Phenotypic characterization and genomic analysis of a Salmonella phage L223 for biocontrol of Salmonella spp. in poultry.

The escalating incidence of foodborne salmonellosis poses a significant global threat to food safety and public health. As antibiotic resistance in Salmonella continues to rise, there is growing interest in bacteriophages as potential alternatives. In this study, we isolated, characterized, and evaluated the biocontrol efficacy of lytic phage L223 in chicken meat. Phage L223 demonstrated robust stability across a broad range of temperatures (20-70 °C) and pH levels (2-11) and exhibited a restricted host range targeting Salmonella spp., notably Salmonella Typhimurium and Salmonella Enteritidis. Characterization of L223 revealed a short latent period of 30 min and a substantial burst size of 515 PFU/cell. Genomic analysis classified L223 within the Caudoviricetes class, Guernseyvirinae subfamily and Jerseyvirus genus, with a dsDNA genome size of 44,321 bp and 47.9% GC content, featuring 72 coding sequences devoid of antimicrobial resistance, virulence factors, toxins, and tRNA genes. Application of L223 significantly (p < 0.005) reduced Salmonella Typhimurium ATCC 14,028 counts by 1.24, 2.17, and 1.55 log CFU/piece after 2, 4, and 6 h of incubation, respectively, in experimentally contaminated chicken breast samples. These findings highlight the potential of Salmonella phage L223 as a promising biocontrol agent for mitigating Salmonella contamination in food products, emphasizing its relevance for enhancing food safety protocols.

Bacteriophage Removal from Infected Salmonella Cultures.

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.

Isolation, whole genome sequencing and application of a broad-spectrum Salmonella phage.

Salmonella is considered as one of the most common zoonotic /foodborne pathogens in the world. The application of bacteriophages as novel antibacterial agents in food substrates has become an emerging strategy. Bacteriophages have the potential to control Salmonella contamination.We have isolated and characterized a broad-spectrum Salmonella phage, SP154, which can lyse 9 serotypes, including S. Enteritidis, S. Typhimurium, S. Pullorum, S. Arizonae, S. Dublin, S. Cholerasuis, S. Chester, S. 1, 4, [5], 12: i: -, and S. Derby, accounting for 81.9% of 144 isolates. SP154 showed a short latent period (40 min) and a high burst size (with the first rapid burst size at 107 PFUs/cell and the second rapid burst size at approximately 40 PFUs/cell). Furthermore, SP154 activity has higher survival rates across various environmental conditions, including pH 4.0-12.0 and temperatures ranging from 4 to 50 °C for 60 min, making it suitable for diverse food processing and storage applications. Significant reductions in live Salmonella were observed in different foods matrices such as milk and chicken meat, with a decrease of up to 1.9 log10 CFU/mL in milk contamination and a 1 log10 CFU/mL reduction in chicken meat. Whole genome sequencing analysis revealed that SP154 belongs to the genus Ithacavirus, subfamily Humphriesvirinae, within the family Schitoviridae. Phylogenetic analysis based on the terminase large subunit supported this classification, although an alternate tree using the tail spike protein gene suggested affiliation with the genus Kuttervirus, underscoring the limitations of relying on a single gene for phylogenetic inference. Importantly, no virulence or antibiotic resistance genes were detected in SP154. Our research highlights the potential of using SP154 for biocontrol of Salmonella in the food industry.

Tactical terminase: How a Salmonella prophage navigates oxidative stress.

Stress-induced prophages commonly "jump ship" by inducing lysis via the host SOS response. In a recent work, Uppalapati et al. reports an alternate, stress-selective strategy. Instead of promoting lysis, the Salmonella Gifsy-1 prophage arrests growth specifically when the SOS response coincides with oxidative stress.

What's on a prophage: analysis of Salmonella spp. prophages identifies a diverse range of cargo with multiple virulence- and metabolism-associated functions.

