Metagenomic profile of gut microbiota in children during cholera and recovery

Abstract

Background: The diverse bacterial communities colonizing the gut (gastrointestinal tract) of infants as commensal flora, which play an important role in nutrient absorption and determining the state of health, are known to alter due to diarrhea.

Method: Bacterial community dynamics in children suffering from cholera and during recovery period were examined in the present study by employing metagenomic tool, followed by DNA sequencing and analysis. For this, bacterial community DNA was extracted from fecal samples of nine clinically confirmed cholera children (age 2-3 years) at day 0 (acute cholera), day 2 (antibiotic therapy), day 7 and, and day 28, and the variable region of 16S rRNA genes were amplified by universal primer PCR.

Results: 454 parallel sequencing of the amplified DNA followed by similarity search of the sequenced data against an rRNA database allowed us to identify V. cholerae, the cause of cholera, in all nine children at day 0, and as predominant species in six children, accounting for 35% of the total gut microbiota on an average in all the nine children. The relative abundance (mean ± sem %) of bacteria belonging to phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, was 55 ± 7, 18 ± 4, 13 ± 4, and 8 ± 4, respectively, at day 0, while these values were 12 ± 4, 43 ± 4, 33 ± 3, and 12 ± 2, respectively, at day 28. As antibiotic therapy began, V. cholerae count declined significantly (p< 0.001) and was found only in four children at day 2 and two children at day 7 with the relative abundance of 3.7% and 0.01%, respectively, which continued up to day 28 in the two children. Compared to acute cholera condition (day 0), the relative abundance of Escherichia coli, Enterococcus, and Veillonella increased at day 2 (antibiotic therapy) while Bifidobacterium, Bacteroides, and Ruminococcus decreased.

Conclusion: Cholera results expulsion of major commensal bacteria of phyla Bacteroidetes, Firmicutes, and Actinobacteria, and increase of harmful Proteobacteria to colonize the gut during acute and convalescence states. The observed microbiota disruption might explain the prevalent malnutrition in children of Bangladesh where diarrheal diseases are endemic.

Figure 1

Relative abundance (percentage) of the sequences showing the best hit to V. cholerae (Vc) and Escherichia coli (Ec) on different time points in children with cholera. The data is relative abundance in total gut microbiota of all the patients.

Figure 2

Dominant bacterial groups in the gut of children with acute cholera at the taxonomic level of phylum. Alphabets in the bar are described as follows: A, Actinobacteriaceae; B, Bacteroidaceae; E, Enterobacteriaceae; P, Prevotellaceae; R, Ruminococcaceae; S, Streptococcaceae; V, Vibrionaceae.

Figure 3

Venn diagram showing the shift of commensal and pathogenic bacteria of the top ten bacterial families (by relative abundance) during recovery process in the gut of children with cholera. Day 0, acute cholera; Day 2, antibiotics administrated; and Day 7 to Day 28, recovery period.

Figure 4

Comparison of relative abundance of dominant bacterial groups in children (n=9) with cholera on different time periods at the taxonomic level of family.

Figure 5

Relative abundance (percentage of sequences) of the dominant bacterial phyla in the gut of children with cholera on different time points.