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Page 4 of 19 Kok et al. Microbiome Res Rep 2023;2:30 https://dx.doi.org/10.20517/mrr.2023.16

[23]
Primers used were :
AFB-F 5’-CTT-GTG-TTT-CTT-TCG-GGA-GAC-GCC-A-3’
AFB-R 5’-TCT-TAG-AGT-GCC-CAC-CTC-TGC-G-3’

Bacterial DNA sequencing and assembly
DNA was either sent for sequencing at MicrobesNG (Birmingham, UK) or MiGS (Pittsburgh, PA, USA) for
complete genome Illumina sequencing. Sequencing was performed by MicrobesNG by preparing genomic
DNA libraries with Nextera XT Library Prep Kit (Illumina, San Diego, USA). Libraries were then sequenced
on an lllumina NovaSeq 6000 (Illumina, San Diego, USA) using a 250 bp paired-end protocol
(www.microbesng.com). Sequencing was performed by MiGS on the NextSeq 2000 platform (Illumina Inc.,
San Diego, CA, USA) using a paired-end library (www.seqcenter.com). Genomes were assembled using
[29]
SPAdes 3.15.3 [24,25] and then annotated using either RAST 1.073 [26-28] or Prokka 1.14.5 . Average coverage
was 30x with 157-219 contigs assembled.

Processing soil/hive samples for phages
Soil samples were processed as previously described . Only one pass through a 0.45 μm sterile syringe filter
[17]
was performed. The resulting filtrate was used as a starting material for enrichment. Enrichments were a
combination of 1 mL of starting material, 100 μL of each of eight P. larvae bacterial isolates, 8 mL mBHI and
o
0.4% glucose. These were incubated for 48 h at 37 C, shaken at 100 rpm. After 48 h, enrichments were
centrifuged at 3,200 g for 15 min, and filter sterilized to 0.45 μm. The resulting supernatants were assayed
for phage presence by 3 μL spots on double-layer agar containing one of the P. larvae bacterial isolates.

Phage plaque purification
Phages underwent three rounds of purification. Plaques were picked off a double-agar plate using a 200 μL
pipette tip; the tip was put in 100 μL of BHI and pipetted up and down to remove phage particles. This
lysate was used to inoculate the next double-agar plate.

Creation of lysates
To create phage lysates, 10 plaque plates with the highest number of individual plaques were flooded with
8 mL of BHI. Plates were left to sit at room temperature for 2 h. At the end of 2 h, plates were swirled, and
the lysate was removed. Lysates were filtered with a 0.45 μm filter and pooled in a 50 mL falcon tube. Titers
[30]
were increased using the Rapid Adaptive Mutation of Phage RAMP-UP protocol .
Phage DNA extraction and sequencing
Phage DNA was extracted using a modified zinc chloride precipitation method . Modifications included
[31]
the addition of 1 µL Proteinase K (20 mg/mL), incubated at 37 °C for 10 min after the resuspension in TES
buffer (0.1M Tris-HCl, pH 8, 0.1M EDTA, 0.3% SDS) step. Tubes were left overnight on ice after
isopropanol was added. 1 µL of pure glycogen was added to each tube at the beginning of Day 2 before the
centrifugation step to aid in pelleting of DNA. DNA pellets were resuspended in 50 µL nuclease-free water.

Phage genomes were sequenced and annotated as previously described . Briefly, phage genomes were
[30]
assembled using Geneious 9.05 (Auckland, New Zealand) (https://www.geneious.com); assembled genomes
[32]
were then run through Phage Commander to identify all genes. Genomes were manually checked using
DNA Master and as previously described .
[33]
[34]
Host range testing
The ability of phages to infect each isolate was assessed by 3 μL spots of each phage lysate on double-layer
agar containing 500 μL of bacterial lawn. Each P. larvae bacterial isolate was tested separately. The majority
of spot tests showed the presence of individual plaques owing to low phage titers during this testing.