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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 β-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 β-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., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

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

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

The head of Salmonella virus SPN3US is composed of ~50 different proteins and is unusual because within its packaged genome there is a mass (>40 MDa) of ejection or E proteins that enter the Salmonella cell. The assembly mechanisms of this complex structure are poorly understood. Previous studies showed that eight proteins in the mature SPN3US head had been cleaved by the prohead protease. In this study, we present the characterization of SPN3US prohead protease mutants using transmission electron microscopy and mass spectrometry. In the absence of the prohead protease, SPN3US head formation was severely impeded and proheads accumulated on the Salmonella inner membrane. This impediment is indicative of proteolysis being necessary for the release and subsequent DNA packaging of proheads in the wild-type phage. Proteomic analyses of gp245- proheads that the normal proteolytic processing of head proteins had not occurred. Assays of a recombinant, truncated form of the protease found it was active, leading us to hypothesize that the C -terminal propeptide has a role in targeting the protease into the prohead core. Our findings provide new evidence regarding the essential role of proteolysis for correct head assembly in this remarkable parasite.

Salmonella Enteritidis (S. Enteritidis), which could cause human disease and death by consuming the contaminated food, is an important zoonotic pathogen. With the rapid increase of antibiotic resistance all over the world, bacteriophage-based bio-control has gradually attracted public attention widely. In order to find a suitable phage treating S. Enteritidis infection, four phages infecting S. Enteritidis were isolated from poultry fecal samples. Host range showed that four phages had a broad-host-range to Salmonella isolates. The morphological analysis illustrated that all of those phages were classified as the Myoviridae family. The one-step growth curve indicated that bacteriophage BPSELC-1 has a short latent period of about 10 min and a large burst size of 500 pfu/cell in comparison to the other three phages. Then phage BPSELC-1 was sequenced and conducted in vitro experiment. The genome of phage BPSELC-1 is 86,996 bp in size and has 140 putative genes containing structure proteins-encoding genes, tRNA genes and DNA replication or nucleotide metabolism genes. Importantly, no known virulence-associated, antibiotic and lysogeny-related genes were identified in the genome of BPSELC-1. In vitro experiment of phage treatment pointed out that the number of viable S. Enteritidis ATCC 13076 was reduced by 5.9×log 10 at MOI of 10 2 after 4 h. To the best of our knowledge, the phage BPSELC-1 exhibited higher efficiency in S. Enteritidis treatment compared to previous studies. Moreover, it is promising to be used as a broad-spectrum candidate against Salmonella infections in commercial owing to its broad-host-range., Competing Interests: Declaration of competing interest No competing financial interests exist., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 10 7 -10 8 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.

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 × 10 5  cfu/mL and achieved more than 6.58 and 5.97 log unit reductions in cultures containing 5 × 10 6  cfu/mL and 5 × 10 7  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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The aim of this study was to identify and characterize the SE-P3, P16, P37, and P47 phages infecting Salmonella Enteritidis. Transmission electron microscopy analysis showed that the SE phages belonged to the Myoviridae or Siphoviridae family and had plaque sizes between 0.622 ± 0.027 and 1.630 ± 0.036 mm in diameter. sefC, pefA, spvC, sopE, and gipA virulent gene regions were absent in their genome and their calculated genome sizes were between 35.9 and 37.8 kbp. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the protein profiles of each phage were different. The SE phages had a short latent period (10-20 min), large burst size (76-356 PFU/cell), and a short burst time (25-35 min). The multiplicity of infection values and mutant frequency of the phages were 0.01-0.0001 and 10 -7 , respectively. They were very infective against their host bacteria when applied at 20°C, 30°C, or 37°C and adsorbed to their host cells by 96.20-97.65% in the first 5 min of incubation, and also Ca 2+ ions did not have a significant effect on their adsorption. The SE phages were resistant to wide pH ranges and high temperatures. These results indicate that the SE phages are good candidates as therapeutic and biocontrol agents against foodborne pathogenic S. Enteritidis., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Salmonella Enteritidis remains a major threat for food safety. To take efforts to develop phage-based biocontrol for S. Enteritidis contamination in food, in this study, the phages against S. Enteritidis were isolated from sewage samples, characterized by host range assays, DNA restriction enzyme pattern analyses, and transmission electron microscope observations, and tested for antibacterial activity in food; some potent phages were further characterized by bioinformatic analyses. Results showed that based on the plaque quality and host range, seven lytic phages targeting S. Enteritidis were selected, considered as seven distinct phages through DNA physical maps, and classified as Myoviridae or Siphoviridae family by morphologic observations; the combined use of such seven strain phages as a "food additive" could succeed in controlling the artificial S. Enteritidis contamination in the different physical forms of food at a range of temperatures; by bioinformatic analyses, both selected phage BPS 11 Q 3 and BPS 15 Q 2 seemed to be newfound obligate lytic phage strains with no indications for any potentially harmful genes in their genomes. In conclusion, our results showed a potential of isolated phages as food additives for controlling S. Enteritidis contamination in some salmonellosis outbreak-associated food vehicles, and there could be minimized potential risk associated with using BPS 11 Q 3 and BPS 15 Q 2 in food.

