Sensitive detection of viable Cronobacter sakazakii by bioluminescent reporter phage emitting stable signals with truncated holin

As outbreaks of foodborne illness caused by the opportunistic pathogen Cronobacter sakazakii (Cs) continue to occur, particularly in infants consuming powdered infant formula (PIF), the need for sensitive, rapid, and easy-to-use detection of Cs from food and food processing environments is increasing. Here, we developed bioluminescent reporter bacteriophages for viable Cs-specific, substrate-free, rapid detection by introducing luciferase and its corresponding substrate-providing enzyme complex into the virulent phage ΦC01. Although the reporter phage ΦC01_lux, constructed by replacing non-essential genes for phage infectivity with a luxCDABE reporter operon, produced bioluminescence upon Cs infection, the emitted signal was quickly decayed due to the superior bacteriolytic activity of ΦC01. By truncating the membrane pore-forming protein holin and thus limiting its function, the bacterial lysis was delayed and the resultant engineered reporter phage ΦC01_lux_Δhol could produce a more stable and reliable bioluminescent signal. Accordingly, ΦC01_lux_Δhol was able to detect at least an average of 2 CFU/ml of Cs artificially contaminated PIF and Sunsik and food contact surface models within a total of 7 h of assays, including 5 h of pre-enrichment for Cs amplification. The sensitive, easy-to-use, and specific detection of live Cs with the developed reporter phage could be applied as a novel complementary tool for monitoring Cs in food and food-related environments for food safety and public health.