The aim of this study was to critically analyze the effects of hydrogen peroxide on growth and survival of bacterial cells in order to prove or disprove its purported role as a main component responsible for the antibacterial activity of honey. Using the sensitive peroxide/peroxidase assay, broth microdilution assay and DNA degradation assays, the quantitative relationships between the content of H2O2 and honey’s antibacterial activity was established. The results showed that: (A) the average H2O2 content in honey was over 900-fold lower than that observed in disinfectants that kills bacteria on contact. (B) A supplementation of bacterial cultures with H2O2 inhibited E. coli and B. subtilis growth in a concentration-dependent manner, with minimal inhibitory concentrations (MIC90) values of 1.25 mM/107 cfu/ml and 2.5 mM/107 cfu/ml for E. coli and B. subtilis, respectively. In contrast, the MIC90 of honey against E. coli correlated with honey H2O2 content of 2.5 mM, and growth inhibition of B. subtilis by honey did not correlate with honey H2O2 levels at all. (C) A supplementation of bacterial cultures with H2O2 caused a concentration-dependent degradation of bacterial DNA, with the minimum DNA degrading concentration occurring at 2.5 mM H2O2. DNA degradation by honey occurred at lower than ≤2.5 mM concentration of honey H2O2 suggested an enhancing effect of other honey components. (D) Honeys with low H2O2 content were unable to cleave DNA but the addition of H2O2 restored this activity. The DNase-like activity was heat-resistant but catalase-sensitive indicating that H2O2 participated in the oxidative DNA damage. We concluded that the honey H2O2 was involved in oxidative damage causing bacterial growth inhibition and DNA degradation, but these effects were modulated by other honey components.

The ability of agriculture to continually provide food to a growing world population is of crucial importance. Bacterial diseases of plants and animals have continually reduced production since the advent of crop cultivation and animal husbandry practices. Antibiotics have been used extensively to mitigate these losses. The rise of antimicrobial resistant (AMR) bacteria, however, together with consumers’ calls for antibiotic-free products, presents problems that threaten sustainable agriculture. Bacteriophages (phages) are proposed as bacterial population control alternatives to antibiotics. Their unique properties make them highly promising but challenging antimicrobials. The use of phages in agriculture also presents a number of unique challenges. This mini-review summarizes recent development and perspectives of phages used as antimicrobial agents in plant and animal agriculture at the farm level. The main pathogens and their adjoining phage therapies are discussed.

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and Podoviridae.Erwinia amylovora, a member of the Enterobacteriaceae, is the causal organism of fire blight, a serious disease of the pome fruit (19-21). The disease-causing organism is currently controlled by the antibiotic streptomycin and pruning. Another control measure became apparent when bacteriophages that were able to infect E. amylovora and an avirulent yellow bacterium commonly found in the orchard ecosystem were discovered (8, 12). The yellow bacterium was subsequently identified as Pantoea agglomerans (ϭErwinia herbicola). Erskine (11) recognized that E. amylovora phages may play an important role in the epidemiology of fire blight and proposed the use of phages released from the yellow saprophytic bacterium (lysogenic antagonists) as biological control agents (11).Richie (16, 17) isolated E. amylovora phages from aerial portions of fire blight-infected trees by using as a host strain E. amylovora 110 Rif (16). The phages, named PEa1 and PEa7, belonged to two distinct groups based on chemical and physical data. Recently, E. amylovora phages were collected from orchards with fire blight symptoms and were characterized by plaque morphology, PCR, restriction fragment polymorphisms, pulse-field gel electrophoresis, and host range studies (18).The objective of this work was to estimate the diversity of bacteriophages collected from orchards in southern Ontario that had ac...

