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Page 16 of 19 Kok et al. Microbiome Res Rep 2023;2:30 https://dx.doi.org/10.20517/mrr.2023.16

These results, taken together with previous studies, show the importance of testing a variety of phage
cocktails to find the most effective combination of phages, regardless of their individual host ranges. All
[49]
P. larvae phages described to date have genomic features which suggest they are temperate . Despite this
observation, Brady et al. produced a phage cocktail containing three P. larvae phages and tested it on
beehives at risk of AFB infection. This cocktail was able to protect the beehives for at least four months
[18]
post-application . These results indicate that even if a temperate lifecycle is genomically indicated, phages
can work effectively as a protective measure against AFB. Our future work will, however, include extensive
in vitro and field trials to confirm that there are no unintended negative consequences to the use of the
phages. In addition, we plan to investigate adaptive laboratory evolution as a method for selecting phages
that have lost the capacity for temperate life cycles.

This study shows promising results and forms the beginning of the work needed to find a solution to
prophylactically protect honey bees in New Zealand from the destructive disease known as AFB. Further
work will need to be undertaken to completely understand the characteristics of the phages, their
persistence in the environment, cross-resistance of P. larvae strains to phages, and appropriate delivery
mechanisms. In order to bring about a prophylactic solution for beekeepers, we will also need to undertake
in vitro testing on honeybee larvae and large-scale field trials . This work has been made possible by the
[11]
collective efforts of the beekeepers of New Zealand, and we will continue to honour their contributions by
pursuing this project further.

DECLARATIONS
Acknowledgments
We wish to thank the many beekeepers in New Zealand who contributed soil and bee debris samples which
made this work possible. We thank Richard Hall and Hayley Pragert from the ApiWellBeing project, and
the many helpful people from both AsureQuality, Apiculture NZ, and the National American Foulbrood
Pest Management Plan for support of this project. Last but not least, our sincerest thanks to Barry Foster for
his support and kindness.

Authors’ contributions
Conceptualization: Hendrickson HL, Kok DN
Methodology: Kok DN
Formal analysis: Kok DN
Investigation: Kok DN, Zhou D
Writing - original draft preparation: Kok DN
Writing - review and editing: Kok DN, Zhou D, Tsourkas PK, Hendrickson HL
Visualization: Kok DN
Supervision: Hendrickson HL
Project administration: Hendrickson HL
Funding acquisition: Hendrickson HL

Availability of data and materials
The authors confirm that the genomes used for this work are publicly available, and their accession
numbers are listed in Tables 1 and 4.

Financial support and sponsorship
This research was funded by AGMARDT, grant number AIGITINQ-000301, The Sustainable Food & Fibre
Futures Fund, grant number 405604, and Apiculture NZs Honey Trust.