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Kok et al. Microbiome Res Rep 2023;2:30 https://dx.doi.org/10.20517/mrr.2023.16 Page 15 of 19

as phenotypically different. A more thorough analysis of these genomes and their relationships to the global
P. larvae phages is in preparation.

Host range experiments revealed nine distinct infection patterns, which included a subset of six bacterial
strains that were only able to be infected by three phages and two recalcitrant bacterial strains that were not
infected by any of the phages discovered in New Zealand to date. Overall, there was a 93% host range
coverage for our collection of P. larvae phages. In similar host-range experiments with P. larvae phages,
Yost et al. found a 100% host-range coverage when testing 29 phages on 11 P. larvae bacterial strains . In
[17]
another experiment, Brady et al. tested 39 P. larvae specific phages on 59 bacterial strains and also found a
100% host range coverage . Efforts are ongoing to find phages that can lyse the final resistant strains that
[18]
are present in New Zealand. We do not currently know if phages found overseas have the ability to lyse the
resistant P. larvae strains.

Four cocktails were formulated and tested against each of the four P. larvae isolates before the genomes
were known. Cocktails One and Three both contained Phage Callan and Cocktails Two and Four both
contained Phage Dash, as these were two of the phages that were able to infect six bacterial strains resistant
to all other phages.

In vitro testing using these four cocktails varied, with two cocktails standing out as the most effective at
killing three of the bacterial strains. One P. larvae strain, Pl-P1627, was completely resistant to all cocktails.
This was to be expected as this strain was not infected by any of our individual phages. In this limited
instance, we did not see any evidence of emergent infectivity above and beyond that of the individual
phages present in the cocktail.

In our study, cocktails Two and Four were more effective than cocktails One and Three against P. larvae
strains Pl-2006, Pl-2017 and W19-08100. In New Zealand, the likelihood of P. larvae infections is 0.0032 .
[55]
Therefore, an ideal cocktail should be highly effective against each of the prevalent P. larvae strains, but it
need not be effective against infection by multiple strains simultaneously at the level of a hive.

Interestingly, cocktails One and Four had a predicted Breadth of activity of 50% (2/4 phages were able to
1
infect at least 2/4 bacterial strains), while cocktails Two and Three had a 75% Breadth of activity. This
1
suggests that the quality of the phage cocktails tested here cannot be attributed to the Breadth of activity
1
alone. The cause of the difference in outcomes of these phage cocktails remains to be investigated.
We observed host-dependent phage antagonism in our cocktails. When phage Callan was used in place of
phage Dash on P. larvae W19-08100. This was surprising because phage Callan is not able to form plaques
on this host. There are at least two possible explanations for this phenomenon. It may imply that the effect
of the presence of Callan in the cocktail is the result of a direct host response preventing cocktail members
from infection as a result of phage Callan DNA in the cytoplasm. Another possible mechanism is that the
phage Callan lysate contains some effector which changes the susceptibility of this host to other phage
cocktail members. Several studies have shown that phages within phage cocktails can have an antagonistic
relationship. Forti et al found combining six phages into a cocktail to lyse Pseudomonas aeruginosa showed
a lower host range than what had been predicted based on individual phage host ranges . Another study
[71]
testing different phage cocktails on Escherichia coli O157 showed that not all combinations of phages were
as effective as others and phage antagonism was common in certain cocktails . Our results suggest that
[72]
there can be both host dependence on these antagonistic effects and that non-plaque forming phages can
induce host resistance to infection. These preliminary results warrant further investigation.