Engineered Reporter Phages for Rapid Bioluminescence-Based Detection and Differentiation of Viable<i>Listeria</i>Cells

Abstract

The pathogen <i>Listeria monocytogenes</i> causes listeriosis, a severe foodborne disease associated with high mortality. Rapid and sensitive methods are required for specific detection of this pathogen during food production. Bioluminescence-based reporter bacteriophages are genetically engineered viruses that infect their host cells with high specificity and transduce a heterologous luciferase gene whose activity can be detected with high sensitivity to indicate the presence of viable target cells. Here, we use synthetic biology for <i>de novo</i> genome assembly and activation as well as CRISPR-Cas-assisted phage engineering to construct a set of reporter phages for the detection and differentiation of viable <i>Listeria</i> cells. Based on a single phage backbone, we compare the performance of four reporter phages that encode different crustacean, cnidarian, and bacterial luciferases. From this panel of reporter proteins, nanoluciferase (NLuc) was identified as a superior enzyme and was subsequently introduced into the genomes of a broad host range phage (A511) and two serovar 1/2- and serovar 4b/6a-specific <i>Listeria</i> phages (A006 and A500, respectively). The broad-range NLuc-based phage A511::<i>nluc</i>_CPS detects one CFU of <i>L. monocytogenes</i> in 25 g of artificially contaminated milk, cold cuts, and lettuce within less than 24 h. In addition, this reporter phage successfully detected <i>Listeria</i> spp. in potentially contaminated natural food samples without producing false-positive or false-negative results. Finally, A006::<i>nluc</i> and A500::<i>nluc</i> enable serovar-specific <i>Listeria</i> diagnostics. In conclusion, these NLuc-based reporter phages enable rapid, ultrasensitive detection and differentiation of viable <i>Listeria</i> cells using a simple protocol that is 72 h faster than culture-dependent approaches.<b>IMPORTANCE</b> Culture-dependent methods are the gold standard for sensitive and specific detection of pathogenic bacteria within the food production chain. In contrast to molecular approaches, these methods detect viable cells, which is a key advantage for foods generated from heat-inactivated source material. However, culture-based diagnostics are typically much slower than molecular or proteomic strategies. Reporter phage assays combine the best of both worlds and allow for near online assessment of microbial safety because phage replication is extremely fast, highly target specific, and restricted to metabolically active host cells. In addition, reporter phage assays are inexpensive and do not require highly trained personnel, facilitating their on-site implementation. The reporter phages presented in this study not only allow for rapid detection but also enable an early estimation of the potential virulence of <i>Listeria</i> isolates from food production and processing sites.