Presence of phage-plasmids in multiple serovars of Salmonella enterica.

Evidence is accumulating in the literature that the horizontal spread of antimicrobial resistance (AMR) genes mediated by bacteriophages and bacteriophage-like plasmid (phage-plasmid) elements is much more common than previously envisioned. For instance, we recently identified and characterized a circular P1-like phage-plasmid harbouring a bla CTX-M-15 gene conferring extended-spectrum beta-lactamase (ESBL) resistance in Salmonella enterica serovar Typhi. As the prevalence and epidemiological relevance of such mechanisms has never been systematically assessed in Enterobacterales, in this study we carried out a follow-up retrospective analysis of UK Salmonella isolates previously sequenced as part of routine surveillance protocols between 2016 and 2021. Using a high-throughput bioinformatics pipeline we screened 47 784 isolates for the presence of the P1 lytic replication gene repL, identifying 226 positive isolates from 25 serovars and demonstrating that phage-plasmid elements are more frequent than previously thought. The affinity for phage-plasmids appears highly serovar-dependent, with several serovars being more likely hosts than others; most of the positive isolates (170/226) belonged to S. Typhimurium ST34 and ST19. The phage-plasmids ranged between 85.8 and 98.2 kb in size, with an average length of 92.1 kb; detailed analysis indicated a high amount of diversity in gene content and genomic architecture. In total, 132 phage-plasmids had the p0111 plasmid replication type, and 94 the IncY type; phylogenetic analysis indicated that both horizontal and vertical gene transmission mechanisms are likely to be involved in phage-plasmid propagation. Finally, phage-plasmids were present in isolates that were resistant and non-resistant to antimicrobials. In addition to providing a first comprehensive view of the presence of phage-plasmids in Salmonella, our work highlights the need for a better surveillance and understanding of phage-plasmids as AMR carriers, especially through their characterization with long-read sequencing.

Prophage terminase with tRNase activity sensitizes Salmonella enterica to oxidative stress.

Phage viruses shape the evolution and virulence of their bacterial hosts. The Salmonella enterica genome encodes several stress-inducible prophages. The Gifsy-1 prophage terminase protein, whose canonical function is to process phage DNA for packaging in the virus head, unexpectedly acts as a transfer ribonuclease (tRNase) under oxidative stress, cleaving the anticodon loop of tRNALeu. The ensuing RNA fragmentation compromises bacterial translation, intracellular survival, and recovery from oxidative stress in the vertebrate host. S. enterica adapts to this transfer RNA (tRNA) fragmentation by transcribing the RNA repair Rtc system. The counterintuitive translational arrest provided by tRNA cleavage may subvert prophage mobilization and give the host an opportunity for repair as a way of maintaining bacterial genome integrity and ultimately survival in animals.

Collateral sensitivity increases the efficacy of a rationally designed bacteriophage combination to control Salmonella enterica.

The ability of virulent bacteriophages to lyse bacteria influences bacterial evolution, fitness, and population structure. 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 isolated 12 bacteriophages termed SPLA phage which infect the foodborne pathogen Salmonella enterica. To determine phage host range, a diverse collection of Enterobacteriaceae and Salmonella enterica was used and genes involved in infection by six SPLA phages were identified using Salmonella Typhimurium strain ST4/74. Candidate host receptors included lipopolysaccharide (LPS), cellulose, and BtuB. Lipopolysaccharide was identified as a susceptibility factor for phage SPLA1a and mutations in LPS biosynthesis genes spontaneously emerged during culture with S. Typhimurium. Conversely, LPS was a resistance factor for phage SPLA5b which suggested that emergence of LPS mutations in culture with SPLA1a represented collateral sensitivity to SPLA5b. We show that bacteria-phage co-culture with SPLA1a and SPLA5b was more successful in limiting the emergence of phage resistance compared to single phage co-culture. Identification of host susceptibility and resistance genes and understanding infection dynamics are critical steps in the rationale design of phage cocktails against specific bacterial pathogens.IMPORTANCEAs antibiotic resistance continues to emerge in bacterial pathogens, bacterial viruses (phage) represent a potential alternative or adjunct to antibiotics. One challenge for their implementation is the predisposition of bacteria to rapidly acquire resistance to phages. We describe a functional genomics approach to identify mechanisms of susceptibility and resistance for newly isolated phages that infect and lyse Salmonella enterica and use this information to identify phage combinations that exploit collateral sensitivity, thus increasing efficacy. Collateral sensitivity is a phenomenon where resistance to one class of antibiotics increases sensitivity to a second class of antibiotics. We report a functional genomics approach to rationally design a phage combination with a collateral sensitivity dynamic which resulted in increased efficacy. Considering such evolutionary trade-offs has the potential to manipulate the outcome of phage therapy in favor of resolving infection without selecting for escape mutants and is applicable to other virus-host interactions.

