Regarding the lack of in

Regarding the lack of in vivo susceptibility of E. coli to coliphages, several factors need consideration. Prominent are physical barriers for phages to reach E. coli (increased peristalsis in diarrhea, reduced phage diffusion rate in mucus layer, pathogen protection in biofilms) or slow growth rate of bacterial rsv in the stationary phase (Brüssow, 2013). It will be important to study E. coli-phage interaction in vivo with animal experiments. However, E. coli does not induce diarrhea in small laboratory animals and infections with Citrobacter rodentium in mice, commonly used as a surrogate, are not a suitable model system since it induces a colitis (Collins et al., 2014). Experiments in animals like piglets or calves which suffer naturally from E. coli infection in the small intestine and diarrhea (Fairbrother et al., 2005) are clearly needed. Another important limitation of our study is the question whether our observations in the fecal samples represent what is happening within the gut. From an analytical viewpoint it would be desirable to analyze bacteria and phages in samples from the upper small intestine. ETEC and EPEC infections occur in the upper parts of the gut and the bacteria excreted with stool might not any longer be in the same growth phase as in the small intestine (stationary vs. logarithmic growth). However, such sampling is invasive and ethically difficult to justify in a normally self-limiting disease in children. Again, dissection experiments in experimentally infected and phage-treated pigs might provide the needed insight. In fact, data from pig have supported the “kill-the-winner” model of phage–bacterium population dynamics also for the gut ecosystem (Allen et al., 2011) suggesting that high titers of target bacteria are needed for PT. ETEC showed only a short-lived stool peak titer which corresponds to recent reports describing in adult cholera patients from Bangladesh an even shorter peak titer of V. cholerae maintained for just 6h (Hsiao et al., 2014). Curiously, the peak titer of ETEC occurred on the second day of hospitalization as if ETEC was not the cause for hospital admission. In fact, the role of E. coli as a pathogen has recently been challenged for its low pathogenicity index. Only certain E. coli pathotypes in selected age groups of children and geographical areas were by this criterion significantly linked with diarrhea (Taniuchi et al., 2013; Liu et al., 2014; Kotloff et al., 2013). In fact, E. coli diarrhea in children from developing countries is increasingly considered as a polymicrobial infection (Taniuchi et al., 2013; Kotloff et al., 2013) as was also seen in the present study. Microbiota analysis by 16S rRNA gene sequencing confirmed that only a small fraction of the stool microbiota from children hospitalized with a microbiologically diagnosed E. coli diarrhea were represented by Escherichia. At admission, the dominant stool bacteria were fecal streptococci independently whether ETEC, EAEC or non-E. coli diarrhea cases were considered. Comparison with healthy local controls suggests a marked streptococcal dysbiosis in the acute phase of diarrhea. Notably, the relative abundance of streptococci was positively correlated with stool output, it predicted the length of diarrhea duration and in EAEC or non-E. coli diarrhea it decreased with the resolution of diarrhea. Is a fecal streptococcal dysbiosis thus a potential biomarker for diarrhea? Since all these observations apply to the average of the studied population and not to individuals, who showed great variation, this marker is of limited use for the clinical microbiologist. However, fecal streptococci diagnosed as S. salivarius also represented a prominent part of the acute diarrhea microbiota in adult cholera patients from Bangladesh (Hsiao et al., 2014). E. coli diarrhea in adults and children from Bangladesh showed an ordered gut microbiota succession and fecal streptococci belonged to this sequence (David et al., 2015). In the GEMS survey children from Bangladesh displayed a significant increase in fecal streptococci during diarrhea. This was, although at lower level, also observed in African children (Pop et al., 2014). Are fecal streptococci therefore candidate new enteropathogens as suggested by Chinese researchers (Jin et al., 2013)? This is unlikely for four reasons: first, using effluents from ileostoma patients, fecal streptococci of the S. bovis complex have been identified as typical small intestine commensal in humans (Booijink et al., 2010). Fecal streptococci are fast-growing, efficient fermenters of simple carbohydrates (van den Bogert et al., 2013) possibly allowing a transient outgrowth when competing gut bacteria are purged from the gut by increased peristalsis in diarrhea. Second, no diarrhea-relevant virulence genes were detected in the three sequenced genomes of fecal streptococci isolated from diarrhea patients. Notably, a biologically proven virulence (pilus) gene identified in a S. gallolyticus isolated from an endocarditis patient (Danne et al., 2011) was not detected in the stool isolates. Third, an increased abundance of S. gallolyticus was also seen in colon cancer patients (Boleij and Tjalsma, 2013). Bacteria of the S. gallolyticus species complex might adhere to the chronically (colon cancer) or transiently (diarrhea) injured gut mucosa. Interestingly, the replacement of the streptococci by fecal bifidobacteria occurs with the same kinetics as the repair of the gut mucosa with newly synthesized enterocytes. Finally, the analysis of the fecal microbial community types showed that elevated fecal Streptococcus abundance had no pathogenic potential as long as they are counter-balanced by an elevated Bifidobacterium abundance. This could mean that our understanding of diarrhea etiology will have to change in the future: not only need quantitative pathogen titers to be determined, pathogen constellations and pathogens interactions with commensal bacteria need to be considered for the understanding of diarrhea. In our dataset, loss of bifidobacteria was in fact the best biomarker for diarrhea.