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Kok et al. Microbiome Res Rep 2023;2:30 https://dx.doi.org/10.20517/mrr.2023.16 Page 11 of 19

Figure 5. Host range of Paenibacillus larvae (P. larvae) phages on P. larvae bacterial isolates from New Zealand. Grey boxes indicate cell
lysis and white boxes indicate no cell lysis has occurred. NB: In some instances, spot clearing was observed, but plaques were not. This
is explicit in Supplementary Figure 2.

None of the phages were capable of lysing all 30 bacterial isolates, but they were able to lyse between 57% to
87%. Bacterial isolatets Pl-P1627 and W19_08094, both isolated from the Otago region, are not lysed by any
of the phages found in New Zealand to date. Pl-P1627 belongs to a different multilocus sequence type than
the other seven bacterial strains identified and sequenced in this paper, as well as having 12 unique spacer
sequences within its CRISPR arrays.

Phages Dash, Lilo, and Callan can infect P. larvae strains W19_08078, W19_08082, W19_08091,
PFR-Pl-2006, W19_07957, and W19_08023 which are not lysed by any other phage. W19_08099 and
W19_08100 are not able to be infected by these three phages, which otherwise infect all non-resistant
bacteria. P. larvae isolates Pl-F1A, Pl-F2B, Pl-WAI, Pl-2017, Pl-TP, Pl-CHCH, W19_07823, and W19_07831
are lysed by all phages in this study. P. larvae isolate W19_08100 is lysed by nine of the phages. Phages
TonyLawson77, Bob, and Rosalind have the smallest ranges of infectivity and are only capable of lysing 57%
of the strains.

Cocktail formulation and in vitro testing
Initially, four cocktails were formulated based on the host range of the phages to ensure coverage of as many
bacterial strains as possible [Table 5]. We note that at this time, the phage genome sequences were not yet
known. The incidence of American foulbrood is low and must be reported by law in New Zealand; the
likelihood of a colony being infected with P. larvae in New Zealand is 0.0032 , so the chance of more than
[55]
one strain infecting a single hive in New Zealand is extremely low (~0.00001024). Our phage cocktail design,
therefore, focused on covering the breadth of strains that could infect a colony [Table 4].