The first complete genome sequence for a myoviridal bacteriophage, ⌽Ea21-4, infecting Erwinia amylovora, Erwinia pyrifoliae, and Pantoea agglomerans strains has been determined. The unique sequence of this terminally redundant, circularly permuted genome is 84,576 bp. The ⌽Ea21-4 genome has a GC content of 43.8% and contains 117 putative protein-coding genes and 26 tRNA genes. ⌽Ea21-4 is the first phage in which a precisely conserved rho-independent terminator has been found dispersed throughout the genome, with 24 copies in all. Also notable in the ⌽Ea21-4 genome are the presence of tRNAs with six-and nine-base anticodon loops, the absence of a small packaging terminase subunit, and the presence of nadV, a principle component of the NAD ؉ salvage pathway, which has been found in only a few phage genomes to date. ⌽Ea21-4 is the first reported Felix O1-like phage genome; 56% of the predicted ⌽Ea21-4 proteins share homology with those of the Salmonella phage. Apart from this similarity to Felix O1, the ⌽Ea21-4 genome appears to be substantially different, both globally and locally, from previously reported sequences. A total of 43 of the 117 genes are unique to ⌽Ea21-4, and 32 of the Felix O1-like genes do not appear in any phage genome sequences other than ⌽Ea21-4 and Felix O1. N-terminal sequencing and matrix-assisted laser desorption ionization-time of flight analysis resulted in the identification of five ⌽Ea21-4 genes coding for virion structural proteins, including the major capsid protein.Members of the Erwinia genus and of the closely related genera Pectobacterium, Pantoea, and Brennaria are responsible for a variety of blight, wilt, and soft rot diseases of plants (19,(22)(23)(24). Bacteriophages, or simply "phages," have been proposed as a means of suppressing the populations of these pathogens and thereby preventing some of these diseases (17,33,53), and yet very little sequence information is available for the phages infecting these genera. Such information is critical to the development of molecular tracking tools that can reveal both the ecological interactions and the environmental fates of phages in a therapeutic context.In the case of Erwinia amylovora, the causative agent of fire blight, many infecting phages have been described, encompassing all three families of Caudovirales, the tailed phages (13,18,33,54,59,74). The diversity of these phages, particularly those described by Gill et al. (18), is extensive, but sequence information is limited to the unpublished genome of Era103 (GenBank accession no. NC_009014) and a 3.3-kb region of the Ea1h genome (26). Both of these phages are members of the Podoviridae (54,74). No sequence data have previously been described for E. amylovora phages belonging to the Siphoviridae or the Myoviridae. While the precise nature of their evolutionary and taxonomic relationships has been complicated by processes such as horizontal gene transfer, there are usually substantial differences among the proteomes of phages in different families (49,56).To address this lack...

Erwinia amylovora bacteriophages (phages) belonging to the Myoviridae and Podoviridae families demonstrated a preference for either high-exopolysaccharide-producing (HEP) or low-exopolysaccharide-producing (LEP) bacterial hosts when grown on artificial medium without or with sugar supplementation. Myoviridae phages produced clear plaques on LEP hosts and turbid plaques on HEP hosts. The reverse preference was demonstrated by most Podoviridae phages, where clear plaques were seen on HEP hosts. Efficiency of plating (EOP) was determined by comparing phage growth on the original isolation host to the that on the LEP or HEP host. Nine of 10 Myoviridae phages showed highest EOPs on LEP hosts, and 8 of 11 Podoviridae phages had highest EOPs on HEP hosts. Increasing the production of EPS on sugar-supplemented medium or decreasing production by knocking out the synthesis of amylovoran or levan, the two EPSs produced by E. amylovora, indicated that these components play crucial roles in phage infection. Amylovoran was virtually essential for proliferation of most Podoviridae phages when phage population growth was compared to the wild type. Decreased levan production resulted in a significant reduction of progeny from phages in the Myoviridae family. Thus, Podoviridae phages are adapted to hosts that produce high levels of exopolysaccharides and are dependent on host-produced amylovoran for pathogenesis. Myoviridae phages are adapted to hosts that produce lower levels of exopolysaccharides and host-produced levan.