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Background The rise of nature-based ecotourism in the past decade has introduced unprecedented challenges in managing the increasing interaction between humans and animals. The potential transmission of antibiotic resistant microbes between humans and non-human primate populations is a concern due to their genetic similarity. Malaysia is well known for hotspots of wildlife diversity where non-human primates like monkeys and orangutans have become popular tourist attractions. In this study, we assessed the prevalence of antimicrobial resistant Staphylococcus aureus, Enterococcus species, and other Enterobacteriaceae in the faeces of human (HS) and two non-human primates (NHP) in Malaysia, the Long-tailed macaque (Macaca fascicularis, MF) and Silvered leaf monkey (Trachypithecus cristatus, TC). In addition, the faecal bacterial composition was profiled to evaluate the potential association between antibiotic resistant profiles and composition of gut microbiota. Results We tested the isolated bacteria using a selection of antibiotics. The results showed that both the number of antibiotic resistant strains and resistance level were higher in humans than NHPs. Overall, the composition of gut microbiome and pattern of antibiotic resistance showed that there was higher similarity between MF and TC, the two NHPs, than with HS. In addition, samples with higher levels of antibiotic resistance showed lower bacterial richness. Homo sapiens had the lowest bacterial diversity and yet it had higher abundance of Bacteroides. In contrast, NHPs displayed higher bacterial richness and greater prevalence of Firmicutes such as Ruminococceae and Oscillospira. Conclusion Higher antibiotic susceptibility in NHPs is likely related to low direct exposure to antibiotics. The lack of resistance may also suggest limited antimicrobial resistance transmission between humans and NHP. Nonetheless, continued monitoring over a long period will help mitigate the risk of anthropozoonosis and zooanthroponosis.

Background Singapore and Malaysia are neighbouring countries with similar ethnic make-up: Chinese, Indian, and Malays. However, Singapore is a high-income economy while Malaysia is a middle-income economy. Such a difference has resulted in some levels of dichotomy in diet and lifestyles. In this study, we sought to investigate the gut-ethnic variation across the two neighbouring countries with varying levels of economic development. Methods A total of 439 relatively healthy Malaysian (n=190) and Singaporean (n=249) adults (>18) were included, comprising Chinese (n=240), Indian (n=74), Malay (n=40), and the indigenous Jakun community (n=85). The sequences were processed with DADA2 and annotated using the SILVA database. Results Country of origin explained the most variation in the gut microbiota (PERMANOVA, Pseudo-F=80.798, R2=0.156, p=0.001; IDDF2021-ABS-0150 Figure 1. Ordination). Importantly, ethnicity was still significantly associated with the gut microbiota even after adjusting for the country, age and sex (PERMANOVA, Pseudo-F=4.206, R2=0.019, p=0.001). Several taxa were found to be differentially abundant across ethnicity (ANCOM-BC, q Conclusions Gut-ethnic differences persist across geographical regions, which was likely due to similar lifestyle and cultural practices by individuals sharing similar ancestry. These signals provide potential biomarkers on the role of the gut microbiota in the aetiology of the unequal disease burdens affecting the different ethnic groups in this multicultural region.

