Characterization and whole-genome sequencing of broad-host-range Salmonella-specific bacteriophages for bio-control.

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×log10 at MOI of 102 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.

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens.

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of < 0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full-length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane.

National outbreak of Salmonella Java phage type 3b variant 9 infection using parallel case-control and case-case study designs, United Kingdom, July to October 2010.

Between July and October 2010, a national outbreak comprising 136 cases of Salmonella Java phage type 3b variant 9 was identified by the Health Protection Agency. Most cases were female. Cases had a median age of 39.5 years and lived in London, the South East and East of England. Parallel case­control and case­case study designs were undertaken to test the generated hypotheses. The case­case study aimed to examine if the infection was associated with eating food items purchased from commercial catering settings, and the reference group comprised non-travel related cases of S. Enteritidis infected during the same time period as the cases. The case­control study was designed to examine if the infection was associated with specific food items purchased from commercial catering settings, and recruited case-nominated controls. However, in response to poor recruitment we adapted our methods to investigate food exposures in the same way. Results of epidemiological investigations are compatible with salad vegetables as the potential source, but no common suppliers of salad were identified and no organisms were isolated from environmental and food samples. Limitations in the case­control study highlight the potential value of using a combination of epidemiological methods to investigate outbreaks.

Investigation of Salmonella Phage-Bacteria Infection Profiles: Network Structure Reveals a Gradient of Target-Range from Generalist to Specialist Phage Clones in Nested Subsets.

Bacteriophages that lyse Salmonella enterica are potential tools to target and control Salmonella infections. Investigating the host range of Salmonella phages is a key to understand their impact on bacterial ecology, coevolution and inform their use in intervention strategies. Virus-host infection networks have been used to characterize the "predator-prey" interactions between phages and bacteria and provide insights into host range and specificity. Here, we characterize the target-range and infection profiles of 13 Salmonella phage clones against a diverse set of 141 Salmonella strains. The environmental source and taxonomy contributed to the observed infection profiles, and genetically proximal phages shared similar infection profiles. Using in vitro infection data, we analyzed the structure of the Salmonella phage-bacteria infection network. The network has a non-random nested organization and weak modularity suggesting a gradient of target-range from generalist to specialist species with nested subsets, which are also observed within and across the different phage infection profile groups. Our results have implications for our understanding of the coevolutionary mechanisms shaping the ecological interactions between Salmonella phages and their bacterial hosts and can inform strategies for targeting Salmonella enterica with specific phage preparations.

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.

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.

Salmonella Infantis and Salmonella Enteritidis specific bacteriophages isolated form poultry faeces as a complementary tool for cleaning and disinfection against Salmonella.

Salmonellosis represents an important public health concern. Several authors point out the inefficiency of the cleaning and disinfection protocols to remove the bacteria from the field. For this reason, innovative techniques, as bacteriophages, could be implemented to control the bacteria. The main objectives of this study were to assess the effect of bacteriophages against Salmonella Infantis and Salmonella Enteritidis on farm surfaces, and to evaluate bacteriophage procedure application as sanitiser against Salmonella in field conditions. Thus, most prevalent serovars in poultry production were selected (Salmonella Infantis and Salmonella Enteritidis) to contaminate farm facilities. Then, two specific bacteriophages isolated from poultry faeces were applied against them. Results showed Salmonella Infantis and Salmonella Enteritidis decreased of 4.55 log10CFU/mL and 3.85 log10CFU/mL, respectively; the maximum reduction in Salmonella was the 5th day, after 108 PFU/mL and 103 PFU/mL bacteriophage application. These results highlight bacteriophages as a promising tool together with cleaning and disinfection.

Evaluation of Salmonella-lytic properties of bacteriophages isolated from commercial broiler houses.

