Abstract
The pathogen Listeria monocytogenes 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 de novo 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 Listeria 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 Listeria phages (A006 and A500, respectively). The broad-range NLuc-based phage A511::nlucCPS detects one CFU of L. monocytogenes in 25 g of artificially contaminated milk, cold cuts, and lettuce within less than 24 h. In addition, this reporter phage successfully detected Listeria spp. in potentially contaminated natural food samples without producing false-positive or false-negative results. Finally, A006::nluc and A500::nluc enable serovar-specific Listeria diagnostics. In conclusion, these NLuc-based reporter phages enable rapid, ultrasensitive detection and differentiation of viable Listeria cells using a simple protocol that is 72 h faster than culture-dependent approaches.IMPORTANCE 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 Listeria isolates from food production and processing sites.
Highlights
The pathogen Listeria monocytogenes causes listeriosis, a severe foodborne disease associated with high mortality
Listeria monocytogenes is an opportunistic, Gram-positive pathogen that can cause listeriosis, a severe foodborne disease associated with very high mortality rates (20 to 30%) among the young, old, pregnant, and immunocompromised (YOPI) risk group [1,2,3]
We found that A511::nlucCPS produced a robust signal (Ͼ103 Relative light units (RLUs)) from all positive samples, and the reporter phage-derived results correlated 100% with the results aem.asm.org 6
Summary
The pathogen Listeria monocytogenes causes listeriosis, a severe foodborne disease associated with high mortality. The broad-range NLuc-based phage A511::nlucCPS detects one CFU of L. monocytogenes in 25 g of artificially contaminated milk, cold cuts, and lettuce within less than 24 h This reporter phage successfully detected Listeria spp. in potentially contaminated natural food samples without producing false-positive or falsenegative results. A006::nluc and A500::nluc enable serovar-specific Listeria diagnostics These NLuc-based reporter phages enable rapid, ultrasensitive detection and differentiation of viable Listeria cells using a simple protocol that is 72 h faster than culture-dependent approaches. PCR-based, immunological, or mass spectrometry-based protocols are much faster but cannot differentiate between dead and viable cells, which is an important prerequisite when processed products are tested These methods often require trained personnel and expensive machinery and are frequently compromised by food matrix effects, possibly leading to either false-positive or false-negative results [9]. Luciferase-based reporter phages are very sensitive and, following short preenrichments of less than 24 h in selective media, can detect cell numbers as low as 10 CFU/g of food [13]
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