The gain of mobile elements, such as prophages, can introduce cargo to the recipient bacterium that could facilitate its persistence in or expansion to a new environment, such as a host. While previous studies have focused on identifying and characterizing the genetic diversity of prophages, analyses characterizing the cargo that prophages carry have not been extensively explored. We characterized prophage regions from 303 Salmonella spp. genomes (representing 254 unique serovars) to assess the distribution of prophages in diverse Salmonella. On average, prophages accounted for 3.7% (0.1%-8.8%) of the total genomic content of each isolate. Prophage regions annotated as Gifsy 1 and Salmon Fels 1 were the most commonly identified intact prophages, suggesting that they are common throughout the Salmonella genus. Among 21,687 total coding sequences (CDSs) from intact prophage regions in subsp. enterica genomes, 7.5% (median; range: 1.1%-47.6%) were categorized as having a function not related to prophage integration or phage structure, some of which could potentially provide a functional attribute to the host Salmonella cell. These predicted functions could be broadly categorized into CDSs involved in: (i) modification of cell surface structures (i.e., glycosyltransferases); (ii) modulation of host responses (e.g., SodC/SodA, SopE, ArtAB, and typhoid toxin); (iii) conferring resistance to heavy metals and antimicrobials; (iv) metabolism of carbohydrates, amino acids, and nucleotides; and (v) DNA replication, repair, and regulation. Overall, our systematic analysis of prophage cargo highlights a broader role for prophage cargo in influencing the metabolic, virulence, and resistance characteristics of Salmonella. IMPORTANCE: Lysogenic bacteriophages (phages) can integrate their genome into a bacterial host's genome, potentially introducing genetic elements that can affect the fitness of the host bacterium. The functions of prophage-encoded genes are important to understand as these genes could be mobilized and transferred to a new host. Using a large genomic dataset representing >300 isolates from all known subspecies and species of Salmonella, our study contributes important new findings on the distribution of prophages and the types of cargo that diverse Salmonella prophages carry. We identified a number of coding sequences (CDSs) annotated as having cell surface-modifying attributes, suggesting that prophages may have played an important role in shaping Salmonella's diverse surface antigen repertoire. Furthermore, our characterization of prophages suggests that they play a broader role in facilitating the acquisition and transfer of CDSs associated with metabolism, DNA replication and repair, virulence factors, and to a lesser extent, antimicrobial resistance.

Genetic recombination-mediated evolutionary interactions between phages of potential industrial importance and prophages of their hosts within or across the domains of Escherichia, Listeria, Salmonella, Campylobacter, and Staphylococcus.

BACKGROUND: The in-depth understanding of the role of lateral genetic transfer (LGT) in phage-prophage interactions is essential to rationalizing phage applications for human and animal therapy, as well as for food and environmental safety. This in silico study aimed to detect LGT between phages of potential industrial importance and their hosts. METHODS: A large array of genetic recombination detection algorithms, implemented in SplitsTree and RDP4, was applied to detect LGT between various Escherichia, Listeria, Salmonella, Campylobacter, Staphylococcus, Pseudomonas, and Vibrio phages and their hosts. PHASTER and RAST were employed respectively to identify prophages across the host genome and to annotate LGT-affected genes with unknown functions. PhageAI was used to gain deeper insights into the life cycle history of recombined phages. RESULTS: The split decomposition inferences (bootstrap values: 91.3-100; fit: 91.433-100), coupled with the Phi (0.0-2.836E-12) and RDP4 (P being well below 0.05) statistics, provided strong evidence for LGT between certain Escherichia, Listeria, Salmonella, and Campylobacter virulent phages and prophages of their hosts. The LGT events entailed mainly the phage genes encoding for hypothetical proteins, while some of these genetic loci appeared to have been affected even by intergeneric recombination in specific E. coli and S. enterica virulent phages when interacting with their host prophages. Moreover, it is shown that certain L. monocytogenes virulent phages could serve at least as the donors of the gene loci, involved in encoding for the basal promoter specificity factor, for L. monocytogenes. In contrast, the large genetic clusters were determined to have been simultaneously exchanged by many S. aureus prophages and some Staphylococcus temperate phages proposed earlier as potential therapeutic candidates (in their native or modified state). The above genetic clusters were found to encompass multiple genes encoding for various proteins, such as e.g., phage tail proteins, the capsid and scaffold proteins, holins, and transcriptional terminator proteins. CONCLUSIONS: It is suggested that phage-prophage interactions, mediated by LGT (including intergeneric recombination), can have a far-reaching impact on the co-evolutionary trajectories of industrial phages and their hosts especially when excessively present across microbially rich environments.

