Oxygen restriction induces a viable but non-culturable population in bacteria

This study examined whether anoxic conditioning induces viable but non-culturable (VBNC) bacteria during biofilm growth and if reactive oxygen species (ROS) contribute to this loss of culturability. A significant subpopulation of VBNC Pseudomonas aeruginosa was induced by anoxic conditioning, ranging from 5 to 90 % of the total population, in both planktonic and biofilm models. Anoxic conditioning also induced VBNC subpopulations of Staphylococcus aureus and Staphylococcus epidermidis. Resuscitation of the VBNC population was achieved by substituting 10 mM NO3- as an alternative electron acceptor or, in the case of P. aeruginosa, by adding sodium pyruvate as a ROS scavenger during normoxic incubation. Bacterial detection in clinical samples was improved by supplementing 10 mM NO3- to LB plates and incubating under anoxic conditions. These results demonstrate that habituation to an infectious anoxic micro-environment complicates diagnostic culturing of bacteria, especially in the case of chronic infections where oxygen is restricted due to the host immune response.