Broad Host Range Bacteriophages Research Articles

Cell-free synthesis of the Salmonella specific broad host range bacteriophage, felixO1

Phage-based biocontrol of foodborne Salmonella is limited by the requisite use of Salmonella to propagate the phages. This limitation can be circumvented by producing Salmonella phages using a cell-free gene expression system (CFE) with a non-pathogenic chassis. Here, we produce the Salmonella phage felixO1 using an E. coli-based CFE system.

Broad host range bacteriophage, EscoHU1, infecting Escherichia coli O157:H7 and Salmonella enterica: Characterization, comparative genomics, and applications in food safety

Shiga toxin-producing Escherichia coli and Salmonella enterica are important pathogens worldwide. Bacteriophages can be effectively used to reduce the incidence of foodborne pathogens. The host recognition systems of phages are highly specific, with the host range of a common phage being limited to the species or strain level. Here, we characterized a novel broad-host-range phage, EscoHU1, that infects several bacterial species, including E. coli and S. enterica, and evaluated its antimicrobial potential to inhibit E. coli O157:H7 and S. Typhimurium growth in food systems. The adsorption of EscoHU1 on E. coli was faster than that on S. Typhimurium; however, the one-step growth curves of EscoHU1 in both species were similar. Genomic analysis of EscoHU1 revealed that this phage has long direct terminal repeats at both ends of the genome, and phylogenetic analysis of the terminase large subunit confirmed that EscoHU1 belongs to the genus Epseptimavirus. Comparative analysis of structural proteins indicated a diversity of proteins related to the host range (receptor-binding proteins and L-shaped tail fibers). Challenge tests using beef and milk demonstrated the antimicrobial effects of EscoHU1 in inhibiting the growth of E. coli O157:H7 and S. Typhimurium in the food system. However, the antimicrobial effect of EscoHU1 on S. Typhimurium was lower than that on E. coli O157:H7. These results suggest that the novel broad-host-range phage EscoHU1 may serve as an effective antimicrobial agent to reduce food poisoning caused by E. coli O157:H7 and S. Typhimurium.

SGP-C: A Broad Host Range Temperate Bacteriophage; Against Salmonella gallinarum.

Salmonella gallinarum is a poultry restricted-pathogen causing fowl-typhoid disease in adult birds with mortality rates up-to 80% and exhibit resistance against commonly used antibiotics. In this current study, a temperate broad host range bacteriophage SGP-C was isolated against S. gallinarum from poultry digesta. It showed infection ability in all the 15 tested field strains of S. gallinarum. The SGP-C phage produced circular, turbid plaques with alternate rings. Its optimum activity was observed at pH 7.0 and 37–42°C, with a latent period of 45 min and burst size of 187 virions/bacterial cell. The SGP-C lysogens, SGPC-L5 and SGPC-L6 exhibited super-infection immunity against the same phage, an already reported feature of lysogens. A virulence index of 0.5 and 0.001 as MV50 of SGP-C suggests its moderate virulence. The genome of SGP-C found circular double stranded DNA of 42 Kbp with 50.04% GC content, which encodes 63 ORFs. The presence of repressor gene at ORF49, and absence of tRNA sequence in SGP-C genome indicates its lysogenic nature. Furthermore, from NGS analysis of lysogens we propose that SGP-C genome might exist either as an episome, or both as integrated and temporary episome in the host cell and warrants further studies. Phylogenetic analysis revealed its similarity with Salmonella temperate phages belonging to family Siphoviridae. The encoded proteins by SGP-C genome have not showed homology with any known toxin and virulence factor. Although plenty of lytic bacteriophages against this pathogen are already reported, to our knowledge SGP-C is the first lysogenic phage against S. gallinarum reported so far.

