Genomics of Salmonella phage ΦStp1: candidate bacteriophage for biocontrol.

Multidrug-resistant Salmonella causing Salmonellosis is a food-borne pathogen and hence a public health hazard. Alternatives to antibiotics, such as phages, are possible solutions to this increasing drug resistance. In this context, several Salmonella phages were isolated and characterized. This paper describes the physiochemical and whole genome characterization of one such bacteriophage, ΦStp1, which efficiently infects serovars Salmonella Enteritidis and Salmonella Typhimurium. Morphological observations by transmission electron microscopy and phylogenetic analysis using terminase gene classified ΦStp1 to family Siphoviridae, closely resembling 'T5 like phage' morpho-types. With a maximum adsorption time of 50 min, ΦStp1 latent period was 30 min with 37 phages/cell burst size. ΦStp1 draft genome sequenced by shotgun method comprised 112,149 bp in 3 contigs with 37.99% GC content, 168 predicted ORFs, and 15 tRNAs. Genes involved in host shut down, DNA replication, regulation, nucleotide metabolism, lysis, and morphogenesis were also noted. The study not only provided an insight into the characteristics of phage genome, but also information about proteins encoded by bacteriophages, therefore contributing to understanding phage diversity. Sequence analysis also proved the absence of virulence and lysogeny-related genes, which only went to confirm ΦStp1 as a promising therapeutic agent against Salmonella infections.

Core lipopolysaccharide-specific phage SSU5 as an Auxiliary Component of a Phage Cocktail for Salmonella biocontrol.

Salmonella spp. are among the major food-borne pathogens that cause mild diarrhea to severe bacteremia. The use of bacteriophages to control various food-borne pathogens, including Salmonella, has emerged as a promising alternative to traditional chemotherapy. We isolated the Siphoviridae family phage SSU5, which can infect only rough strains of Salmonella. The blocking of SSU5 adsorption by periodate treatment of host Salmonella cells and spotting and adsorption assays with mutants that contain various truncations in their lipopolysaccharide (LPS) cores revealed that the outer core region of the LPS is a receptor of SSU5. SSU5 could infect O-antigen (O-Ag)-deficient Salmonella mutants that developed by challenging of O-Ag-specific phages, and consequently, it delayed the emergence of the phage-resistant Salmonella population in broth culture when treated together with phages using O-Ag as a receptor. Therefore, these results suggested that phage SSU5 would be a promising auxiliary component of a phage cocktail to control rough strains of Salmonella enterica serovar Typhimurium, which might emerge as resistant mutants upon infection by phages using O-Ag as a receptor.

wksl3, a New biocontrol agent for Salmonella enterica serovars enteritidis and typhimurium in foods: characterization, application, sequence analysis, and oral acute toxicity study.

Of the Salmonella enterica serovars, S. Enteritidis and S. Typhimurium are responsible for most of the Salmonella outbreaks implicated in the consumption of contaminated foods in the Republic of Korea. Because of the widespread occurrence of antimicrobial-resistant Salmonella in foods and food processing environments, bacteriophages have recently surfaced as an alternative biocontrol tool. In this study, we isolated a virulent bacteriophage (wksl3) that could specifically infect S. Enteritidis, S. Typhimurium, and several additional serovars. Transmission electron microscopy revealed that phage wksl3 belongs to the family Siphoviridae. Complete genome sequence analysis and bioinformatic analysis revealed that the DNA of phage wksl3 is composed of 42,766 bp with 64 open reading frames. Since it does not encode any phage lysogeny factors, toxins, pathogen-related genes, or food-borne allergens, phage wksl3 may be considered a virulent phage with no side effects. Analysis of genetic similarities between phage wksl3 and four of its relatives (SS3e, vB_SenS-Ent1, SE2, and SETP3) allowed wksl3 to be categorized as a SETP3-like phage. A single-dose test of oral toxicity with BALB/c mice resulted in no abnormal clinical observations. Moreover, phage application to chicken skin at 8°C resulted in an about 2.5-log reduction in the number of Salmonella bacteria during the test period. The strong, stable lytic activity, the significant reduction of the number of S. Enteritidis bacteria after application to food, and the lack of clinical symptoms of this phage suggest that wksl3 may be a useful agent for the protection of foods against S. Enteritidis and S. Typhimurium contamination.