Background The gut microbiome plays an instrumental role in cardiovascular, metabolic and gastrointestinal health (Valdes et al. 2018). In Traditional Chinese Medicine (TCM), one’s health status can be described by nine body constitutions (BCs) which are broadly classified as ‘Neutral’, indicating optimal health, or ‘Unbalanced’, indicating suboptimal health. Similar to the gut microbiome, BCs are strongly influenced by lifestyle and diet. We evaluated the gut microbial composition of Neutral, Qi-Deficiency (QD), Phlegm-dampness (PD) and Damp Heat (DH) subjects. The latter three are unbalanced BCs, with QD associated with heart disease (Zhu et al. 2017), PD with obesity and metabolic syndrome (Wang et al. 2013), and DH with gastrointestinal disease (Liang et al. 2020). Methods Stool samples were collected from 211 healthy subjects and gut microbial composition was profiled using 16S rRNA amplicon sequencing of the V3-V4 regions. BCs were determined using the Constitution in Chinese Medicine Questionnaire (Wang, 2005). Alpha diversity of QD, PD and DH was inferred and compared against that of Neutral using the Kruskal-Wallis test. Differentially abundant bacterial phyla, family and species were identified by calculating Log2FoldChange of taxa in the unbalanced BCs compared to Neutral. Results There were 135 Neutral, 59 QD, 24 PD and 12 DH subjects. 16 participants exhibited two or more unbalanced BCs. PD exhibited significantly lower alpha diversity (Shannon, Simpson) than Neutral (p Conclusions Varying TCM BCs exhibit unique microbiota signatures. Participants with unbalanced BCs are associated with greater dysbiosis compared to participants with Neutral BC. The lower alpha diversity in PD is consistent with that in obese and metabolic syndrome patients (Stanislawski et al. 2019; Oh et al. 2021). Streptococcus gallolyticus, a species associated with gastrointestinal malignancy (Chand et al. 2016), was significantly higher in all three unbalanced BCs and thus may serve as a potential biomarker for identifying patients with unbalanced BCs.

Background The impact of non-dietary lifestyle factors on the gut microbiota, such as physical activity, has not been frequently reported. Recent studies have suggested that exercise can increase the number of beneficial microbial species which enhance short-chain fatty acid synthesis and carbohydrate metabolism. Therefore, we investigated the relationship between physical activity and gut microbiome composition within a cohort of multi-ethnic healthy Singaporeans. Methods 257 individuals completed the Global Physical Activity Questionnaire (GPAQ) (Armstrong et al. 2006) and Food Frequency Questionnaire (FFQ) (Whitton et al. 2017), to obtain quantitative measures of their physical activity, nutrition and dietary habits, respectively. They also provided paired stool samples, which underwent 16S rRNA V3-4 amplicon sequencing. Community and individual microbial features associated with physical activity were assessed using microbial ecology measures such as α-diversity and principal coordinate analysis (PCA), as well as, multivariate sparse Partial Least Squares analyses, whilst confounding for nutritional and dietary habits. Results Using the GPAQ, 57% of the individuals were deemed to have an active lifestyle. Active individuals have increased microbial α-diversity, although not statistically significant. Unsupervised PCA analysis identified a subset of active individuals with distinct microbiome signatures, whereby these individuals, compared to those with sedentary lifestyles, have increased abundances of Prevotella spp., which is in keeping with their increased fibre intake. Sedentary lifestyle individuals were found to have increased abundance of Lachnoclostridium spp., whereby phylogenetic similar microbial features (such as Roseburia and Subdoligranulum spp.) were significantly correlated with more frequent eating out at hawker stalls and fast-food establishments (IDDF2021-ABS-0169 Figure 1. Correlation between gut microbiome with (a) nutrient intake; and (b) food habit). Studying the metabolic potential of the microbiome, active individuals had increased abundances of gene content related to energy production, whereby sedentary individuals had increased abundances of gene content related to degradation of aromatic compounds. Conclusions Our study reaffirms that increased physical activity is associated with increased microbial diversity and beneficial microbial features. However, the differences in gut microbiota associated with physical activity may be due to differences in dietary intake and practices, rather than the exercise itself. Further studies investigating the link of gut microbiome and physical activity should control and standardize the dietary intake of participants.

Ethnicity is consistently reported as a strong determinant of human gut microbiota. However, the bulk of these studies are from Western countries, where microbiota variations are mainly driven by relatively recent migration events. Malaysia is a multicultural society, but differences in gut microbiota persist across ethnicities. We hypothesized that migrant ethnic groups continue to share fundamental gut traits with the population in the country of origin due to shared cultural practices despite subsequent geographical separation. To test this hypothesis, the 16S rRNA gene amplicons from 16 studies comprising three major ethnic groups in Malaysia were analysed, covering 636 Chinese, 248 Indian and 123 Malay individuals from four countries (China, India, Indonesia and Malaysia). A confounder-adjusted permutational multivariate analysis of variance (PERMANOVA) detected a significant association between ethnicity and the gut microbiota (PERMANOVA R 2=0.005, pseudo-F=2.643, P=0.001). A sparse partial least squares – discriminant analysis model trained using the gut microbiota of individuals from China, India and Indonesia (representation of Chinese, Indian and Malay ethnic group, respectively) showed a better-than-random performance in classifying Malaysian of Chinese descent, although the performance for Indian and Malay were modest (true prediction rate, Chinese=0.60, Indian=0.49, Malay=0.44). Separately, differential abundance analysis singled out Ligilactobacillus as being elevated in Indians. We postulate that despite the strong influence of geographical factors on the gut microbiota, cultural similarity due to a shared ethnic origin drives the presence of a shared gut microbiota composition. The interplay of these factors will likely depend on the circumstances of particular groups of migrants.