Genomic analysis and characterization of bacteriophage vB_SpuS_NX263 infecting Salmonella enterica subsp. enterica serovar Pullorum.

In this study, a new Salmonella phage, NX263, was isolated from sewage. This phage could lyse 90.57% (48/53) of the bacterial strains tested and showed good activity over a wide range of temperature (up to 60°C) and pH (5-10). Phylogenetic analysis showed that it should be classified as a member of the genus Skatevirus. The genome of phage NX263 is 46,574 bp in length with a GC content of 45.52%. It contains 89 open reading frames and two tRNA genes. No lysogeny, drug resistance, or virulence-associated genes were identified in the genome sequence, suggesting that this phage could potentially be used to treat Salmonella Pullorum infections.

Bacteriophage-Resistant Salmonella rissen: An In Vitro Mitigated Inflammatory Response.

Non-typhoid Salmonella (NTS) represents one of the major causes of foodborne diseases, which are made worse by the increasing emergence of antibiotic resistance. Thus, NTS are a significant and common public health concern. The purpose of this study is to investigate whether selection for phage-resistance alters bacterial phenotype, making this approach suitable for candidate vaccine preparation. We therefore compared two strains of Salmonella enterica serovar Rissen: RR (the phage-resistant strain) and RW (the phage-sensitive strain) in order to investigate a potential cost associated with the bacterium virulence. We tested the ability of both RR and RW to infect phagocytic and non-phagocytic cell lines, the activity of virulence factors associated with the main Type-3 secretory system (T3SS), as well as the canonic inflammatory mediators. The mutant RR strain-compared to the wildtype RW strain-induced in the host a weaker innate immune response. We suggest that the mitigated inflammatory response very likely is due to structural modifications of the lipopolysaccharide (LPS). Our results indicate that phage-resistance might be exploited as a means for the development of LPS-based antibacterial vaccines.

Efficacy of Salmonella Bacteriophage S1 Delivered and Released by Alginate Beads in a Chicken Model of Infection.

Modern bacteriophage encapsulation methods based on polymers such as alginate have been developed recently for their use in phage therapy for veterinary purposes. In birds, it has been proven that using this delivery system allows the release of the bacteriophage in the small intestine, the site of infection by Salmonella spp. This work designed an approach for phage therapy using encapsulation by ionotropic gelation of the lytic bacteriophage S1 for Salmonella enterica in 2% w/v alginate beads using 2% w/v calcium chloride as crosslinking agent. This formulation resulted in beads with an average size of 3.73 ± 0.04 mm and an encapsulation efficiency of 70%. In vitro, the beads protected the bacteriophages from pH 3 and released them at higher pH. To confirm that this would protect the bacteriophages from gastrointestinal pH changes, we tested the phage infectivity in vivo assay. Using a model chicken (Gallus gallus domesticus) infected with Salmonella Enteritidis, we confirmed that after 3 h of the beads delivery, infective phages were present in the chicken's duodenal and caecal sections. This study demonstrates that our phage formulation is an effective system for release and delivery of bacteriophage S1 against Salmonella Enteritidis with potential use in the poultry sector.