Isolation and characterization of a rare waterborne lytic phage of Salmonella enterica serovar Paratyphi B.

A lytic phage of Salmonella serovar Paratyphi B, named φSPB, was isolated from surface waters of the Pavana River in India. Phage φSPB is a member of the Podoviridae family and is morphologically similar to the 7-11 phages of the C3 morphotype of tailed phages, characterized by a very long, cigar-shaped head. The head measured approximately 153 × 57 nm, and the tail size was 12 × 7 nm. The phage was stable over a wide range of pH (4-9) and temperature (4-40 °C). The adsorption rate constant was 4.7 × 10(-10). Latent and eclipse periods were 10 and 15 min, respectively, and the burst size was 100 plaque-forming units/infected cell after 25 min at 37 °C. The phage DNA was 59 kb in size. Ten major proteins were observed on SDS-PAGE, although some of these proteins could be bacterial contaminants. This is the first report of Salmonella enterica subsp. enterica serovar Paratyphi B phage of C3 morphotype from India that has many unique features, such as high replication potential, short replication time, and stability over a wide range of pH and temperature, making it a promising biocontrol agent against the drug-resistant strains of Salmonella Paratyphi B.

Genetic relationships of phage types and single nucleotide polymorphism typing of Salmonella enterica Serovar Typhimurium.

Salmonella enterica serovar Typhimurium is one of the leading causes of gastroenteritis in humans. Phage typing has been used for the epidemiological surveillance of S. Typhimurium for over 4 decades. However, knowledge of the evolutionary relationships between phage types is very limited. In this study, we used single nucleotide polymorphisms (SNPs) as molecular markers to determine the relationships between common S. Typhimurium phage types. Forty-four SNPs, including 24 identified in a previous study and 20 from 6 available whole-genome sequences, were used to analyze 215 S. Typhimurium isolates belonging to 45 phage types. Altogether, 215 isolates and 6 genome strains were differentiated into 33 SNP profiles and four distinctive phylogenetic clusters. Fourteen phage types, including DT9, one of the most common phage types in Australia, were differentiated into multiple SNP profiles. These SNP profiles were distributed into different phylogenetic clusters, indicating that they have arisen independently multiple times. This finding suggests that phage typing may not be useful for long-term epidemiological studies over long periods (years) and diverse localities (different countries or continents). SNP typing provided a discriminative power similar to that of phage typing. However, 12 SNP profiles contained more than one phage type, and more SNPs would be needed for further differentiation. SNP typing should be considered as a replacement for phage typing for the identification of S. Typhimurium strains.

Complete genome sequence of Salmonella bacteriophage SPN3US.

Salmonella bacteriophage SPN3US was isolated from a chicken fecal sample. It is a virulent phage belonging to the Myoviridae family and showing effective inhibition of Salmonella enterica and a few Escherichia coli O157:H7 strains. Here we announce the completely sequenced first genome of a Salmonella phage using flagella as receptors. It is the largest genome among Salmonella phages sequenced to date, and major findings from its annotation are described.

Significance of the bacteriophage treatment schedule in reducing Salmonella colonization of poultry.

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_Phi20, UAB_Phi78, and UAB_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.

Salmonella Typhimurium-specific bacteriophage ΦSH19 and the origins of species specificity in the Vi01-like phage family.

