Gut microbiota play an important role in nutrition and healthy living of children. In this study, altered bowel movement particularly high purging in acute watery diarrhea due to cholera was found to be a basis for both qualitative and quantitative changes in the gut microbiota of affected children. Data presented in this study clearly showed that cholera partially washes out the gut resulting in loss of important commensal bacteria, which presumably creates room for harmful proteobacteria to colonize in disproportionately larger proportion and settle down in the gut, a scenario that we have observed in malnourished children of Bangladesh (Monira et al., 2011).
A wide variety of microorganisms including some human pathogens do not respond to artificial media when culturing methods are employed. Although non-culturable state has been established as a survival strategy, a great majority of intestinal microbiota are obligate anaerobic and extremely oxygen-sensitive in nature. Because of the complex nutritional requirements, recent estimates of in vitro culturability of bacteria were shown to range from 15 - 58% [10, 11]. In the present study, we investigated bacterial community dynamics in fecal samples of children suffering from acute watery diarrhea (cholera) using culture-independent molecular (metagenomic) tools. Pyrosequencing and analyses of the amplified 16S rDNA allowed us to estimate the relative abundance of major bacterial community in gut. As previously demonstrated, this is a well-adapted method to detect pathogenic agents of diarrhea  as well as to determine the commensal gut microbiota in well nourished and malnourished children . As observed in the present study, during acute phase of infection, V. cholerae was found to be the predominant species, followed by some other bacterial species, which were less abundant and may presumably be due to expulsion of the major gut microbiota resulted by purging in cholera. This appears to be in agreement with data presented by several culture-based studies (aerobic and anaerobic) showing the reductions of bacterial species in watery stools of severely purging adult patients with cholera [7, 14].
It is now widely understood that the gut bacteria can influence the energy absorption from foods and the state of health [15, 16]. Although information about strictly anaerobic (commensal) microbiota in the gut of children with acute watery diarrhea (cholera) is lacking, according to a recent quantitative PCR-based study , bacteria belonging to Bacteroides-Prevotella-Porphyromonas group, Eubacterium rectale, L. acidophilus and F. prausnitzii groups were low during diarrhea compared to recovery state. Moreover, Bacteroides, Bifidobacterium, Lactobacillus, and Eubacterium were shown to be less abundant in gut during diarrhea [9, 18]. The data presented in this study demonstrate a clear picture of the gut bacterial communities in cholera and its recovery process, showing a decreasing trend of the bacteria belonging to the family Bacteroidaceae, Prevotellaceae, Bifidobacteriaceae, Clostridiaceae, Faecalibacteriaceae, Megamonas, Megasphaera etc., in children suffering from cholera. This is presumed to be due to the observed overgrowth of the invading pathogen, V. cholerae, and the resulted alteration of the strictly anaerobic environment that prevails in the normal human gut.
Cholera patients are routinely treated with a 1–3 day course of effective antibiotics [19, 20] along with rehydration therapy, to shorten illness and reduce both rapid water loss [19, 21] and the period of V. choleare excretion. However, antimicrobial agents often inhibit sensitive population causing shift in the intestinal microbial populations, with a decrease in anaerobic bacteria and a concomitant increase in aerobic bacteria [22, 23]. Likewise, the relative abundance of four strictly anaerobic bacteria namely Bacteroides, Clostridium, Faecalibacterium, and Bifidobacterium, which were found in the gut of six children during acute cholera in the present study, reduced significantly during the period of antibiotic therapy (data not shown). Antibiotics namely, cephalosporins and some penicillins were shown to affect the gut microbiota and facilitate colonization of the gut by Enterococcus especially the ones that are resistant to vancomycin . Although the antimicrobial susceptibility pattern of gut microbiota was not determined in the present study, an increase of the Enterococcus was evident during antibiotic receiving period when E. coli flourished rapidly and became the most abundant bacteria, suggesting the role of antibiotic in facilitating the resistant lineages of E. coli to selectively colonize the gut, as shown recently in pigs fed with performance-enhancing antibiotics . The other possibility of this sudden increase of E. coli and Enterococcus at day 2 might be that the total bacterial number in the gut, including these two groups of bacteria, decreased during administration of antibiotics, but the abundance of E. coli and Enterococcus increased relatively because they were either less or not susceptible to those antibiotics than other gut bacteria.
We observed substantial inter individual variations of the bacterial genera in children with cholera from day 0 to the end of the study period. These children were randomly selected from those attending the outpatient department of the Dhaka Hospital and on the basis of inclusion criteria, acute watery diarrhea (cholera). They came from different localities and were consuming different diets. As diet and metabolism have a profound relation with indigenous bacterial populations [26–28], it is possible that these children might possess individual microbiota inventory related to the nature of diets. In our study we have observed that only three bacterial genera e.g., Vibrio, Escherichia, and Streptococcu s were common in all 9 children during acute cholera when complete data set was analyzed. Although Escherichia and Streptococcus were present among the gut bacterial community after one month, their relative abundance was very low compared to other bacteria namely, Bifidobacterium, Veillonella, Faecalibacterium, Eubacterium, Prevotella, Ruminococcus, Clostridium, Enterococcus, and Megamonas, which were the most abundant gut microbiota in all tested children at day 28. Now the question is, why the type and abundance of restored gut microbiota were so divergent among the hospitalized children, considering that they received similar care in terms of volumes of rehydration solution and type of semisolid and solid foods. Besides, the prolonged presence in trace amount of pathogenic Vibrio among the commensal bacteria of two children during the process of recovery at day 28 was interesting in the present study. Our study was limited up to 28 days, and so, it is not understood whether the pathogenic Vibrio in the two children eventually settled down as commensal among the gut bacteria. If this occurs, it is plausible to propose healthy human carriers for cholera and other pathogenic bacteria, although further study is needed to understand more about this. Nevertheless, the floral diversity observed in the post-cholera children in the present study suggests that the type of colonic flora may be determined by as yet unknown factor(s) that differ between individuals.
Since it is becoming increasingly evident that gut microbiota help prevent disease [29, 30] and have profound influence on nutrition and health , the observed lower abundance of anaerobic bacteria in the gut of children during cholera and convalescence might have negative impact on post-cholera recovery process. This is presumable because each of the component bacteria of the gut community has their important role to play namely by digesting food that allows their host to optimally absorb nutrients and energy. Therefore, the observed lower abundance of major commensal bacteria during cholera and convalescence might affect the normal growth of children leading to growth stunting, considering that the bacterial diversity in the gut is positively linked with the status of health . The results of the lower abundance of major commensal bacteria observed during cholera and convalescence in the present study appear in agreement with our previous study in which we observed that the concentration of short chain fatty acids (SCFAs), the metabolic products of gut bacterial fermentation, was significantly lower in children suffering from cholera . The major limitation of the present study may be that, we worked with a small number of children with cholera. Nevertheless, the data presented in this study on metagenomes of the gut using high throughput pyrosequencing is the first of its kind, which provide a snapshot of the bacterial community in children during cholera and its recovery. While future study designed with a large cohort of cholera patients of different age groups remains an important area of interests, post-cholera intervention study to accelerate the restoration of commensal gut microbiota seems to be of great public health significance.