Clozapine Efficacy and Adverse Drug Reactions Among a Nationwide Study of 1021 Australians Prescribed Clozapine: The ClozaGene Study.

BACKGROUND AND HYPOTHESIS: The ClozaGene Study is a nationwide cohort of adults who have been treated with clozapine. While clozapine is indicated in the management of treatment-resistant schizophrenia, it is associated with a considerable adverse drug reaction (ADR) burden, and not all patients achieve adequate symptomatic response. The current study focuses on self-reported experiences of clozapine use and response, clozapine-associated ADRs, and mental health comorbidity. STUDY DESIGN: A total of 1021 participants (41.0% female; aged 46.2 ±â€…10.6 years [range 18-66]) were recruited via a mail-out based on prescriptions for clozapine. Participants completed a self-report questionnaire. STUDY RESULTS: Most participants (90.1%, n = 912) were living with schizophrenia while 41.5% reported a lifetime diagnosis of depression, 15.6% bipolar disorder, and 8.1% schizoaffective disorder. Clozapine was currently prescribed to 944 (92.5%) participants and 37.8% of these participants self-reported currently taking additional antipsychotic medication. Nearly 3 quarters of participants living with schizophrenia reported that clozapine helped control their schizophrenia symptoms moderately to very well. The most commonly reported ADRs were sialorrhea (80.3%), weight gain (71.0%), constipation (56.9%), and sedation (52.8%). The prevalence of clozapine cessation due to clozapine-induced myocarditis and neutropenia was 1% and 0.4%, respectively. CONCLUSIONS: Our findings highlight the high rate of psychotic and metabolic symptoms and ADRs among adults prescribed clozapine in the general Australian population. Future genomic analyses will focus on identifying genetic variants influencing clozapine treatment response and side effects.

Sex Differences Between Female and Male Individuals in Antipsychotic Efficacy and Adverse Effects in the Treatment of Schizophrenia.

BACKGROUND AND OBJECTIVE: Antipsychotics are core treatments for people living with psychotic disorders. Understanding individualised factors that influence both efficacy and adverse responses will improve outcomes. The objective of this study was to examine sex differences in antipsychotic-related efficacy and tolerability. METHODS: This was a secondary analysis of data from phase 1 and 1a of Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE); participants with schizophrenia were randomly assigned to double-blinded treatment with oral olanzapine, quetiapine, risperidone, ziprasidone or perphenazine. Measures included Positive and Negative Syndrome Scale (PANSS), Clinical Global Impressions (CGI) scale and Calgary Depression Rating Scale, as well as self-reported side effects, medication compliance, dosage, weight measurements and various blood parameters. RESULTS: There were 1460 participants including 380 female and 1080 male individuals. Very few differences existed between male and female participants in response, adverse reactions, compliance or antipsychotic dosage. However, significantly more female participants than male participants reported constipation (28% vs 16%), dry mouth (50% vs 38%), gynecomastia/galactorrhea (11% vs 3%), incontinence/nocturia (16% vs 8%) and self reported weight gain (37% vs 24%) [all p < 0.001]. Within the risperidone treatment group, there was a significantly greater increase in prolactin levels (p < 0.001) among female participants (n = 61) than male participants (n = 159). No overall differences in clinician-rated measures, weight gain or other laboratory indicators were found. CONCLUSIONS: While overall sex differences were limited across efficacy and tolerability for antipsychotic treatment, there were some specific findings with risperidone. Further examination of sex differences within antipsychotic trials will be important to improve efficacy and reduce adverse responses across as well as individualising care for people with schizophrenia.

Evaluating the epidemiology of clozapine-associated neutropenia among people on clozapine across Australia and Aotearoa New Zealand: a retrospective cohort study.

