Bacteriophages LIMElight and LIMEzero of Pantoea agglomerans, belonging to the "phiKMV-like viruses".
ABSTRACT: Pantoea agglomerans is a common soil bacterium used in the biocontrol of fungi and bacteria but is also an opportunistic human pathogen. It has been described extensively in this context, but knowledge of bacteriophages infecting this species is limited. Bacteriophages LIMEzero and LIMElight of P. agglomerans are lytic phages, isolated from soil samples, belonging to the Podoviridae and are the first Pantoea phages of this family to be described. The double-stranded DNA (dsDNA) genomes (43,032 bp and 44,546 bp, respectively) encode 57 and 55 open reading frames (ORFs). Based on the presence of an RNA polymerase in their genomes and their overall genome architecture, these phages should be classified in the subfamily of the Autographivirinae, within the genus of the "phiKMV-like viruses." Phylogenetic analysis of all the sequenced members of the Autographivirinae supports the classification of phages LIMElight and LIMEzero as members of the "phiKMV-like viruses" and corroborates the subdivision into the different genera. These data expand the knowledge of Pantoea phages and illustrate the wide host diversity of phages within the "phiKMV-like viruses."
Project description:Bacteriophages are viruses capable of recognizing with high specificity, propagating inside of, and destroying their bacterial hosts. The phage lytic life cycle makes phages attractive as tools to selectively kill pathogenic bacteria with minimal impact on the surrounding microbiome. To effectively harness the potential of phages in therapy, it is critical to understand the phage-host dynamics and how these interactions can change in complex populations. Our model examined the interactions between the plant pathogen Erwinia amylovora, the antagonistic epiphyte Pantoea agglomerans, and the bacteriophages that infect and kill both species. P. agglomerans strains are used as a phage carrier; their role is to deliver and propagate the bacteriophages on the plant surface prior to the arrival of the pathogen. Using liquid cultures, the populations of the pathogen, carrier, and phages were tracked over time with quantitative real-time PCR. The jumbo Myoviridae phage ?Ea35-70 synergized with both the Myoviridae ?Ea21-4 and Podoviridae ?Ea46-1-A1 and was most effective in combination at reducing E. amylovora growth over 24 h. Phage ?Ea35-70, however, also reduced the growth of P. agglomerans. Phage cocktails of ?Ea21-4, ?Ea46-1-A1, and ?Ea35-70 at multiplicities of infections (MOIs) of 10, 1, and 0.01, respectively, no longer inhibited growth of P. agglomerans. When this cocktail was grown with P. agglomerans for 8 h prior to pathogen introduction, pathogen growth was reduced by over four log units over 24 h. These findings present a novel approach to study complex phage-host dynamics that can be exploited to create more effective phage-based therapies.
Project description:Lytic bacteriophages are in development as biological control agents for the prevention of fire blight disease caused by Erwinia amylovora. Temperate phages should be excluded as biologicals since lysogeny produces the dual risks of host resistance to phage attack and the transduction of virulence determinants between bacteria. The extent of lysogeny was estimated in wild populations of E.?amylovora and Pantoea agglomerans with real-time polymerase chain reaction primers developed to detect E.?amylovora phages belonging to the Myoviridae and Podoviridae families. Pantoea agglomerans, an orchard epiphyte, is easily infected by Erwinia spp. phages, and it serves as a carrier in the development of the phage-mediated biological control agent. Screening of 161 E.?amylovora isolates from 16 distinct geographical areas in North America, Europe, North Africa and New Zealand and 82 P.?agglomerans isolates from southern Ontario, Canada showed that none possessed prophage. Unstable phage resistant clones or lysogens were produced under laboratory conditions. Additionally, a stable lysogen was recovered from infection of bacterial isolate Ea110R with Podoviridae phage ?Ea35-20. These laboratory observations suggested that while lysogeny is possible in E.?amylovora, it is rare or absent in natural populations, and there is a minimal risk associated with lysogenic conversion and transduction by Erwinia spp. phages.
