Mutual Exclusion of Methanobrevibacter Species in the Human Gut Microbiota Facilitates Directed Cultivation of a Candidatus Methanobrevibacter Intestini Representative.

Methanogenic Archaea (methanogens) are a phylogenetically diverse group of microorganisms and are considered to be the most abundant archaeal representatives in the human gut. However, the gut methanogen diversity of human populations in many global regions remains poorly investigated. Here, we report the abundance and diversity of gut methanogenic Archaea in a multi-ethnic cohort of healthy Singaporeans by using a concerted approach of metagenomic sequencing, 16S rRNA gene amplicon sequencing, and quantitative PCR. Our results indicate a mutual exclusion of Methanobrevibacter species, i.e., the highly prevalent Methanobrevibacter smithii and the less prevalent Candidatus Methanobrevibacter intestini in more than 80% of the samples when using an amplicon sequencing-based approach. Leveraging on this finding, we were able to select a fecal sample to isolate a representative strain, TLL-48-HuF1, for Candidatus Methanobrevibacter intestini. The analyzed physiological parameters of M. smithii DSM 861T and strain TLL-48-HuF1 suggest high similarity of the two species. Comparative genome analysis and the mutual exclusion of the Methanobrevibacter species indicate potentially different niche adaptation strategies in the human host, which may support the designation of Candidatus M. intestini as a novel species. IMPORTANCE Methanogens are important hydrogen consumers in the gut and are associated with differing host health. Here, we determine the prevalence and abundance of archaeal species in the guts of a multi-ethnic cohort of healthy Singapore residents. While Methanobrevibacter smithii is the most prevalent and abundant methanogen in the human gut of local subjects, the recently proposed Candidatus Methanobrevibacter intestini is the abundant methanogen in a minority of individuals that harbor them. The observed potential mutual exclusion of M. smithii and Ca. M. intestini provides further support to the proposal that the two physiologically similar strains may belong to different Methanobrevibacter species.

Extended-Spectrum ß-Lactamase-Producing and mcr-1-Positive Escherichia coli from the Gut Microbiota of Healthy Singaporeans.

Multidrug-resistant (MDR) Escherichia coli strains that carry extended-spectrum ß-lactamases (ESBLs) or colistin resistance gene mcr-1 have been identified in the human gut at an increasing incidence worldwide. In this study, we isolated and characterized MDR Enterobacteriaceae from the gut microbiota of healthy Singaporeans and show that the detection rates for ESBL-producing and mcr-positive Enterobacteriaceae are 25.7% (28/109) and 7.3% (8/109), respectively. Whole-genome sequencing analysis of the 37 E. coli isolates assigned them into 25 sequence types and 6 different phylogroups, suggesting that the MDR E. coli gut colonizers are highly diverse. We then analyzed the genetic context of the resistance genes and found that composite transposons played important roles in the cotransfer of blaCTX-M-15/55 and qnrS1, as well as the acquisition of mcr-1. Furthermore, comparative genomic analysis showed that 12 of the 37 MDR E. coli isolates showed high similarity to ESBL-producing E. coli isolates from raw meat products in local markets. By analyzing the core genome single nucleotide polymorphisms (SNPs) shared by these isolates, we identified possible clonal transmission of an MDR E. coli clone between human and raw meat, as well as a group of highly similar IncI2 (Delta) plasmids that might be responsible for the dissemination of mcr-1 in a much wider geographic region. Together, these results suggest that antibiotic resistance may be transmitted between different environmental settings by the expansion of MDR E. coli clones, as well as by the dissemination of resistance plasmids. IMPORTANCE The human gut can harbor both antibiotic-resistant and virulent Escherichia coli which may subsequently cause infections. In this study, we found that multidrug-resistant (MDR) E. coli isolates from the gut of healthy Singaporeans carry a diverse range of antibiotic resistance mechanisms and virulence factor genes and are highly diverse. By comparing their genomes with the extended-spectrum ß-lactamase (ESBL)-producing E. coli isolates from raw meat products that were sampled at a similar time from local markets, we detected an MDR E. coli clone that was possibly transmitted between humans and raw meat products. Furthermore, we also found that a group of resistance plasmids might be responsible for the dissemination of colistin resistance gene mcr-1 in Singapore, Malaysia, and Europe. Our findings call for better countermeasures to block the transmission of antibiotic resistance.

Genome-wide identification of cis DNA methylation quantitative trait loci in three Southeast Asian Populations.

