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Page 6 of 19 Kok et al. Microbiome Res Rep 2023;2:30 https://dx.doi.org/10.20517/mrr.2023.16

Table 1. Paenibacillus larvae bacterial strains isolated from New Zealand
No. of Size range contigs
P. larvae strain Isolation location MLST ST GC% Accession No.
contigs (Kbp)
Pl-WAI Wellington 18 44.2% 219 0.128-218 JARDRH000000000
Pl-TP Rotorua 18 44.1% 157 0.128-250 JARDRJ000000000
Pl-CHCH Christchurch 18 44.1% 163 0.128-218 JARDRI000000000
PFR-Pl-2017 Auckland 18 44.1% 176 0.128-218 JARDRG000000000
PFR-Pl-2006 Hamilton 18 44.1% 185 0.128-218 JARDAI000000000
Pl-F1A North Canterbury 18 44.1% 175 0.5-191 JARDRL000000000
Pl-F2B South Canterbury 18 44.0% 167 0.5-191 JARDRM000000000
Pl-P1627 Queenstown 23 44.1% 171 0.5-195 JARDRK000000000

P. Larvae: Paenibacillus larvae.

DNA was extracted from each of the bacterial isolates and submitted for genome sequencing. Genomes
were assembled using SPAdes 3.15.3 [24,25] and then annotated using either RAST 1.073 [26-28] or Prokka
1.14.5 . The resulting genome assemblies had a range of 157 to 219 contigs, with sizes varying from 0.12 to
[29]
218 Kbp. These P. larvae strains had GC contents of 44.0% to 44.2% [Table 1].

Multilocus sequence typing (MLST) was undertaken using PubMLST . MLST for P. larvae consists of the
[35]
following seven housekeeping genes: ftsA (cell division protein), clpC (catabolite control protein A), glpT
(glycerol-3-phosphate permease), glpF (glycerol uptake facilitator protein), rpoB (RNA polymerase beta
subunit), Natrans (forward sodium dependant transporter), and sigF (sporulation sigma factor F) as these
[36]
offered the most diversity between genomes tested . Seven of the New Zealand isolates belonged to the 18
MLST ST and one belonged to 23 MLST ST [Table 1]. MLST can also be used to distinguish between the
ERIC I genotype and the ERIC II genotype. MLST 18 and MLST 23 both belong to the ERIC I genotype [36-38] .

We used CRISPRFinder to look for detectable CRISPR systems in these eight isolates . Seven of the isolates
[39]
contained four CRISPR arrays and one isolate contained five. The total number of spacers within the
CRISPR arrays for each isolate varied from 15-25 spacers [Table 2]. Across all eight isolates, 29 unique
spacers were observed, Pl-P1627 contained 12 unique spacers that were not found in any of the other
isolates.

We also used DefenseFinder [40,41] to search for known anti-phage systems in our bacterial strains. All eight
isolates contained the same seven anti-phage systems: both a type I and II restriction-modification
system , a Gao_let system , two Cas systems (CAS_Class1-Subtype-III-B and CAS_Class1-Subtype-I-
[42]
[43]
[45]
[44]
B) , a Wadjet_III system , and a Mokosh_TypeII system .
[46]
Finally, we used Phaster [47,48] to identify prophages contained within the genomes. Phaster designates
prophages as either intact, questionable or incomplete by comparing them to a NCBI database of complete
viral genomes. Potential prophage regions are then given a completeness score; this score is calculated on
the proportion of phage genes in the identified region. An intact prophage has a score > 90, a questionable
prophage has a score between 70-90, and an incomplete prophage has a score < 70. All isolates contained at
least one intact prophage, with six containing two intact prophages. The intact prophages were genomically
[49]
similar to P. larvae Phage Harrison and Phage Vegas . All genomes also contained 3-4 questionable
prophages and 6-9 incomplete prophages [Table 3].