Single and combined application of bacteriophage and cinnamon oils against pathogenic Listeria monocytogenes in milk and smoked salmon.

Nowadays, the discovery of alternative natural antimicrobial substances such as bacteriophages, essential oils, and other physical and chemical agents is developing in the food industry. In this study, nine bacteriophages were isolated from various parts of raw chickens and exhibited lytic activities against L. monocytogenes and various Listeria spp. The characterization of phage vB_LmoS-PLM9 was stable at 4 to 50 °C and pH range from 4 to 10. Phage vB_LmoS-PLM9 had a circular, double-stranded genomic DNA with 38,345 bp having endolysin but no antibiotic resistance or virulence genes. Among the eight essential oils tested at 10 %, cinnamon bark, and cassia oils showed the strongest antilisterial activities. The combined use of phage vB_LmoS-PLM9 and cinnamon oils indicated higher efficiency than single treatments. The combination of phage (MOI of 10) and both cinnamon oils (0.03 %) reduced the viable counts of L. monocytogenes and inhibited the regrowth of resistant cell populations in broth at 30 °C. Furthermore, treatment with the combination of phage (MOI of 100) and cinnamon oil (0.125 %) was effective in milk, especially at 4 °C by reducing the viable count to less than lower limit of detection. These results suggest combining phage and cinnamon oil is a potential approach for controlling L. monocytogenes in milk.

A Bacteriophage Cocktail Significantly Reduces Listeria monocytogenes without Deleterious Impact on the Commensal Gut Microbiota under Simulated Gastrointestinal Conditions.

In this study, we examined the effect of a bacteriophage cocktail (tentatively designated as the Foodborne Outbreak Pill (FOP)) on the levels of Listeria monocytogenes in simulated small intestine, large intestine, and Caco-2 model systems. We found that FOP survival during simulated passage of the upper gastrointestinal was dependent on stomach pH, and that FOP robustly inhibited L. monocytogenes levels with effectiveness comparable to antibiotic treatment (ampicillin) under simulated ilium and colon conditions. The FOP did not inhibit the commensal bacteria, whereas ampicillin treatment led to dysbiosis-like conditions. The FOP was also more effective than an antibiotic in protecting Caco-2 cells from adhesion and invasion by L. monocytogenes (5-log reduction vs. 1-log reduction) while not triggering an inflammatory response. Our data suggested that the FOP may provide a robust protection against L. monocytogenes should the bacterium enter the human gastrointestinal tract (e.g., by consumption of contaminated food), without deleterious impact on the commensal bacteria.

Non-coding RNA regulates phage sensitivity in Listeria monocytogenes.

Non-coding RNAs (ncRNAs) have gained increasing attention as their diverse roles in virulence and environmental stress in Listeria monocytogenes have become clearer. The ncRNA rliB is an atypical member of the CRISPR family, conserved at the same genomic locus in all analyzed L. monocytogenes genomes and also in other Listeria species. In this study, rliB defective mutants (Lm3-22-ΔrliB) were constructed by homologous recombination. The growth cycle of Lm3-22-ΔrliB mutants was slower than that of wild-type Lm3-22. The sensitivity of Lm3-22-ΔrliB to the Listeria phage vB-LmoM-SH3-3 was significantly increased, and the efficiency of plaque formation was enhanced by 128 fold. Compared with wild type, the adhesion and invasion of Lm3-22-ΔrliB decreased significantly (9.3% and 1.33%, respectively). After 4 hours of infection, the proliferation of Lm3-22-ΔrliB in RAW264.7 cells also decreased significantly. Transcription level of invasion-related surface proteins showed that the internalin genes lmo0610 and lm0514, and the peptidoglycan binding protein gene lmo1799 in Lm3-22-ΔrliB were significantly increased. In addition, after interaction with phage, the transcription levels of inlA, lmo0610, lmo1799, lmo2085, and lmo0514 in Lm3-22-ΔrliB cells were significantly upregulated, while inlB was downregulated, compared with Lm3-22 control group with phage treatment. Therefore, rliB deletion effectively regulated the interaction between Listeria and phage, weaken its invasion ability, and provided a new theoretical basis for biocontrol of phage.

Glucose Decoration on Wall Teichoic Acid Is Required for Phage Adsorption and InlB-Mediated Virulence in Listeria ivanovii.

Listeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall teichoic acid [WTA]) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene liv1070, which encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention on the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting that the trade-off between phage resistance and virulence attenuation may be a general feature in the genus Listeria. IMPORTANCE Listeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants and occasionally in immunocompromised humans. Recent investigations revealed that in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently results in disconnection of one of the bacterium's major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria, and the host may be a feature common to the genus Listeria.

Control of the Serine Integrase Reaction: Roles of the Coiled-Coil and Helix E Regions in DNA Site Synapsis and Recombination.

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA-binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn directs the relative trajectories of the CC motifs on each subunit of the att-bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal-length CC motifs and 14 residues surrounding the tip where the pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggest that molecular interactions between CC motif tips may differ in integrative (attP × attB) and excisive (attL × attR) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminately, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after the initial assembly of the integrase synaptic tetramer. IMPORTANCE The robust and exquisitely regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage and, in the case of the A118 prophage, are an important virulence factor of Listeria monocytogenes. The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

Characterization of a Novel Group of Listeria Phages That Target Serotype 4b Listeria monocytogenes.

Listeria monocytogenes serotype 4b strains are the most prevalent clinical isolates and are widely found in food processing environments. Bacteriophages are natural viral predators of bacteria and are a promising biocontrol agent for L. monocytogenes. The aims of this study were to characterize phages that specifically infect serotype 4b strains and to assess their ability to inhibit the growth of serotype 4b strains. Out of 120 wild Listeria phages, nine phages were selected based on their strong lytic activity against the model serotype 4b strain F2365. These nine phages can be divided into two groups based on their morphological characteristics and host range. Comparison to previously characterized phage genomes revealed one of these groups qualifies to be defined as a novel species. Phages LP-020, LP-027, and LP-094 were selected as representatives of these two groups of phages for further characterization through one-step growth curve and inhibition of serotype 4b L. monocytogenes experiments. Listeria phages that target serotype 4b showed an inhibitory effect on the growth of F2365 and other serotype 4 strains and may be useful for biocontrol of L.monocytogenes in food processing environments.

Gene gain and loss and recombination shape evolution of Listeria bacteriophages of the genus Pecentumvirus.

Listeria monocytogenes is an important food-borne pathogen and its bacteriophages are promising tools for its control in food and surfaces. Listeria bacteriophages belonging to the genus Pecentumvirus of the family Herelleviridae are strictly lytic, have a contractile tail and a large double stranded DNA genome (mean of 135.4 kb). We report the isolation and genome sequences of two new Pecentumvirus bacteriophages: vB_Lino_VEfB7 and vB_Liva_VAfA18. Twenty-one bacteriophages of this genus have been described and their genomes were used for the study of Pecentumvirus evolution. Analyses showed collinear genomes and gene gain and loss propensity and recombination events were distinctly found in two regions. A large potential recombination event (≈20 kB) was detected in P100 and vB_Liva_VAfA18. Phylogenetic analyses of multi-gene alignments showed that diversification events formed two groups of species distantly related.

Mutant and Recombinant Phages Selected from In Vitro Coevolution Conditions Overcome Phage-Resistant Listeria monocytogenes.

Bacteriophages (phages) are currently available for use by the food industry to control the foodborne pathogen Listeria monocytogenes Although phage biocontrols are effective under specific conditions, their use can select for phage-resistant bacteria that repopulate phage-treated environments. Here, we performed short-term coevolution experiments to investigate the impact of single phages and a two-phage cocktail on the regrowth of phage-resistant L. monocytogenes and the adaptation of the phages to overcome this resistance. We used whole-genome sequencing to identify mutations in the target host that confer phage resistance and in the phages that alter host range. We found that infections with Listeria phages LP-048, LP-125, or a combination of both select for different populations of phage-resistant L. monocytogenes bacteria with different regrowth times. Phages isolated from the end of the coevolution experiments were found to have gained the ability to infect phage-resistant mutants of L. monocytogenes and L. monocytogenes strains previously found to be broadly resistant to phage infection. Phages isolated from coinfected cultures were identified as recombinants of LP-048 and LP-125. Interestingly, recombination events occurred twice independently in a locus encoding two proteins putatively involved in DNA binding. We show that short-term coevolution of phages and their hosts can be utilized to obtain mutant and recombinant phages with adapted host ranges. These laboratory-evolved phages may be useful for limiting the emergence of phage resistance and for targeting strains that show general resistance to wild-type (WT) phages.IMPORTANCEListeria monocytogenes is a life-threatening bacterial foodborne pathogen that can persist in food processing facilities for years. Phages can be used to control L. monocytogenes in food production, but phage-resistant bacterial subpopulations can regrow in phage-treated environments. Coevolution experiments were conducted on a Listeria phage-host system to provide insight into the genetic variation that emerges in both the phage and bacterial host under reciprocal selective pressure. As expected, mutations were identified in both phage and host, but additionally, recombination events were shown to have repeatedly occurred between closely related phages that coinfected L. monocytogenes This study demonstrates that in vitro evolution of phages can be utilized to expand the host range and improve the long-term efficacy of phage-based control of L. monocytogenes This approach may also be applied to other phage-host systems for applications in biocontrol, detection, and phage therapy.