Because of recent interest in bacteriophage therapy in poultry, information regarding the interaction of bacteriophages and potential host bacteria in the environment should be collected. The present studies were initiated with a rather typical commercial broiler integrator within the south-central United States to examine environmental Salmonella levels in two broiler complexes, attempt to isolate Salmonella-lytic bacteriophages, and elucidate a possible reason for differing apparent Salmonella prevalence. Significantly (P < 0.05) less Salmonella was isolated from houses in complex 1 (15/44 [34%] Salmonella-positive drag swabs) as compared to houses in complex 2 (22/24 [92%]). A total of seven Salmonella-lytic bacteriophages were isolated from Salmonella-positive environments, and two bacteriophages were isolated from a single Salmonella-negative house. During the initial bacteriophage isolation, individual bacteriophages did not replicate in the Salmonella host isolated from the same environment, and lysis of additional Salmonella hosts relied on high numbers of bacteriophage to be present. This suggests that the presence of these bacteriophages in the environment of a commercial broiler house had little to no effect on the presence of Salmonella. This study highlights the need to find additional bacteriophage sources, more effective isolation methods, and more innovative approaches to using bacteriophages to treat enteric disease.

Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence.

Bacteriophages, which are the natural predators of bacteria, have re-emerged as an attractive alternative to combat antibiotic resistant bacteria. Phages are highly specific at the species and strain level and measurement of the phage host range plays an important role in utilizing the phage as antimicrobials. The most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6%) of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format.

Comparison of the in vitro pathogenicity of two Salmonella Typhimurium phage types.

The in vitro pathogenicity of Salmonella enterica serovar Typhimurium phage type (pt) 90 and pt 506 (also known as DT 104) isolates from human and porcine origin was studied in adhesion and invasion assays to the human cell line Caco-2 and the porcine cell line IPI-2. Interleukin-8 (IL-8) production by these two cell lines in response to stimulation by the two Salmonella phage types was also measured. Generally, Salmonella Typhimurium pt 506 and pt 90 adhered to and invaded Caco-2 cells and IPI-2 cells equally well. The release of IL-8 by Caco-2 cells or by IPI-2 cells was similar, independent of the Salmonella phage type used for stimulation of the cells. These data suggest that Salmonella Typhimurium pt 90 has a similar ability to cause Salmonella infections as Salmonella Typhimurium DT 104.

Characterization of a broad host-spectrum virulent Salmonella bacteriophage fmb-p1 and its application on duck meat.

The aim of this study was to find a virulent bacteriophage for the biocontrol of Salmonella in duck meat. A broad host-spectrum virulent phage, fmb-p1, was isolated and purified from an duck farm, and its host range was determined to include S. Typhimurium, S. Enteritidis, S. Saintpaul, S. Agona, S. Miami, S. Anatum, S. Heidelberg and S. Paratyphi-C. Electron microscopy and genome sequencing showed that fmb-p1 belongs to the family Siphoviridae. The genome of fmb-p1 is composed of a 43,327-bp double-stranded DNA molecule with 60 open reading frames and a total G+C content of 46.09%. There are no deleterious sequences or genes encoding known harmful products in the phage fmb-p1 genome. Phage fmb-p1 was stable under different temperature (40-75°C), pH (4-10) and NaCl solutions (1-11%). The phage treatment (9.9×109 PFU/cm2) caused a peak reduction in S. Typhimurium of 4.52 log CFU/cm2 in ready-to-eat (RTE) duck meat, whereas potassium sorbate treatment (PS, 2mg/cm2) resulted in a 0.05-0.12 log reduction. Compared to PS treatment, there was significant difference in the S. Typhimurium reduction (P˂0.05) by phage treatment at both 4°C and 25°C. The results suggested that phage could be applied to reduce Salmonella, on commercial poultry products.

Inflammation boosts bacteriophage transfer between Salmonella spp.