Characterization of two novel Salmonella phages having biocontrol potential against Salmonella spp. in gastrointestinal conditions.

Salmonella is a primary enteric pathogen related to the contamination of poultry and other food products in numerous foodborne outbreaks. The continuous emergence of multidrug-resistant bacteria has become a serious issue due to the overuse of antibiotics. Hence, lytic phages are considered alternative biocontrol agents against these bacterial superbugs. Here, two Salmonella phages-S4lw and D5lw-were subjected to genomic and biological characterization and further encapsulated to improve the stability under acidic conditions mimicking gastrointestinal conditions. The two lytic phages, S4lw and D5lw, taxonomically belong to new species under the Guernseyvirinae and Ackermannviridae families, respectively. Each phage showed antimicrobial activities against diverse Salmonella spp., such as S. Enteritidis and S. Typhimurium, achieving 1.7-3.4 log reduction after 2-6 h of treatment. The phage cocktail at a multiplicity of infection (MOI) of 100 or 1000 completely inhibited these Salmonella strains for at least 14 h at 25 °C. Additionally, the bead-encapsulated phage cocktail could withstand low pH and different simulated gut environments for at least 1 h. Overall, the newly isolated phages can potentially mitigate Salmonella spp. under the gastrointestinal environments through encapsulation and may be further applied via oral administration to resolve common antimicrobial resistance issues in the poultry production chain.

Genomic analysis of Salmonella bacteriophages revealed multiple endolysin ORFs and importance of ligand-binding site of receptor-binding protein.

Salmonella is a prevalent foodborne pathogen causing millions of global cases annually. Antimicrobial resistance is a growing public health concern, leading to search for alternatives like bacteriophages. A total of 97 bacteriophages, isolated from cattle farms (n = 48), poultry farms (n = 37), and wastewater (n = 5) samples in Türkiye, were subjected to host-range analysis using 36 Salmonella isolates with 18 different serotypes. The broadest host range belonged to an Infantis phage (MET P1-091), lysing 28 hosts. A total of 10 phages with the widest host range underwent further analysis, revealing seven unique genomes (32-243 kb), including a jumbophage (>200 kb). Except for one with lysogenic properties, none of them harbored virulence or antibiotic resistance genes, making them potential Salmonella reducers in different environments. Examining open reading frames (ORFs) of endolysin enzymes revealed surprising findings: five of seven unique genomes contained multiple endolysin ORFs. Despite sharing same endolysin sequences, phages exhibited significant differences in host range. Detailed analysis unveiled diverse receptor-binding protein sequences, with similar structures but distinct ligand-binding sites. These findings emphasize the importance of ligand-binding sites of receptor-binding proteins. Additionally, bacterial reduction curve and virulence index revealed that Enteritidis phages inhibit bacterial growth even at low concentrations, unlike Infantis and Kentucky phages.

Oral Toxicity Study for Salmonella Killing Lytic Bacteriophage NINP13076 in BALB/c Mice and Its Effect on Probiotic Microbiota.

Viruses that infect bacteria are emerging as attractive biocontrol agents and biopreservatives for foods. Since these bacteriophages kill the target pathogens by lysis and are also consumed along with food, it is essential to evaluate their collateral toxicity on the probiotic gut microbiota. In this study, we examined the acute oral toxicity of a Salmonella phage isolated from sewage in mice. Acute oral administration of the Salmonella phage for five consecutive days did not show any significant pathological changes in the vital organs like lung, kidneys, heart, liver, and intestine. In addition, growth of typical probiotic microbiota remained unaffected even after incubation up to 24 h with the Salmonella phage. The results of this study clearly showed that oral administration of the lytic Salmonella phage did not have any significant adverse effects on the animals, may not harm the probiotic gut microbiota, and are likely to be safe for use in food preservation.

