FIGURE 5.

Challenges remain to the wide-spread application of engineered probiotics. (A) Engineered probiotics must compete with the resident microbial species for nutrients to replicate and then act on pathogens. To compete effectively they must be as well adapted as the resident microbes but must not contribute to overwhelming the resident microbiota. The killing of pathogens must be precise to avoid off-target killing of resident microbiota which would increase dysbiosis. This imposes the limitation that the pathogen must be specifically identified before the application of the engineered probiotic. (B) It is likely that variants of any pathogen will arise that can avoid detection or develop resistance to the mechanism of killing used by the engineered probiotic. Biofilms that are formed by a variety of pathogens that colonize the gut pose a challenge for both detection and killing of the pathogen. (C) Administration of live-cell probiotic vaccines presents the concern of tolerant pathogens resistant to immune cell responses from vaccination. Live cell probiotics can also be recognized by immune cells as foreign microbes and targeted for cell death, thereby decreasing their viability and vaccine actions. (D) Containment of an engineered probiotic is important as it is difficult to predict how the organism will act in different environments and there could be unexpected effects on animals, insects or plants for which the probiotic was not engineered. Even within the planned host environment transfer of genetic material to other organisms is a concern as this can occur through conjugation and direct transfer to bacteria as well as through phage mediated transduction and natural DNA uptake occurring upon cell lysis for any reason.