Bacteriophage biocontrol to fight Listeria outbreaks in seafood.

Listeria monocytogenes is a well-known pathogen responsible for the severe foodborne disease listeriosis. The control of L. monocytogenes occurrence in seafood products and seafood processing environments is an important challenge for the seafood industry and the public health sector. However, bacteriophage biocontrol shows great potential to be used as safety control measure in seafood. This review provides an update on Listeria-specific bacteriophages, focusing on their application as a safe and natural strategy to prevent L. monocytogenes contamination and growth in seafood products and seafood processing environments. Furthermore, the main properties required from bacteriophages intended to be used as biocontrol tools are summarized and emerging strategies to overcome the current limitations are considered. Also, major aspects relevant for bacteriophage production at industrial scale, their access to the market, as well as the current regulatory status of bacteriophage-based solutions for Listeria biocontrol are discussed.

Lineage-specific evolution and gene flow in Listeria monocytogenes are independent of bacteriophages.

Listeria monocytogenes is a foodborne pathogen causing systemic infection with high mortality. To allow efficient tracking of outbreaks a clear definition of the genomic signature of a cluster of related isolates is required, but lineage-specific characteristics call for a more detailed understanding of evolution. In our work, we used core genome MLST (cgMLST) to identify new outbreaks combined to core genome SNP analysis to characterize the population structure and gene flow between lineages. Whilst analysing differences between the four lineages of L. monocytogenes we have detected differences in the recombination rate, and interestingly also divergence in the SNP differences between sub-lineages. In addition, the exchange of core genome variation between the lineages exhibited a distinct pattern, with lineage III being the best donor for horizontal gene transfer. Whilst attempting to link bacteriophage-mediated transduction to observed gene transfer, we found an inverse correlation between phage presence in a lineage and the extent of recombination. Irrespective of the profound differences in recombination rates observed between sub-lineages and lineages, we found that the previously proposed cut-off of 10 allelic differences in cgMLST can be still considered valid for the definition of a foodborne outbreak cluster of L. monocytogenes.

Critical Anti-CRISPR Locus Repression by a Bi-functional Cas9 Inhibitor.

Bacteriophages must rapidly deploy anti-CRISPR proteins (Acrs) to inactivate the RNA-guided nucleases that enforce CRISPR-Cas adaptive immunity in their bacterial hosts. Listeria monocytogenes temperate phages encode up to three anti-Cas9 proteins, with acrIIA1 always present. AcrIIA1 binds and inhibits Cas9 with its C-terminal domain; however, the function of its highly conserved N-terminal domain (NTD) is unknown. Here, we report that the AcrIIA1NTD is a critical transcriptional repressor of the strong anti-CRISPR promoter. A rapid burst of anti-CRISPR transcription occurs during phage infection and the subsequent negative feedback by AcrIIA1NTD is required for optimal phage replication, even in the absence of CRISPR-Cas immunity. In the presence of CRISPR-Cas immunity, full-length AcrIIA1 uses its two-domain architecture to act as a "Cas9 sensor," tuning acr expression according to Cas9 levels. Finally, we identify AcrIIA1NTD homologs in other Firmicutes and demonstrate that they have been co-opted by hosts as "anti-anti-CRISPRs," repressing phage anti-CRISPR deployment.