No studies have investigated the influence of ethnicity in a multi-ethnic middle-income country with a long-standing history of co-habitation. Stool samples from 214 Malaysian community members (46 Malay, 65 Chinese, 49 Indian, and 54 Jakun) were collected. The gut microbiota of the participants was investigated using 16S amplicon sequencing. Ethnicity exhibited the largest effect size across participants (PERMANOVA Pseudo-F = 4.24, R2 = 0.06, p = 0.001). Notably, the influence of ethnicity on the gut microbiota was retained even after controlling for all demographic, dietary factors and other covariates which were significantly associated with the gut microbiome (PERMANOVA Pseudo-F = 1.67, R2 = 0.02, p = 0.002). Our result suggested that lifestyle, dietary, and uncharacterized differences collectively drive the gut microbiota variation across ethnicity, making ethnicity a reliable proxy for both identified and unidentified lifestyle and dietary variation across ethnic groups from the same community.

Emerging evidence has shown a link between the perturbations and development of the gut microbiota in infants with their immediate and long-term health. To better understand the assembly of the gut microbiota in preterm infants, faecal samples were longitudinally collected from the preterm (n = 19) and term (n = 20) infants from birth until month 12. 16S rRNA gene sequencing (n = 141) and metabolomics profiling (n = 141) using nuclear magnetic resonance spectroscopy identified significant differences between groups in various time points. A panel of amino acid metabolites and central metabolism intermediates significantly correlated with the relative abundances of 8 species of bacteria were identified in the preterm group. In contrast, faecal metabolites of term infants had significantly higher levels of metabolites which are commonly found in milk such as fucose and β-hydroxybutyrate. We demonstrated that the early-life factors such as gestational age, birth weight and NICU exposures, exerted a sustained effect to the dynamics of gut microbial composition and metabolism of the neonates up to one year of age. Thus, our findings suggest that intervention at this early time could provide ‘metabolic rescue’ to preterm infants from aberrant initial gut microbial colonisation and succession.

Coffee is rich in antioxidant and has been shown to confer various health benefits. Here, we investigated the effect of single-dose coffee consumption in healthy human subjects. About 30 healthy volunteers were recruited and given a serving of sugar free black coffee. Urine and fecal samples were collected and analyzed. Significant changes in urinary metabolites relating to coffee, gut microbial and host energy metabolisms were observed post-coffee consumption. Clear sex differences were also observed in the urinary metabolic profiles pre- and post-coffee consumption. Sex differences in richness and composition of gut microbiota were observed, however, the effect of single-dose coffee consumption on host gut microbiota were unremarkable. These findings indicated that single-dose coffee consumption affects sex-specific host metabolic responses that relates to gut-microbe and energy metabolism. This study demonstrated the utility of systems biology tools to unravel complexity of host-diet biology and gut microbial responses. PRACTICAL APPLICATIONS: This study demonstrated that integrated systems biology approach enabled efficient extractions of host biochemical and microbial information that allows food industry to ascertain the impact of diet and longitudinal assessment of potential functional food in humans.