Forty strains of Salmonella enterica (S. enterica) subspecies salamae (II), arizonae (IIIa), diarizonae (IIIb), and houtenae (IV) were isolated from human or environmental samples and tested for bacteriophage production. Production of bacteriophages was observed in 15 S. enterica strains (37.5%) belonging to either the subspecies salamae (8 strains) or diarizonae (7 strains). Activity of phages was tested against 52 pathogenic S. enterica subsp. enterica isolates and showed that phages produced by subsp. salamae had broader activity against pathogenic salmonellae compared to phages from the subsp. diarizonae. All 15 phages were analyzed using PCR amplification of phage-specific regions and 9 different amplification profiles were identified. Five phages (SEN1, SEN4, SEN5, SEN22, and SEN34) were completely sequenced and classified as temperate phages. Phages SEN4 and SEN5 were genetically identical, thus representing a single phage type (i.e. SEN4/5). SEN1 and SEN4/5 fit into the group of P2-like phages, while the SEN22 phage showed sequence relatedness to P22-like phages. Interestingly, while phage SEN34 was genetically distantly related to Lambda-like phages (Siphoviridae), it had the morphology of the Myoviridae family. Based on sequence analysis and electron microscopy, phages SEN1 and SEN4/5 were members of the Myoviridae family and phage SEN22 belonged to the Podoviridae family., Competing Interests: The authors have declared that no competing interests exist.

While screening for new antimicrobial agents for multidrug-resistant Salmonella enterica, the novel lytic bacteriophage STP4-a was isolated and characterized. Phage morphology revealed that STP4-a belongs to the family Myoviridae. Bacterial challenge assays showed that different serovars of Salmonella enterica were susceptible to STP4-a infection. The genomic characteristics of STP4-a, containing 159,914 bp of dsDNA with an average GC content of 36.86 %, were determined. Furthermore, the endolysin of STP4-a was expressed and characterized. The novel endolysin, LysSTP4, has hydrolytic activity towards outer-membrane-permeabilized S. enterica and Escherichia coli. These results provide essential information for the development of novel phage-based biocontrol agents against S. enterica.

Two lytic phages, vB&#95;SenM-PA13076 (PA13076) and vB&#95;SenM-PC2184 (PC2184), were isolated from chicken sewage and characterized with host strains Salmonella Enteritidis (SE) ATCC13076 and CVCC2184, respectively. Transmission electron microscopy revealed that they belonged to the family Myoviridae. The lytic abilities of these two phages in liquid culture showed 104 multiplicity of infection (MOI) was the best in inhibiting bacteria, with PC2184 exhibiting more activity than PA13076. The two phages exhibited broad host range within the genus Salmonella. Phage PA13076 and PC2184 had a lytic effect on 222 (71.4%) and 298 (95.8%) of the 311 epidemic Salmonella isolates, respectively. We tested the effectiveness of phage PA13076 and PC2184 as well as a cocktail combination of both in three different foods (chicken breast, pasteurized whole milk and Chinese cabbage) contaminated with SE. Samples were spiked with 1 × 10(4) CFU individual SE or a mixture of strains (ATCC13076 and CVCC2184), then treated with 1 × 10(8) PFU individual phage or a two phage cocktail, and incubated at 4 °C or 25 °C for 5 h. In general, the inhibitory effect of phage and phage cocktail was better at 4 °C than that at 25 °C, whereas the opposite result was observed in Chinese cabbage, and phage cocktail was better than either single phage. A significant reduction in bacterial numbers (1.5-4 log CFU/sample, p < 0.05) was observed in all tested foods. The two phages on the three food samples were relatively stable, especially at 4 ºC, with the phages exhibiting the greatest stability in milk. Our research shows that our phages have potential effectiveness as a bio-control agent of Salmonella in foods.

