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Page 2 of 21 O’Connell et al. Microbiome Res Rep 2023;2:21 https://dx.doi.org/10.20517/mrr.2023.17

Conclusion: The link between genus and subcluster classifications appears robust, as most subclusters can be
assigned a single genus and vice versa. By relating the taxonomic and clustering classification systems, they can be
easily kept up to date to best reflect MP diversity, which could aid the rapid selection of related (or diverse) phages
for research, therapeutic and diagnostic purposes.

Keywords: Mycobacteriophage, taxonomy, ICTV, actinobacteriophage database, novel genera, novel subclusters

INTRODUCTION
The frequency of antibiotic resistance (AR) is increasing at a worrying rate. There has been an increase in
[1,2]
resistant nontuberculosis mycobacteria (NTM) infections, demonstrating varying levels of AR . Although
NTM are typically opportunistic pathogens, treatment failure may lead to stubborn colonisation . For
[2]
example, Mycobacterium avium sbsp. paratuberculosis (MAP) causes chronic gastroenteritis (i.e., Johne’s
disease) in ruminant animals . In order to address the challenges of AR-related mycobacterial infections,
[3]
alternatives to traditional antibiotic regimens are actively being explored. One alternative is
mycobacteriophage (MP) therapy . Recently, human case studies involving effective MP therapy were
[4]
described, the first being the successful treatment of a young cystic fibrosis patient with a chronic AR
Mycobacterium abscessuss pulmonary infection . As the number of isolated and sequenced phages
[5,6]
increases, greater opportunity to create robust MP therapy will arise, either through identifying or
engineering phages capable of infecting NTM.

As the genomics era progresses, sequencing and annotation of phage genomes have become as routine and
vital as phenotypic characterisation (e.g., host range, burst size, and adsorption assays). The ever-increasing
volume of available genomic information and sequence analysis software allows for more in-depth in silico
analyses that may provide valuable insights regarding the potential functionality and taxonomy of phages, as
suggested by Lawrence et al. . By exploring the genomic data further, it may be possible to identify other
[7]
characteristics (e.g., host range, pH tolerance, heat tolerance) shared by phages belonging to the same
taxonomic groups (e.g., those belonging to the same genus, subfamily, or family group) that may greatly aid
the design of phage therapies and diagnostics. However, assumptions made while characterising a new
[8,9]
isolate based on their supposed taxonomy can only be trusted if phage taxonomy is well maintained .

The latest software to be introduced for identifying phage taxonomic relationships is VIRIDIC, which
calculates intergenomic similarities between viral genomes in a pairwise manner, as well as their length ratio
and the aligned genome fraction . The output of the algorithm includes a hierarchical heatmap of the
[10]
similarity scores, which places the most similar genomes together. This heatmap is accompanied by a cluster
table that indicates genus- and species-level relationships based on pre-set thresholds of genomic
[10]
similarity . The default settings of VIRIDIC are set to identify genome groups based on the latest
taxonomic demarcations, i.e., genus threshold of ≥ 70% and species threshold of ≥ 95% [10,11] . During its
development, it was noted that VIRIDIC produced results that most closely supported those of the
traditional BLASTN algorithm, while outperforming other bioinformatic tools with regard to estimating the
relatedness between more distally related genomes . The use of VIRIDIC to identify unknown or outdated
[10]
phage taxonomy has become commonplace (e.g., classifying phages targeting Pseudomonas, Salmonella,
Vibrio, and Bacillus) since its development in 2020, as systems move toward sequence-based classifications,
as predicted by Lawrence et al. in 2002 [7,10,12-17] . With this in mind, the existing taxonomy of publicly available
MP was interrogated to determine whether the current classifications remain accurate or require revisions.
Thus far, global efforts have isolated almost 12,000 MP, and over 2,100 have been fully sequenced, largely as
part of the SEA-PHAGES program. The program initially involved undergrad students undertaking massive