Evolution of Listeria monocytogenes in a Food Processing Plant Involves Limited Single-Nucleotide Substitutions but Considerable Diversification by Gain and Loss of Prophages.

Whole-genome sequencing (WGS) is becoming the standard method for subtyping Listeria monocytogenes Interpretation of WGS data for isolates from foods and associated environments is, however, challenging due to a lack of detailed data on Listeria evolution in processing facilities. Here, we used previously collected WGS data for 40 L. monocytogenes isolates obtained from a cold-smoked salmon processing facility between 1998 and 2015 to probe the L. monocytogenes molecular evolution in this facility, combined with phenotypic assessment of selected isolates. Isolates represented three clusters (1, 2, and 3); cluster 3 isolates (n = 32) were obtained over 18 years. The average mutation rate for cluster 3 was estimated as 1.15 × 10-7 changes per nucleotide per year (∼0.35 changes per genome per year); the most recent common ancestors (MRCAs) of subclusters 3a and 3b were estimated to have occurred around 1958 and 1974, respectively, within the age of the facility, suggesting long-term persistence in this facility. Extensive prophage diversity was observed within subclusters 3a and 3b, which have one shared and six unique prophage profiles for each subcluster (with 16 prophage profiles found among all 40 isolates). The plasmid-borne sanitizer tolerance operon bcrABC was found in all cluster 2 and 3 isolates, while the transposon-borne sanitizer tolerance gene qacH was found in one cluster 1 isolate; presence of these genes was correlated with the ability to survive increased concentrations of sanitizers. Selected isolates showed significant variation in the ability to attach to surfaces, with persistent isolates attaching better than transient isolates at 21°C.IMPORTANCE Knowledge about the genetic evolution of L. monocytogenes in food processing facilities over multiple years is generally lacking. This information is critical to interpret WGS findings involving food or food-associated isolates. This study suggests that L. monocytogenes that persists in processing facilities may evolve with a low single-nucleotide mutation rate mostly driven by negative (i.e., purifying) selection but with rapid diversification of prophages. Hence, isolation of L. monocytogenes with few single-nucleotide polymorphism (SNP) differences in different locations (e.g., supplier plants and receiving plants) is possible, highlighting the importance of epidemiological and detailed isolate metadata for interpreting WGS data in traceback investigation. Our study also shows how advanced WGS data analyses can be used to support root cause analysis efforts and may, for example, pinpoint the time when a persistence event started (which then potentially could be linked to facility changes, introduction of new equipment, etc.).

Post-process treatments are effective strategies to reduce Listeria monocytogenes on the surface of leafy greens: A pilot study.

Growth of L. monocytogenes is among the most important factors affecting the risk of human listeriosis. In ready to eat leafy greens, the use of anti-Listeria treatments represents a good alternative to inhibit growth during storage. Several commercially available antimicrobial agents have been suggested as effective intervention strategies. Among them, phage preparations and bacteriocin-producing strains have shown promising results against L. monocytogenes. In this study, we investigate the efficacy of two commercially available surface treatments, the bacteriophage formulation PhageGuard Listex (Micreos Food Safety B.V., NL) and the bacteriocin-producing culture SafePro® (CHR Hansen, DK) to inactivate L. monocytogenes in fresh-cut curly endive after processing and during storage. Fresh-cut endive was inoculated with a cold-adapted L. monocytogenes cocktail of 6 strains (4.4 ±â€¯0.0 log cfu/g) and treated with the anti-Listeria treatments. The treatments were applied using a spray system at two different places within the processing line, on the conveyor belt and in the centrifuge. A total of 5 different treatments were applied: i) Untreated (CT); ii) PhageGuard Listex on the conveyor belt (Listex_Conveyor); iii) PhageGuard Listex during centrifugation (Listex_Centrifuge); iv) SafePro on the conveyor belt (SafePro_Conveyor); and v) SafePro during centrifugation (SafePro_Centrifuge). Samples were stored 3 days at 5 °C plus 5 days at 8 °C. PhageGuard Listex treatment reduced L. monocytogenes in fresh-cut endive by 2.5 logs, regardless of the place of treatment application (conveyor belt or centrifuge). On the other hand, SafePro only reduced L. monocytogenes by 0.2 and 0.4 logs, at the conveyor belt and centrifuge, respectively. Maximum L. monocytogenes reductions of about 3.5 log units were observed in fresh-cut endive treated with PhageGuard Listex after 3 days of storage. At the end of the shelf life (8 days), the initial trends were maintained and the fresh-cut curly endive treated with PhageGuard Listex showed the lowest L. monocytogenes concentration. However, by the end of the shelf-life, L. monocytogenes showed higher levels (1.3-fold) than immediately after the application of the treatment. One hypothesis could be that L. monocytogenes cells, which were able to survive the anti-Listeria treatments, were also able to proliferate under the specific storage conditions. Based on the obtained results, PhageGuard Listex seems to be a promising decontamination agent for leafy greens aiming to reduce growth of the bacteria but further work is needed.