Broad host range bacteriophages found in rhizosphere soil of a healthy tomato plant in Bulgaria

The urgent need of research of new approaches to control bacterial disease on economical important crops, focuses our attention on bacteriophages as alternative biocontrol agents. Thus, the purpose of this paper is to present the isolation and initial characterization of three bacteriophages (SfXv124t/1, 2 and 3) isolated from rhizosphere soil of a healthy tomato plant in Bulgaria that are capable to lyse three phytopathogenic bacteria. The initial characterization includes determination of: their host range, plaque morphology, optimal storage temperature of pure phage lysates, their sensitivity to UV light, thermal inactivation, optimal multiplicity of infection (MOI) and virion morphology. The obtained results showed that one of the phage isolates was capable to lyse wild strains from three phytopathogenic bacterial species: Xanthomonas vesicatoria, Xanthomonas euvesicatoria and Xanthomonas gardneri, and the two remaining phages were active against X. vesicatoria and X. euvesicatoria. On X. vesicatoria lawn, the phages produced the same plaque types that differed only in their size. Storage at 4 °C for 26 days did not lead to decrease in phage titer as opposed to storage at 28 °C followed by decrease to varying degree for all three phages. The results obtained after exposure of the phage lysates to sunlight (UVA + B) and UVC light in separate experiments showed that UVC had a potent phagocidal effect as after 50 min of exposure there were no viable phages in the samples. UVA an UVB had lethal effect for two of the phage isolates and absolutely no lethal effect for the third one as after 50 min of exposure to sunlight there was no decrease in the initial phage titer. Phage isolates were tested for their thermal inactivation after incubation of pure phage lysates at three different temperatures: 55 °C, 75 °C and 95 °C for a period of 10 and 30 min. The most lethal temperature turned out to be 95 °C as after 10 min there were no viable phages in the samples. Phage isolate SfXv124t/1 was the most susceptible as its titer decreased by 1 lg after 10 min of incubation at 55 °C and by another 1 lg after 30 min. The most thermally resistant isolate was SfXv124t/3 as its titer remained stable after 30 min of incubation at 55 °C and decreased only by lg after incubation at 75 °C for 10 min. The optimal MOI for SfXv124t/3 was 0,01 (tested range 0,01–100) with maximal phage titer, reported at the 24th hour of incubation. TEM micrographs of the same isolates reveals that it belongs to family Podoviridae.

Locally isolated broad host-range bacteriophage kills methicillin-resistant Staphylococcus aureus in an in vivo skin excisional wound model in mice

From the perspective of an ever-increasing multidrug resistance among bacterial pathogens, bacteriophages are receiving renewed interest as potential alternative to antibiotics. We investigated the potential of a locally isolated species-specific phage against Staphylococcus aureus infection in a skin excisional wound model in mice. A significant time-dependent increase (P < 0.05) in wound healing was observed in the phage-treated mice groups. The animals treated with the phage ΦDMSA-2 exhibited a faster re-epithelialization and faster tissue re-modelling. Bacterial load in the infected tissue in all phage-treated groups diminished. The mean ± SD CFU per ml decreased from 3.3 × 108 ± 3.5 × 106 at day 1–1.43 × 103 ± 8.48 × 102 at day 16 (P < 0.05). The highest reduction in the bacterial load was observed in G5 (povidine-iodine treated) and G6 (povidine iodine + phage 107 PFU) groups as no bacterial counts were detected by day 12 in both groups. Interestingly, group G3, which was treated with a lower phage concentration (5 × 106 PFU), resulted in total clearing of the inoculated bacteria by day 16; while bacterial counts were still detected by that time in group G4, which was treated with a higher phage concentration of 107 PFU. Animals from phage-treated group G3 survived 100%, while those from the infected wound control group survived at a rate of 34% at day 9 and reached 0% by the end of day 22 (P < 0.001). The data from this study convincingly showed that phage treatment of the S. aureus-infected wounds resulted in a faster wound healing and a 100% survival of the animals. The results emphasize the utility of locally isolated species-specific phages in treatment against multidrug-resistant MRSA infections.