Bacteriophage transfer (lysogenic conversion) promotes bacterial virulence evolution. There is limited understanding of the factors that determine lysogenic conversion dynamics within infected hosts. A murine Salmonella Typhimurium (STm) diarrhea model was used to study the transfer of SopEΦ, a prophage from STm SL1344, to STm ATCC14028S. Gut inflammation and enteric disease triggered >55% lysogenic conversion of ATCC14028S within 3 days. Without inflammation, SopEΦ transfer was reduced by up to 105-fold. This was because inflammation (e.g., reactive oxygen species, reactive nitrogen species, hypochlorite) triggers the bacterial SOS response, boosts expression of the phage antirepressor Tum, and thereby promotes free phage production and subsequent transfer. Mucosal vaccination prevented a dense intestinal STm population from inducing inflammation and consequently abolished SopEΦ transfer. Vaccination may be a general strategy for blocking pathogen evolution that requires disease-driven transfer of temperate bacteriophages.

Genetic and molecular analysis of GogB, a phage-encoded type III-secreted substrate in Salmonella enterica serovar typhimurium with autonomous expression from its associated phage.

Salmonella enterica serovar Typhimurium is lysogenized by several temperate bacteriophages that encode lysogenic conversion genes, which can act as virulence factors during infection and contribute to the genetic diversity and pathogenic potential of the lysogen. We have investigated the temperate bacteriophage called Gifsy-1 in S.enterica serovar Typhimurium and show here that the product of the gogB gene encoded within this phage shares similarity with proteins from other Gram-negative pathogens. The amino-terminal portion of GogB shares similarity with leucine-rich repeat-containing virulence-associated proteins from other Gram-negative pathogens, whereas the carboxyl-terminal portion of GogB shares similarity with uncharacterized proteins in other pathogens. We show that GogB is secreted by both type III secretion systems encoded in Salmonella Pathogenicity Island-1 (SPI-1) and SPI-2 but translocation into host cells is a SPI-2-mediated process. Once translocated, GogB localizes to the cytoplasm of infected host cells. The genetic regulation of gogB in Salmonella is influenced by the transcriptional activator, SsrB, under SPI-2-inducing conditions, but the modular nature of the gogB gene allows for autonomous expression and type III secretion following horizontal gene transfer into a heterologous pathogen. These data define the first autonomously expressed lysogenic conversion gene within Gifsy-1 that acts as a modular and promiscuous type III-secreted substrate of the infection process.

Computational determination of the effects of virulent Escherichia coli and salmonella bacteriophages on human gut.

BACKGROUND AND OBJECTIVE: Salmonella and Escherichia coli are different types of bacteria that cause food poisoning in humans. In the elderly, infants and people with chronic conditions, it is very dangerous if Salmonella or E. coli gets into the bloodstream and then they must be treated by phage therapy. Treating Salmonella and E. coli by phage therapy affects the gut flora. This research paper presents a system for detecting the effects of virulent E. coli and Salmonella bacteriophages on human gut. METHODS: A method based on Domain-Domain Interactions (DDIs) model is implemented in the proposed system to determine the interactions between the proteins of human gut bacteria and the proteins of bacteriophages that infect virulent E. coli and Salmonella. The system helps gastroenterologists to realize the effect of injecting bacteriophages that infect virulent E. coli and Salmonella on the human gut. RESULTS: By testing the system over Enterobacteria phage 933W, Enterobacteria phage VT2-Sa and Enterobacteria phage P22, it resulted in four interactions between the proteins of the bacteriophages that infect E. coli O157:H7, E. coli O104:H4 and Salmonella typhimurium and the proteins of human gut bacterium strains. CONCLUSION: Several effects were detected such as: antibacterial activity against a number of bacterial species in human gut, regulation of cellular differentiation and organogenesis during gut, lung, and heart development, ammonia assimilation in bacteria, yeasts, and plants, energizing defense system and its function in the detoxification of lipopolysaccharide, and in the prevention of bacterial translocation in human gut.

Bacteriophages with potential to inactivate Salmonella Typhimurium: Use of single phage suspensions and phage cocktails.