PHIDA: A High Throughput Turbidimetric Data Analytic Tool to Compare Host Range Profiles of Bacteriophages Isolated Using Different Enrichment Methods.

Bacteriophages are viruses that infect bacteria and are present in niches where bacteria thrive. In recent years, the suggested application areas of lytic bacteriophage have been expanded to include therapy, biocontrol, detection, sanitation, and remediation. However, phage application is constrained by the phage's host range-the range of bacterial hosts sensitive to the phage and the degree of infection. Even though phage isolation and enrichment techniques are straightforward protocols, the correlation between the enrichment technique and host range profile has not been evaluated. Agar-based methods such as spotting assay and efficiency of plaquing (EOP) are the most used methods to determine the phage host range. These methods, aside from being labor intensive, can lead to subjective and incomplete results as they rely on qualitative observations of the lysis/plaques, do not reflect the lytic activity in liquid culture, and can overestimate the host range. In this study, phages against three bacterial genera were isolated using three different enrichment methods. Host range profiles of the isolated phages were quantitatively determined using a high throughput turbidimetric protocol and the data were analyzed with an accessible analytic tool "PHIDA". Using this tool, the host ranges of 9 Listeria, 14 Salmonella, and 20 Pseudomonas phages isolated with different enrichment methods were quantitatively compared. A high variability in the host range index (HRi) ranging from 0.86-0.63, 0.07-0.24, and 0.00-0.67 for Listeria, Salmonella, and Pseudomonas phages, respectively, was observed. Overall, no direct correlation was found between the phage host range breadth and the enrichment method in any of the three target bacterial genera. The high throughput method and analytics tool developed in this study can be easily adapted to any phage study and can provide a consensus for phage host range determination.

Systematic exploration of Escherichia coli phage-host interactions with the BASEL phage collection.

Bacteriophages, the viruses infecting bacteria, hold great potential for the treatment of multidrug-resistant bacterial infections and other applications due to their unparalleled diversity and recent breakthroughs in their genetic engineering. However, fundamental knowledge of the molecular mechanisms underlying phage-host interactions is mostly confined to a few traditional model systems and did not keep pace with the recent massive expansion of the field. The true potential of molecular biology encoded by these viruses has therefore remained largely untapped, and phages for therapy or other applications are often still selected empirically. We therefore sought to promote a systematic exploration of phage-host interactions by composing a well-assorted library of 68 newly isolated phages infecting the model organism Escherichia coli that we share with the community as the BASEL (BActeriophage SElection for your Laboratory) collection. This collection is largely representative of natural E. coli phage diversity and was intensively characterized phenotypically and genomically alongside 10 well-studied traditional model phages. We experimentally determined essential host receptors of all phages, quantified their sensitivity to 11 defense systems across different layers of bacterial immunity, and matched these results to the phages' host range across a panel of pathogenic enterobacterial strains. Clear patterns in the distribution of phage phenotypes and genomic features highlighted systematic differences in the potency of different immunity systems and suggested the molecular basis of receptor specificity in several phage groups. Our results also indicate strong trade-offs between fitness traits like broad host recognition and resistance to bacterial immunity that might drive the divergent adaptation of different phage groups to specific ecological niches. We envision that the BASEL collection will inspire future work exploring the biology of bacteriophages and their hosts by facilitating the discovery of underlying molecular mechanisms as the basis for an effective translation into biotechnology or therapeutic applications.

Genetic interference exerted by Salmonella-delivered CRISPR/Cas9 significantly reduces the pathological burden caused by Marek's disease virus in chickens.