Bacteriophages as Preservation Agents to Promote Minimally Processed Food Safety

Foodborne disease related to ready to eat products contaminated by foodborne pathogens is a global concern due to its impact on human health and economy worldwide. Minimally processed food like ready to eat fruits and vegetables are not subjected to common preservation processes, additionally, the use of antibiotics and chemicals for pathogens eradication may affect consumers health. Baceriophages or viruses that infect bacteria have been suggested as bio-control tools and advantageous bio-preservation agents to promote minimally processed food safety. Recent studies show a reduction of pathogens after application of phages cocktails on fresh produce. Usefulness of broad-host range bacteriophages as bio-preservation agents needs more investigation.

Molecular Insights into Bacteriophage Evolution toward Its Host.

Bacteriophages (phages), viruses that infect bacteria, are considered to be highly host-specific. To add to the knowledge about the evolution and development of bacteriophage speciation toward its host, we conducted a 21-day experiment with the broad host-range bacteriophage Aquamicrobium phage P14. We incubated the phage, which was previously isolated and enriched with the Alphaproteobacteria Aquamicrobium H14, with the Betaproteobacteria Alcaligenaceae H5. During the experiment, we observed an increase in the phage’s predation efficacy towards Alcaligenaceae H5. Furthermore, genome analysis and the comparison of the bacteriophage’s whole genome indicated that rather than being scattered evenly along the genome, mutations occur in specific regions. In total, 67% of the mutations with a frequency higher than 30% were located in genes that encode tail proteins, which are essential for host recognition and attachment. As control, we incubated the phage with the Alphaproteobacteria Aquamicrobium H8. In both experiments, most of the mutations appeared in the gene encoding the tail fiber protein. However, mutations in the gene encoding the tail tubular protein B were only observed when the phage was incubated with Alcaligenaceae H5. This highlights the phage’s tail as a key player in its adaptation to different hosts. We conclude that mutations in the phage’s genome were mainly located in tail-related regions. Further investigation is needed to fully characterize the adaptation mechanisms of the Aquamicrobium phage P14.

Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu.

The broad host range bacteriophage Mu employs a novel ‘methylcarbamoyl’ modification to protect its DNA from diverse restriction systems of its hosts. The DNA modification is catalyzed by a phage-encoded protein Mom, whose mechanism of action is a mystery. Here, we characterized the co-factor and metal-binding properties of Mom and provide a molecular mechanism to explain ‘methylcarbamoyl’ation of DNA by Mom. Computational analyses revealed a conserved GNAT (GCN5-related N-acetyltransferase) fold in Mom. We demonstrate that Mom binds to acetyl CoA and identify the active site. We discovered that Mom is an iron-binding protein, with loss of Fe2+/3+-binding associated with loss of DNA modification activity. The importance of Fe2+/3+ is highlighted by the colocalization of Fe2+/3+ with acetyl CoA within the Mom active site. Puzzlingly, acid-base mechanisms employed by >309,000 GNAT members identified so far, fail to support methylcarbamoylation of adenine using acetyl CoA. In contrast, free-radical chemistry catalyzed by transition metals like Fe2+/3+ can explain the seemingly challenging reaction, accomplished by collaboration between acetyl CoA and Fe2+/3+. Thus, binding to Fe2+/3+, a small but unprecedented step in the evolution of Mom, allows a giant chemical leap from ordinary acetylation to a novel methylcarbamoylation function, while conserving the overall protein architecture.

