Abstract

Results

Socio-economic and hygiene practice of the study group

Significant differences in the demographics and hygiene habits (Pseudo-F=30.62, P_MC=0.001) were detected among the three studied ethnic groups. A follow-up pairwise comparison showed significant difference between all ethnic pairings (P_MC=0.001). Generally, the Chinese recorded the highest socio-economic status with no family earning less than RM500 per month (Table 1). Additionally, they exhibited the lowest level of parasitic infection rate (5.9%, single infection; 0% mixed infection) and highest level of hygiene consciousness (i.e. all Chinese subjects practiced proper rubbish disposal and defecate as well as taking bath in the bathroom). A slightly lower household income (~8% <RM500) and higher parasitic load (12.5%) was recorded among the Malays. In contrast, Orang Asli was the poorest group with 45% of the families earning less than RM500 per month. Children under this group were all tested positive for different types of parasitic co-infections such as Ascaris, Trichuris, hookworm or/and protozoan parasite infections.

Table 1

Socio-economic characteristics and hygiene level of rural children aged 7–12 in Pangkor and Bota, Perak, Malaysia.

	Malays		Chinese		Orang Asli		Total
	Sum	%	Sum	%	Sum	%
Socioeconomic characteristics
Ethnicity	24	39.3	17	27.9	20	32.8	61
Gender
Male	15	62.5	8	47.1	10	50	33
Female	9	37.5	9	52.9	10	50	28
Income
<RM500	2	8.3	0	0	9	45	11
Family Size≥5 members	18	75	1	5.9	10	50	29
Rear pet(s)	9	14.8	0	0	19	95	28
Hygiene level
Parasitic Infection
Singla	2	8.3	1	5.9	0	0	3
Mixedb	1	4.2	0	0	20	100	21
Pipe water availabilityc	24	100	17	100	16	80	57
Water storage
Plastic bottles	17	70.8	0	0	10	50	27
Bucket	6	25	1	5.9	10	50	17
Cement water tank	1	42	16	94.1	0	0	17
Cover water storage	20	83.3	1	5.9	20	100	41
Bathing locations
Toilet	23	95.8	17	100	0	0	40
River	1	4.2	0	0	20	100	21
Defecation locations
Toilet	24	100	17	100	5	25	46
Bushes/river	0	0	0	0	15	75	15
Proper rubbish disposal	23	95.8	17	100	15	75	55

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^aTrichuris tichiuria only.

^bCombination of at least two of the following: Trichuris trichiura, Iodamoeba butschlii, Blastocystis spp., Entamoeba histolytica, Entamoeba coli, Ascaris lumbricoides, and hookworm.

^cPipe water supply to the household.

Terminal Restriction Fragment Length Polymophism (TRFLP)-inferred faecal bacterial richness and composition

Alpha diversity measures including observed Terminal Restriction Fragments (TRFs) and Shannon Diversity Index suggested that the Chinese harboured a significantly lower faecal bacterial richness in comparison to the Malays and Orang Asli (Table S1). However, no significant differences in TRFs richness and evenness were observed between the two latter groups. We subsequently compared the beta diversity (community overlap) using PERMANOVA and CAP analyses. Both marginal and pairwise PERMANOVA achieved statistical significant at P<0.01, indicating the presence of ethnicity-specific bacterial composition. Consistent result was observed in the CAP ordination (Fig. 1). Interestingly, Orang Asli cluster was found to be more distantly related to Chinese than Malays in CAP1 axis which coincided with the socio-economic status of the subjects.

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Figure 1

Canonical analysis of principal coordinates (CAP) for TRFLP data.

A 67% correct prediction rate was recorded based on the cross validation. Note: Dotted samples were chosen for 16S NGS analysis.

Correlation of social demographic parameters and faecal bacterial diversity

The distribution of the faecal bacterial diversity was significantly modelled using the pre-assigned socio-economic- and hygiene practice-related parameters (Table 2). The former attributed for 15% and the latter 18% of the total variation. When the data was analysed by considering each parameters independently, the step-wise selection algorithm selected “water storage” and “parasitic infection” as the best explanatory parameters for the faecal bacterial community composition. Cumulatively, the two variables explained 12% of the total variation in the model. On the other hand, when we modelled the faecal bacterial composition by splitting the data into each respective ethnic group, “pet” and “water storage” were shown to exert the largest effect on Orang Asli while no parameter was implicated for Chinese and Malay groups.

Table 2

Distance-based linear models of socioeconomic and hygiene characteristics of faecal bacterial composition in children.

