In Vietnam, diarrhea, especially persistent diarrhea, is one of the most common diseases in children, while a significant proportion of cases are negative with pathogens; thus, there is an urgent need to understand gut bacterial dysbiosis. In this study, bacteria in the fecal samples of five healthy and ten diarrheal children were separated from other residues, then adopted to extract their metagenomic DNA for evaluating their diversity based on V3 and V6–V8 regions and the 16S rRNA gene by PCR-RFLP and PCR-DGGE. As a result, bacterial metagenomic DNAs with high quality, quantity and diversity were successfully extracted using a GeneJET kit and a chemical protocol. A sequence analysis of 73 representative DNA fragments from gels indicated a remarkable bacterial dysbiosis in all groups of diarrhea. Viral diarrhea was characterized by extremely reduced bacterial diversity with the blossom of Bifidobacterium and Streptococcus. Streptococcus was also the most abundant in persistent diarrhea. Beneficial bacteria that may play a role in the self- rebalance in intestinal bacterial communities, such as Bifidobacterium, Lactobacillus, and Enterococcus, were seen in all diarrheal groups, while Bacteroides and Akkermansia muciniphila were seen in the healthy group but absent in the diarrheal groups. This study provides additional evidence for a relationship between intestinal bacterial dysbiosis and diarrhea in children, emphasizing an increase in Streptococcus.

Persistent diarrhea remains a significant cause of morbidity in young children, yet the role of gut microbiota has not been fully clarified. This prospective study evaluated the diversity and predicted functions of the gut microbiota in 30 children aged 6–24 months with persistent diarrhea of unknown etiology (patient group, PG) and 30 healthy controls (healthy group, HG). Nearly full-length 16S rRNA genes from fecal bacterial metagenomic DNA were sequenced and taxonomically annotated. Subsequently, all downstream analyses, including diversity assessment, differential abundance and functional prediction analyses, and data visualization, were performed using R software (version 4.5.0, 2025). The PG showed lower Shannon and higher Simpson indices than the HG (p < 0.05), reflecting reduced microbial diversity. At the phylum level, Firmicutes predominated in the PG, whereas Actinobacteriota, Bacteroidota, and Verrucomicrobiota were more abundant in the HG (|log₂FC| > 1 and FDR < 0.05). At the genus and species levels, the PG exhibited a marked depletion of essential commensals such as Bifidobacterium longum, Faecalibacterium, Lactobacillus, and Eubacterium, alongside an enrichment of opportunistic taxa including Klebsiella, Enterococcus lactis, and Streptococcus spp. (FDR < 0.05). Functional predictions using PICRUSt2 indicated an enrichment of carbohydrate metabolism and reductions in amino acid metabolism, B-vitamin pathways, and the biosynthesis of endogenous antibiotics (FDR < 0.05). These findings suggest that the PG harbors a dysbiotic gut microbiota characterized by reduced diversity, depletion of key commensal taxa, expansion of opportunistic bacteria, and potentially adverse shifts in metabolic functions.

Persistent diarrhea of unknown etiology in children under 2 years of age is a common problem and poses a major challenge for the health sector. However, knowledge of the composition and dysbiosis of the intestinal phageome, phage-associated bacteriome in the persistent diarrhea remains limited. In this study, a process for phage enrichment and metagenomic extraction was developed and applied to recover gut phage metagenomes from 30 healthy children and 30 children with persistent diarrhea for high-throughput sequencing. Taxonomic annotation using Kraken2 revealed that, besides Norwalk virus, Primate bocaparvovirus 1 and Human-associated gemykibivirus 2, phage communities in the diarrhea group showed reduced diversity and contained sample-dependent phages targeting Salmonella enterica, Enterobacter, Shigella flexneri, Clostridioides difficile, Pseudomonas aeruginosa, Streptococcus miti, uropathogenic Escherichia coli and functioned balancing bacterial communities. Bacterial fraction in the metagenomic datasets reflected clear patterns of dysbiosis, including a severe deficiency of beneficial bacteria, an increase in Firmicutes, a marked decline in Actinobacteria, Bacteroidetes, Proteobacteria and sample-dependent enrichment of Enterococcus, Escherichia and Acinetobacter in diarrhea cases. This study, for the first time, investigated the dynamics of gut phageome, phage-associated bacteriome in children with persistent diarrhea of unknown causes in Vietnam, providing new insight for complementary treatment.

Acute diarrhea is a common disease in children under 5 years old and can develop into persistent diarrhea, greatly affecting the children's health. Although advanced techniques had been used to diagnose and detect common pathogens in hospitals, however, 40% of cases are negative for the pathogens. In this study, to investigate dominant bacteria in stool samples of three persistent-diarrheal children with unidentified pathogenic agents, the V3 and V68 regions of the 16S rRNA gene were amplified from fecal bacterial metagenomic DNA, separated on DGGE gel, and the dominant DNA bands were sequenced. As a result, the V3 and V68 regions of bacteria in persistent diarrheal children were less diverse and different from the corresponding DNA bands of the indicator strains. Sequence analysis and similarity comparison of six DNA bands of V3 regions and seven DNA bands of V68 regions showed that two V3 sequences (of 160 bp) derived from two samples were novel and did not match any genes from the non-redundant database, but they shared 93.75% similarity to each other. The four V3 sequences left derived from all three samples were the most similar (94.53-100%) with the corresponding genes of Brevibacterium. Six of the seven V68 sequences derived from dominant DNA bands of all three samples were the most similar (from 99.4% to 100%) to the corresponding genes of referent strains belonging to the genus Enterococcus. In sample D3, a sequence of the V68 region possessed 100% identity to the E. faecalis ATCC 19433 strain. This is the first study report that Brevibacterium was the dominant bacteria in the gastrointestinal microflora of children with persistent diarrhea although the bacterial genus has been reported to cause dangerous diseases in humans with immunodeficiency.