Immunization with Salmonella Abortusequi phage lysate protects guinea pig against the virulent challenge of SAE-742

Salmonella Abortusequi causes important clinical diseases in horses possibly leading to abortion. In the present investigation, the protective efficacy of both plain and aluminum hydroxide gel adjuvanted phage lysate was evaluated in guinea pig model. Broad host range bacteriophage PIZ-SAE-2, was characterized and used for generation of lysates. Three different lysate batches, produced through separate cycles and characterized, were pooled together for immunization study. Plain and adjuvanted phage lysate preparations elicited both humoral and cellmediated immunity. The adjuvanted lysate at a dose of 50 μl elicited the highest protective efficacy against direct challenge at 28th DPI. Thus, the present study describes a new method of bacterial inactivation for producing a new class of better & safe immunprophylactic agents. This is the first report of producing an inactivated vaccine candidate using a new approach against equine salmonellosis.

Isolation and Characterization of Novel Broad Host Range Bacteriophages of Vibrio cholerae O1 from Bengal.

Objectives:We have isolated a total of five newer cholera phages which are novel broad host range to incorporate with the existing phage typing schemes for an extended typing scheme.Materials and Methods:These newly isolated phages were well characterized including the electron micrograph. A total of 300 Vibrio cholerae strains were isolated from the different endemic region in India were included in phage typing study.Results:These phages were found different from the existing phages. Electron microscopic results showed that the phages belonged to myophage and podophage group. Characterization of the phages based on pH, temperature, and organic solvent sensitivity showed differences among the phages used in this study. All the strains of Vibrio O1 were typeable (100%) with the five set of cholera phages. Of these, 40% strains were clustered under Type-1.Conclusion:The newer Vibrio phages are novel and broad host range and will be useful to incorporate with the existing phage typing system for more precisely discriminate the strains of Vibrio cholerae.

Assessment of biofilm removal capacity of a broad host range bacteriophage JHP against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an efficient biofilm-dwelling microbial pathogen, associated with nosocomial infections. These biofilm-associated infections are resistant to antibiotics and immune defenses, therefore pose major problem against their treatment. This scenario demands alternative therapeutic regimens, and bacteriophage therapy is one among potential strategies for clinical management of multiple drug resistance. In this investigation, the efficacy of a bacteriophage, JHP, is evaluated to eradicate P. aeruginosa biofilms. Growth kinetics of P. aeruginosa biofilm revealed that the highest cell density biofilm (1.5 × 1016 CFU/mL) was established within the polystyrene microtiter plate at 72 h post inoculation. Pseudomonas aeruginosa biofilms of different ages, treated with JHP (0.6 MOI) for different post-infection durations, reduced biomass from 2 to 4.5 logs (60-90%). JHP treatment before biofilm development reduced the bacterial load up to 9 logs (>95% bacterial load reduction) as compared with untreated control, which highlights its potential to prevent biofilm formation in indwelling medical devices. Combinations of JHP with other phages or antibiotics could be an efficient alternative for P. aeruginosa biofilm removal in clinical and industrial settings.

Genome Analysis of a Novel Broad Host Range Proteobacteria Phage Isolated from a Bioreactor Treating Industrial Wastewater.

Bacteriophages are viruses that infect bacteria, and consequently they have a major impact on the development of a microbial population. In this study, the genome of a novel broad host range bacteriophage, Aquamicrobium phage P14, isolated from a wastewater treatment plant, was analyzed. The Aquamicrobium phage P14 was found to infect members of different Proteobacteria classes (Alphaproteobacteria and Betaproteobacteria). This phage contains a 40,551 bp long genome and 60% of its genes had blastx hits. Furthermore, the bacteriophage was found to share more than 50% of its genes with several podoviruses and has the same gene order as other polyvalent bacteriophages. The results obtained in this study led to the conclusion that indeed general features of the genome of the Aquamicrobium phage P14 are shared with other broad host range bacteriophages, however further analysis of the genome is needed in order to identify the specific mechanisms which enable the bacteriophage to infect both Alphaproteobacteria and Betaproteobacteria.

More Is Better: Selecting for Broad Host Range Bacteriophages.

