Bacteriophage compositions

Abstract

A method of identifying and / or isolating one or more bacteriophages that display lytic activity against at least one target bacterial genus, species, or strain. The method includes the creation of a transposon mutant library using at least one bacterial strain from said genus or species and identifying the genes involved in resistance and / or susceptibility by exposing said library to at least one or more bacteriophages.

Description

[0001]Bacteriophage Compositions The present invention relates to the identification, isolation, application, characterisation of bacteriophages and process of bacteriophage infection and design of bacteriophage mixtures or cocktails. Bacteriophages (Phages) are viruses which infect and lyse bacteria. Phages may be present in every niche in which bacteria thrive and are predicted to play crucial roles in population dynamics and bacterial evolution (4). Bacteriophages infect their hosts by binding to specific receptors located on the cell surface of a susceptible host. In Gram Negative bacteria, well characterised receptors include Outer Membrane Proteins (OMPs), Lipopolysacharide (LPS) and flagella antigens (5). Once bound, the phage injects its genetic material into the bacterium and, in the case of lytic phages, hijacks the hosts cellular machinery to produce new virions (6). The end point of infection is the translation of a phage encoded enzyme which lyses the bacterial cell to release newly assembled phage progeny (7). The number of strains of bacteria which a bacteriophage can successfully infect is commonly referred to as the host range. Phages typically have a narrow host range, in which they only infect a specific bacterium and its close relatives. This is due to many factors including the specificity of phage encoded receptor binding proteins as well as a diverse array of host phage defence mechanisms (8). Bacteriophages have many applications and have been commonplace in molecular biology for techniques such as horizontal gene transfer by transduction (9), diagnostics (10) and bacterial typing (11). More recently, the ability of lytic phages to kill bacteria has renewed interest for their use in both Phage therapy and as methods in agriculture and food processing (12, 13). This has, in part, been due to the emergence of difficult to treat, antibiotic resistant bacterial infections. However, the use of phages in this context is difficult, due to the rapid emergence of phage resistance and the narrow host range of bacteriophages. To overcome these limitations, phages are often pooled together to form a ‘phage cocktail’ to both extend host range and limit the emergence of resistance. Selection of Phages to be included in a phage cocktail should be based on a robust understanding of the bacteriophage host range, targeted receptor and mechanisms of host resistance (14). However, data is often limited, leading to poor cocktail design and failed applications (15). In a first aspect of the invention there is provided a method of identifying and / or isolating one or more bacteriophages that display lytic activity against at least one target bacterial genus, species, or strain said method including the creation of a transposon mutant library using at least one bacterial strain from said genus or species and identifying the genes involved in resistance and / or susceptibility by exposing said library to at least one or more bacteriophages. Preferably the bacteriophages identified are used as one or more components of a phage cocktail or bacteriophage mixture effective against one or more bacterial species or strains. In a preferred embodiment the method includes the isolation of two or more phages and the use of a whole genome functional screen fitness assay to identify all host genes involved in infection by said isolated phages. In a preferred embodiment the transposon mutant library is prepared using established methods and the role of specific genes in sensitivity and to phage assessed using TraDIS. Typically TraDIS is used to establish both host susceptibility and resistance genes involved in infection by bacteriophages. In one embodiment using the receptor information from the TraDIS screen combinations of bacteriophages are identified that work more effectively together than other phage combinations. Typically improved efficacy includes increased lytic activity and / or reduced host resistance. Typically relatively large or small sets of bacteriophages can be isolated from one or more different environments and / or samples. An advantage of the present invention is that relatively small set of bacteriophages can be uti lised. Further typically the bacteriophages are isolated from food enrichments, environmental water samples, wastewater effluent and / or the like. In one embodiment TraDIS is used to identify receptors for each bacteriophage. In a preferred embodiment the method includes the step of selecting one or more bacterial strains. Typically a plurality of strains are used. Further typically a plurality of strains are used for the enrichment of environmental samples for the isolation of bacteriophages. Strains may be taken from a single species. In one embodiment one or more serovars are selected from the species selected. In one embodiment the strains are isolated from food products. In one embodiment the includes the step of bacterial culture and / or allelic exchange. Typically the strains are cultured in LB (Luria Bertani) broth or on 1.5% LB Agar plates. Further typically, where appropriate, and / or required, cultures were supplemented with Kanamycin (50 µg / ml) or Hygromycin (75µg / ml). In one embodiment construction of any mutant strains was performed by one-step inactivation using a method previously described by Datsenko and Wanner (23). The skilled person will recognise that other methods of mutation can be used. Typically PCR products were generated using primers. The primers typically used are l isted (below). In one embodiment the primers are amplified. Typically the aphII gene from plasmid pKD4 is used and tagged it with 50bp regions which were homologous to the target for mutagenesis at both ends. In one embodiment bacteriophage samples from food are enriched in buffered peptone water (BPW) and incubation. Typically to isolate bacteriophages, the sample is centrifuged and the supernatant filtered. Further typically an aliquot of filtered sample is added to LB Broth which is inoculated with exponential phase bacterial culture. Typically enrichments are centrifuged and filtered. Further typically samples are checked for the presence of bacteriophages using the double-agar overlay method. In a preferred embodiment method includes the steps of bacteriophage nucleic acid extraction, sequencing and / or bioinformatic analysis. Typically increasing the titre of bacteriophages is performed using subsequent liquid enrichments. Further typically high titre (substantially at least 109PFU / ml) lysates are used for Nucleic Acid extraction. In one embodiment aliquots of each phage are DNase treated. Typically DNase treated samples are precipitated and resuspended. In one embodiment nucleic acid purification is performed with proteinase digestion. Typically NexteraXT libraries are prepared following the manufacturer’s protocols (Illumina, USA) and sequenced using the Illumina NextSeq500 platform, although the skilled person will appreciate that any sequencing