Acquisition, loss, and replication of functional modules promote the genetic diversity of Salmonella bacteriophages.

Owing to the threats that Salmonella poses to public health and the abuse of antimicrobials, bacteriophage therapy against Salmonella is experiencing a resurgence. Although several phages have been reported as safe and efficient for controlling Salmonella, the genetic diversity and relatedness among Salmonella phages remain poorly understood. In this study, whole-genome sequences of 91 Salmonella bacteriophages were obtained from the National Center for Biological Information genome database. Phylogenetic analysis, mosaic structure comparisons, gene content analysis, and orthologue group clustering were performed. Phylogenetic analysis revealed four singletons and two major lineages (I-II), including five subdividing clades, of which Salmonella phages belonging to morphologically distinct families were clustered in the same clade. Chimeric structures (n = 31), holin genes (n = 18), lysin genes (n = 66), DNA packaging genes (n = 55), and DNA metabolism genes (n = 24) were present in these phages. Moreover, phages from different subdivided clusters harboured distinct genes associated with host cell lysis, DNA packaging, and DNA metabolism. Notably, phages belonging to morphologically distinct families shared common orthologue groups. Although several functional modules of phages SS1 and SE16 shared > 99% nucleotide sequence identity with phages SI2 and SI23, the major differences between these phages were the absence and replication of functional modules. The data obtained herein revealed the genetic diversity of Salmonella phages at genomic, structural, and gene content levels. The genetic diversity of Salmonella phages is likely owing to the acquisition, loss, and replication of functional modules.

Newly Isolated Virulent Salmophages for Biocontrol of Multidrug-Resistant Salmonella in Ready-to-Eat Plant-Based Food.

Due to irrational antibiotic stewardship, an increase in the incidence of multidrug resistance of bacteria has been observed recently. Therefore, the search for new therapeutic methods for pathogen infection treatment seems to be necessary. One of the possibilities is the utilization of bacteriophages (phages)-the natural enemies of bacteria. Thus, this study is aimed at the genomic and functional characterization of two newly isolated phages targeting MDR Salmonella enterica strains and their efficacy in salmonellosis biocontrol in raw carrot-apple juice. The Salmonella phage vB_Sen-IAFB3829 (Salmonella phage strain KKP 3829) and Salmonella phage vB_Sen-IAFB3830 (Salmonella phage strain KKP 3830) were isolated against S. I (6,8:l,-:1,7) strain KKP 1762 and S. Typhimurium strain KKP 3080 host strains, respectively. Based on the transmission electron microscopy (TEM) and whole-genome sequencing (WGS) analyses, the viruses were identified as members of tailed bacteriophages from the Caudoviricetes class. Genome sequencing revealed that these phages have linear double-stranded DNA and sizes of 58,992 bp (vB_Sen-IAFB3829) and 50,514 bp (vB_Sen-IAFB3830). Phages retained their activity in a wide range of temperatures (from -20 °C to 60 °C) and active acidity values (pH from 3 to 11). The exposure of phages to UV radiation significantly decreased their activity in proportion to the exposure time. The application of phages to the food matrices significantly reduced the level of Salmonella contamination compared to the control. Genome analysis showed that both phages do not encode virulence or toxin genes and can be classified as virulent bacteriophages. Virulent characteristics and no possible pathogen factors make examined phages feasible to be potential candidates for food biocontrol.

Research Note: Therapeutic effect of a Salmonella phage combination on chicks infected with Salmonella Typhimurium.

