Academic journal article Journal of Health Population and Nutrition

16S rRNA Gene-Targeted TTGE in Determining Diversity of Gut Microbiota during Acute Diarrhoea and Convalescence

Academic journal article Journal of Health Population and Nutrition

16S rRNA Gene-Targeted TTGE in Determining Diversity of Gut Microbiota during Acute Diarrhoea and Convalescence

Article excerpt

INTRODUCTION

The human gastrointestinal tract consists of an extremely complex ecosystem of a diverse microbiota, which is a positive health asset for humans and plays a vital role in the healthy functioning of their human hosts. The gut habitat is highly anoxic, and anaerobic bacteria outnumber the aerobic bacteria by a factor between 103and 105 (1). Evidence suggests that these commensal microorganisms have important metabolic roles, trophic functions, and protective effects. Moreover, these microbiota exhibit a series of important enzyme activities (2). During the onset of diarrhoea, the anaerobic environment is disrupted due to the high rate of purging, and the commensal microbiota are expelled and replaced by aerobic and pathogenic bacteria. Previous studies have examined the gastrointesti- nal microbiota in acute cholera (3,4) and in acute diarrhoea cases (5-7), using conventional culture techniques, microscopy, and biochemical tests, showing that the faecal anaerobic bacteria were significantly reduced in number during acute disease leading to aerobic bacteria to increase several folds. The major anaerobic bacterial group, including Bacteroides, Clostridium, Bifidobacterium, Lactobacillus, and Eubacterium, was found 3-4 times lower in acute cholera and diarrhoea patients. Therefore, the restoration of appropriate commensal gut micro- biota, which is the key to recovery following acute diarrhoea, is very important.

The predominant species of human intestinal microbiota are non-culturable because of their obligate anaerobic and extremely oxygen-sensitive nature. Besides, due to the complex nutritional requirements and lack of beneficial effects that they receive from complex host-microbe and microbe- microbe interaction, recent estimates of culturability of bacteria range from only 15% to 58% in the laboratory even when excellent bacteriological culture methods are used (8,9). The metagenomic technique such as temporal temperature gradient gel electrophoresis (TTGE) is an excellent new tool for the analysis of the gut microbiota diversity without depending on the culture method. In TTGE, the variable regions of the 16S rRNA gene are amplified using universal primer PCR, and the amplified DNAs are then separated based on sequence specificity to determine the microbial diversity in the human gut (10-12). Currently, the molecular techniques are being commonly employed to determine the community dynamics in the diverse ecosystems, such as biofilms (13), soils (14), ocean depths (15), hot springs (16), fermented foods (17), and the gut of humans and other animals (18,19).

Many factors, such as age, geographic locations, diets, pH, and bile acid, including intestinal infections, determine the nature and composition of resident bacterial populations in the colon (20). The designing of an appropriate therapeutic intervention involves a clear understanding of the composition of microbiota and their metabolic functions in the gut. In acute diarrhoea, the gut of patients is washed out due to purging and frequent loss of loose stools. As a result, the gut commensal microbiota are likely to be less abundant than those of age-matched healthy children. The aim of the study was to analyze faecal samples of children to determine the extent of loss of gut bacteria during acute diarrhoea and convalescence, using a culture-independent molecular tool-TTGE.

MATERIALS AND METHODS

Patients

The study was conducted among children suffering from diarrhoea admitted to an ongoing clinical study of icddr,b during August 2008-May 2009. The children were aged 7-18 months (median 12 months). Faecal samples were collected from 21 children at day 0 (after enrollment and before any medication), at day 1 (after 24 hours), day 2, day 3, and day 7. As a standard management, they received glucose-based oral rehydration solution for rehydration and also received the usual antibiotic therapy, i.e. a parenteral ampicillin dose of 100 mg/ kg in four to two equally-divided doses for five days. …

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