Gut virome profile in healthy Saudi children

Abstract

Background:

The role of viruses is well known in health and disease. The aim of this report was to describe the profile of viruses in the gut of healthy Saudi children.

Methods:

In 20 randomly selected school age children from Riyadh, stool samples were collected in cryovials and stored at −80° C. At the time of analysis, the samples were sent by express mail in a temperature-controlled container to the laboratory in the USA, Viral DNA was isolated and shotgun metagenomic sequencing was performed. The abundance of each organism was expressed as an average relative percentage across the viral phylogenetic tree from phyla to species.

Results:

The median age of the children was 11.3 (range 6.8–15.4) years, and 35% were males. Caudovirales were the most abundant bacteriophage order (77%) and Siphoviridae, Myoviridae, and Podoviridae families predominated, accounting for 41%, 25%, and 11%, respectively. Among the viral bacteriophage species, the most abundant were the Enterobacteria phages.

Conclusion:

The profile and abundance of the gut virome in healthy Saudi children reveal important differences from the literature. Further studies from different populations with larger sample sizes are needed to understand the role of gut viruses in the pathogenesis of disease in general and in the response to fecal microbiota therapy in particular.

INTRODUCTION

The gut virome includes all the nucleic acids (DNA and RNA) of the virus-like particles. Quantitatively, the virome is at least equal to bacteria, and may outnumber bacterial cells in the gut.[1,2] The virome is dominated by bacteriophages which are viruses that infect bacteria. Bacteriophages can be lytic or lysogenic.[3] Lytic viruses penetrate bacteria and control the genetic replication to produce virions that are released and may infect new bacteria. Lysogenic viruses integrate into the genome of bacteria without lysing (killing) them. Thus, the ability of phages to transfer genes from one host to another can lead to increased diversity of viral species, increased antibiotic resistance, and/or induction of virulence factors in the host bacteria.[4] Other phages may alter the antigenicity of their hosts by modifying the O-antigen component. In the era of fecal microbiota therapy (FMT), studies on the role of the viral component of fecal samples of healthy donors and their effects on the response to FMT are needed.[5,6] Accordingly, the characterization of the virome profile in health is the first step. As with the bacterial component of the microbiome, the genetic makeup of an individual’s virome is influenced by diet, nutrition status, health, socioeconomic group, geographical location, age, lifestyle, season, and medication.[7,8,9] Studies on the role of dietary lifestyle in the gut virome profile suggested variations between populations with different dietary lifestyles, indicating the need for studies from different populations.[8,9,10,11] In this study, we used shotgun metagenomic DNA sequencing (untargeted sequencing) of purified viral samples from healthy children.[12] The objective was to characterize the profile of bacteriophages and DNA eukaryotic viruses in a cohort of healthy Saudi children, a Middle eastern population. RNA eukaryotic viruses were not analyzed.

SUBJECTS AND METHODS

The study population

The study was performed at King Khalid University Hospital, King Saud University Medical City, King Saud University; and King Fahad Medical City Children Hospital, Ministry of Health, Riyadh, the Kingdom of Saudi Arabia (KSA). Stool samples were collected from healthy schoolchildren taken from a larger random sample recruited for a mass screening study.[13] The children were on a normal family diet at the time of sample collection.

Sample collection and storage

Stool samples were collected in cryovials and stored at −80°C. At the time of analysis, the samples were sent by express mail in a temperature-controlled container filled with dry ice until delivery, to the laboratory where metagenomic, bioinformatics, and statistical analyses were performed (CosmosID, Rockville, MD, USA).

DNA isolation and sequencing

DNA was isolated using the DNeasy PowerSoil DNA kit (Qiagen, Hilden, Germany), with each process done according to the manufacturer’s instructions. Isolated viral DNA was quantified by Qubit (Thermo Fisher Scientific, Waltham, MA, USA).

DNA libraries were prepared using the Illumina Nextera XT library preparation kit, according to the manufacturer’s protocol. Library quantity and quality were assessed with Qubit and Tapestation (Agilent Technologies, Santa, Clara, CA, USA). Libraries were then sequenced on an HiSeq platform (2 × 150 bp; Illumina, San Diego, CA, USA).

Bioinformatic and abundance analysis

Unassembled sequencing reads were directly analyzed with the CosmosID bioinformatics platform (CosmosID Inc., Rockville, MD, USA) described elsewhere for microbiome analysis and quantification of each organism’s relative abundance.[14,15,16,17] Briefly, the system uses curated genome databases and a high-performance data-mining algorithm that rapidly disambiguates hundreds of millions of metagenomic sequence reads into the discrete microorganisms engendering the sequences.

The abundance of each organism was calculated and expressed as an average relative percentage across the viral phylogenetic tree from phyla to species.

