Development of a high-resolution multiplex qPCR method to profile microbial consortia in spaceflight water recovery systems

A key component of life support on the International Space Station (ISS) is the Water Recovery System (WRS), which recycles and disinfects urine, other wastewater, and humidity condensate for use as potable water. A resident mixed-species bacterial population has persisted in the WRS, upstream from the disinfection components, despite various microbial control methods. Five bacterial species (<i>Pseudomonas aeruginosa, Burkholderia contaminans</i>, <i>Methylobacterium fujisawaense, Ralstonia insidiosa,</i> and <i>Cupriavidus metallidurans</i>) have been regularly isolated from the WRS and have a propensity to form biofilms, which can reduce susceptibility to antimicrobial treatments. WRS organisms have been associated with biofouling and potential corrosion of valves and filtration components. Currently, microbial monitoring on the ISS requires samples to be collected and sent back to Earth for culture-based analysis, a process that can take months, given the constraints of spacecraft scheduling. Culture-independent monitoring could be established on the ISS and other spacecraft, thereby enabling rapid estimates of microbial population density and individual species' susceptibility to disinfection. This technology will be critical in future missions beyond low Earth orbit, when access to reference labs is not possible. Here, we report on the development and validation of a multiplex quantitative Polymerase Chain Reaction (qPCR) method to identify and quantify members of a five species model biofilm community acquired from the ISS. Species-specific primers and probes detect organisms to an estimated detection limit of 10⁴-10⁶ CFU and detect the specific targeting of <i>C. metallidurans</i> by ampicillin when overall population levels were not significantly changed.