Project description:Bacteriophage KP34 is a novel virus belonging to the subfamily Autographivirinae lytic for extended-spectrum ?-lactamase-producing Klebsiella pneumoniae strains. Its biological features, morphology, susceptibility to chemical and physical agents, burst size, host specificity and activity spectrum were determined. As a potential antibacterial agent used in therapy, KP34 molecular features including genome sequence and protein composition were examined. Phylogenetic analyses and clustering of KP34 phage genome sequences revealed its clear relationships with "phiKMV-like viruses". Simultaneously, whole-genome analyses permitted clustering and classification of all phages, with completely sequenced genomes, belonging to the Podoviridae.
Project description:Lytic Pseudomonas aeruginosa phages LKD16 and LKA1 were locally isolated and morphologically classified as Podoviridae. While LKD16 adsorbs weakly to its host, LKA1 shows efficient adsorption (ka = 3.9 x 10(-9) ml min(-1)). LKA1, however, displays a narrow host range on clinical P. aeruginosa strains compared to LKD16. Genome analysis of LKD16 (43,200 bp) and LKA1 (41,593 bp) revealed that both phages have linear double-stranded DNA genomes with direct terminal repeats of 428 and 298 bp and encode 54 and 56 genes, respectively. The majority of the predicted structural proteins were experimentally confirmed as part of the phage particle using mass spectrometry. Phage LKD16 is closely related to bacteriophage phiKMV (83% overall DNA homology), allowing a more thoughtful gene annotation of both genomes. In contrast, LKA1 is more distantly related, lacking significant DNA homology and showing protein similarity to phiKMV in 48% of its gene products. The early region of the LKA1 genome has diverged strongly from phiKMV and LKD16, and intriguing differences in tail fiber genes of LKD16 and LKA1 likely reflect the observed discrepancy in infection-related properties. Nonetheless, general genome organization is clearly conserved among phiKMV, LKD16, and LKA1. The three phages carry a single-subunit RNA polymerase gene adjacent to the structural genome region, a feature which distinguishes them from other members of the T7 supergroup. Therefore, we propose that phiKMV represents an independent and widespread group of lytic P. aeruginosa phages within the T7 supergroup.
Project description:A novel siphovirus, vB_PagS_MED16 (MED16) was isolated in Lithuania using <i>Pantoea agglomerans</i> strain BSL for the phage propagation. The double-stranded DNA genome of MED16 (46,103 bp) contains 73 predicted open reading frames (ORFs) encoding proteins, but no tRNA. Our comparative sequence analysis revealed that 26 of these ORFs code for unique proteins that have no reliable identity when compared to database entries. Based on phylogenetic analysis, MED16 represents a new genus with siphovirus morphology. In total, 35 MED16 ORFs were given a putative functional annotation, including those coding for the proteins responsible for virion morphogenesis, phage-host interactions, and DNA metabolism. In addition, a gene encoding a preQ<sub>0</sub> DNA deoxyribosyltransferase (DpdA) is present in the genome of MED16 and the LC-MS/MS analysis indicates 2'-deoxy-7-amido-7-deazaguanosine (dADG)-modified phage DNA, which, to our knowledge, has never been experimentally validated in genomes of <i>Pantoea</i> phages. Thus, the data presented in this study provide new information on <i>Pantoea</i>-infecting viruses and offer novel insights into the diversity of DNA modifications in bacteriophages.
Project description:A cold-adapted siphovirus, vB_PagS_AAS23 (AAS23) was isolated in Lithuania using the <i>Pantoea agglomerans</i> strain AUR for the phage propagation. The double-stranded DNA genome of AAS23 (51,170 bp) contains 92 probable protein encoding genes, and no genes for tRNA. A comparative sequence analysis revealed that 25 of all AAS23 open reading frames (ORFs) code for unique proteins that have no reliable identity to database entries. Based on the phylogenetic analysis, AAS23 has no close relationship to other viruses publicly available to date and represents a single species of the genus <i>Sauletekiovirus</i> within the family <i>Drexlerviridae</i>. The phage is able to form plaques in bacterial lawns even at 4 °C and demonstrates a depolymerase activity. Thus, the data presented in this study not only provides the information on <i>Pantoea</i>-infecting bacteriophages, but also offers novel insights into the diversity of cold-adapted viruses and their potential to be used as biocontrol agents.
