The study investigated the possible effects of diarrhoea on the diversity of the gut microbiota in children with acute diarrhoea, using a culture-independent metagenomic tool—TTGE. The results presented herein demonstrate a transient reduction in the bacterial diversity that took place after the onset of acute diarrhoea in children. This concurs with results published in recent culture-based studies in which children with diarrhoea were shown to reduce the number of bacterial species (5
). The results of the present study are comparable with a cholera study in which the gut microbiota were analyzed in children suffering from acute cholera using TTGE, and the lower number of bands [7.7±2.6 (mean±standard deviation)] was observed, indicating the lower bacterial diversity in the acute cholera phase (24
In the present study, children suffering from diarrhoea received an appropriate rehydration therapy together with antimicrobial therapy and continued to be fed as per the guidelines of the World Health Organization. Although antimicrobials exert a beneficial effect by eliminating pathogenic agents, thereby controlling infections, the oral administration of antimicrobial agents often causes a shift in the intestinal microbial populations, with a decrease in anaerobic bacteria and a concomitant increase in aerobic bacteria (25
). In the present study, the bacterial diversity in diarrhoea patients, as reflected by the number of different TTGE bands, did not change significantly even after 24 hours of the initiation of antibiotic therapy. However, when the distance values between day 0 and day 1 were analyzed, significant alterations in the bacterial species pattern were observed, suggesting that the antibiotic therapy might have helped the resistant community to selectively flourish at the same time eliminating those microbiota that were sensitive to the drug used, although such shifting was not corroborated in the overall microbiota diversity in the gut as observed at day 0 and day 1.
In our study, no consistency was observed in the banding patterns that occurred in the majority of the patients at day 0 or at day 1, or the following days. The high inter-individual variation in the banding patterns that we observed at day 0 may presumably be due to the differences in their living conditions, health and immune status, and the genetic make-up. However, the band number at day 0 (acute diarrhoea) was enumerated and considered the baseline, and the changes occurring over time in the following days were taken into account as variation. Although a dominant band (bands parallel to E. coli
in the reference lane) reflecting selective proliferation of a particular species was observed during day 1-3, the overall TTGE profiles for the antibiotic therapy period were more or less static, except that the banding positions were not consistent for every patient that we tested. By contrast, this pattern changed again when the antibiotic therapy ended, and a significant bacterial diversity in terms of numbers and positioning of the dominant bands was observed at day 7. One possible reason for this observation could be that fewer bacterial species could have proliferated in the presence of antibiotics during the clinical period when the sensitive population must have been eliminated/suppressed. This supposition is supported by results of a recent culture-based study in which administration of ampicillin to humans was shown to induce β-lactamase production by various intestinal microorganisms, including Enterobacteriaceae and some members of the Bacteroides fragilis
group, which allowed simultaneous multiplication of some otherwise ampicillin-sensitive bacteria, such as Clostridium difficile
In the present study, the TTGE profiles of diarrhoeal and non-diarrhoeal children varied greatly in terms of species richness as revealed by the number of different TTGE bands representing different species. These differences in the numbers of bands were in agreement with the values published in an earlier study (29
) which analyzed the influence of host genetics on the predominant bacteria in the faecal microbiota of identical twins, fraternal twins, and unrelated pairs, aged four months to 10 years, showing that the numbers of TTGE bands varied from 8 to 28. The data of the present study may be further substantiated by results of another study in which non-cholera control subjects aged 24-32 months possessed TTGE bands that varied from 20 to 24, showing that these values were almost double when compared with the diversity of microbiota in the gut of children suffering from cholera (24
This study has limitations as the number of samples was low. Another limitation was the sampling timeframe because one week was not enough for the displaced microbiota to be restored in the gut of post-diarrhoeal children. We believe that follow-up of the patients for an extended period of time would be needed to get a clearer picture of status of the gut microbiota in post-diarrhoeal children. Although the identification of TTGE bands representing different species would be interesting, in the present study, the intensity of the majority of the bands was very low and was not suitable for the purpose of purification of DNA by cutting TTGE bands for sequencing and analyses. Again, the positions of bands varied greatly in day 1 to day 3; so, extensive cloning and sequencing of dominant bands during treatment were also not suitable.
The results of this study clearly showed that the bacterial diversity of the gut microbiota was significantly reduced during acute diarrhoea and that the diversity did not reach the level of non-diarrhoeal control children after one week. It is presumed that nutritional and appropriate therapeutic intervention during the post-diarrhoeal phase might be beneficial for achieving a higher magnitude of the diversity of the gut microbiota similar to that found in non-diarrhoeal children in a shorter duration.