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Page 12 of 19 Kok et al. Microbiome Res Rep 2023;2:30 https://dx.doi.org/10.20517/mrr.2023.16

Table 5. The phages contained within the four cocktails
Cocktail one Callan Logan ApiWellbeing Freya
Cocktail two Dash AJG77 UtuhinaGold_ NHScienceFair
Zacery
Cocktail three Callan Ted FutureBee BarryFoster_
Benicio
Cocktail four Dash LunBun Carlos Bloomfield

The initial set of cocktails, One to Four, were capable of lysing 93% of all P. larvae strains in our collection
(28/30). Ultimately, each cocktail had a 70%-73% breadth of activity across our 30 bacterial isolates. The
breadth of activity is calculated as the susceptibility of a pathogen to at least two phages in the cocktail .
[56]
Higher breadth is an indication of the cocktail's ability to mitigate phage resistance in the pathogen.

Once the four cocktails were decided upon, they were tested against four bacterial strains chosen to
represent the types of P. larvae present in New Zealand; the bacterial strains selected were Pl-2017, Pl-2006,
W19-08100, and Pl-P1627 [Figure 6A]. P. larvae Pl-P1627 was chosen as a negative control as it is not
infected by the P. larvae phages in our collection to date.

All four cocktails showed good activity on strain Pl-2017, with cocktails Two and Four showing slightly
better lysis potential. Cocktails Two and Four were very effective on strain Pl-2006, while cocktails One and
Three only showed adequate lysis at the highest concentration of phages. On strain W19-08100, cocktails
One, Two and Four all showed some lysis, while cocktail Three showed very little lysis potential on the
susceptible P. larvae isolates. P. larvae Pl-P1627 was not infected by the cockails.

Ultimately, we sequenced the phage genomes and found that despite their excellent activity, phage cocktails
Two and Four contained phage Dash, a phage with a large Plx1 toxin encoded in the genome. The host
range of phage Dash was, however, very similar to that of phage Callan. These two Harrison cluster phages
shared 91% of their genes and Callan does not contain the Plx1 toxin. We therefore tested the capacity of
cocktails Two and Four with phage Callan on the same P. larvae strains [Supplementary Figure 3 and
Figure 6B]. To our surprise, this seemingly small substitution did not retain the activity of the original
cocktails Two and Four. Replacing phage Dash with the safer phage, phage Callan reduced the capacity of
the cocktails to lyse these strains. This phenomenon warrants further investigation and suggests that phage-
phage interactions are coming into play.

DISCUSSION
In this study, we have set out to lay the groundwork for using phages as a prophylactic against the
devastating pathogen P. larvae in the New Zealand apiculture industry. Our work began with the isolation
and preliminary sequencing of a set of eight novel P. larvae strains from across New Zealand. These
P. larvae bacterial strains were directly isolated from bee larvae or beehives with clinical signs of AFB. This
allowed us to start the only collection of P. larvae bacterial strains available in New Zealand at the time.
Previous New Zealand work had suggested that two separate ERIC genotypes (I and II) were present . To
[20]
take a cursory look, these P. larvae isolates were sequenced to between 157 to 219 contigs and were found to
belong to either the 18 or 23 MLST. MLST 18 and MLST 23 are consistent with the ERIC I genotype [36-38] . To
the best of our knowledge, there is no complete genome sequence data suggesting the presence of the ERIC
II genotype in New Zealand today.