BACKGROUND: Whole genome sequencing of bacteriophages suitable for biocontrol of pathogens in food products is a pre-requisite to any phage-based intervention procedure. Trials involving the biosanitization of Salmonella Typhimurium in the pig production environment identified one such candidate, ΦSH19. RESULTS: This phage was sequenced and analysis of its 157,785 bp circular dsDNA genome revealed a number of interesting features. ΦSH19 constitutes another member of the recently-proposed Myoviridae Vi01-like family of phages, containing S. Typhi-specific Vi01 and Shigella-specific SboM-AG3. At the nucleotide level ΦSH19 is highly similar to phage Vi01 (80-98% pairwise identity over the length of the genome), with the major differences lying in the region associated with host-range determination. Analyses of the proteins encoded within this region by ΦSH19 revealed a cluster of three putative tail spikes. Of the three tail spikes, two have protein domains associated with the pectate lyase family of proteins (Tsp2) and P22 tail spike family (Tsp3) with the prospect that these enable Salmonella O antigen degradation. Tail spike proteins of Vi01 and SboM-AG3 are predicted to contain conserved right-handed parallel ß-helical structures but the internal protein domains are varied allowing different host specificities. CONCLUSIONS: The addition or exchange of tail spike protein modules is a major contributor to host range determination in the Vi01-like phage family.

Specific detection of Salmonella enterica and Escherichia coli strains by using ELISA with bacteriophages as recognition agents.

The use of bacteriophages, instead of antibodies, in the ELISA-based detection of bacterial strains was tested. This procedure appeared to be efficient, and specific strains of Salmonella enterica and Escherichia coli could be detected. The sensitivity of the assay was about 10(5) bacterial cells/well (10(6)/ml), which is comparable with or outperforms other ELISA tests detecting intact bacterial cells without an enrichment step. The specificity of the assay depends on the kind of bacteriophage used. We conclude that the use of bacteriophages in the detection and identification of bacteria by an ELISA-based method can be an alternative to the use of specific antibodies. The advantages of the use of bacteriophages are their environmental abundance (and, thus, a possibility to isolate various phages with different specificities) and the availability of methods for obtaining large amounts of phage lysates, which are simple, rapid, cheap, and easy.

The evolution and distribution of phage ST160 within Salmonella enterica serotype Typhimurium.

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.

Characterization and Application of a Lytic Phage D10 against Multidrug-Resistant Salmonella.

Salmonella is a widely distributed foodborne pathogen that is a serious threat to human health. The accelerated development of drug resistance and the increased demand for natural foods invoke new biocontrol agents to limit contamination by multidrug-resistant (MDR) Salmonella strains. In this study, a lytic Salmonella phage named D10 was characterized at the biological and genomic levels. D10 possesses a short latent period (10 min) and a large burst size (163 PFU/cell), as well as adequate stability under a range of pH conditions and moderate thermal tolerance. D10 effectively lysed different MDR Salmonella serovars and repressed their dynamic growth in the medium. Genomic analysis disclosed that D10 is a new member of the Siphoviridae family and lacks the genes implicated in lysogeny, pathogenicity, or antibiotic resistance. A three-ingredient phage cocktail was then developed by mixing D10 with previously identified myovirus D1-2 and podovirus Pu20. The cocktail significantly reduced the count of MDR strains in liquid eggs, regardless of the temperature applied (4 and 25 °C). These results suggest that phage D10 is a promising tool to prevent food contamination by MDR Salmonella.

Use of a mixture of bacteriophages for biological control of Salmonella enterica strains in compost.

Bacteriophages specific to Salmonella strains were isolated from sewage effluent and characterized. A five-strain bacteriophage mixture was applied to dairy manure compost inoculated with Salmonella enterica serotype Typhimurium. Bacteriophage treatment resulted in a greater than 2-log-unit reduction of Salmonella within 4 h at all moisture levels compared to the controls.

Exploiting the role of TolC in pathogenicity: identification of a bacteriophage for eradication of Salmonella serovars from poultry.