Antibiotic treatment failure is increasingly encountered for the emergence of pandrug-resistant isolates, including the prototypical broad-host-range Salmonella enterica serovar (S.) Typhimurium, which mainly transmitted to humans through poultry products. In this study we explored the therapeutic potential of a Salmonella phage composition containing a virulent phage and a nonproductive phage that does not produce progeny phage against chicks infected with a pandrug-resistant S. Typhimurium strain of avian origin. After approximately 107 CFU of S. Typhimurium strain ST149 were administrated to chicks by intraperitoneal injection, the phage combination (∼108 PFU) was gavaged at 8-h, 32-h, and 54-h postinfection. At d 10 postinfection, phage treatment completely protected chicks from Salmonella-induced death compared to 91.7% survival in the Salmonella challenge group. In addition, phage treatment also greatly reduced the bacterial load in various organs, with Salmonella colonization levels decreasing more significantly in spleen and bursa than in liver and cecal contents, possibly due to higher phage titers in these immune organs. However, phages could not alleviate the decreased body weight gain and the enlargement of spleen and bursa of infected chicks. Further examination of the bacterial flora in the cecal contents of chicks found that S. Typhimurium infection caused a remarkable decrease in abundance of Clostridia vadin BB60 group and Mollicutes RF39 (the dominant genus in chicks), making Lactobacillus the dominate genus. Although phage treatment partially restored the decline of Clostridia vadin BB60 group and Mollicutes RF39 and increased abundance of Lactobacillus caused by S. Typhimurium infection, Fournierella that may aggravate intestinal inflammation became the major genus, followed by increased Escherichia-Shigella as the second dominate bacterial genus. These results suggested that successive phage treatment modulated the structural composition and abundance of bacterial communities, but failed to normalize the intestinal microbiome disrupted by S. Typhimurium infection. Phages need to be combined with other means to control the spread of S. Typhimurium in poultry.

Temperate phage influence virulence and biofilm-forming of Salmonella Typhimurium and enhance the ability to contaminate food product.

Salmonella is a food-borne zoonotic pathogen that threatens food safety and public health security. Temperate phages can influence bacterial virulence and phenotype and play an important role in bacterial evolution. However, most studies on Salmonella temperate phages focus on prophage induced by bacteria, with few reports on Salmonella temperate phages isolated in the environment. Moreover, whether temperate phages drive bacterial virulence and biofilm formation in food and animal models remains unknown. In this study, Salmonella temperate phage vB_Sal_PHB48 was isolated from sewage. TEM and phylogenetic analysis indicated that phage PHB48 belongs to the Myoviridae family. Additionally, Salmonella Typhimurium integrating PHB48 was screened and designated as Sal013+. Whole genome sequencing revealed that the integration site was specific and we confirmed that the integration of PHB48 did not change the O-antigen and coding sequences of Sal013. Our in vitro and in vivo studies showed that the integration of PHB48 could significantly enhance the virulence and biofilm formation of S. Typhimurium. More importantly, the integration of PHB48 significantly improved the colonization and contamination ability of bacteria in food samples. In conclusion, we isolated Salmonella temperate phage directly from the environment and systematically clarified that PHB48 enhanced the virulence and biofilm-forming ability of Salmonella. In addition, we found that PHB48 increased the colonization and contamination ability of Salmonella in food samples. These results indicated that the highly pathogenic Salmonella induced by temperate phage was more harmful to food matrices and public health security. Our results could enhance the understanding of the evolutionary relationship between bacteriophages and bacteria, and raise public awareness of large-scale outbreaks resulting from Salmonella virulence enhancement in food industry.

The Broad-Spectrum Endolysin LySP2 Improves Chick Survival after Salmonella Pullorum Infection.

Salmonella pullorum causes typical "Bacillary White Diarrhea" and loss of appetite in chicks, which leads to the death of chicks in severe cases; thus, it is still a critical issue in China. Antibiotics are conventional medicines used for Salmonella infections; however, due to the extensive long-term use and even abuse of antibiotics, drug resistance becomes increasingly severe, making treating pullorum disease more difficult. Most of the endolysins are hydrolytic enzymes produced by bacteriophages to cleave the host's cell wall during the final stage of the lytic cycle. A virulent bacteriophage, YSP2, of Salmonella was isolated in a previous study. A Pichia pastoris expression strain that can express the Salmonella bacteriophage endolysin was constructed efficiently, and the Gram-negative bacteriophage endolysin, LySP2, was obtained in this study. Compared with the parental phage YSP2, which can only lyse Salmonella, LySP2 can lyse Salmonella and Escherichia. The survival rate of Salmonella-infected chicks treated with LySP2 can reach up to 70% and reduce Salmonella abundance in the liver and intestine. The treatment group showed that LySP2 significantly improved the health of infected chicks and alleviated organ damage caused by Salmonella infection. In this study, the Salmonella bacteriophage endolysin was expressed efficiently by Pichia pastoris, and the endolysin LySP2 showed good potential for the treatment of pullorum disease caused by Salmonella pullorum.