BACKGROUND: Clozapine is associated with the risk of serious neutropenia. However, this risk might decrease over time, meaning that indefinite absolute neutrophil count (ANC) monitoring could be unnecessary. We aimed to determine the epidemiology and timing of clozapine-associated neutropenia outcomes, to investigate variables that might contribute to the odds of neutropenia, and to determine risk of competing neutropenic events during clozapine treatment. METHODS: We performed a retrospective analysis of the Australian and New Zealand Viatris Pharmacovigilance system (one of two monitoring databases for these two countries) between June 6, 1990, and Oct 25, 2022. Patients were excluded from analysis if they commenced clozapine before 1990, did not have a haematology test within 2 weeks of commencement date, or had no follow-up. We measured minor neutropenia (ANC 1·0-1·5 × 109 per L) and serious neutropenia (ANC <1·0 × 109 per L) leading to cessation of clozapine within 6 weeks of the neutropenic event. We determined the rates of minor and serious neutropenia and calculated odds ratios (ORs) for the likelihood of neutropenia leading to cessation. For serious neutropenia leading to cessation, we used time-to-event to calculate rolling weekly averages and to perform competing risk analysis of outcomes using Cox proportional hazards models and a Fine-Gray subdistribution hazards regression model. For the subset of data where information on previous clozapine use was available, we did an analysis for participants who did and did not have previous clozapine exposure. FINDINGS: We included 26 630 people, with 2·6 million ANC values. Within the total cohort, 17 585 people (66%) were male, 9025 (33·9%) female, and 20 (0·1%) other gender, and the mean age was 36·1 years (SD 13·7). We did not have data on race or ethnicity. Of the 26 630 people taking clozapine, 1146 (4·3%) had minor neutropenia, 313 (1·2%) had serious neutropenia leading to cessation, and 223 (0·8%) had serious neutropenia unrelated to clozapine without cessation. In people with no previous exposure to clozapine (n=15 973), the cumulative incidence of serious neutropenia leading to cessation was 0·9% at 18 weeks and 1·4% at 2 years; the weekly incidence rate for serious neutropenia leading to cessation peaked at 9 weeks (0·128%) and fell to a rolling average weekly incidence of 0·001% by 2 years. For minor neutropenia, the cumulative incidence was 1·7% at 18 weeks and 3·5% at 2 years; the weekly incidence rate peaked at 9 weeks (0·218%) and fell to a stable rolling average of 0·01%. The median time to a serious neutropenic event leading to cessation was 17 weeks (IQR 9·96-102). Previous clozapine exposure reduced the risk of serious neutropenia leading to cessation (OR 0·19, 95% CI 0·12-0·31; p <0·0001). INTERPRETATION: Most serious neutropenia leading to clozapine cessation occurs within 18 weeks of treatment and becomes negligible after 2 years. Weekly haematological monitoring after the first 18 weeks could be safely reduced to once every 4 weeks and ceased after 2 years unless clinically indicated. Clozapine retrial after interruption with 2 cumulative years of unremarkable testing might not require further haematological monitoring. A serious neutropenia ANC threshold of ≤1·0 × 109 per L could be used in more jurisdictions. FUNDING: None.

Prevalence of frailty in severe mental illness: findings from the UK Biobank.

BACKGROUND: Severe mental illness (SMI) is associated with significant morbidity. Frailty combines biological ageing, comorbidity and psychosocial factors and can predict adverse health outcomes. Emerging evidence indicates that frailty is higher in individuals with SMI than in the general population, although studies have been limited by sample size. AIMS: To describe the prevalence of frailty in people with SMI in a large cohort using three different frailty measures and examine the impact of demographic and sociodemographic variables. METHOD: The UK Biobank survey data, which included individuals aged 37-73 years from England, Scotland and Wales from 2006 to 2010, with linked in-patient hospital episodes, were utilised. The prevalence of frailty in individuals with and without SMI was assessed through three frailty measures: frailty index, physical frailty phenotype (PFP) and Hospital Frailty Risk Score (HFRS). Stratified analysis and dichotomous logistic regression were conducted. RESULTS: A frailty index could be calculated for 99.5% of the 502 412 UK Biobank participants and demonstrated greater prevalence of frailty in women and an increase with age. The prevalence of frailty for those with SMI was 3.19% (95% CI 3.0-3.4), 4.2% (95% CI 3.8-4.7) and 18% (95% CI 15-23) using the frailty index, PFP and HFRS respectively. The prevalence ratio was between 3 and 18 times higher than in those without SMI. CONCLUSIONS: As a measure, frailty captures the known increase in morbidity associated with SMI and may potentially allow for earlier identification of those who will benefit from targeted interventions.