Using a screening procedure, three bacteriophages, ST27, ST29, and ST35, were identified with selective activity for Salmonella enterica serovar Typhimurium (SL1344) but not SL1344 tolC::aph. Overproduction of TolC led to a lower efficiency of plating (EOP), further suggesting that TolC was the target receptor. Activity against other serovars of Salmonella was observed but not against other species of Enterobacteriaceae. This study provides proof of principle that bacteriophages can be active against the outer membrane protein of tripartite resistance-nodulation-division (RND) efflux pumps and so could be used to reduce the numbers of Salmonella cells in animals reared for food production.

Selection and characterization of a multivalent Salmonella phage and its production in a nonpathogenic Escherichia coli strain.

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 therapy to reduce preprocessing Salmonella infections in market-weight swine.

Contamination of meat products with food-borne pathogens usually results from the carcass coming in contact with the feces of an infected animal during processing. In the case of Salmonella, pigs can become colonized with the organism during transport and lairage from contaminated trailers and holding pens, resulting in increased pathogen shedding just prior to processing. Increased shedding, in turn, amplifies the likelihood of carcass contamination by magnifying the amount of bacteria that enters the processing facility. We conducted a series of experiments to test whether phage therapy could limit Salmonella infections at this crucial period. In a preliminary experiment done with small pigs (3 to 4 weeks old; 30 to 40 lb), administration of an anti-Salmonella phage cocktail at the time of inoculation with Salmonella enterica serovar Typhimurium reduced Salmonella colonization by 99.0 to 99.9% (2- to 3-log reduction) in the tonsils, ileum, and cecum. To test the efficacy of phage therapy in a production-like setting, we inoculated four market-weight pigs (in three replicates) with Salmonella enterica serovar Typhimurium and allowed the challenged pigs to contaminate a holding pen for 48 h. Sixteen naïve pigs were randomly split into two groups which received either the anti-Salmonella phage cocktail or a mock treatment. Both groups of pigs were comingled with the challenged pigs in the contaminated pen. Treatment with the anti-Salmonella phage cocktail significantly reduced cecal Salmonella concentrations (95%; P<0.05) while also reducing (numerically) ileal Salmonella concentrations (90%; P=0.06). Additional in vitro studies showed that the phage cocktail was also lytic against several non-Typhimurium serovars.

Salmonella Enteritidis bacteriophage candidates for phage therapy of poultry.

AIMS: Salmonella is a worldwide foodborne pathogen causing acute enteric infections in humans. In the recent years, the use of bacteriophages has been suggested as a possible tool to combat this zoonotic pathogen in poultry farms. This work aims to isolate and perform comparative studies of a group of phages active against a collection of specific Salmonella Enteritidis strains from Portugal and England. Also, suitable phage candidates for therapy of poultry will be selected. METHODS AND RESULTS: The Salm. Enteritidis strains studied were shown to have a significantly high occurrence of defective (cryptic) prophages; however, no live phages were found in the strains. Bacteriophages isolated from different environments lysed all except one of the tested Salm. Enteritidis strains. The bacteriophages studied were divided into different groups according to their genetic homology, RFLP profiles and phenotypic features, and most of them showed no DNA homology with the bacterial hosts. The bacteriophage lytic efficacy proved to be highly dependent on the propagation host strain. CONCLUSIONS: Despite the evidences shown in this work that the Salm. Enteritidis strains used did not produce viable phages, we have confirmed that some phages, when grown on particular hosts, behaved as complexes of phages. This is most likely because of the presence of inactive phage-related genomes (or their parts) in the bacterial strains which are capable of being reactivated or which can recombine with lytic phages. Furthermore, changes of the bacterial hosts used for maintenance of phages must be avoided as these can drastically modify the parameters of the phage preparations, including host range and lytic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows that the optimal host and growth conditions must be carefully studied and selected for the production of each bacteriophage candidate for animal therapy.

Occurrence of Salmonella-specific bacteriophages in swine feces collected from commercial farms.

