Highlights d A high-throughput screen identifies key Salmonella genes used in initial gut colonization d Host food and microbiota provide luminal aspartate and malate for Salmonella respiration d The DcuABC transporters pump aspartate and malate into the Salmonella cell d Aspartate and malate conversion into fumarate fuels growth by H 2 /fumarate respiration

Fast and reliable detection of bacterial pathogens in clinical samples, contaminated food products, and water supplies can drastically improve clinical outcomes and reduce the socio-economic impact of disease. As natural predators of bacteria, bacteriophages (phages) have evolved to bind their hosts with unparalleled specificity and to rapidly deliver and replicate their viral genome. Not surprisingly, phages and phage-encoded proteins have been used to develop a vast repertoire of diagnostic assays, many of which outperform conventional culture-based and molecular detection methods. While intact phages or phage-encoded affinity proteins can be used to capture bacteria, most phage-inspired detection systems harness viral genome delivery and amplification: to this end, suitable phages are genetically reprogrammed to deliver heterologous reporter genes, whose activity is typically detected through enzymatic substrate conversion to indicate the presence of a viable host cell. Infection with such engineered reporter phages typically leads to a rapid burst of reporter protein production that enables highly sensitive detection. In this review, we highlight recent advances in infection-based detection methods, present guidelines for reporter phage construction, outline technical aspects of reporter phage engineering, and discuss some of the advantages and pitfalls of phage-based pathogen detection. Recent improvements in reporter phage construction and engineering further substantiate the potential of these highly evolved nanomachines as rapid and inexpensive detection systems to replace or complement traditional diagnostic approaches.

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.

Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have used a microtiter plate-based protocol to screen a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk. Eight suitable PGH constructs were identified by this approach, and their efficacies against S. aureus in heat-treated milk were compared by time-kill assays. The two most active enzymes (lysostaphin and CHAPK_CWT-LST) reduced S. aureus numbers in milk to undetectable levels within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The PGH combination completely eradicated S. aureus from milk: no more bacteria were detected within 24 h after the addition of the enzymes, corresponding to a reduction of >9 log units from the level in the control. Efficacy was also retained at different inoculum levels (3 log versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas lysostaphin retained its activity, reducing bacterial numbers by >3.5 log units within 3 h. IMPORTANCE Staphylococci, and S. aureus in particular, are a major cause of bovine mastitis, an inflammation of the mammary gland in cows that is associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell walls and are refractory to resistance development; thus, they have promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk among a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and thus support their further characterization in animal models.

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