In acute cases of E coli diarrhea

In acute cases of E. coli diarrhea, the barrier function of the gut mucosa in the small intestine is compromised. When the pathogen is lysed by phage, in vivo released endotoxins and enterotoxins can thus potentially cross into the circulation. However, no systemic reaction to endotoxin was observed (Yang et al., 2010) which is not surprising since no significant in vivo replication of the phage was seen, hence no significant lysis of EZ Cap Reagent GG with concomitant toxin release had occurred. While oral coliphages as virions are safe in diarrhea patients, it is still unclear whether oral phages are safe when acting as infectious virus lysing the E. coli target cell in the gut of diarrhea patients. Neither the T4-like nor the Russian coliphage cocktail showed a clinical benefit over standard care in terms of stool output, stool frequency or rehydration. This observation should be interpreted with caution since it could still represent a false-negative outcome because at interim analysis only half of the planned patients were enrolled. The reported trial was thus not powered to detect a treatment effect, but only to assess safety issues and to gain a mechanistic insight into PT. Notably, we did not observe a substantial in vivo replication of the orally applied phage which is a basic requirement for PT. Despite a careful screening, only 60% of the 120 enrolled patients harbored an E. coli pathogen in the stool. E. coli is thus a less frequent cause of diarrhea (18% of all screened patients) than we had anticipated for Bangladeshi children (36% of all screened patients) (Albert et al., 1999). Furthermore, even in microbiologically defined E. coli diarrhea less than 5% of the total fecal bacteria were E. coli. When only ETEC pathogens were counted, a median titer of 105cfu ETEC per g stool was detected. With an estimated in vivo burst size of 10 (Brüssow, 2013), no phage amplification beyond 106 pfu/g stool was therefore expected, which is close to the observed peak fecal phage titers. That a microbiologically defined E. coli diarrhea was not characterized by high intestinal pathogen titers can also be deduced from the observation that ETEC and EAEC infections in the present study showed only 10-fold higher fecal E. coli titers than diarrhea patients lacking an E. coli pathogen. Likewise, careful quantitative PCR analysis in stools from Peruvian children with EPEC diarrhea showed titers of 3×105EPEC/g stool which were just 10-fold higher than EPEC titers in healthy controls (Barletta et al., 2011). Since the in vitro replication threshold for T4 phage on a laboratory indicator E. coli strain was determined to 103cfu/ml (Wiggins and Alexander, 1985), the pathogenic E. coli titer in confirmed cases of E. coli diarrhea might simply not be high enough to sustain phage replication in vivo. From our data it is impossible to predict what percentage of the diarrheal episodes is caused by E. coli. Only a minority of the screened children had an E. coli diarrhea by standard tests. Even less showed a high fecal abundance of E. coli by molecular tests and stool titrations. We cannot deduce from our data whether a low abundance bacterial group could be responsible for diarrhea in children. However, to overcome limitations for future phage therapy trials, we strongly recommend to control first and foremost whether the targeted pathogen occurs with sufficient titers to support in vivo phage replication. There are still other limitations which have to be considered when interpreting the negative clinical outcome of the present study. Only half of the patients contained phage-sensitive E. coli colonies in the stool. The Russian phage cocktail was obviously not customized to the regional patients and we had to adapt our T4 phage cocktail to the ecological situation in Bangladesh to achieve a reasonable coverage (Bourdin et al., 2014b). Since the phages were isolated several years before the start of the trial, a constant adaption of the phage cocktail to the prevailing ecological situation is needed. However, it is not clear whether phage coverage was indeed the limiting factor in the current trial since the phage susceptibility of the stool isolates did not correlate with fecal phage titers. Both phage preparations were given without antacid. Buffering of gastric acidity by a slightly alkaline mineral water was perhaps too weak and an additional dose of bicarbonate as recommended by the commercial supplier might have increased phage transit through the stomach. Since it would also increase the risk of nosocomial cross-infection in a diarrhea hospital, such a treatment was excluded for ethical reasons. However, we do not believe that pharmaceutic formulation of the phage was a limiting factor in the current trial since children have a higher (more alkaline) stomach pH than adults. Indeed, in previous trials sensitive biologicals were given to children with diarrhea (antibodies, probiotics), which were recovered intact from the feces (Hilpert et al., 1987) or achieved clinical effects in Bangladesh (Sarker et al., 1998, 2005). However, a higher oral phage dose might be desirable since children in Bangladesh showed a less good fecal bioavailability of phage than adults from Switzerland (Bruttin and Brüssow, 2005) suggesting some loss during gastrointestinal passage.