Isolation, identification and some characteristics of two lytic bacteriophages against Salmonella enterica serovar Paratyphi B and S. enterica serovar Typhimurium from various food sources.

Salmonellosis is an important worldwide food-borne disease. Increasing resistance to Salmonella spp. has been reported in recent years, and now the prevalence of multidrug-resistant Salmonella spp. is a worldwide problem. This necessitates alternative approaches like phage therapy. This study aimed to isolate bacteriophages specific for Salmonella enterica serovar Paratyphi B and S. enterica serovar Typhimurium isolated from different sources (chicken meat, beef and eggshells). The antibiotic resistance profiles of the bacteria were determined by phenotypic and genotypic methods. The prevalence of extended-spectrum ß-lactamase genes was examined by polymerase chain reaction. In total, 75% of the isolated Salmonella strains were resistant to tetracycline, whereas 70% of them were resistant to azithromycin. All of the isolates from beef were resistant to nalidixic acid. The most common extended-spectrum ß-lactamase genes among the isolates were blaSHV (15%) followed by blaTEM (10%) and blaCTX (5%). Two specific bacteriophages were isolated and characterized. The host range for vB_SparS-ui was Salmonella Paratyphi B, S. enterica serovar Paratyphi A and S. enterica, while that for vB_StyS-sam phage was Salmonella Typhimurium and S. enterica serovar Enteritidis. The characteristics of the isolated phages indicate that they are proper candidates to be used to control some foodstuff contaminations and also phage therapy of infected animals.

Bi- and Multi-directional Gene Transfer in the Natural Populations of Polyvalent Bacteriophages, and Their Host Species Spectrum Representing Foodborne Versus Other Human and/or Animal Pathogens.

Unraveling the trends of phage-host versus phage-phage coevolution is critical for avoiding possible undesirable outcomes from the use of phage preparations intended for therapeutic, food safety or environmental safety purposes. We aimed to investigate a phenomenon of intergeneric recombination and its trajectories across the natural populations of phages predominantly linked to foodborne pathogens. The results from the recombination analyses, using a large array of the recombination detection algorithms imbedded in SplitsTree, RDP4, and Simplot software packages, provided strong evidence (fit: 100; P ≤ 0.014) for both bi- and multi-directional intergeneric recombination of the genetic loci involved collectively in phage morphogenesis, host specificity, virulence, replication, and persistence. Intergeneric recombination was determined to occur not only among conspecifics of the virulent versus temperate phages but also between the phages with these different lifestyles. The recombining polyvalent phages were suggested to interact with fairly large host species networks, including sometimes genetically very distinct species, such as e.g., Salmonella enterica and/or Escherichia coli versus Staphylococcus aureus or Yersinia pestis. Further studies are needed to understand whether phage-driven intergeneric recombination can lead to undesirable changes of intestinal and other microbiota in humans and animals.

Bacteriophages vB_Sen-TO17 and vB_Sen-E22, Newly Isolated Viruses from Chicken Feces, Specific for Several Salmonella enterica Strains.

Two newly discovered bacteriophages, isolated from chicken feces and infecting Salmonella enterica strains, are described in this report. These phages have been named vB_Sen-TO17 and vB_Sen-E22, and we present their molecular and functional characterization. Both studied viruses are able to infect several S. enterica strains and develop lytically, but their specific host ranges differ significantly. Electron microscopic analyses of virions have been performed, and full genome sequences were determined and characterized, along with molecular phylogenetic studies. Genomes of vB_Sen-TO17 (ds DNA of 41,658 bp) and vB_Sen-E22 (dsDNA of 108,987 bp) are devoid of homologs of any known or putative gene coding for toxins or any other proteins potentially deleterious for eukaryotic cells. Both phages adsorbed efficiently (>95% adsorbed virions) within 10 min at 42 °C (resembling chicken body temperature) on cells of most tested host strains. Kinetics of lytic development of vB_Sen-TO17 and vB_Sen-E22, determined in one-step growth experiments, indicated that development is complete within 30-40 min at 42 °C, whereas burst sizes vary from 9 to 79 progeny phages per cell for vB_Sen-TO17 and from 18 to 64 for vB_Sen-E22, depending on the host strain. Virions of both phages were relatively stable (from several percent to almost 100% survivability) under various conditions, including acidic and alkaline pH values (from 3 to 12), temperatures from -80 °C to 60 °C, 70% ethanol, chloroform, and 10% DMSO. These characteristics of vB_Sen-TO17 and vB_Sen-E22 indicate that these phages might be considered in further studies on phage therapy, particularly in attempts to eliminate S. enterica from chicken intestine.