Bacteriophages are viruses that infect bacteria. In this perspective, we discuss several aspects of a characteristic feature of bacteriophages, their host range. Each phage has its own particular host range, the range of bacteria that it can infect. While some phages can only infect one or a few bacterial strains, other phages can infect many species or even bacteria from different genera. Different methods for determining host range may give different results, reflecting the multiple mechanisms bacteria have to resist phage infection and reflecting the different steps of infection each method depends on. This makes defining host range difficult. Another difficulty in describing host range arises from the inconsistent use of the words “narrow” and especially “broad” when describing the breadth of the host range. Nearly all bacteriophages have been isolated using a single host strain of bacteria. While this procedure is fairly standard, it may more likely produce narrow rather than broad host range phage. Our results and those of others suggest that using multiple host strains during isolation can more reliably produce broader host range phages. This challenges the common belief that most bacteriophages have a narrow host range. We highlight the implications of this for several areas that are affected by host range including horizontal gene transfer and phage therapy.

Salmonella Host Range of Bacteriophages That Infect Multiple Genera

Conventionally, bacteriophages are considered viruses capable of amplification only in a narrow range of closely related bacteria. Presently, we selected bacteriophages with the ability to infect more than 1 bacterial genus. Initially, wild-type bacteriophages were selected for ability to form plaques in Salmonella enteritidis agar overlays. For determination of host specificity, a pool of 44 bacteriophages was combined with each bacterial isolate in tryptic soy broth. This mixture was incubated with fresh bacterial culture and media for 4 sequential passes, and the resulting bacteriophage titer was determined using S. enteritidis. One Klebsiella and 3 different Escherichia isolates successfully amplified some bacteriophage(s) from the S. enteritidis-selected bacteriophage pool (experiment 1). Amplification of bacteriophages in each species was confirmed by the formation of increased plaque forming units in a tryptic soy agar overlay with the enteric (alternative host) bacteria, Klebsiella or Escherichia (experiment 2). Two selected bacteriophages, confirmed to amplify in Escherichia or Klebsiella, were further evaluated for ability to amplify in 10 different Salmonella serovars by amplification in broth culture (experiment 3). One had the ability to amplify in 6 different Salmonella serovars, and the other had the ability to amplify in 2 different Salmonella serovars. These experiments suggest that bacteriophage host range is not always genera-restricted and that selection of subpopulations of bacteriophages capable of amplification in alternative genera may provide a tool for selection of broad host-range bacteriophages for the pathogen of interest. Selection of non-pathogenic host isolates to support replication of Salmonella bacteriophages may allow improved safety for bacteriophage application to poultry because this would reduce the necessity for 100% purification of the bacteriophages(s) from resistant host bacteria.

Restriction of bacteriophage plaque formation in Streptomyces spp

Several Streptomyces species that produce restriction endonucleases were characterized for their ability to propagate 10 different broad host range bacteriophages. Each species displayed a different pattern of plaque formation. A restrictionless mutant of S. albus G allowed plaque formation by all 10 phages, whereas the wild-type strain showed plaques with only 2 phages. DNA isolated from three of the phages was analyzed for the presence of restriction sites for Streptomyces species-encoded enzymes, and a very strong correlation was established between the failure to form plaques on Streptomyces species that produced particular restriction enzymes and the presence of the corresponding restriction sites in the phage DNA. Also, the phages that lacked restriction sites in their DNA generally formed plaques on the corresponding restriction endonuclease-producing hosts at high efficiency. The DNAs from the three phages analyzed also generally contained either many or no restriction sites for the Streptomyces species-produced enzymes, suggesting a strong evolutionary trend to either eliminate all or tolerate many restriction sites. The data indicate that restriction plays a major role in host range determination for Streptomyces phages. Analysis of bacteriophage host ranges of many other uncharacterized Streptomyces hosts has identified four relatively nonrestricting hosts, at least two of which may be suitable hosts for gene cloning. The data also suggest that several restriction systems remain to be identified in the genus Streptomyces.