In this study the isolation and characterization of three bacteriophages (ST4, L13, and SG3) infecting Salmonella gallinarum were carried out. They were further tested for their in vivo efficacy in phage therapy. All three phages belong to the Siphoviridae family with isometric heads and non-contractile tails. They have a broad host range among serovars of Salmonella enterica. The burst sizes were observed to be 1670, 80, and 28 for ST4, L13, and SG3, respectively. The in vivo efficacy of the phages was tested in chickens. Layer chickens were challenged with S. gallinarum, whereas contact chickens were cohabited without direct challenge. Each bacteriophage was orally inoculated in the form of feed additives. Mortality was observed and S. gallinarum was periodically re-isolated from the livers, spleens, and cecums of the chickens. Bacterial re-isolation from the organs and mortality decreased significantly in both challenged and contact chickens treated with the bacteriophages compared with untreated chickens serving as the control. The three bacteriophages may be effective alternatives to antibiotics for the control of fowl typhoid disease in chickens.

A novel flagellatropic phage of Salmonella enterica serovar Typhimurium, called iEPS5, was isolated and characterized. iEPS5 has an icosahedral head and a long noncontractile tail with a tail fiber. Genome sequencing revealed a double-stranded DNA of 59,254 bp having 73 open reading frames (ORFs). To identify the receptor for iEPS5, Tn5 transposon insertion mutants of S. Typhimurium SL1344 that were resistant to the phage were isolated. All of the phage-resistant mutants were found to have mutations in genes involved in flagellar formation, suggesting that the flagellum is the adsorption target of this phage. Analysis of phage infection using the ΔmotA mutant, which is flagellated but nonmotile, demonstrated the requirement of flagellar rotation for iEPS5 infection. Further analysis of phage infection using the ΔcheY mutant revealed that iEPS5 could infect host bacteria only when the flagellum is rotating counterclockwise (CCW). These results suggested that the CCW-rotating flagellar filament is essential for phage adsorption and required for successful infection by iEPS5. In contrast to the well-studied flagellatropic phage Chi, iEPS5 cannot infect the ΔfliK mutant that makes a polyhook without a flagellar filament, suggesting that these two flagellatropic phages utilize different infection mechanisms. Here, we present evidence that iEPS5 injects its DNA into the flagellar filament for infection by assessing DNA transfer from SYBR gold-labeled iEPS5 to the host bacteria.

Salmonella enterica subsp. enterica serovar Enteritidis is a major causative agent of gastroenteritis with contaminated eggs and chicken meat being the major source of infection. Phages are seriously being considered as a safe and cheaper alternative to antibiotics. The intestinal content of chicken was used as source for isolating phages. Phage designated as ΦSP-1 was selected for the study. Transmission electron microscopy (TEM) of phage ΦSP-1 revealed that it belonged to family Podoviridae. The optimal multiplicity of infection (MOI) was 5 phages/cell. Latent and rise period were calculated to be 30 and 55 minutes respectively, while burst size was 44 phages/bacterial cell. The genome size of ΦSP-1 was estimated to be 86 kb from pulsed-field gel electrophoresis analysis (PFGE). The effect of different physical and chemical parameters like temperature, pH, salinity and CaCl₂ were analyzed to optimize the conditions for large scale production of phages and to check the viability of ΦSP-1 under different physiochemical conditions. A temperature of 40 °C, pH 8 and 0.25 M NaCl were found to be optimum for phage adsorption and it was able to survive up to a temperature of 50 °C for 3 min. Capability to survive under hostile environmental conditions, absence of virulence genes in genome and genus specificity suggest suitability of ΦSP-1 to be used as a biocontrol agent., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We suggest a bacteriophage genus, "Viunalikevirus", as a new genus within the family Myoviridae. To date, this genus includes seven sequenced members: Salmonella phages ViI, SFP10 and ΦSH19; Escherichia phages CBA120 and PhaxI; Shigella phage phiSboM-AG3; and Dickeya phage LIMEstone1. Their shared myovirus morphology, with comparable head sizes and tail dimensions, and genome organization are considered distinguishing features. They appear to have conserved regulatory sequences, a horizontally acquired tRNA set and the probable substitution of an alternate base for thymine in the DNA. A close examination of the tail spike region in the DNA revealed four distinct tail spike proteins, an arrangement which might lead to the umbrella-like structures of the tails visible on electron micrographs. These properties set the suggested genus apart from the recently ratified subfamily Tevenvirinae, although a significant evolutionary relationship can be observed.

Salmonella remains the major cause of food-borne diseases worldwide, with chickens known to be the main reservoir for this zoonotic pathogen. Among the many approaches to reducing Salmonella colonization of broilers, bacteriophage offers several advantages. In this study, three bacteriophages (UAB&#95;Phi20, UAB&#95;Phi78, and UAB&#95;Phi87) obtained from our collection that exhibited a broad host range against Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium were characterized with respect to morphology, genome size, and restriction patterns. A cocktail composed of the three bacteriophages was more effective in promoting the lysis of S. Enteritidis and S. Typhimurium cultures than any of the three bacteriophages alone. In addition, the cocktail was able to lyse the Salmonella enterica serovars Virchow, Hadar, and Infantis. The effectiveness of the bacteriophage cocktail in reducing the concentration of S. Typhimurium was tested in two animal models using different treatment schedules. In the mouse model, 50% survival was obtained when the cocktail was administered simultaneously with bacterial infection and again at 6, 24, and 30 h postinfection. Likewise, in the White Leghorn chicken specific-pathogen-free (SPF) model, the best results, defined as a reduction of Salmonella concentration in the chicken cecum, were obtained when the bacteriophage cocktail was administered 1 day before or just after bacterial infection and then again on different days postinfection. Our results show that frequent treatment of the chickens with bacteriophage, and especially prior to colonization of the intestinal tract by Salmonella, is required to achieve effective bacterial reduction over time.

