Introduction: We previously demonstrated that phosphate [Pi] depletion develops within the mucus layer of the distal intestine in mice during surgical injury and activates a lethal phenotype in Pseudomonas aeruginosa via conserved phosphate response regulation. The aims of the present study were to determine whether other intestinal pathogens also activate their virulence in response to [Pi] depletion and to determine if they display molecular evidence of exposure to [Pi] depletion in vivo.

Methods: Stool samples were consecutively collected from 12 ICU patients yielding 60 microbial isolates that were identified by culture and profiled for antibiotic resistance. Isolates were then tested for their ability to express virulence in response to [Pi] limitation using C. elegans nematodes as a screening tool. C. elegans, which feed on microbes as their main food source, were transferred onto high cell density microbial lawns growing on high (25 mM) and low (0.1 mM) phosphate media and tracked for known behavioral responses associated with microbial virulence expression such as colony avoidance, reduced progeny formation, and mortality. We also performed electron microscopy of recovered strains to identify if phosphate sensing protein PstS was expressed on bacterial membranes as a function of phosphate scavenging under phosphate limited conditions. Finally to assess if microbes are exposed to low [Pi] when present in the human intestine during critical illness, human stool filtrates were assayed by ELISA for bacterial proteins involved in phosphate sensing (i.e PstS).

Results: Pathogens typically associated with gut derived sepsis were isolated from stool of critically ill patients including C. albicans, K. pneumoniae, P. aeruginosa, and S. marcescens. There was predominance (~70%) of multi-drug resistance (MDR) among isolates. Phosphate dependent mortality against C. elegans was observed for all pathogens isolated, with C. albicans and MDR P. aeruginosa displaying the greatest killing effect (p<0.01, data not shown). Nematodes fed on phosphate depleted lawns of MDR K. pneumoniae and S. marcescens expressed colony avoidance behavior and reduced progeny formation with both phenotypes disappearing when bacterial strains were grown under [Pi] sufficient conditions (p <0.001, data not shown). PstS was identified to be present on outer membrane surface in certain bacterial strains recovered from stool of ICU patients demonstrating the overactivation of phosphate-scavenging system. Finally proteins found to be secreted by isolated strains of P. aeruginosa under low Pi conditions (PstS PA5369, PstS PA0688, PA0681) were detected to be several fold increased in the stool filtrates of critically ill patients providing compelling evidence that intestinal pathogens are exposed to low [Pi] in vivo.

Conclusion: These data provide the first clinical evidence that, despite aggressive daily protocols to prevent and treat hypophosphatemia with supplemental phosphate, the intestinal tract of critically ill patients can be depleted of phosphate- a condition which has the potential to shift a wide variety of its colonizing flora to express enhanced virulence and a potentially lethal phenotype. Strategies to maintain local intestinal phosphate sufficiency during critical illness may have important therapeutic non-antibiotic implications for infection/sepsis prevention.