Pectobacterium atrosepticum Phage vB_PatP_CB5: A Member of the Proposed Genus 'Phimunavirus'.

Pectobacterium atrosepticum is a phytopathogen of economic importance as it is the causative agent of potato blackleg and soft rot. Here we describe the Pectobacterium phage vB_PatP_CB5 (abbreviated as CB5), which specifically infects the bacterium. The bacteriophage is characterized in detail and TEM micrographs indicate that it belongs to the Podoviridae family. CB5 shares significant pairwise nucleotide identity (≥80%) with P. atrosepticum phages φM1, Peat1, and PP90 and also shares common genome organization. Phylograms constructed using conserved proteins and whole-genome comparison-based amino acid sequences show that these phages form a distinct clade within the Autographivirinae. They also possess conserved RNA polymerase recognition and specificity loop sequences. Their lysis cassette resembles that of KP34virus, containing in sequential order a U-spanin, a holin, and a signal⁻arrest⁻release (SAR) endolysin. However, they share low pairwise nucleotide identity with the type phage of the KP34virus genus, Klebsiella phage KP34. In addition, phage KP34 does not possess several conserved proteins associated with these P. atrosepticum phages. As such, we propose the allocation of phages CB5, Peat1, φM1, and PP90 to a separate new genus designated Phimunavirus.

Genomic characteristics of vB_PpaP_PP74, a T7-like Autographivirinae bacteriophage infecting a potato pathogen of the newly proposed species Pectobacterium parmentieri.

Bacteriophage vB_PpaP_PP74 (PP74) is a novel virulent phage that infects members of the species Pectobacterium parmentieri, a newly established species of soft-rot-causing bacteria in the family Pectobacteriaceae, derived from potato-specific Pectobacterium wasabiae. vB_PpaP_PP74 was identified as a member of the family Podoviridae by transmission electron microscopy. The phage has a 39,790-bp dsDNA genome containing 50 open reading frames (ORFs). Because of the absence of genes encoding toxins or lysogeny factors, PP74 may be considered a candidate phage for pathogen biocontrol applications. The genome layout is similar to genomes of T7-like phages within the subfamily Autographivirinae, and therefore, functions can be attributed to most of ORFs. However, the closest nucleotide sequence homologs of phage PP74 are unclassified Escherichia phages. Based on phylogenetic analysis, vB_PpaP_PP74 is a sensu lato T7-like phage, but it forms a distant subgenus group together with homologous enterobacterial phages.

Isolation and characterization of a novel bacteriophage infecting Vibrio alginolyticus.

Vibrio alginolyticus is a common marine bacterium implicated in disease outbreaks in marine farmed fish and invertebrates. Due to the inappropriate use of antibiotics in aquaculture, alternative therapies have been proposed. One of the most promising options is the use of lytic bacteriophages to control pathogenic bacteria. This work describes the isolation and characterization of a lytic phage (VEN) against a V. alginolyticus strain (V2) isolated from a disease outbreak in common dentex (Dentex dentex) cultured at the Hellenic Centre for Marine Research (HCMR) in Crete, Greece. The bacteriophage is morphologically similar to phages from Podoviridae family and remained stable for 1 year at 4 °C and over 1 h when kept at 50 °C. VEN was able to lyse the host bacteria at several multiplicity of infection (MOI) (0.1-100) in liquid cultures. However, it was unable to infect other V. alginolyticus strains. Its genome consists of 44,603 bp with a GC content of 43.5%, while sequence analysis revealed the presence of 54 potential ORFs with a T7-like genomic organization. Almost 65% of the predicted ORFs presented homology with proteins of the vibriophages Vc1 and phi-A318 infecting Vibrio cyclitrophicus and Vibrio alginolyticus, respectively. Phylogenetic analysis applying the amino acid sequence of the large terminase subunit confirmed the close relationship of these phages. Furthermore, the comparison of the RNA polymerase of these phages revealed that the motifs A, B and C related to the catalytic activity and the recognition loop related to promotor identification were also conserved. VEN has an obligate lytic life cycle demonstrated by experimental data and genomic analysis. These results suggest that VEN may provide a good candidate to control recurrent diseases caused by V. alginolyticus at HCMR.

Characterization of five newly isolated bacteriophages active against Pseudomonas aeruginosa clinical strains.

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections, especially in patients with immunodeficiency. It exhibits multiple mechanisms of resistance, including efflux pumps, antibiotic modifying enzymes and limited membrane permeability. The primary reason for the development of novel therapeutics for P. aeruginosa infections is the declining efficacy of conventional antibiotic therapy. These clinical problems caused a revitalization of interest in bacteriophages, which are highly specific and have very effective antibacterial activity as well as several other advantages over traditional antimicrobial agents. Above all, so far, no serious or irreversible side effects of phage therapy have been described. Five newly purified P. aeruginosa phages named vB_PaeM_WP1, vB_PaeM_WP2, vB_PaeM_WP3, vB_PaeM_WP4 and vB_PaeP_WP5 have been characterized as potential candidates for use in phage therapy. They are representatives of the Myoviridae and Podoviridae families. Their host range, genome size, structural proteins and stability in various physical and chemical conditions were tested. The results of these preliminary investigations indicate that the newly isolated bacteriophages may be considered for use in phagotherapy.