Salmonella is one of the leading causes of human foodborne illness and is associated with swine production. Bacteriophages are naturally occurring viruses that prey on bacteria and have been suggested as a potential intervention strategy to reduce Salmonella levels in food animals on the farm and in the lairage period. If phages are to be used to improve food safety, then we must understand the incidence and natural ecology of both phages and their hosts in the intestinal environment. This study investigates the incidence of phages that are active against Salmonella spp. in the feces of commercial finishing swine. Fecal samples (n = 60) were collected from each of 10 commercial swine finishing operations. Samples were collected from 10 randomly selected pens throughout each operation; a total of 600 fecal samples were collected. Salmonella spp. were found in 7.3% (44/600) of the fecal samples. Bacteriophages were isolated from fecal samples through two parallel methods: (1) initial enrichment in Salmonella Typhimurium; (2) initial enrichment in Escherichia coli B (an indicator strain), followed by direct spot testing against Salmonella Typhimurium. Bacteriophages active against Salmonella Typhimurium were isolated from 1% (6/600) of the individual fecal samples when initially enriched in Salmonella Typhimurium, but E. coli B-killing phages were isolated from 48.3% (290/600) of the fecal samples and only two of these phages infected Salmonella Typhimurium on secondary plating. Collectively, our results indicate that bacteriophages are widespread in commercial swine, but those capable of killing Salmonella Typhimurium may be present at relatively low population levels. These results indicate that phages (predator) populations may vary along with Salmonella (prey) populations; and that phages could potentially be used as a food safety pathogen reduction strategy in swine.

Sanitation of faeces from source-separating dry toilets using urea.

AIM: To develop a reliable and simple method to produce safe fertilizers from human excreta using urea for sanitation of faeces. METHODS AND RESULTS: Urea was added to faecal matter (17% dry matter) at concentrations of 0.5-2% (w/w) and inactivation of Salmonella enterica subspecies 1 serovar Typhimurium (Salm. Typhimurium), Enterococcus spp. and the Salm. Typhimurium bacteriophage 28B was monitored at 14, 24 and 34 degrees C. Urea additions enhanced inactivation and inactivation rates were positively related to increasing NH(3) (aq) concentration and temperature. Salm. Typhimurium was the most sensitive of the organisms studied, while Enterococcus spp. showed more persistence, especially at lower temperatures. The bacteriophage was the most resistant organism studied. CONCLUSIONS: Salmonella reduction levels that meet requirements for safe reuse of faeces as fertilizer (i.e. 6 log(10) reduction) can be achieved for 1% urea within 2 months at 14 degrees C or within 1 week at 24 degrees C and 34 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The relationships between organism inactivation rates and temperature, ammonia and pH were identified. Urea treatment proved to be a robust and efficient option for safe recycling of plant nutrients.

[Rapid detection of salmonella in food by using fluorescently labeled phage O-I].

OBJECTIVE: To develop a rapid detection method for Salmonella in food by using specific Salmonella-phage O-I. METHODS: One hundred bacteria strains and 120 food sample isolates were infected using fluorescently labeled O-I phage genome with SYBR gold stain (a nucleic acid dye, 1 x working solution), then were observed under epi-fluorescence microscopy. The sensitivity of the method was tested. RESULTS: Among the 100 strains infected with O-I/SYBR gold stain, 40 Salmonella strains exhibited rod fluorescence. Other bacteria including 10 Proteus, 20 Shigella, 20 E. coli and 10 Staphylococcus did not exhibit this feature The sensitivity of detecting Salmonella was 10 CFU/100 microL. The detection for 120 food samples by using the O-I/SYBR gold stain had similar results to those by using the biochemical method. CONCLUSION: Fluorescent-labeled O-I phage could rapidly, sensitively and specifically detect Salmonella species in food samples.

Molecular characterization of the Salmonella enterica serovar Typhi Vi-typing bacteriophage E1.

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.