The Lytic Activity of Bacteriophage ZCSE9 against Salmonella enterica and Its Synergistic Effects with Kanamycin.

Salmonella, the causative agent of several diseases in humans and animals, including salmonellosis, septicemia, typhoid fever, and fowl typhoid, poses a serious threat to global public health and food safety. Globally, reports of therapeutic failures are increasing because of the increase in bacterial antibiotic resistance. Thus, this work highlights the combined phage-antibiotic therapy as a promising approach to combating bacterial resistance. In this manner, the phage ZCSE9 was isolated, and the morphology, host infectivity, killing curve, combination with kanamycin, and genome analysis of this phage were all examined. Morphologically, phage ZCSE9 is a siphovirus with a relatively broad host range. In addition, the phage can tolerate high temperatures until 80 °C with one log reduction and a basic environment (pH 11) without a significant decline. Furthermore, the phage prevents bacterial growth in the planktonic state, according to the results of the time-killing curve. Moreover, using the phage at MOI 0.1 with kanamycin against five different Salmonella serotypes reduces the required antibiotics to inhibit the growth of the bacteria. Comparative genomics and phylogenetic analysis suggested that phage ZCSE9, along with its close relatives Salmonella phages vB_SenS_AG11 and wksl3, belongs to the genus Jerseyvirus. In conclusion, phage ZCSE9 and kanamycin form a robust heterologous antibacterial combination that enhances the effectiveness of a phage-only approach for combating Salmonella.

Isolation, characterization, and genome analysis of a broad host range Salmonella phage vB_SenS_TUMS_E4: a candidate bacteriophage for biocontrol.

Salmonella enteritidis is one of the most important foodborne pathogens that cause numerous outbreaks worldwide. Some strains of Salmonella have become progressively resistant to antibiotics, so they could represent a critical threat to public health and have led to the use of alternative therapeutic approaches like phage therapy. In this study, a lytic phage, vB_SenS_TUMS_E4 (E4), was isolated from poultry effluent and characterized to evaluate its potential and efficacy for bio-controlling S. enteritidis in foods. Transmission electron microscopy revealed that E4 has a siphovirus morphotype, with an isometric head and non-contractile tail. Determining the host range showed that this phage can effectively infect different motile as well as non-motile Salmonella enterica serovars. The biological characteristics of E4 showed that it has a short latent period of about 15 min and a large burst size of 287 PFU/cell, and is also significantly stable in a broad range of pHs and temperatures. The E4 whole genome contains 43,018 bp and encodes 60 coding sequences (CDSs) but no tRNA genes. Bioinformatics analysis revealed that the genome of E4 lacks any genes related to lysogeny behavior, antibiotic resistance, toxins, or virulence factors. The efficacy of phage E4 as a bio-control agent was assessed in various foodstuffs inoculated with S. enteritidis at 4°C and 25°C, and the resulting data indicated that it could eradicate S. enteritidis after a very short time of 15 min. The findings of the present study showed that E4 is a hopeful candidate as a bio-control agent against S. enteritidis and has the potential to be used in various foodstuffs.

Phenotypic Characterization and Comparative Genomic Analysis of Novel Salmonella Bacteriophages Isolated from a Tropical Rainforest.

