeal segment (P1) of Cubitermes orthognathus represented almost exclusively gram-positive bacteria with low G+C content (LGC bacteria). In the posterior gut segments, their proportion decreased progressively, and the clone libraries comprised a variety of phyla, including the Cytophaga-Flexibacter-Bacteroides group, various subgroups of Proteobacteria, and the spirochetes. Phylogenetic analysis revealed that many of the clones clustered with sequences from the guts of other termites, and some even formed clusters containing only clones from C. orthognathus. The abundance and axial distribution of major phylogenetic groups in the gut of Cubitermes ugandensis were determined by fluorescence in situ hybridization with group-specific oligonucleotide probes. While the results were generally in good agreement with those of the clonal analysis, direct counts with probes specific for the Planctomycetales revealed a severe underestimation of representatives of this phylum in the clone libraries. Results obtained with newly designed FISH probes directed against two clusters of LGC clones from C. orthognathus indicated that the clones were restricted to specific gut regions. A molecular fingerprinting analysis published in a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6018-6024, 2003) corroborated the presence of compartment-specific bacterial communities in the gut of different Cubitermes species.

Axial dynamics, stability, and interspecies similarity of bacterial community structure in the highly compartmentalized gut of soil-feeding termites (Cubitermes spp.).

The highly compartmentalized gut of soil-feeding termites is characterized by pronounced axial dynamics in physicochemical conditions and microbial processes. In a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6007-6017, 2003), we demonstrated that the variety of physicochemical conditions in the different gut compartments of Cubitermes spp. is reflected in the diversity of the respective intestinal microbial communities. Here, we used molecular fingerprints of 16S rRNA genes of the bacterial community, obtained by terminal restriction fragment length polymorphism (T-RFLP) analysis, to describe the axial dynamics of the bacterial community structure in the different gut sections. Comparison of the T-RFLP profiles with the predicted terminal restriction fragments of the clones in clone libraries of the gut segments in Cubitermes orthognathus confirmed that all hindgut sections harbored distinct bacterial communities. Morisita indices of community similarity, calculated by comparing the different patterns, revealed large differences between the bacterial communities of soil, gut, and nest material and also among the individual gut sections. By contrast, comparison of the homologous gut segments of different Cubitermes species indicated that the three termite species investigated possessed a similar, gut-specific microbiota that remained comparatively stable even during several months of maintenance in the laboratory.

Isolation and characterization of new strains of methanogens from cold terrestrial habitats.

Five strains of methanogenic archaea (MT, MS, MM, MSP, ZB) were isolated from permanently and periodically cold terrestrial habitats. Physiological and morphological studies, as well as phylogenetic analyses of the new isolates were performed. Based on sequences of the 16S rRNA and methyl-coenzyme M reductase a-subunit (mcrA) genes all new isolates are closely related to known mesophilic and psychrotolerant methanogens. Both, phylogenetic analyses and phenotypic properties allow to classify strains MT, MS, and MM as members of the genus Methanosarcina. Strain MT is a new ecotype of Methanosarcina mazei, whereas strains MM and MS are very similar to each other and can be assigned to the recently described psychrotolerant species Methanosarcina lacustris. The hydrogenotrophic strain MSP is a new ecotype of the genus Methanocorpusculum. The obligately methylotrophic strain ZB is closely related to Methanomethylovorans hollandica and can be classified as new ecotype of this species. All new isolates, including the strains from permanently cold environments, are not true psychrophiles according to their growth temperature characteristics. In spite of the ability of all isolates to grow at temperatures as low as 1-5 degrees C, all of them have their growth optima in the range of moderate temperatures (25-35 degrees C). Thus, they can be regarded as psychrotolerant organisms. Psychrotolerant methanogens are thought to play an important role in methane production in both, habitats under seasonal temperature variations or from permanently cold areas.