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Kok et al. Microbiome Res Rep 2023;2:30 https://dx.doi.org/10.20517/mrr.2023.16 Page 3 of 19

Honeybees are primarily kept in New Zealand for the production of high-value honey, like mānuka
(Leptospermum scoparium) and rātā (Metrosideros robusta), not for their pollination services. As a result,
[11]
honeybee colonies are often located in rough terrain or on private property . New Zealand is also roughly
the size of Great Britain or Japan, so sampling from across this geographic space was a technical challenge.
Accessing honeybee colonies and their detritus to search for phages would have been extremely onerous for
our science team. We therefore chose to use a community science approach to phage discovery. This
approach simplified our sampling regime, but more importantly, it allowed us to begin to communicate the
value of our project directly to the beekeepers through an infographic, face-to-face interactions, and
speaking at local and national stakeholder meetings.

Herein, we describe how we developed a collection of novel P. larvae bacterial isolates and used these to
discover P. larvae phages native to New Zealand. We briefly describe their genome sequences, report phage
host ranges, and design phage cocktails in addition to performing in vitro testing of several phage cocktails.
This work forms the groundwork to develop an approach to protecting beehives using New Zealand native
phages that can be applied to protect hives against infection by a devastating bacterial pathogen that is
affecting this industry globally.

MATERIALS AND METHODS
Isolation of Paenibacillus larvae
[21]
P. larvae was isolated by swabbing suspected brood frames and wiping swabs on MYPGP with Nalidixic
o
acid (10 μg/mL) and Pipemidic acid (10 μg/mL) plates. Plates were incubated at 37 C for 3-5 days until
colonies had formed. Single colonies were picked and purified by single colony isolation on another
o
MYPGP plate. Subsequently single colonies were picked and grown in liquid MYPGP for 48 h at 37 C and
shaken at 100 rpm, then frozen at -80 C.
o

Sporulation of Paenibacillus larvae for microscopy
To produce bacterial spores for microscopy, a 10-fold dilution series of P. larvae PFR-Pl-2006 bacterial
culture was spread onto several MYPGP agar plates. Plates were incubated at 37 C for 6-7 days and plates
o
exhibiting individual colonies were selected. After incubation, spores were removed from the plates by
washing with 5 mL cold sterile water. Water was added to the plate, and the surface of the plate was gently
scraped with a sterile inoculation loop to loosen spores. Water and spores were then removed from the
plates using a syringe and transferred into Eppendorf tubes. The spore suspension was concentrated via
o
centrifugation (12,000 × g, 15 min, 4 C). After centrifugation, the supernatant was discarded, and the spore
pellet was resuspended in 1 mL of cold water. This step was repeated three times. The final spore pellets
o [22]
from all tubes were resuspended in a total volume of 2 mL cold water. Spores were stored at 4 C .

Bacterial DNA extraction and 16S rRNA PCR
Bacterial DNA was extracted from overnight cultures in mBHI (Oxoid CM1135B) broth using the
commercially available Promega Wizard Genomic DNA Purification kit (www.promega.com/protocols/).
The protocol for gram-positive bacteria was followed. A PCR mix was prepared with each tube containing a
final volume of 50 μL. The amplification conditions were 95 C (3 min) followed by 30 cycles of 93 C
o
o
o
(1 min), 55 C (30 s), and 72 C (1 min); and a final cycle of 72 C for 5 min. PCR products were visualized
o
o
on a 1% agarose gels run at 120 volts for 30 min.