method could be used. Typically raw Illumina sequencing reads were subject to quality control using fastP (26). Reads passing quality control are used for de novo assembly using the SPAdes based assembler Shovill (27, 28). Further typically reads were mapped to assembled contigs using BWA-mem (29). Similar mapping was used to make host reads to phage genomes to check for lysogenic induction. A Proteomic tree based of genome wide sequence similarity calculated by tBLASTx was produced using VIPtree (30). Viron morphology and genera predictions were made based on closest relatives using PhageAI (31). In one embodiment the includes the steps of bacteriophage host range and growth curve analysis. Typically bacterial strains were grown in LB broth and initial inoculum introduced to each well of a microtiter plate. Further typically bacteriophages were inoculated into wells to achieve a multiplicity of infection (MOI) of 1. In one embodiment the phages are immediately placed into a microplate reader. Typically the plate is incubated and bacterial growth curves plotted using mean baseline corrected Optical Density readings. Area under the curve (AUC) scores were calculated using the previously published method by Xie et al (32). Frequency of Resistance to phages in monotherapy and combination therapy is assessed using the same method with technical replicates. The method used to calculate difference in Area under the Curve is presented in Equation 1 and 2. 24 OD + OD Equation 1-2 – Method used to quantify growth curve data into area under the curve (AUC) scores. In one embodiment construction of TraDIS mutant library includes the preparation of transposomes by mixing purified transposon DNA with a carrier such as glycerol and transposase. Typically for preparation of electrocompetent cells a culture was first prepared in LB broth and incubated. Further typically aliquots of cells grown to stationary phase were inoculated into broth and incubated until the optical density (measured at 600nm) reached 0.20-0.25. Cells at the required optical density are harvested by centrifugation. Typically the resulting cells are washed and resuspended. A portion of electrocompetent bacterial cells were mixed with nuclease free water and TypeOne Restriction Inhibitor (Lucigen) and transposome, further typically on ice. Following electroporation, Cells are resuspended. Further typically, for enumeration, aliquots of transformants were plated on LB agar containing kanamycin. Remaining amount of transformants may be plated in equal aliquots onto LB agar plates containing kanamycin. In one embodiment the plates are incubated and colonies enumerated the following day. Typically resulting colonies are harvested and combined. Further typically a plurality of electroporations are conducted in order to generate a large transposon mutant library. A master mix library stock was created by taking aliquots of each batch and mixing thoroughly. In one embodiment the includes the step of the selection of TraDIS library using Bacteriophages. Typically the Transposon mutant library is challenged with a number of isolated bacteriophages. Further typically a pool of the prepared library is cultured in non-selective conditions. Sub-cultures are prepared with each phage added and incubated. After selection, Genomic DNA is extracted as previously described. In the method the preparation of TraDIS library DNA for Sequencing includes the step of genomic DNA is normalised and tagmented. Fragmented DNA was purified and DNA is amplified by PCR using biotinylated primers specific to the transposon and primers for the tagmented ends of DNA. PCR products were purified again incubated. Typically a subsequent PCR step using barcoded sequencing primers allows for the pooling of samples. Streptavidin beads are magnetically removed from the PCR products which are further purified and size-selected. Typically DNA libraries are quantified and reads were analysed by mapping to a reference genome. In a second aspect of the invention there is provided a method of determining the constituents of a phage cocktail or bacteriophage mixture effective against one or more bacterial species or strains, said method including the isolation of two or more phages and the use of a genomics based fitness assay to identify all host genes involved in infection by said isolated phages. Preferably TraDIS is used to one or more host receptors that likely contribute to the success of a combination of phages as biological control agents. In a third aspect of the invention there is provided a method for exposing a collateral sensitivity mechanism of bacteriophages infecting one or more bacteria by whole genome functional screening of bacteria to identify genes involved in sensitivity to one or more bacteriophages. In a further aspect of the invention there is provided a method to establish both host susceptibility, for example receptors, and / or resistance genes involved in infection by bacteriophages, wherein the method uses a TraDIS screen to identify combinations of bacteriophages which work more effectively together than other phage combinations or single phages. Typically improved efficacy includes increased lytic activity and reduced host resistance. Specific embodiment of the invention are now described with reference to the following figures wherein: Figure 1 shows a proteomic tree of viral genome sequences based on genome-wide sequence similarities of viruses infecting gammaproteobacteria generated by VipTree, with Viral Family annotation based on Inphared. Salmonella Phage Isolates (•) coloured by viral genera: Berlinvirus (red), Seoulvirus (orange), Phikzvirus (yellow), Tequintavirus (green), Seunavirus (Lightblue), Rosemountvirus (Purple) and Lederbergvirus (darkblue). Icons indicate predicted phage morphology. Figures 2a-c show Host Range of 12 Salmonella Phages against 36 bacterial strains determined by microtiter plate liquid assay. A) Maximum likelihood trees of bacterial strains used for host range analysis based on sequence variation (SNPs) in the: 16s rRNA sequence (2ai) and core genome (2aii and 2aii). B) Heat map showing liquid assay virulence score (LAVS) of each phage-host combination generated using Equation 1 and 2. C) Violin plots of LAVS scores of each phage against non- Salmonella (Top), Salmonella serovars (middle) and Salmonella Typhimurium (bottom). Figures 3a-f show volcano plots showing changes in the Insertions within genes in phage treated conditions relative to non-phage treated control. Log fold change in insertions (x-axis) is plotted against significance (y-axis) using -log10(q-value). Each graph shows gene hits for treatment with different phage: A = SPLA1a, B = SPLA1b, C = SPLA2, D = SPLA5b, E = SPLA5c and F = SPLA11. Blue points show a significant decrease in insertions. Red points show a significant increase in insertions. Figures 4a-c show the role of Glycosyltransferase gtrB in Phage Resistance to SPLA5c. A) Genomic Context of gtrABC in Salmonella Typhimurium ST4 / 74. B) Transposon Insertion Sites mapped to Salmonella Typhimurium 4 / 74 in the gtrABC operon in the presence of SPLA1a, SPLA5c and non-Phage Treated control. C) Growth of WT and ΔgtrB strains in the presence and absence of SPLA1a (top) and SPLA5b (Bottom). Figures 5a-b show the growth of Salmonella Typhimurium ST4 / 74 in the presence of SPLA1a and SPLA5b in Monotherapy and Combination therapy. A) Heatmap of end point optical