Resumen de Detection and Verification of the Viable but Nonculturable (VBNC) State of Escherichia coliO157: H7 and Listeria monocytogenesUsing Flow Cytometry and Standard Plating
George Kwabena Afari, Yen-Con Hung
The use of electrolyzed oxidizing (EO) water to inactivate microorganisms on foods has been extensively studied and shown to be effective. However, the prospect of the formation of “viable but nonculturable” (VBNC) cells in pathogens after low free chlorine concentration (FCC) treatments under high organic loads presents safety concerns. This study investigated the effect of EO water FCC on inducing Escherichia coliO157:H7 and Listeria monocytogenesinto the VBNC state and studied possible resuscitation triggering procedures of the VBNC cells. A 5‐strain cocktail of each pathogen (106colony forming units [CFU]/mL) was exposed to EO water (FCC of 20, 10, 5, 2.5, 1.25, 0.625 mg/L) and allowed to stand for 1 and 5 min, followed by the addition of neutralizing broth. Treated samples were plated on nonselective agar and analyzed using flow cytometry. For resuscitation, samples treated with identified VBNC induction conditions were exposed to elevated temperatures (37 °C) as well as addition of sodium pyruvate (SP) and Tween® 20 (T20) solutions. The initial culturing procedures suggested complete inactivation of both pathogens at 2.5 and 1.25 mg/L FCC in the growth medium. However, flow cytometry profiles showed VBNC cells were present. Subjecting samples to the recovery procedures further proved that VBNC E. coliO157:H7 can be resuscitated after exposure to SP and T20 at 37 °C, while L. monocytogenesdid not resuscitate. These findings show that treating pathogens at low FCC can induce the VBNC state, and culturability of E. coliO157:H7 can be restored under appropriate conditions. VBNC induction conditions for foodborne pathogens during chlorine washing treatment were determined in a broth system and the information can serve as a basis for future studies that address the prevention of VBNC formation during produce wash treatments.