Salmonella infections across the globe are becoming more challenging to control due to the emergence of multidrug-resistant (MDR) strains. Lytic phages may be suitable alternatives for treating these multidrug-resistant Salmonella infections. Most Salmonella phages to date were collected from human-impacted environments. To further explore the Salmonella phage space, and to potentially identify phages with novel characteristics, we characterized Salmonella-specific phages isolated from the Penang National Park, a conserved rainforest. Four phages with a broad lytic spectrum (kills >5 Salmonella serovars) were further characterized; they have isometric heads and cone-shaped tails, and genomes of ~39,900 bp, encoding 49 CDSs. As the genomes share a <95% sequence similarity to known genomes, the phages were classified as a new species within the genus Kayfunavirus. Interestingly, the phages displayed obvious differences in their lytic spectrum and pH stability, despite having a high sequence similarity (~99% ANI). Subsequent analysis revealed that the phages differed in the nucleotide sequence in the tail spike proteins, tail tubular proteins, and portal proteins, suggesting that the SNPs were responsible for their differing phenotypes. Our findings highlight the diversity of novel Salmonella bacteriophages from rainforest regions, which can be explored as an antimicrobial agent against MDR-Salmonella strains.

Application of the lytic bacteriophage Rostam to control Salmonella enteritidis in eggs.

Foodborne Salmonella enteritidis infections place human health at risk, driven by regular outbreaks and individual cases by different contaminated food materials. This study was conducted to characterize and employ a single bacteriophage as a potential biocontrol agent. Phage Rostam was isolated, characterized and then applied as biocontrol agent against S. enteritidis in liquid whole eggs and eggshell. Rostam is a novel myovirus belonging to the Rosemountvirus genus and active against Escherichia coli and Salmonella spp. Rostam is stable in a pH range from 4 to 10, a salt concentration of 1-9 %, whereas UV radiation gradually reduces phage stability, and its 53 kb genome sequence indicates this phage does not contain known toxins or lysogeny-associated genes. Its latent period is short with a burst size of 151 PFU/cell, under standard growth conditions. Killing curves indicate that at higher multiplicities of infection (MOI), the reduction in S. enteritidis count is more pronounced. Phage Rostam (MOI 10,000) reduces S. enteritidis growth to below the detection limit at 4 °C in both liquid whole eggs and on the eggshell within 24 h. Due to its high lytic activity and stability in relevant conditions, Rostam has the potential to be an efficient biopreservative for egg and egg products.

Comparative Genomics of a Polyvalent Escherichia-Salmonella Phage fp01 and In Silico Analysis of Its Receptor Binding Protein and Conserved Enterobacteriaceae Phage Receptor.

The polyvalent bacteriophage fp01, isolated from wastewater in Valparaiso, Chile, was described to have lytic activity across bacterial species, including Escherichia coli and Salmonella enterica serovars. Due to its polyvalent nature, the bacteriophage fp01 has potential applications in the biomedical, food and agricultural industries. Also, fundamental aspects of polyvalent bacteriophage biology are unknown. In this study, we sequenced and described the complete genome of the polyvalent phage fp01 (MH745368.2) using long- (MinION, Nanopore) and short-reads (MiSeq, Illumina) sequencing. The bacteriophage fp01 genome has 109,515 bp, double-stranded DNA with an average G+C content of 39%, and 158 coding sequences (CDSs). Phage fp01 has genes with high similarity to Escherichia coli, Salmonella enterica, and Shigella sp. phages. Phylogenetic analyses indicated that the phage fp01 is a new Tequintavirus fp01 specie. Receptor binding protein gp108 was identified as potentially responsible for fp01 polyvalent characteristics, which binds to conserved amino acid regions of the FhuA receptor of Enterobacteriaceae.

Anti-Salmonella polyvinyl alcohol coating containing a virulent phage PBSE191 and its application on chicken eggshell.

The use of antibiotics in the food industry is avoided due to the increase of antibiotic-resistant bacteria. Therefore, the bacteriophage is emerging as an alternative agent. Here, we characterized the Salmonella Enteritidis phage PBSE191 and applied it to a polyvinyl alcohol (PVA) film. Transmission electron microscopic analysis revealed that it belonged to the Caudoviricetes class, with an icosahedral head and flexible tails. The phage showed rapid and strong lytic activity within 1 h. It was active against a broad range of Salmonella isolates, including six serotypes. In 25 min, 99 % of the initial population was adsorbed to the bacterial cell surface. The phage was also applied to 10 % (w/v) PVA films and coatings, which were then characterized in terms of phage stability and antibacterial performance, both in vitro and in foods. The phage remained stable in the 10 % (w/v) PVA solution containing 20 % (w/w, based on PVA weight) sorbitol (PVAS20), indicating that the phage was stable under dry conditions and strongly released in the polymer. Furthermore, significant bacterial cell reduction (2.0 × 105 CFU/film within 2 h) was observed in the phage-containing PVAS20 films. In addition, the PBSE191-containing PVAS20 coating on the chicken eggshell surface showed significant anti-Salmonella efficiency (about 2 log CFU reduction) within 24 h. Overall, the PBSE191 phage possesses a high potential as a biocontrol agent for use as an additive, or as an active antibacterial packaging to improve food safety against Salmonella contamination.