Marginal Tests (Grouped variables)
Group	SS(trace)	Pseudo-F	P	Prop.
Socioeconomic	16346	1.6534	0.014	0.155
Hygiene	19159	1.6836	0.012	0.181
Marginal Tests (Individual variables)*
Variable	SS(trace)	Pseudo-F	P	Prop.	Group
Water storage	8651.9	5.2806	0.001	0.082	H
Cover water storage	7830	4.7387	0.001	0.074	H
Parasitic infection	4793.4	2.8133	0.014	0.046	H
Rear pet	4855.4	2.8515	0.014	0.046	S
Bathing locations	4261.5	2.488	0.018	0.040	H
Ethnicity	4178.6	2.4376	0.024	0.040	S
Family size	4416.9	2.5827	0.024	0.042	S
Defecation locations	3353.1	1.9402	0.044	0.032	H
Sequential Tests
Overall
Variable	AIC	SS(trace)	Pseudo-F	P	Prop.	Cumul.	res.df
Water Storage	453.66	8651.9	5.28	0.001	0.08	0.08	59
Parastic infection	453.25	4071.8	2.55	0.01	0.04	0.12	58
Orang Asli
Variable	AIC	SS(trace)	Pseudo-F	P	Prop.	Cumul.	res.df
Pet	148.64	4359.8	2.84	0.049	0.14	0.14	18
Water Storage	148.43	2893.7	1.99	0.067	0.09	0.23	17
Malays/Chinese
Variable	None

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^*Only elements with significant effect are included.

Pattern in taxonomic composition inferred using 16S rDNA-based Next Generation Sequencing

In order to examine the taxonomic composition of the faecal sample in detail, 6 replicates from each group were randomly selected for 16S pyrosequencing (see Fig. 1). From a total of 73,934 filtered sequences, 4,787 unique sequences were detected. Sample coverage for all samples was in the range of 97–99% (Fig. S1). Using a 97% sequence homology cut-off, significantly higher OTUs were detected in Orang Asli samples as opposed to Malays and Chinese (Table 1). Additionally, the alpha diversity measures applied here suggested a greater sequence richness and evenness in Orang Asli samples in comparison to others. The beta diversity measures including AMOVA (Table S3) and PCO (Fig. 2) further suggested that the Orang Asli encompassed a distinct community structure and composition. Overall, Firmicutes and Bacteroidetes were the dominated phyla while Feacabacterium and Prevotella are the dominant genera in the faeces of the studied cohort (Fig. 3, Fig. S2). However, the differences in faecal microbiota between Orang Asli against Chinese and Malay children can be attributed to the differences in Aeromonadales, and unclassified order under Bacteroidetes and Deltaproteobacteria, and unclassified genus under Ruminococcaceae (Table 3). Interestingly, no significant difference in bacterial taxonomic composition was detected between Malays and Chinese despite relative higher socioeconomic status and hygiene awareness in the latter in comparison to the former.

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Figure 2

Principle coordinate analysis of 16S NGS sequences distribution (square root transformed) based on

(a) Sorensen (membership) and (b) Bray-Curtis similarity (composition) index.

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Figure 3

Bacterial phyla distribution inferred based on 16S-based pyrosequencing.

The distribution was obtained through sub-sampling of 2503 sequences (the lowest obtained sequences in a sample) across all samples.

Table 3

Taxonomic identity of gut microbial taxa showing significant difference in abundance across Malay, Chinese and Orang Asli children.

	Differential abundance taxonomic unit Phylum/Order/Genus
Malays VS Chinese	None
Orang Asli VS Malays	Phylum	Mean in Orang Asli	Mean in Malays	p-value	q-value
	unclassified bacteria	0.072	0.008	0.005	0.002
	Spirochaetes	0.036	0	0.017	0.004
	Order	Mean in Orang Asli	Mean in Malays	p-value	q-value
	unclassified Bacteroidetes	0.116	0.004	<0.001	0.009
	unclassified deltaproteobacteria	0.001	0	0.001	0.010
	Bacteroidales	0.143	0.301	0.002	0.014
	Aeromonadales	0.005	0	0.005	0.021
	unclassified bacteria	0.067	0.007	0.011	0.041
	unclassified Clostridia	0.001	0	0.014	0.041
	Selenomonadales	0.006	0.014	0.019	0.050
	Genus	Mean in Orang Asli	Mean in Malays	p-value	q-value
	unclassified Ruminococcaceae	0.200	0.037	0	0
Orang Asli VS Chinese	Phylum
	None
	Order	Mean in Orang Asli	Mean in Chinese	p-value	q-value
	unclassified Bacteroidetes	0.116	<0.001	<0.001	0.004
	unclassified deltaproteobacteria	0.001	0	0.001	0.021
	Aeromonadales	0.005	0	0.002	0.052
	Genus	Mean in OrangAsli	Mean in Chinese	p-value	q-value
	unclassified Ruminococcaceae	0.200	0.029	0	0
	unclassified Clostridiales	0.083	0.002	<0.001	0.010
	unclassified Bacteroidetes	0.119	0	0.001	0.041
	Alistipes	0	0.049	0.001	0.042

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Note: only taxonomic units with p-value and q-value≤0.05 were reported.