density (OD) at 600nm after 18 hours of treatment with SPLA1a (Top), SPLA5b (Middle) and SPLA1a and SPLA5b (Bottom). B) Example growth curves of infection by SPLA1a (I-III) SPLA5b (IV-VI) and SPLA1a and SPLA5b (VII-IX). Figures 6a-d show the collateral sensitivity dynamic of Bacteriophages SPLA1a and SPLA5b. A) Plaque assays using SPLA1a (left) and SPLA5b (Right) against LPS and BtuB Mutants. B) Genotype of SPLA1a insensitive mutant compared to ST4 / 74 WT strain. C) Growth curve analysis of Wildtype (Red) and ΔrfaK (Green) when treated with no phages (Left), SPLA1a (Middle) and SPLA5b (Right). C) Schematic of the proposed Collateral sensitivity mechanism observed with SPLA1a (Yellow Phage) and SPLA5b (Green Phage). Figures 7a-7b show comparison results of Salmonella shedding in mice between non phage treated mice, mice treated with single phage preparations and phage cocktail treated mice. Bacteriophages are the most abundant biological entity on Earth and are found in all ecosystems where bacteria are present. Their ability to lyse susceptible bacterial hosts influences many aspects of bacterial evolution, fitness, and population structure. Despite the significant impact bacteriophages have on bacterial ecology, very little is known about the interaction between bacteriophages and their hosts, outside of work on model phages. Knowledge of both host susceptibility and resistance factors is crucial for the successful application of bacteriophages as biological control agents in clinical therapy, food processing and agriculture. In this study, we present the isolation of twelve Bacteriophages which infect the foodborne pathogen Salmonella enterica . The host range of each bacteriophage against a diverse set of S. enterica strains is presented. A Genomics based fitness assay (TraDIS) was used to identify all host genes involved in infection by newly isolated phages. TraDIS identified different host receptors, Lipopolysaccharide for SPLA1a and BtuB (a Vitamin B12 transporter) for SPLA5b which likely contributed to the success of combination therapy. Using two phages (SPLA1a and SPLA5b) we that combination therapy was more successful in limiting the emergence of phage resistance compared to monotherapy. Using single isogenic LPS mutants, we show that a lack of O-antigen increases the sensitivity to SPLA5b and demonstrate a Collateral sensitivity dynamic when these bacteriophages are used in combination therapy. Identification of infection dynamics and host resistance and susceptibility factors is critical step in rationale design of Phage cocktails against specific bacterial pathogens. Non-Typhoidal Salmonella (NTS) remains an important pathogen which poses a significant threat to human and livestock health, and the economy. Enteric NTS is estimated to cause 93.8 million diarrheal illnesses and 155,000 deaths each year (1), and an estimated economic cost exceeding 3 billion Euros annually in the European Union alone (2). Many of these infections are caused by the consumption of contaminated food products. In addition, increases in antibiotic resistance of Salmonella isolates makes treatment far more difficult – with extensively drug resistant (XDR) strains increasing in prevalence (3). There is an urgent need for new methods for the treatment and prevention of Salmonella infection – with bacteriophages a potential new tool to prevent the contamination of food or as an alternative to antibiotics for therapy. Bacteriophages (Phages) are viruses which infect and lyse bacteria. Phages are present in almost every niche in which bacteria thrive and are predicted to play crucial roles in population dynamics and bacterial evolution (4). Bacteriophages infect their hosts by binding to specific receptors located on the cell surface of a susceptible host. In Gram Negative bacteria, well characterised receptors include Outer Membrane Proteins (OMPs), Lipopolysacharide (LPS) and flagella antigens (5). Once bound, the phage injects its genetic material into the bacterium and, in the case of lytic hijacks the hosts cellular machinery to produce new virions (6). The end point of infection is the translation of a phage encoded enzyme which lyses the bacterial cell to release newly assembled phage progeny (7). The number of strains of bacteria which a bacteriophage can successfully infect is commonly referred to as the host range. Phages typically have a narrow host range, in which they only infect a specific bacterium and its close relatives. This is due to many factors including the specificity of phage encoded receptor binding proteins as well as a diverse array of host phage defence mechanisms (8). Bacteriophages have many applications and have been commonplace in molecular biology for techniques such as horizontal gene transfer by transduction (9), diagnostics (10) and phage typing (11). More recently, the ability of lytic phages to kill bacteria to kill bacteria has renewed interest for their use in both Phage therapy and as intervention methods in agriculture and food processing (12, 13). This has, in part, been due to the emergence of difficult to treat, antibiotic resistant bacterial infections. However, the use of phages in this context is difficult, due to the rapid emergence of phage resistance and the narrow host range of bacteriophages. To overcome these limitations, phages are often pooled together to form a ‘phage cocktail’ to both extend host range and limit the emergence of resistance. Selection of Phages to be included in a phage cocktail should be based on a robust understanding of the bacteriophage host range, targeted receptor and mechanisms of host resistance (14). However, data is often limited, leading to poor cocktail design and failed applications (15). This study uses transposon directed insertion site sequencing (TraDIS), a genome wide genomics based fitness assay to simultaneously assay all non-essential host genes for their involvement under selective TraDIS has previously been used to identify genes involved in antibiotic stress (16) bile resistance (17) and biofilm formation (18). Transposon mutagenesis has also been used to identify receptors for T4 (19), λ (20) T2, T6 and T7 Phages (21). However much of this work has been limited to the use of well characterised model phages. To address this, we present the isolation and genomic characterisation of twelve bacteriophages from the environment which all display lytic activity against the foodborne pathogen, Salmonella enterica. Using a high-throughput, microtiter plate- based method, we determine the host range and virulence of each bacteriophage against a collection of bacteria found in food – Including diverse Salmonella strains. Using a saturating transposon mutant library prepared using Salmonella enterica sv Typhimurium strain ST4 / 74 we identify host genes involved in resistance and susceptibility to six newly isolated bacteriophages. Using Single Isogenic mutants of genes identified by TraDIS we identify a collateral sensitivity dynamic, where resistance to infection by one phage, SPLA1a, leads to increased sensitivity to a different phage, SPLA5b. This dynamic contributes to increased virulence when used together as a phage cocktail and limits host resistance occurrence. MATERIALS AND METHODS Selection of Bacterial Strains. In total, 12 strains were used for the enrichment of environmental samples for the isolation of bacteriophages. All Strains were Salmonella enterica . The