Page 63 - Read Online

Kok et al. Microbiome Res Rep 2023;2:30 https://dx.doi.org/10.20517/mrr.2023.16 Page 5 of 19

Figure 1. Paenibacillus larvae bacterial strains (A) Locations of the eight isolated Paenibacillus larvae (P. larvae) bacterial strains;
(B) Vegetative form of P. larvae PFR-Pl-2006; (C) Spore form of P. larvae PFR-Pl-2006. Scale bar: 5 μm.

In vitro cocktail assays
Phage titers were normalized to 1 × 10 PFU/mL and 50 μL of each phage selected was combined into a
8
8
o
cocktail. Bacterial cultures were grown in BHI for 48 h at 37 C to ~1 × 10 CFU/mL. The bacteria were
serially diluted up to a 10 dilution and 20 μL was aliquoted into 96-well plates containing 90 μL 2 × BHI
-6
3
and 90 μL BHI. The phage cocktails were also serially diluted such that each row contained from 10 to 10
7
PFU total phage. 20 μL of the phage cocktail was added to each well of the plate. Plates were incubated,
shaking at 37 C for 24 h. Aliquots of 3 μL were spotted onto BHI plates and incubated for three to four days
o
o
at 37 C to observe CFU.
RESULTS
Isolating P. larvae from infected colony material
Previous work suggested that a curated collection of P. larvae isolates from New Zealand had been
characterized . Further investigation revealed that the existing collection had been destroyed (P. Lester
[20]
private communication). Therefore, a new collection of representative P. larvae strains was needed.
AsureQuality, a New Zealand government-approved testing facility, provided us with swabs of brood
frames or infected larvae material and whole brood frames from beehives suspected of AFB infection.
Potential isolates were cultured on semi-selective MYPGP agar plates in order to obtain single colonies.
Ultimately, eight P. larvae strains were isolated from around New Zealand, with each isolate coming from a
different location [Figure 1A and Table 1]. P. larvae is a filamentous (2.5-5 μm by 0.5-0.8 μm), spore-
forming, gram-positive bacterium [Figure 1B and C] . Isolates were confirmed to be P. larvae by positive
[6]
amplification with 16S rRNA PCR primers .
[23]