Characteristics of a Series of Three Bacteriophages Infecting Salmonella enterica Strains.

Molecular and functional characterization of a series of three bacteriophages, vB_SenM-1, vB_SenM-2, and vB_SenS-3, infecting various Salmonella enterica serovars and strains is presented. All these phages were able to develop lytically while not forming prophages. Moreover, they were able to survive at pH 3. The phages revealed different host ranges within serovars and strains of S. enterica, different adsorption rates on host cells, and different lytic growth kinetics at various temperatures (in the range of 25 to 42 °C). They efficiently reduced the number of cells in the bacterial biofilm and decreased the biofilm mass. Whole genome sequences of these phages have been determined and analyzed, including their phylogenetic relationships. In conclusion, we have demonstrated detailed characterization of a series of three bacteriophages, vB_SenM-1, vB_SenM-2, and vB_SenS-3, which reveal favorable features in light of their potential use in phage therapy of humans and animals, as well as for food protection purposes.

Evaluation of the efficiency of using Salmonella Kentucky and Escherichia coli O119 bacteriophages in the treatment and prevention of salmonellosis and colibacillosis in broiler chickens.

Phage therapy is considered an alternative modality in the treatment of different bacterial diseases. However, their therapeutic and preventive roles against infections caused by Salmonella Kentucky and Escherichia coli O119 were of little attention. In this study, two phages were isolated, characterized and assessed for their potential therapeutic and preventive roles against S. Kentucky and E. coli O119 infections in broilers. Commercial 1-day-old arboacres broiler chicks were assigned to seven groups: Group Ӏ was as a negative control, groups (П and Ш) were assigned as positive controls by the challenge of S. Kentucky and E. coli O119, respectively. The remaining four groups (IV, V, VI and VII) were administrated with five repeated phage doses to determine the effect of multiple doses. Phages were administrated in groups (IV and VI) after challenging with S. Kentucky and E. coli O119, respectively to assess their therapeutic role; moreover, their preventive role was evaluated through administration in groups (V and VII) before challenging with S. Kentucky and E. coli O119, respectively. Sampling was done from different organs at three time points and revealed that phage-treated groups had lower colony forming units of S. Kentucky and E. coli. Our results suggest that bacteriophages are efficient in the treatment and prevention of salmonellosis and colibacillosis in broiler farms.

Comparative genomic analysis of 142 bacteriophages infecting Salmonella enterica subsp. enterica.