A guide to support trainee's success in the psychotherapy written case submission.

OBJECTIVE: Many trainees find the Psychotherapy Written Case (PWC) requirement of the Royal Australian and New Zealand College of Psychiatrists training program challenging. The skills developed and assessed through this experience are critical to the competencies expected of a psychiatrist. However, the process of psychodynamic psychotherapy is often dramatically different from the expectations associated with early clinical placements in acute psychiatric settings. To support trainees in achieving success in the PWC, a guide to the written report was developed based on a review of existing resources and various stakeholder perspectives. CONCLUSIONS: The submission should reflect a training case rather than an idealised or fictionalised story attempting to demonstrate the therapist's competence. The PWC submission must meet the requirements of a general psychiatric report and provide a considered reflection on the experience of the novice therapist.

A 40-year study of child maltreatment over the early life course predicting psychiatric morbidity, using linked birth cohort and administrative health data: protocol for the Childhood Adversity and Lifetime Morbidity (CALM) study.

BACKGROUND: Child maltreatment is a major public health issue worldwide. Retrospective studies show a strong association between self-reported child maltreatment and poor mental and physical health problems. Prospective studies that use reports to statutory agencies are less common, and comparisons of self- and agency-reported abuse in the same cohort even rarer. AIMS: This project will link state-wide administrative health data with prospective birth cohort data (N = 7223) from Brisbane in Queensland, Australia (including notifications to child protection agencies), to compare psychiatric outcomes in adulthood of agency- and self-reported child maltreatment while minimising attrition bias. METHOD: We will compare people with all forms of self- and agency-reported child maltreatment to the rest of the cohort, adjusting for confounding in logistic, Cox or multiple regression models based on whether outcomes are categorical or continuous. Outcomes will be hospital admissions, emergency department presentations or community/out-patient contacts for ICD-10 psychiatric diagnoses, suicidal ideation and self-harm as recorded in the relevant administrative databases. CONCLUSIONS: This study will track the life course outcomes of adults after having experienced child maltreatment, and so provide an evidence-based understanding of the long-term health and behavioural consequences of child maltreatment. It will also consider health outcomes that are particularly relevant for adolescents and young adults, especially in relation to prospective notifications to statutory agencies. Additionally, it will identify the overlap and differences in outcome for two different sources of child maltreatment identification in the same cohort.

Optimising plasma clozapine levels to improve treatment response: an individual patient data meta-analysis and receiver operating characteristic curve analysis.

BACKGROUND: Although clozapine is the most efficacious medication for treatment-refractory schizophrenia, not all patients will have an adequate response. Optimising clozapine dose using therapeutic drug monitoring could therefore maximise response. AIMS: Using individual patient data, we undertook a receiver operating characteristic (ROC) curve analysis to determine an optimal therapeutic range for clozapine levels to guide clinical practice. METHOD: We conducted a systematic review of PubMed, PsycINFO and Embase for studies that provided individual participant level data on clozapine levels and response. These data were analysed using ROC curves to determine the prediction performance of plasma clozapine levels for treatment response. RESULTS: We included data on 294 individual participants from nine studies. ROC analysis yielded an area under the curve of 0.612. The clozapine level at the point of optimal diagnostic benefit was 372 ng/mL; at this level, the response sensitivity was 57.3%, and specificity 65.7%. The interquartile range for treatment response was 223-558 ng/mL. There was no improvement in ROC performance with mixed models including patient gender, age or length of trial. Clozapine dose and clozapine concentration to dose ratio did not provide significantly meaningful prediction of response to clozapine. CONCLUSIONS: Clozapine dose should be optimised based on clozapine therapeutic levels. We found that a range between 250 and 550 ng/mL could be recommended, while noting that a level of >350 ng/mL is the most optimal for response. Although some patients may not respond without clozapine levels >550 ng/mL, the benefits should be weighed against the increased risk of adverse drug reactions.

