Endolysins are enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. Due to the absence of an outer membrane in the Gram-positive bacterial cell wall, endolysins can access the peptidoglycan and destroy these organisms when applied externally, making them interesting antimicrobial candidates, particularly in light of increasing bacterial drug resistance. This article reviews the modular structure of these enzymes, in which cell wall binding and catalytic functions are separated, as well as their mechanism of action, lytic activity and potential as antimicrobials. It particularly focuses on molecular engineering as a means of optimizing endolysins for specific applications, highlights new developments that may render these proteins active against Gram-negative and intracellular pathogens and summarizes the most recent applications of endolysins in the fields of medicine, fo...

Food-borne Listeria monocytogenes is a serious threat to human health, and new strategies to combat this opportunistic pathogen in foods are needed. Bacteriophages are natural enemies of bacteria and are suitable candidates for the environmentally friendly biocontrol of these pathogens. In a comprehensive set of experiments, we have evaluated the virulent, broad-host-range phages A511 and P100 for control of L. monocytogenes strains Scott A (serovar 4b) and WSLC 1001 (serovar 1/2a) in different ready-to-eat (RTE) foods known to frequently carry the pathogen. Food samples were spiked with bacteria (1 × 10 ³ CFU/g), phage added thereafter (3 × 10 ⁶ to 3 × 10 ⁸ PFU/g), and samples stored at 6°C for 6 days. In liquid foods, such as chocolate milk and mozzarella cheese brine, bacterial counts rapidly dropped below the level of direct detection. On solid foods (hot dogs, sliced turkey meat, smoked salmon, seafood, sliced cabbage, and lettuce leaves), phages could reduce bacterial counts by up to 5 log units. Variation of the experimental conditions (extended storage over 13 days or storage at 20°C) yielded similar results. In general, the application of more phage particles (3 × 10 ⁸ PFU/g) was more effective than lower doses. The added phages retained most of their infectivity during storage in foods of animal origin, whereas plant material caused inactivation by more than 1 log ₁₀ . In conclusion, our data demonstrate that virulent broad-host-range phages, such as A511 and P100, can be very effective for specific biocontrol of L. monocytogenes in contamination-sensitive RTE foods.

Immobilization and magnetic separation for specific enrichment of microbial cells, such as the pathogen Listeria monocytogenes , depends on the availability of suitable affinity molecules. We report here a novel concept for the immobilization and separation of bacterial cells by replacing antibodies with cell wall-binding domains (CBDs) of bacteriophage-encoded peptidoglycan hydrolases (endolysins). These polypeptide modules very specifically recognize and bind to ligands on the gram-positive cell wall with high affinity. With paramagnetic beads coated with recombinant Listeria phage endolysin-derived CBD molecules, more than 90% of the viable L. monocytogenes cells could be immobilized and recovered from diluted suspensions within 20 to 40 min. Recovery rates were similar for different species and serovars of Listeria and were not affected by the presence of other microorganisms. The CBD-based magnetic separation (CBD-MS) procedure was evaluated for capture and detection of L. monocytogenes from artificially and naturally contaminated food samples. The CBD separation method was shown to be superior to the established standard procedures; it required less time (48 h versus 96 h) and was the more sensitive method. Furthermore, the generalizability of the CBD-MS approach was demonstrated by using specific phage-encoded CBDs specifically recognizing Bacillus cereus and Clostridium perfringens cells, respectively. Altogether, CBD polypeptides represent novel and innovative tools for the binding and capture of bacterial cells, with many possible applications in microbiology and diagnostics.

The host range of bacteriophages defines their impact on bacterial communities and genome diversity. Here, we characterize 94 novel staphylococcal phages from wastewater and establish their host range on a diversified panel of 117 staphylococci from 29 species. Using this high-resolution phage-bacteria interaction matrix, we unveil a multi-species host range as a dominant trait of the isolated staphylococcal phages. Phage genome sequencing shows this pattern to prevail irrespective of taxonomy. Network analysis between phage-infected bacteria reveals that hosts from multiple species, ecosystems, and drug-resistance phenotypes share numerous phages. Lastly, we show that phages throughout this network can package foreign genetic material enclosing an antibiotic resistance marker at various frequencies. Our findings indicate a weak host specialism of the tested phages, and therefore their potential to promote horizontal gene transfer in this environment.

The genus Listeria comprises food-borne pathogens associated with severe infections and a high mortality rate. Endolysins from bacteriophages infecting Listeria are promising tools for both their detection and control. These proteins feature a modular organization, consisting of an N-terminal enzymatically active domain (EAD), which contributes lytic activity, and a C-terminal cell wall binding domain (CBD), which targets the lysin to its substrate. Sequence comparison among 12 different endolysins revealed high diversity among the enzyme's functional domains and allowed classification of their CBDs into two major groups and five subclasses. This diversity is reflected in various binding properties, as determined by cell wall binding assays using CBDs fused to fluorescent marker proteins. Although some proteins exhibited a broad binding range and recognize Listeria strains representing all serovars, others target specific serovars only. The CBDs also differed with respect to the number and distribution of ligands recognized on the cells, as well as their binding affinities. Surface plasmon resonance analysis revealed equilibrium affinities in the pico- to nanomolar ranges for all proteins except CBD006, which is due to an internal truncation. Rapid multiplexed detection and differentiation of Listeria strains in mixed bacterial cultures was possible by combining CBDs of different binding specificities with fluorescent markers of various colors. In addition, cells of different Listeria strains could be recovered from artificially contaminated milk or cheese by CBD-based magnetic separation by using broad-range CBDP40 and subsequently identified after incubation with two differently colored CBD fusion proteins of higher specificity.