Efficient in vivo delivery of a CRISPR/Cas9 plasmid is of paramount importance for effective therapy. Here, we investigated the usability of Salmonella as a plasmid carrier for in vivo therapy against virus-induced cancer using Marek's disease virus (MDV) as a model for study in chickens. A green fluorescent protein-expressing CRISPR/Cas9 plasmid encoding the virulence gene pp38 was constructed against Marek's disease virus. Therapeutic plasmids were transformed into Salmonella carrying lon and sifA gene deletions. The animals in 5 groups were intraperitoneally inoculated with phosphate-buffered saline, vector control, or Salmonella before or after MDV infection, or left uninfected as a naïve control. Therapeutic effectiveness was evaluated by observing disease outcomes and the viral copy number in peripheral blood mononuclear cells. The efficacy of plasmid delivery by Salmonella was 13 ± 1.7% in the spleen and 8.0 ± 1.8% in the liver on the 6th day post-infection. The Salmonella-treated groups showed significant resistance to MDV infection. The maximum effect was observed in the group treated with Salmonella before MDV infection. None of the chickens fully recovered; however, the results suggested that timely delivery of Salmonella could be effective for in vivo CRISPR/Cas9-mediated genetic interference against highly pathogenic MDV. The use of Salmonella in CRISPR systems provides a simpler and more efficient platform for in vivo therapy with CRISPR than the use of conventional in vivo gene delivery methods and warrants further development.

Investigation of Salmonella Phage-Bacteria Infection Profiles: Network Structure Reveals a Gradient of Target-Range from Generalist to Specialist Phage Clones in Nested Subsets.

Bacteriophages that lyse Salmonella enterica are potential tools to target and control Salmonella infections. Investigating the host range of Salmonella phages is a key to understand their impact on bacterial ecology, coevolution and inform their use in intervention strategies. Virus-host infection networks have been used to characterize the "predator-prey" interactions between phages and bacteria and provide insights into host range and specificity. Here, we characterize the target-range and infection profiles of 13 Salmonella phage clones against a diverse set of 141 Salmonella strains. The environmental source and taxonomy contributed to the observed infection profiles, and genetically proximal phages shared similar infection profiles. Using in vitro infection data, we analyzed the structure of the Salmonella phage-bacteria infection network. The network has a non-random nested organization and weak modularity suggesting a gradient of target-range from generalist to specialist species with nested subsets, which are also observed within and across the different phage infection profile groups. Our results have implications for our understanding of the coevolutionary mechanisms shaping the ecological interactions between Salmonella phages and their bacterial hosts and can inform strategies for targeting Salmonella enterica with specific phage preparations.

Mobilization of Tn1721-like structure harboring blaCTX-M-27 between P1-like bacteriophage in Salmonella and plasmids in Escherichia coli in China.

The aim of this study was to explore the characteristics of blaCTX-M-27 carriage and mobilization in Salmonella and Escherichia coli isolates from food-producing animals in China. A total of 2280 E. coli and 229 Salmonella isolates collected from food animals from June 2003 to September 2014 were screened for the presence of blaCTX-M-27 gene. The blaCTX-M-27-positive isolates were typed and plasmid DNA sequenced to determine the genetic context of blaCTX-M-27 and plasmid types present. Bacterial fitness was evaluated by growth curve and plasmid stability in vitro. CTX-M-27-positive E. coli (18, 0.79 %) and Salmonella (34, 14.85 %) were detected. PFGE profiles of CTX-M-27-positive strains revealed a wide variety of genotypes and S. Indiana was the most prevalent serotype. Replicon typing, S1-PFGE and hybridization of CTX-M-27-carrying plasmids confirmed that blaCTX-M-27 gene was located on IncFII (12/18), IncN (4/18), and non-typeable (2/18) plasmids in E. coli and on P1-like bacteriophage (21/34), IncP (4/34), IncFIB (4/34), IncN (2/34), IncHI2 (2/34), and IncA/C (1/34) plasmids in Salmonella. Comparison and analysis of gene context of blaCTX-M-27 in P1-like bacteriophage and plasmids revealed they shared the same structure and contained an identical genetic context with the Tn1721-like structure ΔISEcp1B-blaCTX-M-27-IS903D-iroN-Δmap-Tn1721. In addition, plasmid stability tests indicated that the blaCTX-M-27 P1-like bacteriophage were more stable than plasmids in the absence of cefotaxime selective pressure. These results demonstrate that Tn1721-like transposons harboring CTX-M-27 could be mobilized between different plasmids in E. coli and P1-like bacteriophage disseminated among Salmonella.