Renewal of academic psychiatry without addressing gender equity will render it Jurassic rather than endangered.

While two editorials have raised concerns about the decline in Australian academic psychiatry, for a genuine rejuvenation to ever occur, we will need to re-examine how women can be better included in this important endeavour. While attainment of fellowship has reached gender parity, academic psychiatry has disappointingly lagged, with 80% of its senior leadership roles across Australia and New Zealand still held by men, with a similar situation in the United Kingdom and the United States as well as many other countries. Encouraging women into academic psychiatry is not only critical to progress as a profession but also will help address the current blindness to sex differences in biological psychiatry, as well the social impact of restrictive gender norms and the effects of gender-based violence on mental health. This potentially creates opportunities for significant gains and insights into mental disorders. However, addressing the barriers for women in academia requires tackling the entrenched disparities across salaries, grant funding, publications, teaching responsibilities, keynote invitations and academic promotions alongside the gender-based microaggressions, harassment and tokenism reported by many of our female academics. Many women must grapple with not just a 'second shift' but a 'third shift', making the burden of an academic career unreasonable and burnout more likely. Addressing this is no easy task. The varied research in academic medicine reveals no quick fixes, although promoting gender equity brings significant potential benefits. Areas such as academic psychiatry need to recognise our community's growing discomfort with workplaces that choose to maintain status quo. Gender equity must be a critical part of any quest to revive this important area of practice for our profession.

A Randomized Control Trial of Cognitive Compensatory Training (CCT) and Computerized Interactive Remediation of Cognition-Training for Schizophrenia (CIRCuiTS).

Background: Various modes of delivering cognitive remediation (CR) are effective, but there have been few head-to-head trials of different approaches. This trial aimed to evaluate the relative effectiveness of two different programmes, Cognitive Compensatory Training (CCT) and Computerized Interactive Remediation of Cognition-Training for Schizophrenia (CIRCuiTs). Methods: The study used a single-blind randomized, controlled trial to examine the efficacy and effectiveness of the two therapies. The study aimed to enroll 100 clinically stable patients between the ages of 18 and 65 years who had been diagnosed with a schizophrenia spectrum disorder. Participants were randomized to either the CCT or CIRCuiTs therapy groups. The primary outcome measures were neurocognition using the Brief Assessment of Cognition Scale (BACS) and the Subjective Scale to Investigate Cognition in Schizophrenia (SSTICS). The secondary measure was functional outcomes using the Social Functioning Scale (SFS). Results: There was no group difference in any of the outcome measures post-intervention or at follow-up. Both groups had a small improvement on their SSTICS scores between baseline (M = 30.52 and SD = 14.61) and post-intervention (M = 23.96 and SD = 10.92). Verbal memory scores as measured by list learning improved for both groups between baseline (z = -1.62) and 3-month follow-up (z = -1.03). Both groups improved on the token motor task between baseline (z = -1.38) and post-intervention (z = -0.69). Both groups had a decline in Symbol Coding scores between baseline (z = 0.05) and 3-month follow-up (z = -0.82). Discussion: This underpowered study found no difference in effect between the two approaches studied. If future studies confirm this finding, then it has implications for services where cost and lack of computer technology could pose a barrier in addressing the cognitive domain of schizophrenia spectrum disorders. The final sample size compromised the power of the study to conclusively determine a significant effect.

Epigenome erosion and SOX10 drive neural crest phenotypic mimicry in triple-negative breast cancer.