serovars used were Montevideo, Panama, Mbandaka, Kedougou, Infantis, Derby, Newport, Enteritidis and Typhimurium. Eleven of the Strains were previously isolated from food products in the UK. The remaining strain was an isogenic mutant of Salmonella enterica sv Typhimurium strain ST4 / 74. This strain lacked the Gifsy-1, Gifsy-2, ST46B, SopEΦ and P4-like prophages (22). Strains used for analysis of Bacteriophage host range were termed HRS (Host Range . This collection comprised of 36 strains and was made up of Salmonella enterica as well as other Bacterial strains which were isolated from food products. The summary of these strains can be found in supplementary table 1. Bacterial Culture and Allelic Exchange. Strains were routinely cultured in LB (Luria Bertani) broth or on 1.5% LB Agar plates. Were appropriate, cultures were supplemented with Kanamycin (50 µg / ml) or Hygromycin (75µg / ml). Construction of mutant strains was performed by one-step inactivation using a method previously described by Datsenko and Wanner (23). PCR products were generated using primers listed (Supplementary Table 2). The primers amplified the aphII gene from plasmid pKD4 and tagged it with 50bp reigons which were homologous to the target for mutagenesis at both ends. Allelic exchange was performed in Salmonella enterica sv Typhimurium ST4 / 74 containing pSIM18 (24). Isolation of Bacteriophages. All Environmental Samples were collected between November 2019 and February 2020. Samples included eight wastewater treatment samples, eight retail meat samples, nine lake / river samples and six samples from drains located in a food production factory. Samples from food were enriched in buffered peptone water (BPW) via incubation at 37⁰C for 18hours. In order to isolate bacteriophages, 25ml of sample was centrifuged at 4000 RPM for 10 minutes. The supernatant was fi ltered through a 0.45µM pore size filter. A 5ml aliquot of filtered sample was added to 40ml of 2x LB Broth which was inoculated with 200µl of exponential phase bacterial culture. Enrichments were incubated for 18 hours. Enrichments were centrifuged and filtered as previously described. Samples were checked for the presence of bacteriophages using the double-agar overlay method previously described (25) (21) using individual bacterial strain to identify phage plaques. Bacteriophage Nucleic Acid Extraction, Sequencing and Bioinformatic Analysis. Increasing the titre of bacteriophages was performed using subsequent liquid enrichments. High titre (at least 109PFU / ml) lysates were used for Nucleic Acid extraction. A 1ml aliquot of each phage was DNase treated at 37⁰C for 40 minutes. DNase treated samples were precipitated using polyethylene glycol and resuspended in 200 µl of nuclease free water. Nucleic Acid Purification with Proteinase K digestion was performed using Maxwell® RSC Total Viral Nucleic Acid Purification Kit (Promega, USA). NexteraXT libraries were prepared following the manufactuer’s protocols (Illumina, USA) and sequenced using the Illumina NextSeq500 platform. Raw Illumina sequencing reads were subject to quality control using fastP (26). Reads passing quality control were used for de novo assembly using the SPAdes based assembler Shovill (27, 28). Reads were mapped to assembled contigs using BWA- mem (29). Similar mapping was used to make host reads to phage genomes to check for lysogenic induction. A Proteomic tree based of genome wide sequence similarity calculated by tBLASTx was produced using VIPtree (30). Virion morphology and genera predictions were made based on closest relatives using PhageAI (31). Phylogenetic Reconstruction of Host Range Strains. Genomic DNA from each bacterial strain was sequenced using Illumina Nextseq. For phylogenetic reconstruction of bacterial strains of multiple difference genera, paired end reads were assembled as previously described, and 16s rRNA genes were identified using barrnap (https: / / github.com / tseemann / barrnap). Multiple sequence alignments of 16s performed using MAFFT (32). The alignment was using TrimAL (33). Phylogenetic reconstruction was performed using IQtree (34) with a bootstrap value of 1000. For phylogenetic construction of Salmonella strains, paired end sequences were mapped to the SL1344 reference genome (FQ312003) using the Rapid haploid variant calling and score SNP phylogeney pipeline SNIPPY (https: / / github.com / tseemann / snippy). Maximum likelihood phylogenetic trees was constructed using the multiple sequence alignment by RaxML using the GTRCAT model with bootstrapping value of 100. Bacteriophage Host Range and Growth curve Analysis. Bacterial strains were grown in LB broth for 18 hours at 37⁰C at 200rpm. Overnight Cultures were adjusted to an initial inoculum concentration of 107CFU / ml in each well of a 96-well microtiter plate. Bacteriophages were inoculated into wells to achieve a multiplicity of infection (MOI) of 1. Phages were immediately placed into FluroStar Omega Microplate reader (BMG LABTECH, Germany). Optical Density (600nm) was measured every 15 minutes for 18hours. The plate was incubated at 37 ⁰C with double orbital shaking. Bacterial growth curves were plotted using mean baseline corrected Optical Density readings against time with 3 technical replicates. Area under the curve (AUC) scores were calculated using the previously published method by Xie et al (35). Frequency of Resistance to phages SPLA1a and SPLA5b in monotherapy and combination therapy was assessed using the same method with 96 technical replicates. The method used to calculate difference in Area under the Curve is presented in Equation 1 and 2. 24 OD rea Under the Curve =Σ i+1 + OD A i i=1 2 Equation 1-2 – Method used to quantify growth curve data into area under the curve (AUC) scores. Construction of TraDIS mutant Library. To prepare the transposon mutant library in S. Typhimurium ST4 / 74, transposon DNA, with the tac-like promoter omitted, was amplified by polymerase chain reaction (PCR) using P-Tn5Km-01 and P- Tn5Cm-04 oligonucleotides (sequences available in supplementary table 1). Transposomes were prepared by mixing 2µL purified transposon DNA (100ng / µL) with 2µL 100% glycerol and 4µL EZ-Tn5 transposase (Lucigen). For preparation of electrocompetent cells of S . Typhimurium ST4 / 74, a culture was first prepared in 5mL LB broth and incubated for 18 hours at 37°C in a shaking incubator (200rpm). A 500µL aliquot of cells grown to stationary phase were inoculated into 50mL 2xYT broth and incubated at 37°C until the optical density (measured at 600nm) reached 0.20-0.25. Cells at the required optical density were harvested by at 3500g for 10 minutes at 4°C. The resulting cells were washed three times with 10% glycerol, and resuspended in 600 µL of 10% glycerol. A 60µL portion of electrocompetent bacterial cells were mixed with 2µL sterile nuclease free water (albion), 2 µL TypeOne Restriction Inhibitor (Lucigen) and 0.4µL transposome, on ice. Following electroporation, Cells were immediately resuspended in 1mL S.O.C (prewarmed to 37°C) and recovered at 37°C for 1.5 hours. For enumeration, a 10µL and 100µL aliquot of transformants were plated on LB agar containing kanamycin (50µg / mL). The remaining amount of transformants were plated in equal aliquots onto LB agar plates containing kanamycin (50µg / mL). Plates were incubated at 37°C overnight and colonies enumerated the following day. Resulting colonies were harvested using 1mL LB