BACKGROUND: Bacteriophages are bacterial parasites and are considered the most abundant and diverse biological entities on the planet. Previously we identified 154 prophages from 151 serovars of Salmonella enterica subsp. enterica. A detailed analysis of Salmonella prophage genomics is required given the influence of phages on their bacterial hosts and should provide a broader understanding of Salmonella biology and virulence and contribute to the practical applications of phages as vectors and antibacterial agents. RESULTS: Here we provide a comparative analysis of the full genome sequences of 142 prophages of Salmonella enterica subsp. enterica which is the full complement of the prophages that could be retrieved from public databases. We discovered extensive variation in genome sizes (ranging from 6.4 to 358.7 kb) and guanine plus cytosine (GC) content (ranging from 35.5 to 65.4%) and observed a linear correlation between the genome size and the number of open reading frames (ORFs). We used three approaches to compare the phage genomes. The NUCmer/MUMmer genome alignment tool was used to evaluate linkages and correlations based on nucleotide identity between genomes. Multiple sequence alignment was performed to calculate genome average nucleotide identity using the Kalgin program. Finally, genome synteny was explored using dot plot analysis. We found that 90 phage genome sequences grouped into 17 distinct clusters while the remaining 52 genomes showed no close relationships with the other phage genomes and are identified as singletons. We generated genome maps using nucleotide and amino acid sequences which allowed protein-coding genes to be sorted into phamilies (phams) using the Phamerator software. Out of 5796 total assigned phamilies, one phamily was observed to be dominant and was found in 49 prophages, or 34.5% of the 142 phages in our collection. A majority of the phamilies, 4330 out of 5796 (74.7%), occurred in just one prophage underscoring the high degree of diversity among Salmonella bacteriophages. CONCLUSIONS: Based on nucleotide and amino acid sequences, a high diversity was found among Salmonella bacteriophages which validate the use of prophage sequence analysis as a highly discriminatory subtyping tool for Salmonella. Thorough understanding of the conservation and variation of prophage genomic characteristics will facilitate their rational design and use as tools for bacterial strain construction, vector development and as anti-bacterial agents.

Bacteriophage ZCSE2 is a Potent Antimicrobial Against Salmonella enterica Serovars: Ultrastructure, Genomics and Efficacy.

Developing novel antimicrobials capable of controlling multidrug-resistant bacterial pathogens is essential to restrict the use of antibiotics. Bacteriophages (phages) constitute a major resource that can be harnessed as an alternative to traditional antimicrobial therapies. Phage ZCSE2 was isolated among several others from raw sewage but was distinguished by broad-spectrum activity against Salmonella serovars considered pathogenic to humans and animals. Lytic profiles of ZCSE2 against a panel of Salmonella were determined together with low temperature activity and pH stability. The morphological features of the phage and host infection processes were characterized using a combination of transmission electron and atomic force microscopies. Whole genome sequencing of ZCSE2 produced a complete DNA sequence of 53,965 bp. No known virulence genes were identified in the sequence data, making ZCSE2 a good candidate for phage-mediated biological control purposes. ZCSE2 was further tested against S. Enteritidis in liquid culture and was observed to reduce the target bacterium to below the limits of detection from initial concentrations of 107-108 Colony Forming Units (CFU)/mL. With a broad host-range against pathogenic Salmonella serovars, phage ZCSE2 constitutes a potential tool against a major cause of human and animal disease.

Bacteriophage-Insensitive Mutants of Antimicrobial-Resistant Salmonella Enterica are Altered in their Tetracycline Resistance and Virulence in Caco-2 Intestinal Cells.

Bacteriophages have shown promise as therapeutic alternatives to antibiotics for the control of infectious bacteria, including the human pathogen Salmonella. However, the development of effective phage-based applications requires the elucidation of key interactions between phages and target hosts, particularly since host resistance to phage is inevitable. Little is known about the alteration of host phenotypes following the development of resistance to phage. The aim of this study is to evaluate the antibiotic susceptibility and virulence of a Salmonella isolate following the development of resistance to bacteriophage SI1. We observed enhanced susceptibility to tetracycline and decreased invasion capacity in a differentiated Caco-2 intestinal cell line. Whole genome sequence analysis revealed an array of mutations, most notably, truncations in vgrG1_2, a core gene involved in Type VI secretion and mutations in the lipopolysaccharide, thereby indicating the plausible attachment site of phage SI1. These findings shed light on understanding the underlying mechanism for phage immunity within the host. Importantly, we reveal an associated genetic cost to the bacterial host with developing resistance to phages. Taken together, these results will aid in advancing strategies to delay or eliminate the development of host resistance when designing informed phage-based antimicrobials.

Lytic bacteriophage have diverse indirect effects in a synthetic cross-feeding community.