Non-thermal approach to Listeria monocytogenes inactivation in milk: The combined effect of high pressure, pediocin PA-1 and bacteriophage P100.

Non-thermal food processing and replacement of chemical additives by natural antimicrobials are promising trends in the food industry. The objective of the present work was to evaluate the effect of a process which combines mild high hydrostatic pressure - HHP (200 and 300 MPa, 5 min, 10 °C), phage Listex™ P100 and the bacteriocin pediocin PA-1 as a new non-thermal process for destruction of Listeria monocytogenes (104 CFU mL-1 or 107 CFU mL-1) in milk. For inoculum levels of 104 CFU mL-1, HHP combined with phage P100 eliminated L. monocytogenes immediately after pressurization. When L. monocytogenes was inoculated at levels of 107 CFU mL-1, a synergistic effect between phage P100, pediocin PA-1 and HHP (300 MPa) on the inactivation of L. monocytogenes was observed during storage of milk at 4 °C. For non-pressure treated samples inoculated with phage or pediocin or both, L. monocytogenes counts decreased immediately after biocontrol application, but regrowth was observed in a few samples during storage. Phage particles were stable during refrigerated storage for seven days while pediocin PA-1 remained stable only during three days. Further studies will have to be performed to validate the findings of this work in specific applications (e.g. production of raw milk cheese).

Genomic dissection of the most prevalent Listeria monocytogenes clone, sequence type ST87, in China.

BACKGROUND: Listeria monocytogenes consists of four lineages that occupy a wide variety of ecological niches. Sequence type (ST) 87 (serotype 1/2b), belonging to lineage I, is one of the most common STs isolated from food products, food associated environments and sporadic listeriosis in China. Here, we performed a comparative genomic analysis of the L. monocytogenes ST87 clone by sequencing 71 strains representing a diverse range of sources, different geographical locations and isolation years. RESULTS: The core genome and pan genome of ST87 contained 2667 genes and 3687 genes respectively. Phylogenetic analysis based on core genome SNPs divided the 71 strains into 10 clades. The clinical strains were distributed among multiple clades. Four clades contained strains from multiple geographic regions and showed high genetic diversity. The major gene content variation of ST87 genomes was due to putative prophages, with eleven hotspots of the genome that harbor prophages. All strains carry an intact CRISRP/Cas system. Two major CRISPR spacer profiles were found which were not clustered phylogenetically. A large plasmid of about 90 Kb, which carried heavy metal resistance genes, was found in 32.4% (23/71) of the strains. All ST87 strains harbored the Listeria pathogenicity island (LIPI)-4 and a unique 10-open read frame (ORF) genomic island containing a novel restriction-modification system. CONCLUSION: Whole genome sequence analysis of L. monocytogenes ST87 enabled a clearer understanding of the population structure and the evolutionary history of ST87 L. monocytogenes in China. The novel genetic elements identified may contribute to its virulence and adaptation to different environmental niches. Our findings will be useful for the development of effective strategies for the prevention and treatment of listeriosis caused by this prevalent clone.

Homburgvirus LP-018 Has a Unique Ability to Infect Phage-Resistant Listeria monocytogenes.