Background Probiotics are the most recognised alternative to antibiotic growth promoters (AGPs) for poultry production. They adhere to the intestinal epithelial cells to exert their beneficial effects on the host. However, this is often restricted by indigenous gut microbes that compete for adhesion sites and nutrients. Therefore, phage application has been proposed to reduce the competition of probiotics with gut microbiota. The main objective of this study was to investigate the effects of phages on the gut microbiota and their impact on probiotic efficacy in chickens. Results Four best Escherichia coli lytic phages were selected and characterised. The in vivo trial was conducted to investigate the effects of dietary treatments of phage cocktail at 0.1% (1ɸ) and 0.2% (2ɸ), 0.1% PrimaLac ® probiotic (P) and their combinations (1ɸP, 2ɸP) on growth performance and gut microbiota of chickens. The results from the in vivo trial showed that chickens in the 1ɸP group had significantly (P < 0.05) better body weight (BW), body weight gain (BWG), and feed conversion ratio (FCR), lower serum triglycerides and higher ileal villus height than the control. Chickens in the 1ɸP group also had higher (P < 0.05) ileal villus height, and lower crypt depth than those in the probiotic group. The gut microbiota profiles revealed that 1ɸP supplementation significantly (P < 0.05) reduced E. coli population, increased (P < 0.001) short chain fatty acids (SCFAs) producers, and improved (P < 0.001) gene expressions that are related to carbohydrate and amino acid metabolisms, and nutrient uptake. Conclusions The results from this study showed that 1ɸP treatment could modulate the gut microbiota and enhance growth performance in chickens. This finding could be considered as a potential alternative to AGPs for poultry.

Understanding the basal gut bacterial community structure and the host metabolic composition is pivotal for the interpretation of laboratory treatments designed to answer questions pertinent to host-microbe interactions. In this study, we report for the first time the underlying gut microbiota and systemic metabolic composition in BALB/c mice during the acclimatisation period. Our results showed that stress levels were reduced in the first three days of the study when the animals were subjected to repetitive handling daily but the stress levels were increased when handling was carried out at lower frequencies (weekly). We also observed a strong influence of stress on the host metabolism and commensal compositional variability. In addition, temporal biological compartmental variations in the responses were observed. Based on these results, we suggest that consistency in the frequency and duration of laboratory handling is crucial in murine models to minimise the impact of stress levels on the commensal and host metabolism dynamics. Furthermore, caution is advised in consideration of the temporal delay effect when integrating metagenomics and metabonomics data across different biological matrices (i.e. faeces and urine).

Background Chicken gut microbiota has paramount roles in host performance, health and immunity. Understanding the topological difference in gut microbial community composition is crucial to provide knowledge on the functions of each members of microbiota to the physiological maintenance of the host. The gut microbiota profiling of the chicken was commonly performed previously using culture-dependent and early culture-independent methods which had limited coverage and accuracy. Advances in technology based on next-generation sequencing (NGS), offers unparalleled coverage and depth in determining microbial gut dynamics. Thus, the aim of this study was to investigate the ileal and caecal microbiota development as chicken aged, which is important for future effective gut modulation. Material and methods Ileal and caecal contents of broiler chicken were extracted from 7, 14, 21 and 42-day old chicken. Genomic DNA was then extracted and amplified based on V3 hyper-variable region of 16S rRNA. Bioinformatics, ecological and statistical analyses such as Principal Coordinate Analysis (PCoA) was performed in mothur software and plotted using PRIMER 6. Additional analyses for predicted metagenomes were performed through PICRUSt and STAMP software package based on Greengenes databases. Results A distinctive difference in bacterial communities was observed between ilea and caeca as the chicken aged (P

Gut microbiota plays an important role in mammalian host metabolism and physiological functions. The functions are particularly important in young children where rapid mental and physical developments are taking place. Nevertheless, little is known about the gut microbiome and the factors that contribute to microbial variation in the gut of South East Asian children. Here, we compared the gut bacterial richness and composition of pre-adolescence in Northern Malaysia. Our subjects covered three distinct ethnic groups with relatively narrow range of socioeconomic discrepancy. These included the Malays (n = 24), Chinese (n = 17) and the Orang Asli (indigenous) (n = 20). Our results suggested a strong ethnicity and socioeconomic-linked bacterial diversity. Highest bacterial diversity was detected from the economically deprived indigenous children while the lowest diversity was recorded from the relatively wealthy Chinese children. In addition, predicted functional metagenome profiling suggested an over-representation of pathways pertinent to bacterial colonisation and chemotaxis in the former while the latter exhibited enriched gene pathways related to sugar metabolism.