Characterization of Salmonella Isolates from Various Geographical Regions of the Caucasus and Their Susceptibility to Bacteriophages.

Non-typhoidal Salmonella present a major threat to animal and human health as food-borne infectious agents. We characterized 91 bacterial isolates from Armenia and Georgia in detail, using a suite of assays including conventional microbiological methods, determining antimicrobial susceptibility profiles, matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, serotyping (using the White-Kauffmann-Le Minor scheme) and genotyping (repetitive element sequence-based PCR (rep-PCR)). No less than 61.5% of the isolates were shown to be multidrug-resistant. A new antimicrobial treatment strategy is urgently needed. Phage therapy, the therapeutic use of (bacterio-) phages, the bacterial viruses, to treat bacterial infections, is increasingly put forward as an additional tool for combatting antibiotic resistant infections. Therefore, we used this representative set of well-characterized Salmonella isolates to analyze the therapeutic potential of eleven single phages and selected phage cocktails from the bacteriophage collection of the Eliava Institute (Georgia). All isolates were shown to be susceptible to at least one of the tested phage clones or their combinations. In addition, genome sequencing of these phages revealed them as members of existing phage genera (Felixounavirus, Seunavirus, Viunavirus and Tequintavirus) and did not show genome-based counter indications towards their applicability against non-typhoidal Salmonella in a phage therapy or in an agro-food setting.

Isolation and Characterization of Salmonella Jumbo-Phage pSal-SNUABM-04.

The increasing emergence of antimicrobial resistance has become a global issue. Therefore, many researchers have attempted to develop alternative antibiotics. One promising alternative is bacteriophage. In this study, we focused on a jumbo-phage infecting Salmonella isolated from exotic pet markets. Using a Salmonella strain isolated from reptiles as a host, we isolated and characterized the novel jumbo-bacteriophage pSal-SNUABM-04. This phage was investigated in terms of its morphology, host infectivity, growth and lysis kinetics, and genome. The phage was classified as Myoviridae based on its morphological traits and showed a comparatively wide host range. The lysis efficacy test showed that the phage can inhibit bacterial growth in the planktonic state. Genetic analysis revealed that the phage possesses a 239,626-base pair genome with 280 putative open reading frames, 76 of which have a predicted function and 195 of which have none. By genome comparison with other jumbo phages, the phage was designated as a novel member of Machinavirus composed of Erwnina phages.

Association of host proteins with the broad host range filamentous phage NgoΦ6 of Neisseria gonorrhoeae.

All Neisseria gonorrhoeae strains contain multiple copies of integrated filamentous phage genomes with undefined structures. In this study, we sought to characterize the capsid proteins of filamentous N. gonorrhoeae bacteriophage NgoΦ6 and phagemids propagated in different bacteria. The data demonstrate that purified phage contain phage-encoded structural proteins and bacterial host proteins; host proteins consistently copurified with the phage particles. The bacterial host proteins associated with the phage filament (as identified by mass spectrometry) tended to be one of the predominant outer membrane components of the host strain, plus minor additional host proteins. We were able to copurify a functional ß-lactamase, a phagemid-encoded protein, with phage filaments. We used protein modeling and immunological analysis to identify the major phage encoded structural proteins. The antigenic properties of these proteins depended on the bacterium where the phages were propagated. Polyclonal antibodies against N. gonorrhoeae phage NgoΦ6 recognized phage-encoded proteins if the phage was propagated in N. gonorrhoeae or H. influenzae cells but not if it was propagated in Salmonella or E. coli. We show that the phage filaments isolated from gonococci and Haemophilus are glycosylated, and this may explain the antigenic diversity seen. Taken en toto, the data demonstrate that while the neisserial filamentous phage are similar to other Inovirus with respect to overall genomic organization, their ability to closely associate with host proteins suggests that they have unique surface properties and are secreted by a here-to-fore unknown secretory pathway.

The Mottled Capsid of the Salmonella Giant Phage SPN3US, a Likely Maturation Intermediate with a Novel Internal Shell.