Bacteriophage shape the composition and function of microbial communities. Yet it remains difficult to predict the effect of phage on microbial interactions. Specifically, little is known about how phage influence mutualisms in networks of cross-feeding bacteria. We mathematically modeled the impacts of phage in a synthetic microbial community in which Escherichia coli and Salmonella enterica exchange essential metabolites. In this model, independent phage attack of either species was sufficient to temporarily inhibit both members of the mutualism; however, the evolution of phage resistance facilitated yields similar to those observed in the absence of phage. In laboratory experiments, attack of S. enterica with P22vir phage followed these modeling expectations of delayed community growth with little change in the final yield of bacteria. In contrast, when E. coli was attacked with T7 phage, S. enterica, the nonhost species, reached higher yields compared with no-phage controls. T7 infection increased nonhost yield by releasing consumable cell debris, and by driving evolution of partially resistant E. coli that secreted more carbon. Our results demonstrate that phage can have extensive indirect effects in microbial communities, that the nature of these indirect effects depends on metabolic and evolutionary mechanisms, and that knowing the degree of evolved resistance leads to qualitatively different predictions of bacterial community dynamics in response to phage attack.

Diversity and Host Specificity Revealed by Biological Characterization and Whole Genome Sequencing of Bacteriophages Infecting Salmonella enterica.

Phages infecting members of the opportunistic human pathogen, Salmonella enterica, are widespread in natural environments and offer a potential source of agents that could be used for controlling populations of this bacterium; yet, relatively little is known about these phages. Here we describe the isolation and characterization of 45 phages of Salmonella enterica from disparate geographic locations within British Columbia, Canada. Host-range profiling revealed host-specific patterns of susceptibility and resistance, with several phages identified that have a broad-host range (i.e., able to lyse >40% of bacterial hosts tested). One phage in particular, SE13, is able to lyse 51 out of the 61 Salmonella strains tested. Comparative genomic analyses also revealed an abundance of sequence diversity in the sequenced phages. Alignment of the genomes grouped the phages into 12 clusters with three singletons. Phages within certain clusters exhibited extraordinarily high genome homology (>98% nucleotide identity), yet between clusters, genomes exhibited a span of diversity (<50% nucleotide identity). Alignment of the major capsid protein also supported the clustering pattern observed with alignment of the whole genomes. We further observed associations between genomic relatedness and the site of isolation, as well as genetic elements related to DNA metabolism and host virulence. Our data support the knowledge framework for phage diversity and phage-host interactions that are required for developing phage-based applications for various sectors, including biocontrol, detection and typing.

Morphologic and genomic characterization of a broad host range Salmonella enterica serovar Pullorum lytic phage vB_SPuM_SP116.

For effective use of phages as antimicrobial agents for controlling multidrug resistant S. Pullorum, it is important to understand phage biology. A lytic S. Pullorum phage was isolated and characterized from chicken feces, and its whole genome was sequenced and analyzed. A new lytic phage-vB_SPuM_SP116 (in brief SP116)- isolated and characterized using S. Pullorum SPu-116 as its host belongs to Myoviridae A1 group. Phage SP116 had a lytic effect on 27 of 37 (72.9%) different serotypes of clinical Salmonella strains. It showed a high bactericidal activity in killing all pathogens in cultures containing 5 × 105 cfu/mL and achieved more than 6.58 and 5.97 log unit reductions in cultures containing 5 × 106 cfu/mL and 5 × 107 cfu/mL, respectively. The one-step growth curve showed that the burst size was up to 118 pfu/bacterial cell. Complete genome sequence analysis revealed a linear, double-stranded DNA genome of 87,510 bp with an average G + C content of 38.84%, including 128 predicted open reading frames (ORFs) and 22 tRNA genes. SP116 was classified as a Felix O1 virus based upon the general phage characterization and the genomic information. Regarding its high efficacy in preventing especially S. Pullorum infection and its lack of any bacterial virulence, antimicrobial resistance, and lysogenesis genes, it could be a potential alternative candidate for the treatment of S. Pullorum infections.

Structure and Function of the Branched Receptor-Binding Complex of Bacteriophage CBA120.