Salmonellosis is an internationally important disease of mammals and birds. Unique epidemics in New Zealand in the recent past include two Salmonella serovars: Salmonella enterica subsp. enterica serovar Typhimurium definitive type (DT) 160 (S. Typhimurium DT160) and S. Brandenburg. Although not a major threat internationally, in New Zealand S. Typhimurium DT160 has been the most common serovar isolated from humans, and continues to cause significant losses in wildlife. We have identified DNA differences between the first New Zealand isolate of S. Typhimurium DT160 and the genome-sequenced strain, S. Typhimurium LT2. All the differences could be accounted for in one cryptic phage ST64B, and one novel P22-like phage, ST160. The majority of the ST160 genome is almost identical to phage SE1 but has two regions not found in SE1 which are identical to the P22-like phage ST64T, suggesting that ST160 evolved from SE1 via two recombination events with ST64T. All of the New Zealand isolates of DT160 were identical indicating the clonal spread of this particular Salmonella. Some overseas isolates of S. Typhimurium DT160 differed from the New Zealand strain and contained SE1 phage rather than ST160. ST160 was also identified in New Zealand isolates of S. Typhimurium DT74 and S. Typhimurium RDNC-April06 and in S. Typhimurium DT160 isolates from the USA. The emergence of S. Typhimurium DT160 as a significant pathogen in New Zealand is postulated to have occurred due to the sensitivity of the Salmonella strains to the ST160 phage when S. Typhimurium DT160 first arrived.

We report the selection and amplification of the broad-host-range Salmonella phage phi PVP-SE1 in an alternative nonpathogenic host. The lytic spectrum and the phage DNA restriction profile were not modified upon replication in Escherichia coli Bl21, suggesting the possibility of producing this phage in a nonpathogenic host, contributing to the safety and easier approval of a product based on this Salmonella biocontrol agent.

Phage Gifsy-2, a lambdoid phage infecting Salmonella, has an unusually large composite gene coding for its major capsid protein (mcp) at the C-terminal end, a ClpP-like protease at the N-terminus, and a approximately 200 residue central domain of unknown function but which may have a scaffolding role. This combination of functions on a single coding region is more extensive than those observed in other phages such as HK97 (scaffold-capsid fusion) and lambda (protease-scaffold fusion). To study the structural phenotype of the unique Gifsy-2 capsid gene, we have purified Gifsy-2 particles and visualized capsids and procapsids by cryoelectron microscopy, determining structures to resolutions up to 12A. The capsids have lambdoid T=7 geometry and are well modeled with the atomic structures of HK97 mcp and phage lambda gpD decoration protein. Thus, the unique Gifsy-2 capsid protein gene yields a capsid maturation pathway engaging features from both phages HK97 and lambda., (Copyright 2010 Elsevier Inc. All rights reserved.)

The Lilleengen scheme for typing Salmonella enterica serovar Typhimurium consists of 12 tailed phages. Ten phages are podoviruses and morphologically identical to Salmonella phage P22. Two phages are siphoviruses and identical to flagella-specific phage chi.

Some bacteriophages target potentially pathogenic bacteria by exploiting surface-associated virulence factors as receptors. For example, phage have been identified that exhibit specificity for Vi capsule producing Salmonella enterica serovar Typhi. Here we have characterized the Vi-associated E1-typing bacteriophage using a number of molecular approaches. The absolute requirement for Vi capsule expression for infectivity was demonstrated using different Vi-negative S. enterica derivatives. The phage particles were shown to have an icosahedral head and a long noncontractile tail structure. The genome is 45,362 bp in length with defined capsid and tail regions that exhibit significant homology to the S. enterica transducing phage ES18. Mass spectrometry was used to confirm the presence of a number of hypothetical proteins in the Vi phage E1 particle and demonstrate that a number of phage proteins are modified posttranslationally. The genome of the Vi phage E1 is significantly related to other bacteriophages belonging to the same serovar Typhi phage-typing set, and we demonstrate a role for phage DNA modification in determining host specificity.

Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by > or = 4.2 log10 CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by > or = 2.19 log10 CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.

Out of 177 surveyed bacteriophages, 161 (91%) are tailed and belong to the Myoviridae, Siphoviridae, and Podoviridae families (43, 55, and 59 viruses, respectively). Sixteen filamentous or isometric phages are members of the Inoviridae, Leviviridae, Microviridae, and Tectiviridae families (9%). Many tailed phages belong to established phage genera (P22, T1, T5, and T7), which are widespread in enterobacteria and other Gram-negatives of the Proteobacteria phylum.

Cementing proteins that bind to the virion surface have been described in double-stranded DNA viruses such as herpesvirus, adenovirus, and numerous bacteriophages. The three-dimensional structure of bacteriophage L determined by electron cryo-microscopy reveals binding modes of two cementing proteins-one, called Dec, encoded by phage gene orf134 and the other by an as yet unidentified gene. These two proteins form homotrimers and bind at the quasi 3-fold axes nearest the icosahedral 2-fold axes and at the icosahedral 3-fold vertices, respectively. They do not bind at the quasi 3-fold axes near the icosahedral 5-fold vertices. These observations indicate precise recognition of the two cementing proteins at a subset of the quasi equivalent sites on the phage capsid. Sequence analysis shows striking similarity between the C-terminal portion of phage L Dec protein and five regions in the long tail fiber of a T4-like phage, suggesting functional parallelism between them.

Bacteriophages infecting Salmonella spp. were isolated from sewage using soft agar overlays containing three Salmonella serovars and assessed with regard to their potential to control food-borne salmonellae. Two distinct phages, as defined by plaque morphology, structure and host range, were obtained from a single sample of screened sewage. Phage FGCSSa1 had the broadest host range infecting six of eight Salmonella isolates and neither of two Escherichia coli isolates. Under optimal growth conditions for S. Enteritidis PT160, phage infection resulted in a burst size of 139 PFU but was apparently inactive at a temperature typical of stored foods (5 degrees C), even at multiplicity of infection values in excess of 10 000. While neither isolate had characteristics that would make them candidates for biocontrol of Salmonella spp. in foods, phage FGCSSa1 behaved unusually when grown on two Salmonella serotypes at 37 degrees C in that the addition of phages appeared to retard growth of the host, presumably by the lysis of a fraction of the host cell population.

Bacteriophages (phages) associated with Salmonella were collected from nine swine manure lagoons in Mississippi. Phages were isolated by an enrichment protocol or directly from effluent. For enrichment, chloroform-treated samples were filtered (0.22 mum) and selectively enriched by adding a cocktail of Salmonella strains in trypticase soy broth. After overnight incubation at 35 degrees C, chloroform was added and samples stored at 5 degrees C. Enriched samples were tested by double agar layer (DAL) plaque assay against individual Salmonella isolates. Phage titers of 2.9 x 10(8) to 2.1 x 10(9) plaque forming units (pfu) per mL were produced, but estimation of phage titers in lagoons was not possible. For direct isolation, effluent was clarified by centrifugation, filtered (0.22 microm), and used in DAL plaque assays to select single-plaque isolates for 15 Salmonella strains. Plaque counts varied among Salmonella strains and lagoons. The most sensitive strain for direct phage recovery was ATCC 13311. Phage titers estimated by direct isolation with ATCC 13311 ranged among lagoons from 12 to 148 pfu per mL. In limited host range tests, 66 isolates recovered by the enrichment protocol produced plaques only on Enteritidis and Typhimurium strains of Salmonella and none produced plaques on lagoon isolates of Citrobacter, Escherichia, Proteus, Providencia, or Serratia. Electron microscopy (EM) showed purified enrichment isolates had Podoviridae morphology (tailless 50-nm icosahedral heads with tail spikes). Electron microscopy of clarified concentrated effluent showed 5.5:1 tailless to tailed phages. The isolated phages have potential as typing reagents, specific indicators, and biocontrol agents of Salmonella.

Salmonella phage SPT-1, a member of the Myoviridae family and a relative of phage O1, produces abnormally long tails with coordinate variations of sheath and core length. The length of abnormal tails varies between 140 and 445 nm.

Temperature sensitive mutation in the gene for the 38 kDa minor structural protein of the phage MB78, a virulent phage of Salmonella enterica serovar typhimurium, interferes with phage development at restrictive temperature. Electron microscopy of particles produced at non-permissive temperature indicated that the particles formed are tailless. Two types of particles are seen: (i) empty capsids, which are not perfect icosahedral (ii) icosahedral particles filled with DNA. The gene for the 38 kDa protein is located in the SalIG fragment of the phage genome. Nucleotide sequence of the SalIG fragment of MB78 as well as its temperature sensitive mutant has been determined and analysed. Such analysis indicated that in the mutant the codon GCA has been changed to GTA resulting in substitution of alanine at position 75 of the protein by valine (A75V). This makes the protein thermolabile. Our results suggest that normal functioning of this 38 kDa protein is necessary for attachment of tail fibre to the capsid. Or in other words, this 38 kDa protein is involved in phage morphogenesis.

