Molecular Characterization and Genome Mechanical Features of Two Newly Isolated Polyvalent Bacteriophages Infecting Pseudomonas syringae pv. garcae.

Coffee plants have been targeted by a devastating bacterial disease, a condition known as bacterial blight, caused by the phytopathogen Pseudomonas syringae pv. garcae (Psg). Conventional treatments of coffee plantations affected by the disease involve frequent spraying with copper- and kasugamycin-derived compounds, but they are both highly toxic to the environment and stimulate the appearance of bacterial resistance. Herein, we report the molecular characterization and mechanical features of the genome of two newly isolated (putative polyvalent) lytic phages for Psg. The isolated phages belong to class Caudoviricetes and present a myovirus-like morphotype belonging to the genuses Tequatrovirus (PsgM02F) and Phapecoctavirus (PsgM04F) of the subfamilies Straboviridae (PsgM02F) and Stephanstirmvirinae (PsgM04F), according to recent bacterial viruses' taxonomy, based on their complete genome sequences. The 165,282 bp (PsgM02F) and 151,205 bp (PsgM04F) genomes do not feature any lysogenic-related (integrase) genes and, hence, can safely be assumed to follow a lytic lifestyle. While phage PsgM02F produced a morphogenesis yield of 124 virions per host cell, phage PsgM04F produced only 12 virions per host cell, indicating that they replicate well in Psg with a 50 min latency period. Genome mechanical analyses established a relationship between genome bendability and virion morphogenesis yield within infected host cells.

Characterization and in vitro testing of newly isolated lytic bacteriophages for the biocontrol of Pseudomonas aeruginosa.

Aim: Two lytic phages were isolated using P. aeruginosa DSM19880 as host and fully characterized. Materials & methods: Phages were characterized physicochemically, biologically and genomically. Results & conclusion: Host range analysis revealed that the phages also infect some multidrug-resistant (MDR) P. aeruginosa clinical isolates. Increasing MOI from 1 to 1000 significantly increased phage efficiency and retarded bacteria regrowth, but phage ph0034 (reduction of 7.5 log CFU/ml) was more effective than phage ph0031 (reduction of 5.1 log CFU/ml) after 24 h. Both phages belong to Myoviridae family. Genome sequencing of phages ph0031 and ph0034 showed that they do not carry toxin, virulence, antibiotic resistance and integrase genes. The results obtained are highly relevant in the actual context of bacterial resistance to antibiotics.

The complete genome and comparative analysis of the phage phiC120 infecting multidrug-resistant Escherichia coli and Salmonella strains.

Phages infecting Salmonella and Escherichia coli are promising agents for therapeutics and biological control of these foodborne pathogens, in particular those strains with resistance to several antibiotics. In an effort to assess the potential of the phage phiC120, a virulent phage isolated from horse feces in Mexico, we characterized its morphology, host range and complete genome. Herein, we showed that phiC120 possesses strong lytic activity against several multidrug-resistant E. coli O157: H7 and Salmonella strains, and its morphology indicated that is a member of Myoviridae family. The phiC120 genome is double-stranded DNA and consists of 186,570 bp in length with a 37.6% G + C content. A total of 281 putative open reading frames (ORFs) and two tRNAs were found, where 150 ORFs encoded hypothetical proteins with unknown function. Comparative analysis showed that phiC120 shared high similarity at nucleotide and protein levels with coliphages RB69 and phiE142. Detailed phiC120 analysis revealed that ORF 94 encodes a putative depolymerase, meanwhile genes encoding factors associated with lysogeny, toxins, and antibiotic resistance were absent; however, ORF 95 encodes a putative protein with potential allergenic and pro-inflammatory properties, making needed further studies to guarantee the safety of phiC120 for human use. The characterization of phiC120 expands our knowledge about the biology of coliphages and provides novel insights supporting its potential for the development of phage-based applications to control unwanted bacteria.

Two Phages of the Genera Felixounavirus Subjected to 12 Hour Challenge on Salmonella Infantis Showed Distinct Genotypic and Phenotypic Changes.