Project description:The complete genomes of two virulent phages infecting Citrobacter rodentium are reported here for the first time. Both bacteriophages were isolated from local sewage treatment plant effluents. Genome analyses revealed a close relationship between both phages and allowed their classification as members of the Autographivirinae subfamily in the T7-like genus.
Project description:Bacteriophage PAXYB1 was recently isolated from wastewater samples. This phage was chosen based on its lytic properties against clinical isolates of Pseudomonas aeruginosa (P. aeruginosa). In the present study, characterized PAXYB1, clarified its morphological and lytic properties, and analyzed its complete genome sequence. Based on the morphology of PAXYB1, it is a Podoviridae. The linear GC-rich (62.29%) double-stranded DNA genome of PAXYB1 is 43,337?bp including direct terminal repeats (DTRs) of 468?bp. It contains 60 open reading frames (ORFs) that are all encoded within the same strand. We also showed that PAXYB1 is a virulent phage and a new member of the phiKMV-like phages genus. Twenty-eight out of sixty predicted gene products (gps) showed significant homology to proteins of known function, which were confirmed by analyzing the structural proteome. Altogether, our work identified a novel lytic bacteriophage that lyses P. aeruginosa PAO1 and efficiently infects and kills several clinical isolates of P. aeruginosa. This phage has potential for development as a biological disinfectant to control P. aeruginosa infections.
Project description:Pantoea agglomerans and other Pantoea species cause infections in humans and are also pathogenic to plants, but the diversity of Pantoea strains and their possible association with hosts and disease remain poorly known, and identification of Pantoea species is difficult. We characterized 36 Pantoea strains, including 28 strains of diverse origins initially identified as P. agglomerans, by multilocus gene sequencing based on six protein-coding genes, by biochemical tests, and by antimicrobial susceptibility testing. Phylogenetic analysis and comparison with other species of Enterobacteriaceae revealed that the genus Pantoea is highly diverse. Most strains initially identified as P. agglomerans by use of API 20E strips belonged to a compact sequence cluster together with the type strain, but other strains belonged to diverse phylogenetic branches corresponding to other species of Pantoea or Enterobacteriaceae and to probable novel species. Biochemical characteristics such as fosfomycin resistance and utilization of d-tartrate could differentiate P. agglomerans from other Pantoea species. All 20 strains of P. agglomerans could be distinguished by multilocus sequence typing, revealing the very high discrimination power of this method for strain typing and population structure in this species, which is subdivided into two phylogenetic groups. PCR detection of the repA gene, associated with pathogenicity in plants, was positive in all clinical strains of P. agglomerans, suggesting that clinical and plant-associated strains do not form distinct populations. We provide a multilocus gene sequencing method that is a powerful tool for Pantoea species delineation and identification and for strain tracking.
Project description:We describe the small-scale, laboratory-based, production and quality control of a cocktail, consisting of exclusively lytic bacteriophages, designed for the treatment of Pseudomonas aeruginosa and Staphylococcus aureus infections in burn wound patients. Based on successive selection rounds three bacteriophages were retained from an initial pool of 82 P. aeruginosa and 8 S. aureus bacteriophages, specific for prevalent P. aeruginosa and S. aureus strains in the Burn Centre of the Queen Astrid Military Hospital in Brussels, Belgium. This cocktail, consisting of P. aeruginosa phages 14/1 (Myoviridae) and PNM (Podoviridae) and S. aureus phage ISP (Myoviridae) was produced and purified of endotoxin. Quality control included Stability (shelf life), determination of pyrogenicity, sterility and cytotoxicity, confirmation of the absence of temperate bacteriophages and transmission electron microscopy-based confirmation of the presence of the expected virion morphologic particles as well as of their specific interaction with the target bacteria. Bacteriophage genome and proteome analysis confirmed the lytic nature of the bacteriophages, the absence of toxin-coding genes and showed that the selected phages 14/1, PNM and ISP are close relatives of respectively F8, phiKMV and phage G1. The bacteriophage cocktail is currently being evaluated in a pilot clinical study cleared by a leading Medical Ethical Committee.