Intratumoral heterogeneity is caused by genomic instability and phenotypic plasticity, but how these features co-evolve remains unclear. SOX10 is a neural crest stem cell (NCSC) specifier and candidate mediator of phenotypic plasticity in cancer. We investigated its relevance in breast cancer by immunophenotyping 21 normal breast and 1860 tumour samples. Nuclear SOX10 was detected in normal mammary luminal progenitor cells, the histogenic origin of most TNBCs. In tumours, nuclear SOX10 was almost exclusive to TNBC, and predicted poorer outcome amongst cross-sectional (p = 0.0015, hazard ratio 2.02, n = 224) and metaplastic (p = 0.04, n = 66) cases. To understand SOX10's influence over the transcriptome during the transition from normal to malignant states, we performed a systems-level analysis of co-expression data, de-noising the networks with an eigen-decomposition method. This identified a core module in SOX10's normal mammary epithelial network that becomes rewired to NCSC genes in TNBC. Crucially, this reprogramming was proportional to genome-wide promoter methylation loss, particularly at lineage-specifying CpG-island shores. We propose that the progressive, genome-wide methylation loss in TNBC simulates more primitive epigenome architecture, making cells vulnerable to SOX10-driven reprogramming. This study demonstrates potential utility for SOX10 as a prognostic biomarker in TNBC and provides new insights about developmental phenotypic mimicry-a major contributor to intratumoral heterogeneity.

CoMET: a randomised controlled trial of co-commencement of metformin versus placebo as an adjunctive treatment to attenuate weight gain in patients with schizophrenia newly commenced on clozapine.

BACKGROUND: There is limited evidence on interventions to minimise weight gain at clozapine commencement. We compared the effect of adjunctive metformin versus placebo at clozapine initiation. METHODS: People with schizophrenia commencing on clozapine were randomised to either metformin or placebo for 24 weeks. The primary outcome was difference in the change of body weight. Secondary outcomes included comparative rates of weight gain of more than 5%, overall weight gain/loss, and differences in metabolic and psychosis outcomes. RESULTS: The study was closed prematurely in March 2020 due to COVID-19 restrictions. Ten participants were randomised to each of the metformin and placebo groups. Eight metformin group and five placebo group participants completed the trial and were included in the analysis. The study was insufficiently powered to detect difference between the metformin and placebo groups for the primary outcome of change in weight (0.09 kg vs 2.88 kg, p = 0.231). In terms of secondary outcomes, people in the metformin group were significantly less likely to gain >5% of their body weight (12.5% vs 80%, p = 0.015) and were more likely to lose weight (37.5% vs 0% p = 0.024) compared to placebo. There was no difference between the groups in terms of adverse drug reactions (ADRs). CONCLUSION: While limited by the forced premature closure of the trial due to COVID19, the findings from this randomised controlled trial are promising. Clozapine and metformin co-commencement may be a promising treatment to prevent clozapine-associated weight gain, especially given the low rates of ADRs associated with metformin. This supports the consideration of use of metformin to prevent weight gain in people initiated on clozapine; however, further studies are needed to confirm this finding. TRIAL REGISTRATION: ACTRN12617001547336.

Understanding the drivers of bottlenecks in RANZCP training: modelling and a calculator to determining sustainable trainee intake.

OBJECTIVES: Demand for places in postgraduate psychiatry training programmes has increased over recent years. All systems have capacity limits, and concerns have been raised regarding the sustainability of the current intake. This paper presents a modelling exercise to exploring the presence and strategies to resolve bottleneck in the Queensland training programme. METHODS: Mathematical modelling based on the RANZCP training regulations and the characteristics of the accredited training programme. RESULTS: A training bottleneck was identified which has been impacted by increased training intake, demand for Advanced Training certificates, and location factors. CONCLUSIONS: This investigation raises important questions regarding the future management of postgraduate training in psychiatry in Queensland that may be applicable more widely across Australia and New Zealand. In particular, it highlights the large impact that can result from even small incremental increases in trainees across varying levels of the postgraduate programme and the importance of limiting trainee intake in a manner proportional to the availability of mandatory terms.