The aim of this study was to compare the dynamics of three previously isolated bacteriophages (or phages) individually (phSE-1, phSE-2 and phSE-5) or combined in cocktails of two or three phages (phSE-1/phSE-2, phSE-1/phSE-5, phSE-2/phSE-5 and phSE-1/phSE-2/phSE-5) to control Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) in order to evaluate their potential application during depuration. Phages were assigned to the family Siphoviridae and revealed identical restriction digest profiles, although they showed a different phage adsorption, host range, burst size, explosion time and survival in seawater. The three phages were effective against S. Typhimurium (reduction of ∼2.0 log CFU/mL after 4h treatment). The use of cocktails was not significantly more effective than the use of single phages. A big fraction of the remained bacteria are phage-resistant mutants (frequency of phage-resistant mutants 9.19×10(-5)-5.11×10(-4)) but phage- resistant bacterial mutants was lower for the cocktail phages than for the single phage suspensions and the phage phSE-1 presented the highest rate of resistance and phage phSE-5 the lowest one. The spectral changes of S. Typhimurium resistant and phage-sensitive cells were compared and revealed relevant differences for peaks associated to amide I (1620cm(-1)) and amide II (1515cm(-1)) from proteins and from carbohydrates and phosphates region (1080-1000cm(-1)). Despite the similar efficiency of individual phages, the development of lower resistance indicates that phage cocktails might be the most promising choice to be used during the bivalve depuration to control the transmission of salmonellosis.

Sequence comparison of the genes for immunity, DNA replication, and cell lysis of the P22-related Salmonella phages ES18 and L.

Complementation and hybridization experiments with the generalized transducing Salmonella phages P22, ES18 and L revealed strong similarity between the phages L and P22; the genome of ES18 shows a mosaic structure. About half of its genome, including the early genes, is similar or completely homologous to P22; the other half of the morphologically different ES18 does not show any similarity to P22 nor to E. coli phage lambda. Sequence comparison of the early genes has confirmed that the C-immunity region of ES18 is identical with that of P22, whereas the same region of phage L shows poor (repressor gene) or no similarity. The 5'-terminus of the DNA replication gene 12 of ES18, however, is homologous to the same section of gene O of phage lambda. The lysis genes of ES18 again are identical to those of P22; only gene 15 is mosaic-like and has more similarity to gene Rz of phage lambda. These results will be discussed in terms of the theory of modular genome organization.

A bacteriophage reagent for Salmonella: molecular studies on Felix 01.

Felix 01 (F01) is a bacteriophage originally isolated by Felix and Callow which lyses almost all Salmonella strains and has been widely used as a diagnostic test for this genus. Molecular information about this phage is entirely lacking. In the present study, the DNA of the phage was found to be a double-stranded linear molecule of about 80 kb. 11.5 kb has been sequenced and in this region A + T content is 60%. There are relatively few restriction endonuclease cleavage sites in the native genome and clones show this is due to their absence rather than modification. A restriction map of the genome has been constructed. The ends of the molecule cannot be ligated although they contain 5' phosphates. At least 60% of the genome must encode proteins. In the sequenced portion, many open reading frames exist and these are tightly packed together. These have been examined for homology to published proteins but only 1 to 17 shows similarity to known proteins. F01 is therefore the prototype of a new phage family. On the basis of restriction sites, codon usage and the distribution of nonsense codons in the unused reading frames, a strong case can be made for natural selection that reacts to mRNA structure and function.

Pathogenicity of Salmonella enteritidis phage types 4, 8, and 23 in broiler chicks.

Four hundred fifty day-old Hubbard broiler chicks were subdivided into 15 groups of 30 chicks each. Six groups of chicks received 0.5 ml of broth culture containing 5 x 10(6) colony-forming units (CFU) of Salmonella enteritidis (SE) phage types (PTs) 4, 8, and 23 by crop gavage. Similarly, six other groups received 0.5 ml containing 5 x 10(8) CFU of SE. One group was inoculated with 0.5 ml containing 5 x 10(6) CFU of Salmonella pullorum, and another group received 0.5 ml containing 5 x 10(8) CFU of S. pullorum. A group of 30 chicks were kept as uninoculated controls. Chicks were observed daily for clinical signs and mortality. All birds were weighed at 7, 14, and 21 days postinoculation 21 (DPI). Four chicks were randomly selected from each treatment group, euthanatized, and necropsied at 7 and 14 DPI. Gross lesions were recorded and selected tissues were collected for histopathology. The higher rates of illness and mortality were observed in chicks inoculated with 5 x 10(6) and 5 x 10(8) CFU of S. pullorum, followed by SE PT4 of human origin and SE PT4 of chicken origin. Moderate to high mortality was observed in chicks inoculated with the higher dose of SE isolates that belonged to PT8 and one SE of PT23. Variable mortality was evident in groups inoculated with the lower dose of salmonella. The most consistent gross and histopathologic changes, including fibrinous pericarditis and perihepatitis, were seen in the dead birds from various treatment groups. The lower mean body weights were present in all treatment groups compared with uninoculated controls. No illness or mortality was observed in uninoculated control groups.