The datasets generated during this study are available in the NCBI SRA. Access link: http://www.ncbi.nlm.nih.gov/bioproject/757365.

Ethical approval

This study was approved by the Institutional Review Board of the College of Medicine, King Saud University Riyadh, Kingdom of Saudi Arabia (no. 14/4464/IRB). All children and/or their parents gave informed consent and/or assent for participation in the study.

RESULTS

The study population

Twenty healthy Saudi children were enrolled. The median age was 11.3 (range 6.8–15.4) years, and 35% were males. The weight average and range were 46.9 (20-76) kg and the BMI average and range were 19.8 (12.5-28.0) kg/m². The children were on a normal Saudi family diet dominated by the consumption of rice, bread, red meat, and chicken. In addition, the children frequently consumed fast food and sweetened gaseous drinks but rarely fruit or vegetables.

The abundance of viruses

The profile and abundance in this study were determined by shotgun analysis of the DNA of viral particles only and did not include RNA viruses. Among 206 sequenced taxa, only 24 (11.7%) were not identified in the available database and therefore were designated unidentified. Caudovirales were the most abundant bacteriophage order (77%). The abundance of the top families and genera is illustrated in Figure 1. Among the list of viral families, Siphoviridae, Myoviridae, and Podoviridae families predominated, accounting for 41%, 25%, and 11%, respectively. Similarly, the most abundant genera included Lambdavirus, P2virus, and Nona33virus accounting for 26%, 12%, and 1.5%, respectively [Table 1]. The abundance of all the identified bacteriophage species is shown in Table 2. Among the Enterobacteria phages, the most abundant species were Enterobacteria phage BP-4795, Enterobacteria phage YYZ-2008, Enterobacteria phage mEp460, and Enterobacteria phage P88 accounting for 6.6%, 5.4%, 3.3%, and 3.3%, respectively. The most abundant Escherichia phages included Escherichia phage TL-2011b (2.5%), Escherichia virus P2 (2.4%), Escherichia virus HK022 (2.1%), and Escherichia virus If1 (1.8%), whereas Lactobacillus phage KC5a was the most abundant lactobacillus phage (2.9%). Among Lactococcus phages, Lactococcus phage ul36 was the most abundant (1.7%) and Salmonella phage RE-2010 was the most abundant among the Salmonella phages (1%). Shigella phage SfIV was the most abundant Shigella phage (1.4%) and Streptococcus phage 20617 was the most abundant Streptococcus phage (15%).

Figure 1.

Illustration of the abundance of the top families and genera. Panel (a) shows the predominance of the Siphoviridae family (41%) and the others (23%) cover all other family members with abundance less than 11% each. Panel (b) shows the predominance of the Lambdavirus genera (26%) and the others refer to all other genera with abundance less than 1.5% each

Table 1.

Viral abundance from order to genera level

Level	Organism	Abundance	Level	Organism	Abundance
Order	Caudovirales	0.77	Genera	Lambdavirus	0.26
Family	Geminiviridae	0.0008	Genera	Muvirus	0.001
Family	Inoviridae	0.018	Genera	N15virus	1.40−05
Family	Myoviridae	0.25	Genera	Nona33virus	0.015
Family	Podoviridae	0.11	Genera	P1virus	0.01
Family	Retroviridae	9.95−05	Genera	P22virus	0.014
Family	Siphoviridae	0.41	Genera	P2virus	0.12
Genera	Begomovirus	0.0008	Genera	Pa6virus	4.55−05
Genera	C2virus	0.0001	Genera	Phietavirus	8.90−05
Genera	Cc31virus	0.0012	Genera	Phifelvirus	0.0001
Genera	Epsilon15virus	0.033	Genera	Rb69virus	5.70−05
Genera	Gammaretrovirus	9.95−05	Genera	Sfi11virus	0.004
Genera	Hp1virus	0.0001	Genera	Sfi21dt1virus	0.005
Genera	Jerseyvirus	0.0002	Genera	Tl2011virus	0.006

Table 2.