Listeria phage LP-018 is the only phage from a diverse collection of 120 phages able to form plaques on a phage-resistant Listeria monocytogenes strain lacking rhamnose in its cell wall teichoic acids. The aim of this study was to characterize phage LP-018 and to identify what types of mutations can confer resistance to LP-018. Whole genome sequencing and transmission electron microscopy revealed LP-018 to be a member of the Homburgvirus genus. One-step-growth curve analysis of LP-018 revealed an eclipse period of ~60-90 min and a burst size of ~2 PFU per infected cell. Despite slow growth and small burst size, LP-018 can inhibit the growth of Listeria monocytogenes at a high multiplicity of infection. Ten distinct LP-018-resistant mutants were isolated from infected Listeria monocytogenes 10403S and characterized by whole genome sequencing. In each mutant, a single mutation was identified in either the LMRG_00278 or LMRG_01613 encoding genes. Interesting, LP-018 was able to bind to a representative phage-resistant mutant with a mutation in each gene, suggesting these mutations confer resistance through a mechanism independent of adsorption inhibition. Despite forming plaques on the rhamnose deficient 10403S mutant, LP-018 showed reduced binding efficiency, and we did not observe inhibition of the strain under the conditions tested. Two mutants of LP-018 were also isolated and characterized, one with a single SNP in a gene encoding a BppU domain protein that likely alters its host range. LP-018 is shown to be a unique Listeria phage that, with additional evaluation, may be useful in biocontrol applications that aim to reduce the emergence of phage resistance.

Coordination of cohabiting phage elements supports bacteria-phage cooperation.

Bacterial pathogens often carry multiple prophages and other phage-derived elements within their genome, some of which can produce viral particles in response to stress. Listeria monocytogenes 10403S harbors two phage elements in its chromosome, both of which can trigger bacterial lysis under stress: an active prophage (ϕ10403S) that promotes the virulence of its host and can produce infective virions, and a locus encoding phage tail-like bacteriocins. Here, we show that the two phage elements are co-regulated, with the bacteriocin locus controlling the induction of the prophage and thus its activity as a virulence-associated molecular switch. More specifically, a metalloprotease encoded in the bacteriocin locus is upregulated in response to stress and acts as an anti-repressor for CI-like repressors encoded in each phage element. Our results provide molecular insight into the phenomenon of polylysogeny and its intricate adaptation to complex environments.

The Effect of a Commercially Available Bacteriophage and Bacteriocin on Listeria monocytogenes in Coleslaw.

Changing consumer attitudes show an increased interest in non-chemical antimicrobials in food preservation and safety. This greater interest of consumers in more 'natural' or 'clean-label' food interventions is complicated by concurrent demands for minimally processed, ready-to-eat (RTE) foods with long shelf lives. Two viable interventions are bacteriophage (phage) and bacteriocins, a number of which have already been approved for use in food safety. Listeriosis is a serious foodborne infection which affects at-risk members of the population. Listeriosis incidence has increased between 2008 and 2015 and has a case fatality rate of up to 20% with antibiotic intervention. Here, we tested an intervention to attempt to control a pathogenic Listeria monocytogenes strain in a food model using two of these alternative antimicrobials. Phage P100 on its own had a significant effect on L. monocytogenes ScottA numbers in coleslaw over a 10-day period at 4 °C (p ≤ 0.001). A combination of P100 and Nisaplin® (a commercial formulation of the lantibiotic bacteriocin, nisin) had a significant effect on the pathogen (p ≤ 0.001). P100 and Nisaplin® in combination were more effective than Nisaplin® alone, but not P100 alone.

Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion.

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.

Reprogramming Bacteriophage Host Range through Structure-Guided Design of Chimeric Receptor Binding Proteins.

Bacteriophages provide excellent tools for diagnostics, remediation, and targeted microbiome manipulation, yet isolating viruses with suitable host specificity remains challenging. Using Listeria phage PSA, we present a synthetic biology blueprint for host-range engineering through targeted modification of serovar-specific receptor binding proteins (RBPs). We identify Gp15 as the PSA RBP and construct a synthetic phage library featuring sequence-randomized RBPs, from which host range mutants are isolated and subsequently integrated into a synthetic, polyvalent phage with extended host range. To enable rational design of chimeric RBPs, we determine the crystal structure of the Gp15 receptor-binding carboxyl terminus at 1.7-Å resolution and employ bioinformatics to identify compatible, prophage-encoded RBPs targeting different Listeria serovars. Structure-guided design enables exchange of heterologous RBP head, neck, or shoulder domains to generate chimeric phages with predictable and extended host ranges. These strategies will facilitate the development of phage biologics based on standardized virus scaffolds with tunable host specificities.