broth per plate and combined into a 50mL falcon tube. For storage at -80°C, 50% Glycerol was added to the cells to a final concentration of 15%. In total, 35 electroporation’s were conducted in order to generate a large enough transposon mutant library. A master mix library stock was created by taking aliquots of each batch and mixing thoroughly. The master mix was stored at -20°C until required. For each stress experiment, a 50µL aliquot of S . Typhimurium ST4 / 74 master mix library was used. Whole genome functional screen using transposon directed insertion site sequencing. The transposon mutant library was challenged with six bacteriophages (SPLA1a, SPLA2, SPLA5B, SPLA5c, SPLA11 and SPLA12) that exhibited fully lytic activity when exposed to S. Typhimurium strain ST4 / 74. The Tn5 insertion library was cultured in LB broth at 37oC with shaking for 18 hours. This culture was diluted to 107CFU / ml in 10ml of LB broth containing phage added at an MOI of 10, except SPLA1a that was added to an MOI of 1, and incubated at 37⁰C for 3 Hours. A negative containing with no phage supplementation was included. A 2 ml sample of each culture was then harvested by centrifugation at 2500xg and genomic DNA extracted as previously described. Two independent replicates were carried out. Preparation of TraDIS library DNA for Sequencing. Genomic DNA from the Tn5 insertion library with and without exposure to SPLA phages was diluted to 11.1 ng / µl and tagmented using MuSeek DNA fragment library preparation kit (ThermoFisher, USA). Fragmented DNA was purified using AMPure XP beads (Beckman Coulter, USA). DNA was amplified by PCR using biotinylated primers specific to the transposon and primers for the tagmented ends of DNA. PCR products were purified again using AMPure XP beads and incubated overnight with streptavidin bead (Dynabeads®) to allow for capture of the DNA fragments with the transposon. A Subsequent PCR step using barcoded sequencing primers allowed for the pooling of samples. Streptavidin beads were magnetically removed from the PCR products which were further purified and size-selected using AMPure XP beads. DNA libraries were quantified using Qubit 3.0 (Invitrogen, USA) and Tapestation (Aligent Technologies, USA). Reads were analysed by mapping to the reference genome (Salmonella enterica sv Typhimurium strain ST4 / 74) using BioTraDIS (33). Significant changes in insertions between the phage treated and control conditions were identified as part of the pipeline (p < 0.05). RESULTS Successful Isolation of Bacteriophages lytic against Salmonella enterica. To establish a diverse collection of phage capable of lysis of S. enterica strains we enriched phage from wastewater, river or food samples using a mixture of enrichment strains in broth culture. phage were identified on double agar overlay containing each of the enrichment strains to identify the host S. enterica strain with the greatest sensitivity to lysis. Plaques with distinct morphology were picked and purified 5 further by rounds of enrichment by the preferred host strain. In Total, 12 phages were isolated and designated SPLA phages; SPLA1a, SPLA1b, SPLA2, SPLA3, SPLA4, SPLA5a, SPLA5b, SPLA5c, SPLA9, SPLA10, SPLA11 and SPLA12. Whole genome sequence assembled into a single contiguous sequence for each 10 phage that ranged in size from 40,585 bp to 240,593 bp. Whole genome sequence reads of the S. enterica strains used to enrich the phage failed to align to the assembled phage genomes with the exception of SPLA1b for which sequence reads corresponding to a predicted prophage in S. enterica strain ??? 15 aligned to the full length of the assembled sequence. Table 1 – Summary of Bacteriophages Isolated and used in this Study. Family and Genus Predictions made using PhageAI 20 SPLA phages are diverse, and distributed among different lineages . To investigate the diversity and relationship to phage genome sequences in available databases SPLA phages were placed in a phylogenetic context based on sequence similarity of their predicted proteome sequence. Initial comparison with the Isolation Host Source Genome Size Predicted Family Predicted Genus SPLA4 S.Mbandaka drain 39540 Berlinvirus predicted proteome of ??? indicated that all SPLA phages were closely related to phage known to infect gammaproteobacteria (Supplementary Figure 1). Therefore, to improve clarity the analysis was repeated with only phages of gammaproteobacteria. The SPLA phages were present in clusters on seven deeply rooted lineages that corresponded to Berlinvirus, Seoulvirus, Phikzvirus, Tequintavirus, Seunavirus, Rosemountvirus and Lederbervirus genera (Figure 1). SPLA1a, SPLA3 and SPLA5b were predicted myophages of the genus Seoulvirus with genome sizes of above 200kb and as such classified as Jumbo Phage (34). These Seoulviruses had an average nucleotide sequence identity (ANI) greater than 98.4%. Similarly, SPLA2, SPLA9 and SPLA11 and SPLA12 were of genus Rosemountvirus and exhibited greater than 95.6%% ANI. Phages SPLA4 and SPLA5b were the only phages which are currently assigned to established viral families, Autographiviriae and Demerecviridae respectively, according to the ICTV database (accessed December 2022). SPLA phages are highly specific to Salmonella serotypes. To determine the host range of the SPLA phages, susceptibility of 36 bacterial strains were tested. These bacterial strains included eleven strains of diverse genera of Enterobacterales and 25 S. enterica strains comprising of fourteen different serovars. Strains of Enterobacterales species other than S. enterica exhibited l ittle susceptibility to SPLA phages with the exception of moderate susceptibility of a Hafnia alvei strain, particularly to SPLA2 and SPLA4 phage. Susceptibil ity of S. enterica strains to SPLA phages varied markedly. No one SPLA phage exhibited moderate to high virulence to one or more strains of 14 serotypes tested. Phages SPLA9, SPLA11 and SPLA1a, displayed the broadest host range,with at least moderate virulence (>25 lytic index) for at least ten of the fourteen serotypes tested. Nonetheless, all except SPLA5a, SPLA5b and SPLA1b had at moderate virulence against over half of the serotypes tested (Figure 2 B and C). Despite the close relationship of S . Typhimurium strains relative to strains from distinct serotypes, SPLA phages exhibited similar variability in virulence. Phage SPLA1a was particularly virulent for a broad range of S. Typhimurium strains and SPLA9 and SPLA11 were also moderately virulent for at least eleven of the 12 strains tested. Notably, even very closely related strains exhibited diversity in sensitivity to SPLA phages. For example, S . Typhimurium strains S04698-09 and A53 were both part of the monophasic S . Typhimurium ST34 epidemic clade that emerged in the last three decades, yet these strains exhibited considerable difference in susceptibility to at least five SPLA phages. Furthermore, a S . Typhimurium strain of ST4 / 74 that was genetically modified to remove prophage elements from its genome exhibited a moderate decrease in sensitivity to several SPLA phages. Whole genome functional screen identified host genes involved in infection by SPLA phages. To identify bacterial host genes affecting virulence of infecting SPLA phages, we contructed a saturating transposon insertion mutant library in S . Typhimurium strain ST4 / 74. In a preliminary screen, six SPLA phages (SPLA1a, SPLA1b, SPLA2, SPLA5b, SPLA5c and