Abundance of viral species

No.	Organism	Abundance	No.	Organism	Abundance
1	Bacteroides phage B124-14	0.006	61	Lactobacillus phage LF1	0.001
2	Bacteroides phage B40-8	0.002	62	Lactobacillus phage Lrm1	6.35-05
3	Cronobacter phage ENT39118	1.75-05	63	Lactobacillus phage phiadh	0.0001
4	Cronobacter phage ENT47670	9.00-06	64	Lactobacillus prophage Lj771	0.0002
5	Cronobacter phage phiES15	2.00-05	65	Lactobacillus prophage Lj965	1.35-05
6	Enterobacter virus CC31	0.0008	66	Lactococcus phage 1706	9.50-06
7	Enterobacter virus PG7	0.0004	67	Lactococcus phage 340	1.35-05
8	Enterobacteria phage 933W	0.014	68	Lactococcus phage bIBB29	2.95-05
9	Enterobacteria phage BP-4795	0.066	69	Lactococcus phage bIL170	2.45-05
10	Enterobacteria phage cdtI	0.030	70	Lactococcus phage bIL285	0.001
11	Enterobacteria phage fiAA91-ss	0.024	71	Lactococcus phage bIL286	0.002
12	Enterobacteria phage HK106	0.003	72	Lactococcus phage bIL309	0.001
13	Enterobacteria phage HK140	0.003	73	Lactococcus phage bIL310	0.01
14	Enterobacteria phage HK225	0.001	74	Lactococcus phage bIL311	0.011
15	Enterobacteria phage HK446	0.002	75	Lactococcus phage bIL312	0.006
16	Enterobacteria phage HK542	0.002	76	Lactococcus phage bIL67	9.40-05
17	Enterobacteria phage HK544	0.006	77	Lactococcus phage BK5-T	0.001
18	Enterobacteria phage HK629	0.002	78	Lactococcus phage BM13	0.004
19	Enterobacteria phage HK630	0.002	79	Lactococcus phage c2	2.35-05
20	Enterobacteria phage HK633	0.003	80	Lactococcus phage jm2	5.50-06
21	Enterobacteria phage IME10	0.007	81	Lactococcus phage P008	5.00-06
22	Enterobacteria phage mEp043	0.001	82	Lactococcus phage P335 sensu lat	0.001
23	Enterobacteria phage mEp235	0.0004	83	Lactococcus phage phiLC3	0.001
24	Enterobacteria phage mEp237	0.003	84	Lactococcus phage r1t	0.002
25	Enterobacteria phage mEp460	0.035	85	Lactococcus phage TP901-1	0.006
26	Enterobacteria phage mEpX1	9.35-05	86	Lactococcus phage Tuc2009	0.007
27	Enterobacteria phage mEpX2	0.0004	87	Lactococcus phage ul36	0.017
28	Enterobacteria phage P4	0.023	88	Lambdavirus_u_s	0.0004
29	Enterobacteria phage P88	0.033	89	Leuconostoc phage Lmd1	2.80-05
30	Enterobacteria phage phiP27	0.003	90	Leuconostoc phage P793	5.50-06
31	Enterobacteria phage RB3	2.40-05	91	Leuconostoc phage phiLN03	0.0008
32	Enterobacteria phage SfV	0.015	92	Leuconostoc phage phiLN04	0.001
33	Enterobacteria phage ST104	0.0002	93	Leuconostoc phage phiLN12	4.75-05
34	Enterobacteria phage YYZ-2008	0.054	94	Murine leukemia virus	3.50-06
35	Enterobacterial phage mEp213	7.90-05	95	Pectobacterium phage ZF40	2.15-05
36	Enterobacterial phage mEp234	0.002	96	Phage Gifsy-2	1.50-05
37	Enterobacterial phage mEp390	0.001	97	Propionibacterium virus P1001	4.55-05
38	Escherichia phage D108	0.001	98	Salmonella phage epsilon34	0.003
39	Escherichia phage HK639	0.0001	99	Salmonella phage Fels-1	4.50-05
40	Escherichia phage HK75	0.001	100	Salmonella phage Fels-2	0.001
41	Escherichia phage P13374	0.002	101	Salmonella phage FSL SP-004	0.002
42	Escherichia phage TL-2011b	0.025	102	Salmonella phage g341c	4.00-06
43	Escherichia phage vB_EcoM	1.50-05	103	Salmonella phage HK620	0.004
44	Escherichia virus 933W	0.012	104	Salmonella phage RE-2010	0.01
45	Escherichia virus 9g	3.50-06	105	Salmonella phage SE1	0.0002
46	Escherichia virus HK022	0.021	106	Salmonella phage SE2	0.0002
47	Escherichia virus HK97	0.003	107	Salmonella phage SPN9CC	0.0003
48	Escherichia virus HX01	2.00-05	108	Salmonella phage SSU5	0.008
49	Escherichia virus If1	0.01	109	Salmonella phage ST64B	9.00-05
50	Escherichia virus JS09	2.45-05	110	Salmonella phage vB_SemP_Emek	0.001
51	Escherichia virus Min27	0.001	111	Salmonella phage Vi II-E1	3.50-05
52	Escherichia virus N15	1.40-05	112	Salmonella virus Epsilon15	7.85-05
53	Escherichia virus P1	0.01	113	Salmonella virus P22	0.0002
54	Escherichia virus P2	0.02	114	Salmonella virus PsP3	0.002
55	Escherichia virus phiV10	0.01	115	Salmonella virus SPN1S	3.50-06
56	Escherichia virus RB69	1.25-05	116	Shigella phage Sf6	0.006
57	Escherichia virus TL2011	0.01	117	Shigella phage SfII	0.01
58	Klebsiella phage phiKO2	0.0002	118	Shigella phage SfIV	0.013
59	Lactobacillus phage KC5a	0.03	119	Spleen focus-forming virus	9.60-05
60	Lactobacillus phage Lc-Nu	0.0003	120	Staphylococcus phage Ipla5	8.90-05
121	Streptococcus phage 20617	0.15	138	Streptococcus phage O1205	0.0001
122	Streptococcus phage 2972	6.30-05	139	Streptococcus phage PH 10	0.001
123	Streptococcus phage 315.1	5.25-05	140	Streptococcus phage PH 15	6.70-05
124	Streptococcus phage 315.2	0.0002	141	Streptococcus phage phiBHN167	4.50-06
125	Streptococcus phage 315.6	3.90-05	142	Streptococcus phage Sfi11	0.0007
126	Streptococcus phage 5093	0.0007	143	Streptococcus phage Sfi19	0.0007
127	Streptococcus phage 7201	0.001	144	Streptococcus phage Sfi21	0.0004
128	Streptococcus phage 858	0.001	145	Streptococcus phage SM1	0.0003
129	Streptococcus phage Abc2	0.001	146	Streptococcus phage TP-778L	0.001
130	Streptococcus phage Alq132	0.002	147	Streptococcus phage TP-J34	7.70-05
131	Streptococcus phage DCC1738	0.0002	148	St×2-converting phage 1717	0.083
132	Streptococcus phage DT1	0.001	149	St×2-converting phage 86	0.014
133	Streptococcus phage EJ-1	0.0004	150	Synechococcus phage S-CBS1	1.80-05
136	Streptococcus phage M102	3.70-05	151	Vibrio phage pYD38-A	0.0002
137	Streptococcus phage MM1	0.0001	152	Yersinia phage L-413C	0.046
			153	Watermelon chlorotic stunt virus	0.001