Predicted functional discrepancy between samples

The faecal bacterial composition was used to estimate the functional difference in the children across three ethnic groups. As expected, gut microbiota in Orang Asli children was predicted to perform more unique functions in comparison to Chinese and Malay children (Fig. S3). From a total of 328 KEGG features, 13 were significantly different between Chinese and Malays, 42 between Malays and Orang Asli, and 124 between Chinese and Orang Asli (Fig. 4). Within the selected KEGG functions, we found 32 features shared by Malays and Chinese which were distinct from the Orang Asli. These included pathways related to bacterial colonisation such as bacterial chemotaxis, flagella assembly, and bacterial motility proteins which were also enriched in the Orang Asli. Conversely, elevated protein and fat related metabolism pathways including bile acid biosynthesis, and amino acid and protein metabolism were detected in both Malays and Chinese. Finally, the functional profiles also suggested that the Chinese might have a higher capability in sugar anabolism and catabolism as several features related to the function was significantly higher in Chinese in comparison to the others.

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Figure 4

Pairwise comparison of the predicted functional profiles.

Note: Only statistical significant features (P<0.05) were included in the figure.

Discussion

In this study, we demonstrated that the gut microbial composition of pre-adolescence in Malaysia can be stratified according to the relatively homogenous socioeconomic variation represented by different ethnicity. The pyrosequencing further suggested that the Malays and Chinese gut microbiome shared greater bacterial genetic lineages in comparison to Orang Asli (Fig. 3, Table 3). Interestingly, this pattern coincided with the host genomic characteristic of the studied cohort. Jinam et al.¹⁵ showed that the indigenous Temiar tribe included in this study is genetically distinct to both the Chinese and Malays. Whilst host genetic architecture had been shown to contribute to the development of the individual gut microbiome^16,17, inferring the relationship of host genetic and gut microbial composition is difficult owing to the regionalisation of human population genetics, and the compartmentalisation of socio-demographic characteristics and lifestyles of the population.

In a parallel study focusing on South East Asian children, Mah et al.¹⁸ detected a greater faecal bacterial diversity in children from rural south Thailand while a lower diversity was observed in the urbanised children from Singapore. Similar findings were found to be consistent when the comparison was made between the gut microbiome of urbanised European or American children with those from less developed country such as Bangladesh¹¹, Burkina Faso¹², Malawi⁹ and Venezuela⁹. Our findings are incongruent with these previous findings whereby a higher bacterial diversity was recorded in the Orang Asli in comparison to the Malays and Chinese (Table S1, pyosequencing – all alpha diversity indexes). The Orang Asli was, in general, socially marginalised in Malaysia¹⁹. Approximately 45% of Orang Asli from this study has a household income of <RM500 per month, much lower than the poverty line income of RM764 (US$254) set by the Malaysian government²⁰. A combination of low income and low parental education attainment might have resulted in reduced appreciation to hygiene practices among Orang Asli children in comparison to Malays and Chinese. Based on the DISTLM analysis, both socio-economic- and hygiene-related parameters were found to be significantly related to the gut microbiota diversity (Table 2).

The contribution of gut microbiome dynamic to host immune modulation and responses are well recognised²¹. Various studies had reported strong link between parasitic infection and the changes in gut microbial diversity and community composition. For instance, a significant increase in the Ruminococcaceae was observed in population with whipworm (Trichuris sp) infection as opposed to the normal population¹⁰. Separately, elevated representation of Lactobacillaceae was found in the ileum of mice infected with Heligmosomoides polygyrus²². Hayes et al.²³ established that the commensal microflora may play a part in the physiological development of the parasite in the host environment. Based on these, Lee et al.¹⁰ postulated that the elevated gut microbial diversity in Orang Asli as opposed to urbanised population from United States of America might be associated with the high parasitic infection rate in the former. Our results partially corroborated with this findings as the presence of parasitic infection was found to exert a significant (P=0.011) but small influence (explained ~5% variance) onto the gut microbial diversity. Additionally, we also detected an increased in 16S signature of the unclassified genus affiliated with Ruminococcaceae but not Lactobacillaceae in Orang Asli children.

It is also interesting to note that low gut microbial diversity signature had been detected in individuals with inflammatory bowel disease, metabolic disorder and obesity^5,24,25. Nevertheless, differences detected here is unlikely to be related to obesity as the overall prevalence rate for overweight children is similar for both Malays and Chinese (Malays: 9.3–11.6%; Chinese: 6.6–11.6%)^26,27. No data is available for Orang Asli children but the estimated obesity rate for the adult of the same indigenous tribe (Temiar) is comparable (10.1%)²⁸.

