DEPhT: a novel approach for efficient prophage discovery and precise extraction.

Advances in genome sequencing have produced hundreds of thousands of bacterial genome sequences, many of which have integrated prophages derived from temperate bacteriophages. These prophages play key roles by influencing bacterial metabolism, pathogenicity, antibiotic resistance, and defense against viral attack. However, they vary considerably even among related bacterial strains, and they are challenging to identify computationally and to extract precisely for comparative genomic analyses. Here, we describe DEPhT, a multimodal tool for prophage discovery and extraction. It has three run modes that facilitate rapid screening of large numbers of bacterial genomes, precise extraction of prophage sequences, and prophage annotation. DEPhT uses genomic architectural features that discriminate between phage and bacterial sequences for efficient prophage discovery, and targeted homology searches for precise prophage extraction. DEPhT is designed for prophage discovery in Mycobacterium genomes but can be adapted broadly to other bacteria. We deploy DEPhT to demonstrate that prophages are prevalent in Mycobacterium strains but are absent not only from the few well-characterized Mycobacterium tuberculosis strains, but also are absent from all ∼30 000 sequenced M. tuberculosis strains.

A Bioinformatic Ecosystem for Bacteriophage Genomics: PhaMMSeqs, Phamerator, pdm_utils, PhagesDB, DEPhT, and PhamClust.

The last thirty years have seen a meteoric rise in the number of sequenced bacteriophage genomes, spurred on by both the rise and success of groups working to isolate and characterize phages, and the rapid and significant technological improvements and reduced costs associated with sequencing their genomes. Over the course of these decades, the tools used to glean evolutionary insights from these sequences have grown more complex and sophisticated, and we describe here the suite of computational and bioinformatic tools used extensively by the integrated research-education communities such as SEA-PHAGES and PHIRE, which are jointly responsible for 25% of all complete phage genomes in the RefSeq database. These tools are used to integrate and analyze phage genome data from different sources, for identification and precise extraction of prophages from bacterial genomes, computing "phamilies" of related genes, and displaying the complex nucleotide and amino acid level mosaicism of these genomes. While over 50,000 SEA-PHAGES students have primarily benefitted from these tools, they are freely available for the phage community at large.

PhamClust: a phage genome clustering tool using proteomic equivalence.

IMPORTANCE: Bacteriophage genomes are pervasively mosaic, presenting challenges to describing phage relatedness. Here, we describe PhamClust, a bioinformatic approach for phage genome comparisons that uses a new metric of proteomic equivalence quotient for comparative genomics. PhamClust reliably assorts genomes into groups or clusters of related phages and can subdivide clusters into subclusters. PhamClust is computationally efficient and can readily process thousands of phage genomes. It is also a useful analytic tool for exploring the different types of inter-genome relatedness characteristic of phages in different clusters.

The heterogenous and diverse population of prophages in Mycobacterium genomes.

IMPORTANCE: Mycobacterium species include several human pathogens and mycobacteriophages show potential for therapeutic use to control Mycobacterium infections. However, phage infection profiles vary greatly among Mycobacterium abscessus clinical isolates and phage therapies must be personalized for individual patients. Mycobacterium phage susceptibility is likely determined primarily by accessory parts of bacterial genomes, and we have identified the prophage and phage-related genomic regions across sequenced Mycobacterium strains. The prophages are numerous and diverse, especially in M. abscessus genomes, and provide a potentially rich reservoir of new viruses that can be propagated lytically and used to expand the repertoire of therapeutically useful phages.

Unusual prophages in Mycobacterium abscessus genomes and strain variations in phage susceptibilities.

Mycobacterium abscessus infections are relatively common in patients with cystic fibrosis and are clinically challenging, with frequent intrinsic resistance to antibiotics. Therapeutic treatment with bacteriophages offers some promise but faces many challenges including substantial variation in phage susceptibilities among clinical isolates, and the need to personalize therapies for individual patients. Many strains are not susceptible to any phages or are not efficiently killed by lytic phages, including all smooth colony morphotype strains tested to-date. Here, we analyze a set of new M. abscessus isolates for the genomic relationships, prophage content, spontaneous phage release, and phage susceptibilities. We find that prophages are common in these M. abscessus genomes, but some have unusual arrangements, including tandemly integrated prophages, internal duplications, and they participate in active exchange of polymorphic toxin-immunity cassettes secreted by ESX systems. Relatively few strains are efficiently infected by any mycobacteriophages, and the infection patterns do not reflect the overall phylogenetic relationships of the strains. Characterization of these strains and their phage susceptibility profiles will help to advance the broader application of phage therapies for NTM infections.

PhaMMseqs: a new pipeline for constructing phage gene phamilies using MMseqs2.