Bacteriophages recognize their host cells with the help of tail fiber and tailspike proteins that bind, cleave, or modify certain structures on the cell surface. The spectrum of ligands to which the tail fibers and tailspikes can bind is the primary determinant of the host range. Bacteriophages with multiple tailspike/tail fibers are thought to have a wider host range than their less endowed relatives but the function of these proteins remains poorly understood. Here, we describe the structure, function, and substrate specificity of three tailspike proteins of bacteriophage CBA120-TSP2, TSP3 and TSP4 (orf211 through orf213, respectively). We show that tailspikes TSP2, TSP3 and TSP4 are hydrolases that digest the O157, O77, and O78 Escherichia coli O-antigens, respectively. We demonstrate that recognition of the E. coli O157:H7 host by CBA120 involves binding to and digesting the O157 O-antigen by TSP2. We report the crystal structure of TSP2 in complex with a repeating unit of the O157 O-antigen. We demonstrate that according to the specificity of its tailspikes TSP2, TSP3, and TSP4, CBA120 can infect E. coli O157, O77, and O78, respectively. We also show that CBA120 infects Salmonella enterica serovar Minnesota, and this host range expansion is likely due to the function of TSP1. Finally, we describe the assembly pathway and the architecture of the TSP1-TSP2-TSP3-TSP4 branched complex in CBA120 and its related ViI-like phages.

Isolation, Characterisation and Complete Genome Sequence of a Tequatrovirus Phage, Escherichia phage KIT03, Which Simultaneously Infects Escherichia coli O157:H7 and Salmonella enterica.

Escherichia coli O157:H7 and Salmonella enterica subsp. enterica are the pathogens that frequently cause foodborne illness. Bacteriophage applications have been proposed as effective for preventing food contamination caused by these pathogenic bacteria. Escherichia phage KIT03 was isolated from the soil of a poultry farm in Kyoto, Japan. KIT03 can infect Escherichia coli O157:H7 and Salmonella enterica serotypes Choleraesuis and Enteritidis. One-step growth analysis revealed that KIT03 can propagate within its initial host (E. coli NBRC 3972), E. coli O157:H7 and S. Choleraesuis with an approximate burst size of 39, 51 and 37 phage particles per infected cell, respectively. The morphological type and genome annotation suggested that KIT03 belongs to the family Myoviridae, subfamily Tevenvirinae, genus Tequatrovirus. In vitro challenge tests demonstrated that KIT03 can lyse the tested bacteria and suppress their growth. Based on the susceptibility test and adsorption assay of KIT03 with E. coli K-12 BW25113 mutants, it was proposed that KIT03 may recognise and infect bacteria with a deficient outer core of lipopolysaccharides.

Characterization and genome analysis of a novel bacteriophage vB_SpuP_Spp16 that infects Salmonella enterica serovar pullorum.

A novel virulent bacteriophage vB_SpuP_Spp16 (hereafter designated Spp16) that infects Salmonella enterica serovar pullorum was isolated. Transmission electron microscopy showed that Spp16 possessed an isometric polyhedral head (60 nm in diameter) and a short tail (10 nm in length) belonging to the family Podoviridae. Its complete genome was determined to be 41,832 bp, with a 39.46% GC content by next-generation sequencing. The genome contains 53 proposed open reading frames that are involved in DNA replication and modification, transcriptional regulation, phage structural and packaging proteins and bacterial lysis. No transfer RNA genes were identified. The termini of genome were determined using our previously proposed termini identification method, which suggests that this phage has redundant termini with 421 bp direct terminal repeats. BLASTn analysis revealed the highest sequence similarity with Yersinia phage phi80-18, with a genome coverage of 33% and highest sequence identity of 69%. The phylogenetic analysis indicated that Spp16 forms a distinct branch of the subfamily Autographivirinae. Comparative genomics analysis showed that the phage Spp16 should be regarded as a new subcluster within the GAP227-like cluster in the Autographivirinae subfamily. The phage Spp16 has an obligate lytic life cycle demonstrated by experimental data and genomic analysis. These results suggest that Spp16 may be a proper candidate to control diseases caused by Salmonella enterica serovar pullorum.