Broad-Spectrum Salmonella Phages PSE-D1 and PST-H1 Controls Salmonella in Foods.

Food contamination by Salmonella can lead to serious foodborne diseases that constantly threaten public health. Innovative and effective strategies are needed to control foodborne pathogenic contamination since the incidence of foodborne diseases has increased gradually. In the present study, two broad-spectrum phages named Salmonella phage PSE-D1 and Salmonella phage PST-H1 were isolated from sewage in China. Phages PSE-D1 and PST-H1 were obtained by enrichment with Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) CVCC1806 and Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) CVCC3384, respectively. They were able to lyse Salmonella, E. coli and K. pneumoniae and exhibited broad host range. Further study demonstrated that PSE-D1 and PST-H1 showed high pH and thermal tolerances. Phage PSE-D1 belongs to the Jiaodavirus genus, Tevenvirinae subfamily, while phage PST-H1 belongs to the Jerseyvirus genus, Guernseyvirinae subfamily according to morphology and phylogeny. The results of genome analysis showed that PSE-D1 and PST-H1 lack virulence and drug-resistance genes. The effects of PSE-D1 and PST-H1 on controlling S. Enteritidis CVCC1806 and S. Typhimurium CVCC3384 contamination in three kinds of foods (eggshells, sausages and milk) were further investigated, respectively. Our results showed that, compared to phage-free groups, PSE-D1 and PST-H1 inhibited the growth of their host strain significantly. A significant reduction of host bacteria titers (1.5 and 1.9 log10 CFU/sample, p < 0.001) on eggshells was observed under PSE-D1 and PST-H1 treatments, respectively. Furthermore, administration of PSE-D1 and PST-H1 decreased the counts of bacteria by 1.1 and 1.2 log10 CFU/cm2 (p < 0.001) in sausages as well as 1.5 and 1.8 log10 CFU/mL (p < 0.001) in milk, respectively. Interesting, the bacteriostasis efficacy of both phages exhibited more significantly at 4 °C than that at 28 °C in eggshells and milk and sausages. In sum, the purpose of our research was evaluating the counteracting effect of phage PSE-D1 and PST-H1 on the spread of Salmonella on contaminated foods products. Our results suggested that these two phage-based biocontrol treatments are promising strategies for controlling pathogenic Salmonella contaminated food.

A Combination of Virulent and Non-Productive Phages Synergizes the Immune System against Salmonella Typhimurium Systemic Infection.

Effective phage cocktails consisting of multiple virus types are essential for successful phage therapy against pandrug-resistant pathogens, including Salmonella enterica serovar (S.) Typhimurium. Here we show that a Salmonella phage, F118P13, with non-productive infection and a lytic phage, PLL1, combined to inhibit pandrug-resistant S. Typhimurium growth and significantly limited resistance to phages in vitro. Further, intraperitoneal injection with this unique phage combination completely protected mice from Salmonella-induced death and inhibited bacterial proliferation rapidly in various organs. Furthermore, the phage combination treatment significantly attenuated the inflammatory response, restored the generation of CD4+ T cells repressed by Salmonella, and allowed macrophages and granulocytes to participate in immunophage synergy to promote bacterial clearance. Crucially, the non-productive phage F118P13 is less likely to be cleared by the immune system in vivo, thus providing an alternative to phage cocktail against bacterial infections.

The Combination of Salmonella Phage ST-3 and Antibiotics to Prevent Salmonella Typhimurium In Vitro.

