Isolation and Characterization of Phages Active against Paenibacillus larvae Causing American Foulbrood in Honeybees in Poland.

The aim of this study was the isolation and characterization, including the phage effect on honeybees in laboratory conditions, of phages active against Paenibacillus larvae, the causative agent of American Foulbrood-a highly infective and easily spreading disease occurring in honeybee larva, and subsequently the development of a preparation to prevent and treat this dangerous disease. From the tested material (over 2500 samples) 35 Paenibacillus spp. strains were obtained and used to search for phages. Five phages specific to Paenibacillus were isolated and characterized (ultrastructure, morphology, biological properties, storage stability, and genome sequence). The characteristics were performed to obtain knowledge of their lytic potential and compose the final phage cocktail with high antibacterial potential and intended use of future field application. Preliminary safety studies have also been carried out on healthy bees, which suggest that the phage preparation administered is harmless.

Characterization of CRISPR Spacer and Protospacer Sequences in Paenibacillus larvae and Its Bacteriophages.

The bacterium Paenibacillus larvae is the causative agent of American foulbrood, the most devastating bacterial disease of honeybees. Because P. larvae is antibiotic resistant, phages that infect it are currently used as alternative treatments. However, the acquisition by P. larvae of CRISPR spacer sequences from the phages could be an obstacle to treatment efforts. We searched nine complete genomes of P. larvae strains and identified 714 CRISPR spacer sequences, of which 384 are unique. Of the four epidemiologically important P. larvae strains, three of these have fewer than 20 spacers, while one strain has over 150 spacers. Of the 384 unique spacers, 18 are found as protospacers in the genomes of 49 currently sequenced P. larvae phages. One P. larvae strain does not have any protospacers found in phages, while another has eight. Protospacer distribution in the phages is uneven, with two phages having up to four protospacers, while a third of phages have none. Some phages lack protospacers found in closely related phages due to point mutations, indicating a possible escape mechanism. This study serve a point of reference for future studies on the CRISPR-Cas system in P. larvae as well as for comparative studies of other phage-host systems.

Paenibacillus larvae bacteriophages: obscure past, promising future.

Paenibacillus larvae is a Gram-positive, spore-forming bacterium that is the causative agent of American foulbrood (AFB), the most devastating bacterial disease of the honeybee. P. larvae is antibiotic resistant, complicating treatment efforts. Bacteriophages that target P. larvae are rapidly emerging as a promising treatment. The first P. larvae phages were isolated in the 1950s, but as P. larvae was not antibiotic resistant at the time, interest in them remained scant. Interest in P. larvae phages has grown rapidly since the first P. larvae phage genome was sequenced in 2013. Since then, the number of sequenced P. larvae phage genomes has reached 48 and is set to grow further. All sequenced P. larvae phages encode a conserved N-acetylmuramoyl-l-alanine amidase that is responsible for cleaving the peptidoglycan cell wall of P. larvae. All P. larvae phages also encode either an integrase, excisionase or Cro/CI, indicating that they are temperate. In the last few years, several studies have been published on using P. larvae phages and the P. larvae phage amidase as treatments for AFB. Studies were conducted on infected larvae in vitro and also on hives in the field. The phages have a prophylactic effect, preventing infection, and also a curative effect, helping resolve infection. P. larvae phages have a narrow range, lysing only P. larvae, and are unable to lyse even related Paenibacillus species. P. larvae phages thus appear to be safe to use and effective as treatment for AFB, and interest in them in the coming years will continue to grow.

Identification of the first endolysin Cell Binding Domain (CBD) targeting Paenibacillus larvae.

Bacteriophage endolysins present enormous biotechnological potentials and have been successfully used to control and detect bacterial pathogens. Endolysins targeting Gram-positive bacteria are modular, displaying a cell binding (CBD) and an enzymatically active domain. The CBD of phage endolysins are recognized by their high specificity and host affinity, characteristics that make them promising diagnostic tools. No CBD able to bind Paenibacillus larvae has been identified so far. P. larvae is a Gram-positive spore forming bacteria that causes the American Foulbrood. This highly contagious infection leads to honeybee larvae sepsis and death, resulting in an adverse impact on pollination and on the beekeeping industry. In this work, the first CBD targeting P. larvae was identified and its core binding sequence was investigated. Moreover, it was shown that the domain is highly specific, targeting exclusively P. larvae cells from all ERIC genotypes. The identification of such a domain represents a step forward for the development of effective methods to detect and control this pathogen.

Bacteriophage biodistribution and infectivity from honeybee to bee larvae using a T7 phage model.