SPLA11) had a greater than 50 lytic index for the transposon mutant library and were investigated further. Transposon insertion mutants in the library that changed in abundance during culture with each of the six SPLA phages was determined by TraDIS. Host genes that conferred susceptibility to SPLA phages were identified by insertion mutants that were present in greater relative abundance in transposon mutant libraries exposed to each phage compared to a control that was not exposed to the SPLA phage. These genes had an increase in mapped transposon reads compared to untreated conditions. Some of the most significant genes identified were genes which encode the putative phage receptor. For example, the Vitamin B12 transporter, BtuB, showed a large increase in the number of insertions when treated with SPLA5b (Figure 3D). BtuB is an outermembrane protein (OMP) involved in the active translocation of vitamin B12 into the periplasmic space in gram negative bacteria and is likely to be the receptor for phage SPLA5b. Similarly, many genes involved in the biosynthesis of lipopolysaccharide (LPS) showed increased insertions, suggesting loss of LPS promoted survival of S . Typhimurium strain ST4 / 74 when infected by SPLA1a, SPLA1b and SPLA5c (Figures 4A, 4B and 4E). All 3 Phages showed that mutants with transposon insertions in genes within the rfa and rfb operons – such as the O-antigen ligase, RfaL. On infection with SPLA2, insertions in genes are involved in the biosynthesis of cellulose (yhjU, yhjL, yhjN, yhjQ, yhjR and yhjS) that forms a capsule surrounding the bacterial cell resulted increased survival, suggesting that this may act as a primary receptor for this phage (Figure 3C). We also identified genes with fewer transposon insertions compared to untreated controls following exposure to SPLA phage. Genes with a significant reduction in insertions are putative resistance genes to SPLA phages. For SPLA1a, SPLA 1b, SPLA2, SPLA5c and SPLA11, fewer resistance genes were detected compared to susceptibility genes. Resistance genes included two regulatory genes barA (SPLA2) and oxyR (SPLA5c), rumA involved in methylation of Uracil in 23s ribosomal RNA (SPLA5c), nfi encoding an endonuclease (SPLA5c) and gtrB involved in glycosylation of LPS (SPLA5c and SPLA11). Insertions in genes involved in synthesis of LPS were significantly reduced during SPLA5b infection, suggesting that complete LPS id detrimental to infection by SPLA5b and plays a role in protecting Salmonella from Phage infection. Selection of Cocktail Candidates based on Identification of Resistance genes. Glycosyltransferase (gtr) operons are capable of changing O-serotype by addition of sugars onto basic O- antigen. Despite their high genomic similarity, gtrB was identified to provide resistance to SPLA5c but not SPLA1a. The gtr operon was found to not be prophage encoded and present downstream of the f im operon in Salmonella Typhimurium ST4 / 74. Using Single Isogenic mutants, we demonstrated that gtrB confers a moderate level resistance to SPLA5c, but not SPLA1a and therefore SPLA1a would make a better candidate phage for use in a phage cocktail (Figure 4). Insights into phage infection mechanisms facilitates design of cocktails exhibiting collateral sensitivity. Collateral sensitivity is an evolutionary trade off where resistance to one antimicrobial agent results in increased sensitivity to another. We observed that many of the same LPS biosynthesis genes played contrasting roles during infection with phage SPLA5b and SPLA1a. LPS biosynthesis genes were required for infection by SPLA1a, but conversely acted as resistance genes during infection by SPLA5b. To further investigate this, SPLA phages were used in monotherapy (single phage) and in combination (both phages) as well as a non-phage treated positive control. Ninety-six wells of each phage- Salmonella were carried out. Figure 5 shows host resistance occurred in 96 / 96 wells treated with SPLA5b. When SPLA1a was used in monotherapy, bacterial growth was observed in 10 / 96 wells. When both phages were used in combination, growth was only observed in 1 / 96 wells resulting in a roughly 10-fold reduction in phage resistance. Inactivation of rfaL confers resistance to SPLA1a but increased sensitivity to SPLA5b. Strains in which rfaL or btuB replaced by aphII gene were constructed to investigate their role in sensitivity to SPLA1a and SPLA5b. Both SPLA1a and SPLA5b were able to form plaques on wild-type Salmonella Typhimurium ST4 / 74, although plaques formed by SPLA5b were more turbid than those formed by SPLA1a consistent with a lower virulence of the former. Inactivation of rfaL resulted in complete resistance to SPLA1a, as indicated by the lack of plaques in overlay assays, consistent with O-antigen being the receptor for this phage. In contrast, SPLA5b was still able to infect the rfaL mutant, as expected from the TraDIS data, but plaques were consistently less turbid, suggesting an increase in phage sensitivity. In contrast, Loss of the Vitamin B12 Transporter, by inactivation of the btuB gene, resulted in resistance to SPLA5b as indicated by the lack of plaques. This suggest that BtuB may be the receptor for SPLA5b. Loss of BtuB did not affect susceptibility to SPLA1a (Figure 6A). To further explore this interaction and to determine if loss of LPS resulted in increased susceptibility to SPLA5b, growth curve assays were carried out using S . Typhimurium ST4 / 74 ΔrfaK. The rfaK gene encodes a hexose transferase involved in LPS synthesis. Loss of rfaK does not result in a attenuated growth mutant, shown in figure 6B. No growth was observed when wild-type ST4 / 74 was infected with SPLA1a, however loss of rfaK resulted in the ability to grow under SPLA1a selection. In contrast, growth was observed when wild-type ST4 / 74 was infected with SPLA5b, with the phage causing a extended lag phase compared to non-phage treated conditions. However loss of rfaK resulted in increased phage infection by SPLA5b, compared to wild-type. Growth curves are presented in Figure 6B. It is predicted that resistance SPLA1a, caused by alterations or deletions in the LPS phage receptor would lead to increased resistance to SPLA5b. Therefore, these phages exhibit a collateral sensitivity dynamic, where resistance to once phage leads to increased sensitivity to another. A schematic of this mechanism is displayed in Figure 6C. Wild-type strains are infected well by SPLA1a, but decreased infectivity by SPLA5b is caused by masking of the btuB receptor by LPS. Mutations in LPS, such as ΔrfaL, lead to resistance to SPLA1a due to deletion of the phage receptor and increased sensitivity to SPLA5b as the BtuB receptor is no longer masked by LPS. DISCUSSION Formulation of robust Phage Cocktails will be integral to the success of their applications. Here, we present the isolation of bacteriophages which specifically target Salmonella strains found in food products. We further characterise the phages by testing their host range against thirty-six different bacterial strains. All phages had very limited effects on non-salmonella strains highlighting their specificity and suitability for therapeutic use. Amongst the bacterial strains tested was twelve Salmonella Typhimurium . S . Typhimurium accounts for almost one quarter of all Salmonella isolated from humans as well as 78.2% of pig isolates in 2020 (35). This highlights the need for effective control measures for Salmonella