The novel phage ST-3, capable of infecting the foodborne pathogen Salmonella Typhimurium, was isolated from wastewater. The Biological characters and genome information of ST-3 were analyzed. In the in vitro assay, the phage ST-3 with a MOI of 10 effectively inhibited the growth of Salmonella Typhimurium CGMCC 1.1174 in 6 h. The inhibitory effect of combination phage ST-3 and antibiotics was also studied, the removal rate of planktonic host exposed to ST-3 and levofloxacin hydrochloride at the same time, or to ciprofloxacin followed by ST-3, is higher than that exposed to antibiotic dosing group alone and antibiotic + phage dosing group. The phage ST-3 combined with 0.5 µg/mL levofloxacin hydrochloride resulted in the largest decrease in biofilm biomass at 54%. The phage ST-3 could be a potential agent to control Salmonella Typhimurium growth and provide instruction for use it and antibiotics together.

Expansion of the Plaquing Host Range and Improvement of the Absorption Rate of a T5-like Salmonella Phage by Altering the Long Tail Fibers.

The high host specificity of phages is a real challenge in the therapy applications of the individual phages. This study aimed to edit the long tail fiber proteins (pb1) of a T5-like phage to obtain the engineered phages with expanded plaquing host range. Two T5-like Salmonella phages with high genome sequence homology but different plaquing host ranges, narrow-host range phage vB STyj5-1 (STyj5-1) and wide-host range phage vB BD13 (BD13), were isolated and characterized. The pb1 parts of STyj5-1 were replaced by the corresponding part of BD13 using homologous recombination method to obtain the engineered phages. The alterations of the whole pb1 part or the N-terminal amino acids 1-400 of pb1 of STyj5-1 could expand their plaquing host ranges (from 20 strains to 30 strains) and improve their absorption rates (from 0.28-28.84% to 28.10-99.49%). Besides, the one-step growth curves of these engineered phages with modified pb1 parts were more similar to that of STyj5-1. The burst sizes of phages BD13, STyj5-1 and the engineered phages were 250, 236, 166, and 223 PFU per cell, respectively. The expanded plaquing host range and improved absorption rates of these engineered phages revealed that the pb1 part might be the primary determinant of the host specificities of some T5-like phages. IMPORTANCE Genetic editing can be used to change or expand the host range of phages and have been successfully applied in T2, T4 and other phages to obtain engineered phages. However, there are hardly any similar reports on T5-like phages due to that the determinant regions related to their host ranges have not been completely clarified and the editing of T5-like phages is more difficult compared to other phages. This study attempted and successfully expanded the host range of a narrow-host range T5-like phage (STyj5-1) by exchanging its whole pb1 part or the N-terminal 1-400aa of that part by a broad-host range phage (BD13). These demonstrated the pb1 part might be the primary determinant of the host specificities for some T5-like phages and provided an effective method of extension plaquing host range of these phages.

Salmonella Enteritidis Bacteriophages Isolated from Kenyan Poultry Farms Demonstrate Time-Dependent Stability in Environments Mimicking the Chicken Gastrointestinal Tract.

Multi-drug resistant (MDR) Salmonella enterica Enteritidis is one of the major causes of foodborne illnesses worldwide. This non-typhoidal Salmonella (NTS) serovar is mainly transmitted to humans through poultry products. Bacteriophages (phages) offer an alternative to antibiotics for reducing the incidence of MDR NTS in poultry farms. Phages that survive the harsh environment of the chicken gastrointestinal tract (cGIT), which have low pH, high temperatures, and several enzymes, may have a higher therapeutic or prophylactic potential. In this study, we analysed the stability of 10 different S. Enteritidis phages isolated from Kenyan poultry farms in different pH-adjusted media, incubation temperatures, as well as simulated gastric and intestinal fluids (SGF and SIF, respectively). Furthermore, their ability to persist in water sources available in Kenya, including river, borehole, rain and tap water, was assessed. All phages were relatively stable for 12 h at pHs ranging from 5 to 9 and at temperatures ranging from 25 °C to 42 °C. At pH 3, a loss in viral titre of up to three logs was observed after 3 h of incubation. In SGF, phages were stable for 20 min, after which they started losing infectivity. Phages were relatively stable in SIF for up to 2 h. The efficacy of phages to control Salmonella growth was highly reduced in pH 2- and pH 3-adjusted media and in SGF at pH 2.5, but less affected in SIF at pH 8. River water had the most significant detrimental effect on phages, while the other tested waters had a limited impact on the phages. Our data suggest that these phages may be administered to chickens through drinking water and may survive cGIT to prevent salmonellosis in poultry.