DNA methylation (DNAm) is an epigenetic modification that acts to regulate gene transcription, is essential for cellular processes and plays an important role in complex traits and disease. Variation in DNAm levels is influenced by both genetic and environmental factors. Several studies have examined the extent to which common genetic variation influences DNAm (i.e. mQTLs), however, an improved understanding of mQTLs across diverse human populations is needed to increase their utility in integrative genomic studies in order to further our understanding of complex trait and disease biology. Here, we systematically examine cis-mQTLs in three Southeast Asian populations in the Singapore Integrative Omics (iOmics) Study, comprised of Chinese (n = 93), Indians (n = 83) and Malays (n = 78). A total of 24 851 cis-mQTL probes were associated with at least one SNP in meta- and ethnicity-specific analyses at a stringent significance level. These cis-mQTL probes show significant differences in local SNP heritability between the ethnicities, enrichment in functionally relevant regions using data from the Roadmap Epigenomics Mapping Consortium and are associated with nearby genes and complex traits due to pleiotropy. Importantly, DNAm prediction performance and the replication of cis-mQTLs both within iOmics and between two independent mQTL studies in European and Bangladeshi individuals is best when the genetic distance between the ethnicities is small, with differences in cis-mQTLs likely due to differences in allele frequency and linkage disequilibrium. This study highlights the importance of, and opportunities from, extending investigation of the genetic control of DNAm to Southeast Asian populations.

Emergence of tigecycline- and eravacycline-resistant Tet(X4)-producing Enterobacteriaceae in the gut microbiota of healthy Singaporeans.

OBJECTIVES: The recently discovered tigecycline-inactivating enzyme Tet(X4) can confer high-level tigecycline resistance on its hosts, which makes it a public health concern. This study focused on isolation and screening of Tet(X4)-positive Enterobacteriaceae from the gut microbiota of a cohort of healthy individuals in Singapore. METHODS: MinION and Illumina sequencing was performed to obtain the complete genome sequences of Escherichia coli 2EC1-1 and 94EC. Subsequently, 109 human faecal samples were screened retrospectively for eravacycline-resistant Enterobacteriaceae strains, which were further tested for tet(X4) by PCR. The taxonomy of the isolated strains was determined by 16S rRNA gene PCR and Sanger sequencing. RESULTS: Comparative genomic analysis of E. coli 2EC1-1 and 94EC revealed that both carry tet(X4), which is encoded by IncI1-type plasmids p2EC1-1 and p94EC-2, respectively. Retrospective screening of faecal samples collected from 109 healthy individuals showed that the faecal carriage rate of Tet(X4)-producing Enterobacteriaceae is 10.1% (95% CI = 5.1%-17.3%), suggesting that tet(X4) is widely distributed in the gut microbiota of healthy individuals in Singapore. CONCLUSIONS: To the best of our knowledge, this is the first report on the prevalence of tet(X4) in the gut microbiota of a healthy human cohort, as well as the first description of this resistance mechanism outside of China. Our findings suggest that surveillance of tet(X4) in community settings is vital to monitor the spread of this resistance mechanism.

Establishing multiple omics baselines for three Southeast Asian populations in the Singapore Integrative Omics Study.

The Singapore Integrative Omics Study provides valuable insights on establishing population reference measurement in 364 Chinese, Malay, and Indian individuals. These measurements include > 2.5 millions genetic variants, 21,649 transcripts expression, 282 lipid species quantification, and 284 clinical, lifestyle, and dietary variables. This concept paper introduces the depth of the data resource, and investigates the extent of ethnic variation at these omics and non-omics biomarkers. It is evident that there are specific biomarkers in each of these platforms to differentiate between the ethnicities, and intra-population analyses suggest that Chinese and Indians are the most biologically homogeneous and heterogeneous, respectively, of the three groups. Consistent patterns of correlations between lipid species also suggest the possibility of lipid tagging to simplify future lipidomics assays. The Singapore Integrative Omics Study is expected to allow the characterization of intra-omic and inter-omic correlations within and across all three ethnic groups through a systems biology approach.The Singapore Genome Variation projects characterized the genetics of Singapore's Chinese, Malay, and Indian populations. The Singapore Integrative Omics Study introduced here goes further in providing multi-omic measurements in individuals from these populations, including genetic, transcriptome, lipidome, and lifestyle data, and will facilitate the study of common diseases in Asian communities.