DISCUSSION

Knowledge of the viral profile in healthy individuals is a prerequisite for the study of the role of viruses in disease pathogenesis and etiology. Bacteriophages are the most abundant viruses in humans and infection of bacteria by phages can alter microbiota structure by killing host cells or altering their phenotype, contributing either to the maintenance of intestinal homeostasis or causing microbial imbalance and development of chronic infectious and autoimmune diseases.

To our knowledge, this is the first report on gut viral profiles in healthy Saudi children, a Middle Eastern population who have different cultures and dietary lifestyles than their Western counterparts. Our findings that bacteriophages were the most abundant viruses and Caudovirales were the most abundant order (77%), are consistent with the results of several reviews.[18,19,20,21,22] Interestingly, crAssphages, (cross assembly phage; members of the Caudovirales) were not found in the fecal samples of our children, a finding contrary to reports of the abundance of more than 50% of the human gut samples.[23,24,25] The explanation of this important variation is not clear at present. It is possible that the lack of detection of this virus and others in our sample is related to age, ethnicity, culture, dietary lifestyle, or geographic differences.[26,27] The significance of these new viruses in health or disease is still not clear.[28] Nevertheless, our results are consistent with reports of the predominance of bacteriophages of the Siphoviridae, Podoviridae, and Myoviridae families. Microviridae are less abundant in infants but rise in abundance with age.[29,30] In addition, the profile of phage species in this report is consistent with some studies, reporting that phages of the early bacterial colonizers, including Escherichia, Klebsiella, Enterococcus, Staphylococcus, and Streptococcus species, were some of the most abundant early virome members in children.[31,32]

Similarities with previous reports include the predominance of the bacteriophages Caudovirales order; the Siphoviridae, Podoviridae, and Myoviridae families; the Escherichia, Klebsiella, Enterococcus, Staphylococcus, and Streptococcus species. The most important difference was the lack of cross assembly phage in our study.

Our study has a few limitations including the relatively small sample size which may be acceptable for this first report of the gut virome in Saudi children. In addition, the limitation to DNA viruses is recognized.

In conclusion, the profile and abundance of the intestinal virome in healthy Saudi children reveal similarities and distinctive features as illustrated in the literature. Further studies from different populations with larger sample sizes are needed to advance knowledge of the importance of gut viruses in the pathogenesis of disease in general and their role in the response to FMT in particular.

Financial support and sponsorship

The Deanship of Scientific Research, King Saud University.

Conflicts of interest

There are no conflicts of interest.