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Page 14 of 19 Kok et al. Microbiome Res Rep 2023;2:30 https://dx.doi.org/10.20517/mrr.2023.16

Each strain contained four to five CRISPR arrays with a total of 15 to 25 spacers. P. larvae strains isolated
previously have been found to contain CRISPR arrays as phage defense mechanisms. P. larvae ERIC I
[57]
strains ATCC 9545 and DSM 7030 contained four CRISPR arrays with 17 spacers .

We also used PHASTER to evaluate the sequenced contigs for prophages, and we found each isolate had
one or two intact prophages. In another study, P. larvae ERIC type I strains DSM 25719, MEX14,
[58]
ATCC 9545, and DSM 7030 contained eight, three, five, and five intact prophages, respectively . ATCC
9545 contained prophages similar to the prophages found in our P. larvae isolates.

Seven anti-phage systems were also discovered within our eight bacterial strains. These data suggested to us
that these isolates are encountering an active population of phages in nature and are maintaining a suite of
defense systems to counter infection when they meet. This is common as previous reports suggest that 50%
of bacteria have CRISPR systems and other defense mechanisms such as restriction-modification systems
[59]
[60]
are widely found within prokaryotes .
The ApiWellbeing project, an initiative of the New Zealand Ministry for Primary Industries, generously
gifted us with 22 additional P. larvae isolates from their own recent collection efforts, which brought our
[38]
collection of hosts to 30 . The sequencing and annotation of this collection are underway and will provide
a valuable asset in the future.

[61]
Since the discovery of the first P. larvae phage in 1953 , 69 P. larvae-specific phages have been
found [49,62-69] . Due to the strict biosecurity laws in New Zealand, it is unlikely that non-native phages would
be permitted in the apiculture industry here. We therefore sought to discover a suite of native New Zealand
P. larvae phages to combat AFB.

Previous hunts for P. larvae phages have included samples from soil, bee debris, cosmetics, and bee
wax [62,65,69] . A large-scale hunt across New Zealand was a daunting task for our small team; we, therefore,
approached beekeepers from around New Zealand and received 430 samples of bee debris and soil from
both the North and South Islands. These types of community science phage hunts have been used
previously and there are ongoing community science projects to isolate new phages for
[70]
Pseudomonas aeruginosa . These samples were processed and led to the discovery of 26 independent
phages. Unlike similar efforts overseas [17,62] in which phages have been isolated from infected hives, the
phages discovered herein were reported to have been isolated only from hive material or soil associated with
[62]
healthy hives .
The phage genomes were sequenced to completion, their genes were identified and annotated, and the
genomes were made available publicly [Table 4]. Sequencing and annotation of phage genomes is
particularly important for identifying gene functions that would make the phages unsuitable or unsafe for
therapeutic use. In this study, we found that two phages, Dash and Lilo, contain a dangerous toxin that
confers virulence to P. larvae, thereby ruling out these two phages for future therapy applications.
Unfortunately, at the time the cocktail testing was carried out, these phages had not yet been sequenced, and
the presence of this toxin was therefore unknown.

To determine whether our phage genomes were distinct, we used criteria previously described by
Stamereilers et al. . Phages are usually phenotypically identical if they have an ANI greater than 99.975%.
[19]
We had several groups of phages that had ANI greater than this cut-off value. However, further analysis
showed they all contained at least one amino acid difference, so in these instances, the phages were classed