Typhimurium within the food chain. The most common phage type isolated from pigs is DT193, characterised by resistance to all bacteriophages in the Anderson phage typing scheme (36). The AUC data presented in Figure 2 shows all SPLA phages, apart from SPLA1b and SPLA10, infect this strain well with scores >40. In particular, DT193 was highly susceptible to SPLA1a and SPLA5b with AUC scores of 94 and 91.5 respectively, making these phages particularly promising for controlling Salmonella within the pork production chain. SPLA1a scored high AUC scores for many Typhimurium strains which be due to strains being genetically closely related to each other with limited variation in host receptors and resistance. For SPLA1a, the lowest AUC score was 25.3 for infection against ST313, a Salmonella Typhimurium strain associated with invasive disease in sub- Saharan Africa (37). Other SPLA phages were unable to infect ST313. This strain has an alternative prophage repertoire to other NTS which includes BTP-1 and BTP-5 (38). BTP-1 is known to harbour the phage resistance gene bstA which confers population-level resistance to bacteriophages via abortive infection (39). This could explain the relative phage resistance observed in this strain. In addition to Typhimurium , SPLA phages also infected diverse Salmonella serovars. In particular, SPLA9 achieved high scores against serovars Montevideo, Panama, Infantis and Derby – with scores of 86.8, 93.6, 93.3 and 89.5 respectively. High scores achieved across many diverse Salmonella serovars highlight the potential use for this phage as a broad range treatment for Salmonella. Applications of Phages should use multiple bacteriophages together as a “phage cocktail” in order to limit phage resistance. Previous studies have shown that phage cocktails are effective against many pathogens including Enterococcus , Salmonella and Campylobacter (40-42). Host range analysis highlighted that SPLA1a has a broad host range and SPLA5b has a narrow host range. Using the whole genome functional screen we are able to predict that these phages would work synergistically together and are able to select them even over closely related phages for improved efficacy. We show that bacterial growth is observed in 100% of S.Typhimurium ST4 / 74 cultures treated with narrow host range SPLA5b and 10% of cultures treated with broad host range SPLA1a – Showing that bacterial resistance to these phages occurred readily in culture when used in monotherapy. This shows that these phages cannot be used alone for therapeutic use. When used in a 10-fold decrease in frequency of phage resistance was observed. This is likely due to the phages targeting different receptors which resulted in lower occurrence of phage resistance. A good understanding of the target receptor as well as host genes involved in resistance is critical when designing phage cocktai ls. Infection by bacteriophages is a complex process with host bacteria encoding many genes which can prevent or hinder infection (43). Transposon Directed Insertion site sequencing (TraDIS) is successful in identifying host genes involved in infection by diverse, newly isolated bacteriophages. TraDIS allows the identification of genes which confer both host susceptibility and host resistance. This methodology has previously been used to identify host genes involved in infection by model phages such as for T4 (44), λ (45) T2, T6 and T7 Phages (46). Additionally, it has been used to identify capsule polysaccharides as the receptor for novel phage, RAD2, in Klebsiel la (47) . Understanding the host receptor is important as, along with the complementary receptor binding domain, they represent a major determinant of bacteriophage specificity. Three phages, SPLA1a, SPLA1b and SPLA5c are identified to target Lipopolysaccharide (LPS) as a receptor. A Common mechanism of defence against O-antigen targeting phages is the epigenetic control of O-antigen chain length. Expression of OpvAB leads to shorter O-antigen and enhances phage resistance (48). Additionally, phage selection has previously been shown to give rise to rough, phage resistant Salmonellae (49) .However LPS is also a major virulence factor in Salmonella , and mutations is LPS lead to attenuation (50). Therefore, acquisition of phage resistance is at the evolutionary cost of virulence. Several evolutionary trade-offs have been reported involving phage resistance, including collateral sensitivity to antibiotics (51). TraDIS identified that the success of combination treatment of SPLA1a and could be due to a collateral sensitivity dynamic. As loss of LPS was detrimental to infection by SPLA1a, but beneficial for infection by SPLA5b. Therefore when used in combination, resistance was limited. Using a isogenic mutant with deletion of RfaL, the O-antigen ligase, we confirm that loss of O-antigen enhances resistance to SPLA1a and increases sensitivity to SPLA5b. This could likely be due to the LPS masking the BtuB receptor of SPLA5b. Previous studies have shown that smooth Escherichia coli strains are less susceptible to bacteriophages targeting outer membrane proteins than rough variants (52) suggesting that cell surface polysaccharide blocks bacteriophage receptors. Additionally, O- antigen can also block the LPS inner core receptor used by phage SSU5. Many of the concerns for phage therapy relate to the limited host range and acquisition of phage resistance. TraDIS presents a useful tool for phage biologists as information gained can be used to design better cocktails with receptor diversity to broaden host range and limit the acquisition of phage resistance. Additionally, Identification of the exact genetic determinates of phage susceptibility can be used to help predict which strains a phage or phage cocktail can infect, which is likely to be crucial for successful phage therapies. Turning to figures 7a to 7d which show the results of experiments to determine if oral dosage of phages reduced Salmonella colonisation and / or shedding In vivo. Methods: CD57 / BL6 Mice were split into 5 groups (1. Phage only, 2. Salmonella only, 3. Salmonella and SPLA1a, 4. Salmonella and SPLA5b and 5. Salmonella and SPLA1a and SPLA5b (Cocktail)). On day -1. All mice were treated with 200mg Steptomycin. On day 0, Mice were inoculated by oral gavage with 10^4 CFU Typhimurium Strain ST4 / 74 WITS. After 2 hours, if appropriate, mice were administered bacteriophages by Oral gavage at (10^7 PFU). Fecal pellets were collected on every day. Phage was administered a second time on day 2. Mice were sacrificed on Day 3 with Liver, Mesenteric lymph nodes and Caecums collected. CFU of Salmonella were counted in each organ and in fecal pellets by plating on LB supplemented with Kanamycin. Results: After 1 day. There was no significant difference in Salmonella shedding between non phage treated mice compared to mice treated with single phage preparations. There was a significant reduction in Salmonella shedding when treated with the phage cocktail after 1 day. This difference was maintained throughout the whole experiment. After 3 days, the phage cocktail achieved a significant reduction (>2 log) in Salmonella shedding compared to non treated and was significantly better than single SPLA5b treatment. The phage cocktail significantly reduced colonisation in Liver, Caecum and MLN compared to no treatment. The cocktail performed significantly better than SPLA1a alone in Liver and MLN.