Overall, bacterial phyla distribution was consistent among the three studied ethnic groups. Firmicutes (41–83%) and Bacteroidetes (11–45%) made up the bulk of the total detected 16S rRNA gene sequences (Fig. 3). These two phyla had been shown to be the main constituent of gut flora across different populations of different age and nationality^17,29,30. The Firmicutes/Bacteroidetes ratio of the gut microbes in Malays, Chinese and Orang Asli ranged between 2.0 to 3.5 and was comparable to that of the Italian (2.8) and Korean (2.95) but greater than Mongolian (0.77) and African (0.47). High F/B ratio had been previously associated with obesity and high fat diets^6,12,31. However, recent publications suggested that such link is over-simplified^32,33,34. Owing to the diverse functional capability of each phylum, variation in diets might not induced uniform phylum-wide responses³⁵.

A detailed inspection of the taxonomic composition of the gut flora showed that the Orang Asli children harboured significantly greater Aeromonadales, unclassified order affiliated to Bacteroidetes and Deltaproteobacteria, and unclassified genus under Ruminococcaceae when compared to the Malays and Chinese (Table 3). These lineages included anaerobic relatives such as Ruminobacter amylophilus³⁶, Prevotella ruminicola³⁷, Ruminococcus flavefaciens^37,38 etc that are commonly involved in degradation of dietary fibre into short chain fatty acid (SCFA) in human gut. Indeed, vegetables and grains formed a larger proportion of the Orang Asli diet than that of the Malays and Chinese^39,40. Additionally, higher prevalence of Bacteroidetes (e.g. Prevotella) and Ruminococus spp. were also apparent when comparing the gut microbiome composition of populations under the Eastern/African versus Western lifestyle^9,10,11,12. Interestingly, although the Malays showed a higher frequency in seafood intake than Chinese (70% of the former had fish the day before faecal collection as opposed to 0% in the latter), no significant difference in gut bacterial lineage was detected. Despite the spatial proximity and relatively similar level of district development (rural) of all three ethnicity, the Malays and Chinese children had a higher exposure to westernised and processed diet, coupled with a greater tendency of sedentary lifestyle. In support to previous studies that lifestyle affects gut microbiota diversity and distribution^12,18,41; we further showed that the effect is detectable in populations under similar environments with low level of lifestyle variation. It is noteworthy that 16S rRNA gene signature related to Spirochaetes was only exclusively found in the Orang Asli children. De Filippo et al.¹² suggested that Treponema spp. under Spirochaetes might be part of the gut commensals in rural Africans, who consumed high dietary fibre, in order to degrade undigested complex carbohydrates. Nevertheless, as the taxon was also the causative agent of Yaws disease which was common in Malaysian indigenous population previously⁴², the findings warrant a detail evaluation on the possible re-emergence of the disease in Malaysia.

Based on the predicted metagenome profiles (Fig. 4), it was apparent that the Chinese children harboured significantly more genes related to sugar metabolism as opposed to others. The metabolism of sugar is an important pathway for energy harvesting and essential to physical and mental growth in children. In addition, the gut floras of Chinese and Malays also showed greater capability in fat digestion (i.e. primary and secondary bile acid biosynthesis). The elevated pathways related to sugar and fat metabolism corresponded to higher calories intake of the two ethnic groups in comparison to Orang Asli⁴⁰. It is perhaps surprising however to note that the Chinese children, despite enriched energy metabolism pathways, are generally less obese in the later development stage (adulthood) than the other two groups⁴³. In comparison, pathways related to bacterial proliferation and colonisation were significantly higher in the Orang Asli. Since no obvious disease symptom was observed from the Orang Asli children (e.g. diarrhoea), the signatures might be sourced from non-pathogenic infection of environmental bacteria, given the lower hygiene consciousness of the indigenous population. These environmentally-acquired bacteria might then form part of the natural innate immune responses on gut surfaces⁴⁴.

In conclusion, using a combination of TRFLP profiling and pyrosequencing techniques, we have showed a close relation between the ethnicity related socioeconomic disparity and gut microbial composition in Malaysian children. Such observation might be related to the host genetic and lifestyle variation. We further highlighted the taxonomic and functional distribution differences across the three ethnic groups. As the first targeted report on Malaysian children gut microbiome, our study contributed to the understanding of the least assessed Asian gut microbiota composition. In addition, the results can be used as a baseline to decipher the link between the dynamics of gut microbial and ethnic-related health risk in Malaysia^43,45.

Methods

Additional Information

Accession numbers: The sequences reported in this paper were 393 deposited in the NCBI database under Bioproject PRJNA269514.

How to cite this article: Chong, C. W. et al. Effect of ethnicity and socioeconomic variation to the gut microbiota composition among pre-adolescent in Malaysia. Sci. Rep. 5, 13338; 10.1038/srep13338 (2015).

Supplementary Material

Acknowledgments

Footnotes

References