Bacteriophages (phages) or viruses that specifically infect bacteria have widely been studied as biocontrol agents against animal and plant bacterial diseases. They offer many advantages compared to antibiotics. The American Foulbrood (AFB) is a bacterial disease affecting honeybee larvae caused by Paenibacillus larvae. Phages can be very significant in fighting it mostly due to European restrictions to the use of antibiotics in beekeeping. New phages able to control P. larvae in hives have already been reported with satisfactory results. However, the efficacy and feasibility of administering phages indirectly to larvae through their adult workers only by providing phages in bees' feeders has never been evaluated. This strategy is considered herein the most feasible as far as hive management is concerned. This in vivo study investigated the ability of a phage to reach larvae in an infective state after oral administration to honeybees. The screening (by direct PFU count) and quantification (by quantitative PCR) of the phage in bee organs and in larvae after ingestion allowed us to conclude that despite 104 phages reaching larvae only an average of 32 were available to control the spread of the disease. The fast inactivation of many phages in royal jelly could compromise this therapeutic approach. The protection of phages from hive-derived conditions should be thus considered in further developments for AFB treatment.

Characterization of a new podovirus infecting Paenibacillus larvae.

The Paenibacillus larvae infecting phage API480 (vB_PlaP_API480) is the first reported podovirus for this bacterial species, with an 58 nm icosahedral capsid and a 12 × 8 nm short, non-contractile tail. API480 encodes 77 coding sequences (CDSs) on its 45,026 bp dsDNA genome, of which 47 were confirmed using mass spectrometry. This phage has got very limited genomic and proteomic similarity to any other known ones registered in public databases, including P. larvae phages. Comparative genomics indicates API480 is a new species as it's a singleton with 28 unique proteins. Interestingly, the lysis module is highly conserved among P. larvae phages, containing a predicted endolysin and two putative holins. The well kept overall genomic organisation (from the structural and morphogenetic modules to the host lysis, DNA replication and metabolism related proteins) confirms a common evolutionary ancestor among P. larvae infecting phages. API480 is able to infect 69% of the 61 field strains with an ERIC I genotype, as well as ERIC II strains. Furthermore, this phage is very stable when exposed to high glucose concentrations and to larval gastrointestinal conditions. This highly-specific phage, with its broad lytic activity and stability in hive conditions, might potentially be used in the biocontrol of American Foulbrood (AFB).

Genomic Analysis of 48 Paenibacillus larvae Bacteriophages.

The antibiotic-resistant bacterium Paenibacillus larvae is the causative agent of American foulbrood (AFB), currently the most destructive bacterial disease in honeybees. Phages that infect P. larvae were isolated as early as the 1950s, but it is only in recent years that P. larvae phage genomes have been sequenced and annotated. In this study we analyze the genomes of all 48 currently sequenced P. larvae phage genomes and classify them into four clusters and a singleton. The majority of P. larvae phage genomes are in the 38⁻45 kbp range and use the cohesive ends (cos) DNA-packaging strategy, while a minority have genomes in the 50⁻55 kbp range that use the direct terminal repeat (DTR) DNA-packaging strategy. The DTR phages form a distinct cluster, while the cos phages form three clusters and a singleton. Putative functions were identified for about half of all phage proteins. Structural and assembly proteins are located at the front of the genome and tend to be conserved within clusters, whereas regulatory and replication proteins are located in the middle and rear of the genome and are not conserved, even within clusters. All P. larvae phage genomes contain a conserved N-acetylmuramoyl-l-alanine amidase that serves as an endolysin.

Bacteriophages as an alternative to conventional antibiotic use for the prevention or treatment of Paenibacillus larvae in honeybee hives.

American Foulbrood (AFB) is an infectious disease caused by the bacteria, Paenibacillus larvae. P. larvae phages were isolated and tested to determine each phages' host range amongst 59 field isolate strains of P. larvae. Three phages were selected to create a phage cocktail for the treatment of AFB infections according to the combined phages' ability to lyse all tested strains of bacteria. Studies were performed to demonstrate the safety and efficacy of the phage cocktail treatment as a replacement for traditional antibiotics for the prevention of AFB and the treatment of active infections. Safety verification studies confirmed that the phage cocktail did not adversely affect the rate of bee death even when administered as an overdose. In a comparative study of healthy hives, traditional prophylactic antibiotic treatment experienced a 38±0.7% decrease in overall hive health, which was statistically lower than hive health observed in control hives. Hives treated with phage cocktail decreased 19±0.8%, which was not statistically different than control hives, which decreased by 10±1.0%. In a study of beehives at-risk for a natural infection, 100±0.5% of phage-treated hives were protected from AFB infection, while 80±0.5% of untreated controls became infected. AFB infected hives began with an average Hitchcock score of 2.25 out of 4 and 100±0.5% of the hives recovered completely within two weeks of treatment with phage cocktail. While the n numbers for the latter two studies are small, the results for both the phage protection rate and the phage cure rate were statistically significant (α=0.05). These studies demonstrate the powerful potential of using a phage cocktail against AFB and establish phage therapy as a feasible treatment.