Engineered Reporter Phages for Rapid Bioluminescence-Based Detection and Differentiation of ViableListeriaCells

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.

Reporter Phage-Based Detection of Bacterial Pathogens: Design Guidelines and Recent Developments

“…(D) Released progeny phages [51,52] or bacterial cell content (e.g., ATP, DNA, RNA and bacterial proteins) provide excellent markers for downstream detection of the original bacterial host [53][54][55]. Alternatively, genetically engineered phages encoding reporters such as fluorescent proteins (E) [56][57][58], luciferases (F) [59][60][61][62] or hydrolyzing enzymes (e.g., β-galactosidase) (G) [63,64] are used. These phages express the reporter proteins during host infection to produce an amplifying fluorescent or bioluminescent signal upon the addition of a substrate.…”

“…Similarly, a Listeria ivanovii type II-A CRISPR-Cas system [132] was used for the modification of lytic Listeria phage A511 to create two variants of A511::nluc that transduce bioluminescence into Listeria spp. for detection [62]. While the use of CRISPR-Cas counter selection can greatly improve recombinant phage identification, a current bottleneck is the lack of well-characterized and programmable CRISPR-Cas systems that can be used for reporter phage engineering in different bacterial hosts.…”

“…The first published reporter phage encoding NLuc is E. coli phage ΦV10 for detecting E. coli O157:H7 [102]. Another recent example is the broad host-range, nluc-containing Myovirus A511 (A511::nluc CPS ) that detects a single L. monocytogenes cell in 25 g of various artificially contaminated food samples within less than 24 h. In addition to A511::nluc CPS -mediated detection, other nluc Listeria phages can be used for serovar differentiation of food isolates [62]. Furthermore, Dow et al 2018 used acoustic separation and microfluidics to separate bacteria from blood cells and then employed the NLuc-reporter phage K1E for detection of E. coli [103].…”

“…For instance, expression of NanoLuc from an early promoter controlling the anti-CRISPR locus of Listeria phage A006 provided rapid protein production and early detection during the latent period [90]. Still, many reporter genes are inserted downstream of the strongly expressed but rather late endogenous promoters of the major capsid gene [62,91] the endolysin gene [62], or the tail spike protein [88]. Some examples of recently used constitutive exogenous promoters are P L(L5) for mycobacteria [58,[92][93][94], and PrplU [95] and phi10 T7 [96] for E. coli.…”

“…In addition, various reporter proteins do not require cell lysis or completion of the phage infection cycle to produce a signal. Genome injection and potentially some degree of DNA replication is therefore sufficient [58,62]. In fact, an intact phage genome may not even be required: non-replicative phage particles carrying a luciferase-containing plasmid (fittingly named Smarticles™ (Roche/Geneweave, Los Gatos, CA, USA)) simply transduce the luciferase gene into the target bacteria for detection [100]; however, this approach does remove the possibility of signal amplification from secondary phage infections.…”