Activation of the Ion Channel TRPV4 Induces Epithelial to Mesenchymal Transition in Breast Cancer Cells.

Epithelial to mesenchymal transition (EMT) in cancer is important in therapeutic resistance and invasiveness. Calcium signaling is key to the induction of EMT in breast cancer cells. Although inhibition of specific calcium-permeable ion channels regulates the induction of a sub-set of EMT markers in breast cancer cells, it is still unclear if activation of a specific calcium channel can be a driver for the induction of EMT events. In this study, we exploited the availability of a selective pharmacological activator of the calcium-permeable ion channel TRPV4 to assess the direct role of calcium influx in EMT marker induction. Gene association studies revealed a link between TRPV4 and gene-ontologies associated with EMT and poorer relapse-free survival in lymph node-positive basal breast cancers. TRPV4 was an important component of the calcium influx phase induced in MDA-MB-468 breast cancer cells by the EMT inducer epidermal growth factor (EGF). Pharmacological activation of TRPV4 then drove the induction of a variety of EMT markers in breast cancer cells. These studies demonstrate that calcium influx through specific pathways appears to be sufficient to trigger EMT events.

High-dose antipsychotic therapy and reflective prescribing: development of an online tool for rapid, easy calculation of antipsychotic total daily dose.

OBJECTIVE: Safe and effective antipsychotic prescribing is a fundamental skill in psychiatric practice; however, antipsychotic medications are not without risk. These risks are increased when antipsychotics are prescribed in high doses, with or without polypharmacy. Decision-making regarding antipsychotic prescribing can be hampered by a lack of readily available or easily approachable tools for calculating and interpreting total daily doses, especially when antipsychotic polypharmacy is involved. Our objective was to create an accessible method for calculating antipsychotic total daily dosing. METHODS: We have developed an online calculator for determining antipsychotic total daily dose using information on recommended maximum total daily dosing based on the British National Formulary. RESULTS: This calculator is free, easy to implement and allows for users to input a large variety of possible antipsychotic dosing regimens. CONCLUSIONS: It is hoped that this tool will allow clinicians to readily review their prescribing practice, inform decision-making and improve patient safety outcomes. Further research may be appropriate to determine the impact of this tool on these intended goals.

MicroRNA-196a is regulated by ER and is a prognostic biomarker in ER+ breast cancer.

BACKGROUND: MicroRNAs are potent post-transcriptional regulators involved in all hallmarks of cancer. Mir-196a is transcribed from two loci and has been implicated in a wide range of developmental and pathogenic processes, with targets including Hox, Fox, Cdk inhibitors and annexins. Genetic variants and altered expression of MIR196A are associated with risk and progression of multiple cancers including breast cancer, however little is known about the regulation of the genes encoding this miRNA, nor the impact of variants therein. METHODS: Genomic data and chromatin interaction analysis were used to discover functional promoter and enhancer elements for MIR196A. Expression data were used to associate MIR196A with mechanisms of resistance, breast cancer subtypes and prognosis. RESULTS: Here we demonstrate that MIR196A displays complex and dynamic expression patterns, in part controlled by long-range transcriptional regulation between promoter and enhancer elements bound by ERα. Expression of this miRNA is significantly increased in drug-resistant models of hormone-receptor positive disease. The expression of MIR196A also proves to be a robust prognostic factor for patients with advanced and post-menopausal ER+ disease. CONCLUSION: This work sheds light on the normal and abnormal regulation of MIR196A and provides a novel stratification method for therapeutically resistant breast cancer.

Long-range regulators of the lncRNA HOTAIR enhance its prognostic potential in breast cancer.