Claims

Claims 1. A method of identifying and / or isolating one or more bacteriophages that display lytic activity against at least one target bacterial genus, species, or strain said method including the creation of a transposon mutant library using at least one bacterial strain from said genus or species and identifying the genes involved in resistance and / or susceptibility by exposing said library to at least one or more bacteriophages.

2. A method according to claim 1 wherein the bacteriophages identified are used as one or more components of a phage cocktail or bacteriophage mixture effective against one or more bacterial species or strains.

3. A method according to claim 1 or 2 wherein the method includes the isolation of two or more phages and the use of a whole genome functional screen fitness assay to identify all host genes involved in infection by said isolated phages.

4. A method according to claims 1-3 wherein the role of specific genes in sensitivity and / or resistance to phages is assessed using TraDIS.

5. A method according to claim 4 wherein TraDIS is used to establish both host susceptibility and resistance genes involved in infection by bacteriophages.

6. A method according to claim 5 wherein the receptor information from the TraDIS screen combinations of bacteriophages are identified that work more effectively together than other phage combinations.

7. A method according to 6 wherein improved efficacy includes increased lytic activity and / or reduced host resistance.

8. A method according to any preceding claim wherein a plurality of bacteriophages are isolated from one or more different environments and / or samples.

9. A method according to claim 8 wherein TraDIS is used to identify receptors for each bacteriophage selected from the isolates.

10. A method according to any preceding claim wherein the method includes the step of bacterial culture and / or allelic exchange.

11. A method according to any preceding claim wherein construction of the mutant strains is performed by one-step inactivation.

12. A method according to claim 11 wherein libraries are prepared and sequenced using the Illumina™ platform.

13. A method of determining the constituents of a phage cocktail or bacteriophage mixture effective against one or more bacterial species or strains, said method including the isolation of two or more phages and the use of a genomics based fitness assay according to claim 1 to identify all host genes involved in infection by said isolated phages.

14. A method according to claim 13 wherein TraDIS is used to identify one or more host receptors for susceptibility and / or resistance that contribute to the success of a combination of phages as biological control agents.

15. A phage cocktail or mixture effective against one or more bacterial species or strains, wherein said phages are selected from whole genome functional screening of bacteria according to claim 13.

16. A phage cocktail prepared according to claim 1 or 13 for the treatment of Salmonella infection.

17. A phage cocktail according to claim 16 that includes SPLA1a and / or SPLA5b.

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