The diversity and mosaic architecture of phage genomes present challenges for whole-genome phylogenies and comparative genomics. There are no universally conserved core genes, ∼70% of phage genes are of unknown function, and phage genomes are replete with small (<500 bp) open reading frames. Assembling sequence-related genes into "phamilies" ("phams") based on amino acid sequence similarity simplifies comparative phage genomics and facilitates representations of phage genome mosaicism. With the rapid and substantial increase in the numbers of sequenced phage genomes, computationally efficient pham assembly is needed, together with strategies for including newly sequenced phage genomes. Here, we describe the Python package PhaMMseqs, which uses MMseqs2 for pham assembly, and we evaluate the key parameters for optimal pham assembly of sequence- and functionally related proteins. PhaMMseqs runs efficiently with only modest hardware requirements and integrates with the pdm_utils package for simple genome entry and export of datasets for evolutionary analyses and phage genome map construction.

The Prophage and Plasmid Mobilome as a Likely Driver of Mycobacterium abscessus Diversity.

Mycobacterium abscessus is an emerging pathogen that is often refractory to antibiotic control. Treatment is further complicated by considerable variation among clinical isolates in both their genetic constitution and their clinical manifestations. Here, we show that the prophage and plasmid mobilome is a likely contributor to this variation. Prophages and plasmids are common, abundant, and highly diverse, and code for large repertoires of genes influencing virulence, antibiotic susceptibility, and defense against viral infection. At least 85% of the strains we describe carry one or more prophages, representing at least 17 distinct and diverse sequence "clusters," integrated at 18 different attB locations. The prophages code for 19 distinct configurations of polymorphic toxin and toxin-immunity systems, each with WXG-100 motifs for export through type VII secretion systems. These are located adjacent to attachment junctions, are lysogenically expressed, and are implicated in promoting growth in infected host cells. Although the plethora of prophages and plasmids confounds the understanding of M. abscessus pathogenicity, they also provide an abundance of tools for M. abscessus engineering.IMPORTANCEMycobacterium abscessus is an important emerging pathogen that is challenging to treat with current antibiotic regimens. There is substantial genomic variation in M. abscessus clinical isolates, but little is known about how this influences pathogenicity and in vivo growth. Much of the genomic variation is likely due to the large and varied mobilome, especially a large and diverse array of prophages and plasmids. The prophages are unrelated to previously characterized phages of mycobacteria and code for a diverse array of genes implicated in both viral defense and in vivo growth. Prophage-encoded polymorphic toxin proteins secreted via the type VII secretion system are common and highly varied and likely contribute to strain-specific pathogenesis.

Genome Sequence of Mycobacterium abscessus Phage phiT45-1.

Mycobacteriophage phiT45-1 is a newly isolated bacteriophage spontaneously released from Mycobacterium abscessus strain Taiwan-45 that lytically infects M. abscessus strain BWH-C; phiT45-1 also infects M. abscessus ATCC 19977 but not Mycobacterium smegmatis Phage phiT45-1 has a 43,407-bp genome and carries a polymorphic toxin-immunity cassette associated with type VII secretion systems.

Mycobacterium abscessus Strain Morphotype Determines Phage Susceptibility, the Repertoire of Therapeutically Useful Phages, and Phage Resistance.

Mycobacterium abscessus is an opportunistic pathogen whose treatment is confounded by widespread multidrug resistance. The therapeutic use of bacteriophages against Mycobacterium abscessus infections offers a potential alternative approach, although the spectrum of phage susceptibilities among M. abscessus isolates is not known. We determined the phage infection profiles of 82 M. abscessus recent clinical isolates and find that colony morphotype-rough or smooth-is a key indicator of phage susceptibility. None of the smooth strains are efficiently killed by any phages, whereas 80% of rough strains are infected and efficiently killed by at least one phage. The repertoire of phages available for potential therapy of rough morphotype infections includes those with relatively broad host ranges, host range mutants of Mycobacterium smegmatis phages, and lytically propagated viruses derived from integrated prophages. The rough colony morphotype results from indels in the glycopeptidolipid synthesis genes mps1 and mps2, negating reversion to smooth as a common route to phage resistance. Resistance is thus rare, and although mutations in polyketide synthesis, uvrD2, and rpoZ can confer resistance, these likely also impair survival in vivo The expanded therapeutic repertoire and the resistance profiles show that small cocktails or single phages could be suitable for controlling infections with rough strains.IMPORTANCEMycobacterium abscessus infections in cystic fibrosis patients are challenging to treat due to widespread antibiotic resistance. The therapeutic use of lytic bacteriophages presents a new potential strategy, but the great variation among clinical M. abscessus isolates demands determination of phage susceptibility prior to therapy. Elucidation of the variation in phage infection and factors determining it, expansion of the suite of therapeutic phage candidates, and a greater understanding of phage resistance mechanisms substantially advances the potential for broad implementation of new therapeutic options for M. abscessus infections.