Salmonella Infantis is considered in recent years an emerging Salmonella serovar, as it has been associated with several outbreaks and multidrug resistance phenotypes. Phages appear as a possible alternative strategy to control Salmonella Infantis (SI). The aims of this work were to characterize two phages of the Felixounavirus genus, isolated using the same strain of SI, and to expose them to interact in challenge assays to identify genetic and phenotypic changes generated from these interactions. These two phages have a shared nucleotide identity of 97% and are differentiated by their host range: one phage has a wide host range (lysing 14 serovars), and the other has a narrow host range (lysing 6 serovars). During the 12 h challenge we compared: (1) optical density of SI, (2) proportion of SI survivors from phage-infected cultures, and (3) phage titer. Isolates obtained through the assays were evaluated by efficiency of plating (EOP) and by host-range characterization. Genomic modifications were characterized by evaluation of single nucleotide polymorphisms (SNPs). The optical density (600 nm) of phage-infected SI decreased, as compared to the uninfected control, by an average of 0.7 for SI infected with the wide-host-range (WHR) phage and by 0.3 for SI infected with the narrow-host-range (NHR) phage. WHR phage reached higher phage titer (7 × 1011 PFU/mL), and a lower proportion of SI survivor was obtained from the challenge assay. In SI that interacted with phages, we identified SNPs in two genes (rfaK and rfaB), which are both involved in lipopolysaccharide (LPS) polymerization. Therefore, mutations that could impact potential phage receptors on the host surface were selected by lytic phage exposure. This work demonstrates that the interaction of Salmonella phages (WHR and NHR) with SI for 12 h in vitro leads to emergence of new phenotypic and genotypic traits in both phage and host. This information is crucial for the rational design of phage-based control strategies.

Biological and molecular characterization of a bacteriophage infecting Xanthomonas campestris pv. campestris, isolated from brassica fields.

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot of crucifers. Here, we report a virus that infects Xcc isolated from brassica fields in Brazil. Morphological, molecular and phylogenetic analysis indicated that the isolated virus is a new member of the genus Pbunavirus, family Myoviridae, and we propose the name "Xanthomonas virus XC 2" for this virus. The isolated virus has a narrow host range, infecting only Xcc isolates, and it did not infect unrelated bacteria. These results indicate that the isolated bacteriophage is highly specific for Xcc and may be a potential agent for biological control.

Broad-range lytic bacteriophages that kill Staphylococcus aureus local field strains.

Staphylococcus aureus is a very successful opportunistic pathogen capable of causing a variety of diseases ranging from mild skin infections to life-threatening sepsis, meningitis and pneumonia. Its ability to display numerous virulence mechanisms matches its skill to display resistance to several antibiotics, including ß-lactams, underscoring the fact that new anti-S. aureus drugs are urgently required. In this scenario, the utilization of lytic bacteriophages that kill bacteria in a genus -or even species- specific way, has become an attractive field of study. In this report, we describe the isolation, characterization and sequencing of phages capable of killing S. aureus including methicillin resistant (MRSA) and multi-drug resistant S. aureus local strains from environmental, animal and human origin. Genome sequencing and bio-informatics analysis showed the absence of genes encoding virulence factors, toxins or antibiotic resistance determinants. Of note, there was a high similarity between our set of phages to others described in the literature such as phage K. Considering that reported phages were obtained in different continents, it seems plausible that there is a commonality of genetic features that are needed for optimum, broad host range anti-staphylococcal activity of these related phages. Importantly, the high activity and broad host range of one of our phages underscores its promising value to control the presence of S. aureus in fomites, industry and hospital environments and eventually on animal and human skin. The development of a cocktail of the reported lytic phages active against S. aureus-currently under way- is thus, a sensible strategy against this pathogen.

Characterization of a Vibrio cholerae phage isolated from the coastal water of Peru.

A Vibrio cholerae bacteriophage, family Myoviridae, was isolated from seawater collected from the coastal water of Lima, Peru. Genome size was estimated to be 29 kbp. The temperate phage was specific to V. cholerae and infected 12/13 V. cholerae O1 strains and half of the four non-O1/non-O139 strains tested in this study. Vibrio cholerae O139 strains were resistant to infection and highest infection rates were obtained in low nutrient media amended with NaCl or prepared using seawater as diluent.