Occurrence of Salmonella Enteritidis phage type 29 in Austria: an opportunity to assess the relevance of chicken meat as source of human Salmonella infections.

Assuming that the various phage types of Salmonella Enteritidis (S. Enteritidis) are largely equally virulent, the importance of certain foods as sources of infection for human salmonellosis can be deduced from differences in the distribution of phage types in human and non-human samples. In 2002, S. Enteritidis phage type 29 (PT29) was first isolated from non-human test samples in Austria. S. Enteritidis PT29 accounted for 44 (27.7%) of 159 S. Enteritidis strains, derived from veterinary samples of chicken (e.g. meat, giblets) or chicken habitations (e.g. swabs from the coop and excrement). At the food retail level (chicken meat, chicken liver), five (13.1%) of 38 S. Enteritidis isolates were PT29. The proportion of S. Enteritidis PT29 in human samples was much lower. Only 0.4% (30 human primary isolates) of all S. Enteritidis isolates in the year 2002, and 0.33% (23 human primary isolates) of all human S. Enteritidis strains in 2003 were PT29. In our opinion, the discrepancy between the high prevalence of S. Enteritidis PT29 in broilers and chicken meat and the low number of PT29 cases in humans indicates that chicken meat of Austrian origin is currently only a minor source of human S. Enteritidis infections.

[Some epidemiological features of salmonellosis in veal calves in the Netherlands (author's transl)]. / Enkele epidemiologische aspecten van salmonellose bij mestkalveren in Nederland.

In the Netherlands, salmonellosis in veal calves is caused by S. dublin and S. typhimurium. Strains showing multiple drug resistance are important factors in these cases. Strains of S. typhimurium isolated from dead veal-calves resistance are important factors in these cases. Strains of S. typhimurium isolated from dead veal-calves are confined to a small number of phage types (mainly X 200, X 210, X 193, X ORS). To trace the Salmonella organisms to their origin, faecal samples were collected from one week old calves in markets, in lorries and within 24 hours after their arrival on veal-calf units, and subsequently examined for the presence of Salmonella. Salmonella (47.5 per cent of S. typhimurium, 20 per cent of S. dublin and 32.5 per cent of other serotypes) was isolated from 3.5 per cent of the individual faecal samples of 1,143 calves on seven veal-calf farms (1976-1978). Clinical symptoms caused by S. typhimurium and the other serotypes were not observed on these farms, whereas there were individual cases due to S. dublin. The phage types of S. typhimurium isolated (I 650, VI 260 and II 505) obviously had little if any pathogenic significance in calves. During the period from October 1977 to November 1978. Salmonella was isolated from nearly 6 per cent of 1,880 calves in the markets (S. typhimurium 50 per cent, S. dublin 17 per cent and other serotypes 33 per cent). Well over 25 per cent of the strains of S. typhimurium isolated were of the pathogenic phage types (X 201, X ORS). Salmonella was isolated from well over 30 per cent of eighty-three lorries. It is concluded that markets and lorries are important factors in the epidemiology of salmonellosis in veal calves. However, there also are persistent infections in veal-calf units. Possible routes of infection, in which the veal calf is regarded as the most important source of infection, are discussed. More detailed epidemiological studies are in progress to make it possible to suggest measures by which the incidence of salmonellosis in the Netherlands may be reduced.