The generalized transducing double-stranded DNA bacteriophage ES18 has an icosahedral head and a long noncontractile tail, and it infects both rough and smooth Salmonella enterica strains. We report here the complete 46,900-bp genome nucleotide sequence and provide an analysis of the sequence. Its 79 genes and their organization clearly show that ES18 is a member of the lambda-like (lambdoid) phage group; however, it contains a novel set of genes that program assembly of the virion head. Most of its integration-excision, immunity, Nin region, and lysis genes are nearly identical to those of the short-tailed Salmonella phage P22, while other early genes are nearly identical to Escherichia coli phages lambda and HK97, S. enterica phage ST64T, or a Shigella flexneri prophage. Some of the ES18 late genes are novel, while others are most closely related to phages HK97, lambda, or N15. Thus, the ES18 genome is mosaically related to other lambdoid phages, as is typical for all group members. Analysis of virion DNA showed that it is circularly permuted and about 10% terminally redundant and that initiation of DNA packaging series occurs across an approximately 1-kbp region rather than at a precise location on the genome. This supports a model in which ES18 terminase can move substantial distances along the DNA between recognition and cleavage of DNA destined to be packaged. Bioinformatic analysis of large terminase subunits shows that the different functional classes of phage-encoded terminases can usually be predicted from their amino acid sequence.

Eleven tailed phages are described. They belong to the Myoviridae, Siphoviridae, or Podoviridae families and represent the ViI, T1, T5, Jersey, N4, and P22 species of enterobacterial phages. Morphology is correlated with host range.

Salmonella typhimurium employs the specialized type III secretion system encoded in pathogenicity island 1 (SPI1) to translocate effector proteins into host cells and to modulate host cell signal transduction. The SPI1 type III system and the effector proteins are conserved among all salmonellae and are thought to be acquired by horizontal gene transfer. The genetic mechanisms mediating this horizontal transfer are unknown. Here, we describe that SopE, a SPI1-dependent translocated effector protein, is present in relatively few S. typhimurium isolates. We have isolated a temperate phage that encodes SopE. Phage morphology and DNA hybridization, as well as partial sequence information, suggest that this phage (SopEPhi) is a new member of the P2 family of bacteriophages. By lysogenic conversion this phage can horizontally transfer genes between different S. typhimurium strains. Strikingly, most of the isolates harboring SopEPhi belong to the small group of epidemic strains of S. typhimurium that have been responsible for a large percentage of human and animal salmonellosis and have persisted for a long period of time. Our data suggest that horizontal transfer of type III dependent effector proteins by lysogenic infection with bacteriophages (lysogenic conversion) may provide an efficient mechanism for fine-tuning the interaction of Salmonella spp. with their hosts.

Out of 136 new phages, 80 (59%) are classified into 23 species according to morphology and physicochemical properties. Six new species are described and species beta 4, from a previous classification scheme, is renamed T1. The morphology of 36 phage species is schematically represented.

The lipopolysaccharide (LPS) of the Salmonella cell surface serves as the receptor for a very large number of bacterial viruses. The tailspike protein from these viruses recognizes the LPS as its initial receptor. It is proposed that the study of the P22 and epsilon 34 tailspike proteins could serve as a model for the study of the interaction of proteins with LPS. Toward this end, the tailspike protein of the epsilon 34 phage has been identified. The data suggest similarities between the epsilon 34 tailspike protein and the P22 tailspike protein. Some properties related to the interaction of the phage tailspike with its receptor are reported.

We describe a novel ion exchange chromatographic technique suitable for large-scale preparation of viruses and other biomacromolecular assemblies in highly purified form. The method, which utilizes anion exchange on either of two commercially available cellulose cartridges, is applied to the Escherichia coli bacteriophage PRD1. Viral particles eluted from both QMA and DEAE cartridges retain infectivity and exhibit greater homogeneity of composition, as judged by gel electrophoresis and electron microscopy, than particles purified by rate zonal sucrose gradient centrifugation. The ion exchange protocols are rapid, requiring less than 15 min elution time, and permit retrieval of the purified viral particles at high concentration in aqueous media without centrifugal pelleting. The present method is particularly well suited to the preparation of milligram to decigram quantities of virus, sufficient for many biophysical structural analyses, including investigations by solution spectroscopic and crystal diffraction techniques. The feasibility and advantages of the ion exchange chromatographic procedure are demonstrated by application of laser Raman spectroscopy to ion exchange purified PRD1 virions and subviral assemblies.