Genetic Mining of Newly Isolated Salmophages for Phage Therapy.

Salmonella enterica, a Gram-negative zoonotic bacterium, is mainly a food-borne pathogen and the main cause of diarrhea in humans worldwide. The main reservoirs are found in poultry farms, but they are also found in wild birds. The development of antibiotic resistance in S. enterica species raises concerns about the future of efficient therapies against this pathogen and revives the interest in bacteriophages as a useful therapy against bacterial infections. Here, we aimed to decipher and functionally annotate 10 new Salmonella phage genomes isolated in Spain in the light of phage therapy. We designed a bioinformatic pipeline using available building blocks to de novo assemble genomes and perform syntaxic annotation. We then used genome-wide analyses for taxonomic annotation enabled by vContact2 and VICTOR. We were also particularly interested in improving functional annotation using remote homologies detection and comparisons with the recently published phage-specific PHROG protein database. Finally, we searched for useful functions for phage therapy, such as systems encoded by the phage to circumvent cellular defenses with a particular focus on anti-CRISPR proteins. We, thus, were able to genetically characterize nine virulent phages and one temperate phage and identify putative functions relevant to the formulation of phage cocktails for Salmonella biocontrol.

Working with Phage P22.

Transduction experiments in Escherichia coli and Salmonella are usually performed with virulent phage variants. A widely used P1 mutant, called P1 vir, carries one or more uncharacterized mutations that prevent formation of lysogens. In the case of P22, by far the most frequently used variant is named P22 HT105/1 int-201 This phage has a high transducing (HT) frequency due to a mutant nuclease with lower specificity for the pac sequence. As a result, ∼50% of the P22 HT phage heads carry random transducing fragments of chromosomal DNA. The int mutation reduces the formation of stable lysogens. The basic steps in handling the P22 HT105/1 int-201 phage and in performing transduction experiments in Salmonella are described here.

Crystal structure of the phage-encoded N-acetyltransferase in complex with acetyl-CoA, revealing a novel dimeric arrangement.

Bacteriophages employ diverse mechanisms to facilitate the proliferation of bacteriophages. The Salmonella-infecting phage SPN3US contains a putative N-acetyltransferase, which is widely found in bacteriophages. However, due to low sequence similarity to the N-acetyltransferases from bacteria and eukaryotic cells, the structure and function of phage-encoded acetyltransferases are mainly unknown. This study determines the crystal structure of the putative N-acetyltransferase of SPN3US in complex with acetyl-CoA. The crystal structure showed a novel homodimeric arrangement stabilized by exchanging the C-terminal α-helix within the dimer. The following biochemical analyses suggested that the phage-encoded acetyltransferase might have a very narrow substrate specificity. Further studies are required to reveal the biochemical activity, which would help elucidate the interaction between the phage and host bacteria in controlling pathogenic bacteria.

Salmonella phage akira, infecting selected Salmonella enterica Enteritidis and Typhimurium strains, represents a new lineage of bacteriophages.

Some serovars of Salmonella can cause life-threatening diarrhoeal diseases and bacteriemia. The emergence of multidrug-resistant strains has led to a need for alternative treatments such as phage therapy, which requires available, well-described, diverse, and suitable phages. Phage akira was found to lyse 19 out of 32 Salmonella enterica serovars and farm isolates tested, although plaque formation was observed with only two S. Enteritidis and one S. Typhimurium strain. Phage akira encodes anti-defence genes against type 1 R-M systems, is distinct (<65% nucleotide sequence identity) from related phages and has siphovirus morphology. We propose that akira represents a new genus in the class Caudoviricetes.