Culturable bacterial diversity from a feed water of a reverse osmosis system, evaluation of biofilm formation and biocontrol using phages.

Biofilm formation on reverse osmosis (RO) systems represents a drawback in the application of this technology by different industries, including oil refineries. In RO systems the feed water maybe a source of microbial contamination and thus contributes for the formation of biofilm and consequent biofouling. In this study the planktonic culturable bacterial community was characterized from a feed water of a RO system and their capacities were evaluated to form biofilm in vitro. Bacterial motility and biofilm control were also analysed using phages. As results, diverse Protobacteria, Actinobacteria and Bacteroidetes were identified. Alphaproteobacteria was the predominant group and Brevundimonas, Pseudomonas and Mycobacterium the most abundant genera. Among the 30 isolates, 11 showed at least one type of motility and 11 were classified as good biofilm formers. Additionally, the influence of non-specific bacteriophage in the bacterial biofilms formed in vitro was investigated by action of phages enzymes or phage infection. The vB_AspP-UFV1 (Podoviridae) interfered in biofilm formation of most tested bacteria and may represent a good alternative in biofilm control. These findings provide important information about the bacterial community from the feed water of a RO system that may be used for the development of strategies for biofilm prevention and control in such systems.

Bacteriophages φMR299-2 and φNH-4 can eliminate Pseudomonas aeruginosa in the murine lung and on cystic fibrosis lung airway cells.

UNLABELLED: Pseudomonas aeruginosa is a common cause of infection in the lungs of patients with cystic fibrosis (CF). In addition, biofilm formation and antibiotic resistance of Pseudomonas are major problems that can complicate antibiotic therapy. We evaluated the efficacy of using bacteriophages to kill the pathogen in both biofilms and in the murine lung. We isolated and characterized two phages from a local wastewater treatment plant, a myovirus (φNH-4) and a podovirus (φMR299-2). Both phages were active against clinical isolates of P. aeruginosa. Together, the two phages killed all 9 clinical isolate strains tested, including both mucoid and nonmucoid strains. An equal mixture of the two phages was effective in killing P. aeruginosa NH57388A (mucoid) and P. aeruginosa MR299 (nonmucoid) strains when growing as a biofilm on a cystic fibrosis bronchial epithelial CFBE41o- cell line. Phage titers increased almost 100-fold over a 24-h period, confirming replication of the phage. Furthermore, the phage mix was also effective in killing the pathogen in murine lungs containing 1 × 10(7) to 2 × 10(7) P. aeruginosa. Pseudomonas was effectively cleared (reduced by a magnitude of at least 3 to 4 log units) from murine lungs in 6 h. Our study demonstrates the efficacy of these two phages in killing clinical Pseudomonas isolates in the murine lung or as a biofilm on a pulmonary cell line and supports the growing interest in using phage therapy for the control and treatment of multidrug-resistant Pseudomonas lung infections in CF patients. IMPORTANCE: Given the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment of Pseudomonas infection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can kill Pseudomonas growing on human lung cells and in an animal model of lung infection. The use of bacteriophages is particularly appropriate because the killing agent can replicate on the target cell, generating fresh copies of the bacteriophage. Thus, in the presence of a target, the killing agent multiplies. By using two bacteriophages we can reduce the risk of resistant colonies developing at the site of infection. Bacteriophage therapy is an exciting field, and this study represents an important demonstration of efficacy in validated infection models.

Morphology and general characteristics of bacteriophages infectious to Robinia pseudoacacia mesorhizobia.

Four phages infectious to Mesorhizobium strains were identified in soil samples taken from local Robinia pseudoacacia stands. Based on their polyhedral heads and short noncontractile tails, three of the phages, Mlo30, Mam12, and Mam20, were assigned to group C of Bradley's classification, the Podoviridae family, while phage Mlo1, with its elongated hexagonal head and a long flexible tail represented subgroup B2 bacteriophages, the Siphoviridae family. The phages were homogeneous in respect of their virulence, as they only lysed Mesorhizobium strains, but did not affect strains of Rhizobium or Bradyrhizobium. On the basis of one-step growth experiments, the average virus yield was calculated as approximately 10-25 phage particles for phages Mlo30, Mam12 and Mam20, and as many as 100-120 for phage Mlo1. The rate of phage adsorption to heat-treated cells showed differences in the nature of their receptors, which seemed to be thermal sensitive, thermal resistant, or a combination of the two. Only the receptor for phage Mlo30 was likely to be an LPS molecule, which was supported by a neutralization test. The smooth LPS with O-antigenic chains of the phage-sensitive M. loti strain completely reduced the bactericidal activity of virions at a concentration of 1 µg/ml. The molecular weights of phage DNAs estimated from restriction endonuclease cleavage patterns were in the range from approximately 39 kb for group C phages to approximately 80 kb for B2.