"Giant" phages have genomes of >200 kbp, confined in correspondingly large capsids whose assembly and maturation are still poorly understood. Nevertheless, the first assembly product is likely to be, as in other tailed phages, a procapsid that subsequently matures and packages the DNA. The associated transformations include the cleavage of many proteins by the phage-encoded protease, as well as the thinning and angularization of the capsid. We exploited an amber mutation in the viral protease gene of the Salmonella giant phage SPN3US, which leads to the accumulation of a population of capsids with distinctive properties. Cryo-electron micrographs reveal patterns of internal density different from those of the DNA-filled heads of virions, leading us to call them "mottled capsids". Reconstructions show an outer shell with T = 27 symmetry, an embellishment of the HK97 prototype composed of the major capsid protein, gp75, which is similar to some other giant viruses. The mottled capsid has a T = 1 inner icosahedral shell that is a complex network of loosely connected densities composed mainly of the ejection proteins gp53 and gp54. Segmentation of this inner shell indicated that a number of densities (~12 per asymmetric unit) adopt a "twisted hook" conformation. Large patches of a proteinaceous tetragonal lattice with a 67 Å repeat were also present in the cell lysate. The unexpected nature of these novel inner shell and lattice structures poses questions as to their functions in virion assembly.

Comprehensive Evaluation of the Safety and Efficacy of BAFASAL® Bacteriophage Preparation for the Reduction of Salmonella in the Food Chain.

Bacteriophages are bacterial predators, which are garnering much interest nowadays vis-à-vis the global phenomenon of antimicrobial resistance. Bacteriophage preparations seem to be an alternative to antibiotics, which can be used at all levels of the food production chain. Their safety and efficacy, however, are of public concern. In this study, a detailed evaluation of BAFASAL® preparation was performed. BAFASAL® is a bacteriophage cocktail that reduces Salmonella in poultry farming. In vivo acute and sub-chronic toxicity studies on rats and tolerance study on targeted animals (chicken broiler) conducted according to GLP and OECD guidelines did not reveal any signs of toxicity, which could be associated with BAFASAL® administration. In addition, no evidences of genotoxicity were observed. The tolerance study with 100-times concentrated dose also did not show any statistically significant differences in the assessed parameters. The in vitro crop assay, mimicking normal feed storage and feed application conditions showed that BAFASAL® reduced the number of Salmonella bacteria in experimentally contaminated feed. Moreover, reductions were observed for all examined forms (liquid, powder, spray). Furthermore, the in vivo efficacy study showed that treatment with BAFASAL® significantly decreased Salmonella content in caeca of birds infected with Salmonella Enteritidis. Detailed examination of BAFASAL® in terms of safety and efficacy, adds to the body of evidence that bacteriophages are harmless to animals and effective in the struggle against bacteria.

Phage S144, A New Polyvalent Phage Infecting Salmonella spp. and Cronobacter sakazakii.

Phages are generally considered species- or even strain-specific, yet polyvalent phages are able to infect bacteria from different genera. Here, we characterize the novel polyvalent phage S144, a member of the Loughboroughvirus genus. By screening 211 Enterobacteriaceae strains, we found that phage S144 forms plaques on specific serovars of Salmonella enterica subsp. enterica and on Cronobacter sakazakii. Analysis of phage resistant mutants suggests that the O-antigen of lipopolysaccharide is the phage receptor in both bacterial genera. The S144 genome consists of 53,628 bp and encodes 80 open reading frames (ORFs), but no tRNA genes. In total, 32 ORFs coding for structural proteins were confirmed by ESI-MS/MS analysis, whereas 45 gene products were functionally annotated within DNA metabolism, packaging, nucleotide biosynthesis and phage morphogenesis. Transmission electron microscopy showed that phage S144 is a myovirus, with a prolate head and short tail fibers. The putative S144 tail fiber structure is, overall, similar to the tail fiber of phage Mu and the C-terminus shows amino acid similarity to tail fibers of otherwise unrelated phages infecting Cronobacter. Since all phages in the Loughboroughvirus genus encode tail fibers similar to S144, we suggest that phages in this genus infect Cronobacter sakazakii and are polyvalent.