Predicting response to endocrine therapy and survival in oestrogen receptor positive breast cancer is a significant clinical challenge and novel prognostic biomarkers are needed. Long-range regulators of gene expression are emerging as promising biomarkers and therapeutic targets for human diseases, so we have explored the potential of distal enhancer elements of non-coding RNAs in the prognostication of breast cancer survival. HOTAIR is a long non-coding RNA that is overexpressed, promotes metastasis and is predictive of decreased survival. Here, we describe a long-range transcriptional enhancer of the HOTAIR gene that binds several hormone receptors and associated transcription factors, interacts with the HOTAIR promoter and augments transcription. This enhancer is dependent on Forkhead-Box transcription factors and functionally interacts with a novel alternate HOTAIR promoter. HOTAIR expression is negatively regulated by oestrogen, positively regulated by FOXA1 and FOXM1, and is inversely correlated with oestrogen receptor and directly correlated with FOXM1 in breast tumours. The combination of HOTAIR and FOXM1 enables greater discrimination of endocrine therapy responders and non-responders in patients with oestrogen receptor positive breast cancer. Consistent with this, HOTAIR expression is increased in cell-line models of endocrine resistance. Analysis of breast cancer gene expression data indicates that HOTAIR is co-expressed with FOXA1 and FOXM1 in HER2-enriched tumours, and these factors enhance the prognostic power of HOTAIR in aggressive HER2+ breast tumours. Our study elucidates the transcriptional regulation of HOTAIR, identifies HOTAIR and its regulators as novel biomarkers of patient response to endocrine therapy and corroborates the importance of transcriptional enhancers in cancer.

Molecular diversity of methanogens in fecal samples from captive Sumatran orangutans (Pongo abelii).

Methane emissions have been previously detected from orangutans, but characterization of the diversity of methanogens in this species has yet to be completed. This preliminary study identified methanogen producing microorganims, also called methanogens, present in the feces from a colony of captive Sumatran orangutans at the Perth Zoo. All animals were housed in the same enclosure and were fed primarily a frugivorous diet. Methanogens were detected using a 16S rRNA gene clone library. A total of 207 clones were examined, revealing 37 different methanogen 16S rRNA sequences, or phylotypes. Of these, 31 phylotypes represented by 170 clones had 96.4-100% sequence identity to Methanosphaera stadtmanae, four phylotypes (32 clones) had 95.1-100% sequence identity to Methanobrevibacter smithii, while two phylotypes (five clones) had 95.9-97.7% sequence identity to Methanobacterium beijingense. Overall, five possible new species were identified from the clone library. This represents the first report of Msp. stadtmanae, a methanol utilizer, as the most predominant methanogen in the gastrointestinal tract of animals. This is likely due to the increased availability of methanol from the highly frugivorous diet of the orangutans. Further studies are warranted to properly assess the effects of frugivorous diets on the methanogen population.

Analysis of rumen methanogen diversity in water buffaloes (Bubalus bubalis) under three different diets.

The water buffalo (Bubalus bubalis) is a prominent livestock species for the production of milk and meat in many countries. We investigated the diversity of rumen methanogens in Mediterranean water buffaloes maintained in Brazil under different diets: corn silage, grazing pasture, or sugar cane. A total of 467 clones were isolated from three methanogen 16S rRNA gene clone libraries that each represented a distinct feed type. The 467 clones were assigned to 19 species-level operational taxonomic units (OTUs). Four OTUs were represented in all three libraries, eight OTUs were library-specific, six OTUs were found in only the corn silage and pasture grazing libraries, and one OTU was shared only between pasture grazing and sugar cane libraries. We found that Methanobrevibacter-related sequences were the most abundant in the water buffaloes sampled for our analysis, in contrast to previously reported studies showing that Methanomicrobium mobile-like methanogens were the most abundant methanogens in water buffaloes of Murrah and Surti breeds sampled in India. Considering the worldwide distribution of water buffaloes and the likely wide variety of diets provided, our results combined with studies from other groups support that larger scope analyses of microbiomes for this livestock species would provide great insight into the contribution of geographical location, breed, and diet in determining the population structure of rumen microorganisms.