The typing set includes 27 tailed phages belonging to three families, the Myoviridae (contractile tails, 4 phages), Siphoviridae (long, non-contractile tails, 21 phages) and Podoviridae (short tails, 2 phages). Heads are isometric or elongated. The phages fall into 10 morphological groups, seven of which correspond to known enterobacterial phage species (Jersey, N4, T5, T7, ZG/3a, chi, 16-19) and one to a Rhizobium phage species (CM1). T5-type and T7-type phages are for the first time reported in salmonellae. Two phages produce large amounts of abnormal mottled heads.

The complex double-stranded DNA bacteriophages assemble DNA-free protein shells (procapsids) that subsequently package DNA. In the case of several double-stranded DNA bacteriophages, including P22, packaging is associated with cutting of DNA from the concatemeric molecule that results from replication. The mature intravirion P22 DNA has both non-unique (circularly permuted) ends and a length that is determined by the procapsid. In all known cases, procapsids consist of an outer coat protein, an interior scaffolding protein that assists in the assembly of the coat protein shell, and a ring of 12 identical portal protein subunits through which the DNA is presumed to enter the procapsid. To investigate the role of the portal protein in cutting permuted DNA from concatemers, we have characterized P22 portal protein mutants. The effects of several single amino acid changes in the P22 portal protein on the length of the DNA packaged, the density to which DNA is condensed within the virion, and the outer radius of the capsid have been determined. The results obtained with one mutant (NT5/1a) indicate no change (+/- 0.5%) in the radius of the capsid, but mature DNA that is 4.7% longer and a packing density that is commensurately higher than those of wild-type P22. Thus, the portal protein is part of the gauge that regulates the length and packaging density of DNA in bacteriophage P22. We argue that these findings make models for DNA packaging less likely in which the packing density is a property solely of the coat protein shell or of the DNA itself.

Data from physical and genetic mapping studies show that the vegetative genome of the temperate, Group E 1 Salmonella bacteriophage, epsilon 15, is circularly-permuted. Preliminary evidence suggests that the circular permutation of the epsilon 15 genome is non-random.

The gene 16 protein of the bacteriophage P22 is required as a pilot protein aiding the transfer of DNA from the phage into the Salmonella typhimurium host cell. During assembly 10-20 copies of the 63,000-Da gp 16 protein are incorporated into the procapsid shell prior to DNA packaging. The protein has been purified from isolated procapsids and behaved as a monomer in solution. Upon incubation with purified coat and scaffolding subunits in vitro, it assembled into procapsids with the correct stoichiometry. The addition of physiological quantities of gp 16 resulted in an increased rate of procapsid assembly. Sedimentation of mixtures of coat and gp 16 protein subunits revealed association/dissociation behavior. It is likely that the added gp 16 is acting to stabilize a transient oligomeric coat protein species that functions as the in vitro initiation complex for procapsid assembly.

The tailspike protein of bacteriophage P22 assembles with mature capsids during the final reaction in phage morphogenesis. The gene 9 mutation hmH3034 synthesizes a tailspike protein with a change at amino acid 100 from Asp to Asn. This mutant form of trimeric tailspike protein fails to assemble with capsids in vivo. By using in vitro quantitative tailspike-capsid assembly assays, this mutant tailspike trimer can be shown to assemble with capsids at very high tailspike concentrations. From these assays, we estimate that this single missense mutation decreases by 100-500-fold the affinity of the tailspike for capsids. Furthermore, hmH3034 tailspike protein has a structural defect which makes the mature tailspike trimers sensitive to SDS at room temperature and causes the trimers to "partially unfold." Spontaneously arising intragenic suppressors of the capsid assembly defect have been isolated. All of these suppressors are changes at amino acid 13 of the tailspike protein, which substitute His, Leu or Ser for the wild type amino acid Arg. These hmH3034/sup3034 mutants and the separated sup3034 mutants form fully functional tailspike proteins with assembly activities indistinguishable from wild type while retaining the SDS-sensitive structural defect. From the analysis of the hmH3034 mutant and its suppressors, we propose that in the wild-type tailspike protein, the Asp residue at position 100 and the Arg residue at position 13 form an intrachain or interchain salt bridge which stabilizes the amino terminus of the tailspike protein and that the unneutralized positive charge at amino acid 13 in the hmH3034 protein is the cause of the assembly defect of this protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological and biological properties of Salmonella typhimurium phage IRA were studied. The phage is a member of the Styloviridae family and exhibits a very wide spectrum of lytic activity. Molecular cloning of phage genes whose expression is lethal for the host cell has been performed and consequences of gene expression have been investigated. Expression of recombinant plasmid pKI71 causes structural changes of the cell wall and also degradation of host DNA while plasmid DNA remains intact. Expression of pKI72 blocks normal cell division. The possibility of applying such recombinant clones in marking pathways of microbial contamination in water areas is proposed.

A new phage typing scheme using wild bacteriophages isolated from sewage for phage typing Salmonella bareilly is described. Six hundred and thirty-seven strains of Salm. bareilly could be separated into 11 different phage types using five wild phages. Overall typability was 94.5%. These phages belonged to two different morphotypes. A1 and B1, and showed varying host range.

The rotational diffusion of bacteriophage epsilon 15 was measured before and after virus adsorption to outer membrane vesicles of the host Salmonella anatum. The virus capsid was labeled with eosin isothiocyanate, and the decay of transient dichroism following dye excitation by pulses of plane-polarized light was measured. From the data, the rotational diffusion constant of the unadsorbed virion and its hydrodynamic diameter were estimated and found to be consistent with electron microscopic measurements of the capsid dimensions. Addition of outer membrane vesicles of S. anatum to the virus suspension revealed the immobilization of the virus particles on the membrane surface.

A transducing phage, SE1, which is able to infect Salmonella typhimurium was isolated from a Salmonella enteritidis strain. SE1 is a temperate phage which is heteroimmune with respect to phages P22, L, KB1 and ES18. It is similar in morphology and size to phages P22, L and KB1 and is serologically related to phages P22 and L but not to KB1. Efficiencies of generalized transduction effected by phage SE1 are similar to those for P22HT (int7), a mutant which mediates a high frequency of chromosomal gene transduction. The lengths of chromosomal DNA transduced by SE1 and P22HT (int7) are similar. Furthermore, the SE1 prophage does not exclude the transducing particles from cells it has lysogenized; consequently it is possible to use both SE1 lysogens and non-lysogenic strains as recipients in SE1-mediated transduction experiments, and obtain similar transduction efficiencies. However, the SE1 prophage gives rise to a lysogenic conversion that decreases the rate of adsorption of SE1 and L phages by about 50%, but does not affect adsorption of P22. Altogether these results suggest that phage SE1 may be a useful tool in the genetic manipulation of S. typhimurium.

Using 14C-labeled Salmonella bacterial cells as the substrate, the enzymic and molecular properties of the base-plate parts of phage P22 were studied. The base-plate part consisted of a single protein species which cleaved extensively the O-antigen of Salmonella typhimurium, Salmonelly schottmuellerie and with somewhat slower rate that of Salmonella typhi, releasing oligo-saccharide products with rhamnose at the reducing end. Much less cleavage was observed with a strain of S. typhimurium lysogenic for P22, and no significant reaction with Salmonella anatum, Salmonella newington and Salmonella minneapolis. The base-plate part enzyme was a very heat-stable protein and only 10-20% loss was observed after treatment at 85 degrees C for 5 min. The pH optimum of the enzyme was around 7.5, and the glycosidase activity was not influenced by the ionic strength (25-250 mM( of the medium or the presence of Mg2+. The molecular weight of the base-plate part was 320,000 by sedimentation equilibrium. Dodecylsulphate-acrylamide gel electrophoresis revealed a single band of molecular weight 77,000, indicating that a single base-plate part corresponds to a tetramer of identical subunits. Circular dichroism spectra of P22 base-plate parts showed a major contribution of beta structure. The protein was rich in acidic amino acids, glycine and serine.

The structure of the lipid-containing bacteriophage PR4 was studied using two alkyl imidates, ethyl acetimidate (EAI), a reagent permeant to lipid bilayers and isethionyl acetimidate (IAI), which is impermeant to membranes. The virion is an icosahedral particle consisting of a protein coat surrounding a membrane of phospholipid and protein which in turn encloses the DNA genome. Upon exposure to the permeant reagent, EAI, 50% of the phosphatidylethanolamine (PE) molecules reacted rapidly (half-life less than 10 min). A similar fraction of the PE also reacted with IAI, the impermeant reagent. The remaining half of the PE molecules reacted slowly with EAI (half-life of 80 min) and failed to react with IAI. All of the phage proteins reacted with both EAI and IAI (except a DNA-associated protein which reacted only with EAI). These labeling results indicate that the phage membrane consists of a lipid bilayer and that at least a portion of each phage protein (except the DNA-associated protein) is exposed on the external face of the lipid bilayer. Several of the membrane proteins could be cross-linked either to the phage membrane PE after EAI treatment or to phage phosphatidylglycerol after periodate treatment. The major structural protein of the phage was readily cross-linked to PG but failed to cross-link to PE suggesting that the protein specifically interacts with PG.