## 5 New Technologies to Support the Development of Phage Therapy  
According to the Actinobacteriaphage database, known also as PhagesDB, there are 14,433 mycobacteriaophages with 2618 genomes fully sequenced. For the vast majority of them (14,392 phages), _M. smegmatis_ is the target microorganism, whereas strictly _M. tuberculosis_ is the only host for one mycobacteriophage named DS6A included in the Singleton cluster [58]. A few mycobacteriaophages with a broad host range have been described as effective against both tuberculous and non-tuberculous mycobacteria, such as phages G1, J1, and D1 infecting _M. tuberculosis_, _M. avium_, _M. fortuitum_, and _M. kansasi_ or phages Bxz2, D29, and L5 active towards _M. tuberculosis_, _M. avium_, _M. ulcerans_, _M. fortuitum_, and _M. chelone_[118]. Thus, it is evident that when thinking about phage therapy against tuberculosis, there is a need to use modern molecular methods and technologies for phage engineering to develop the phages efficiently infecting TB strains. Nature has prompted the idea since comparative phage genome analysis reveals their extensive mosaicism. Although the mycobacteriophages are currently grouped into 34 clusters (A-Z, AA-AE) and seven singletons based on genome similarity (phages sharing at least 35% of their genes are grouped within the same cluster), many genes are shared between clusters. This mosaicism probably arises from exchanging genetic modules between phages presented in the same host cell via horizontal gene transfer. There is speculation that this mechanism may be an evolutionary strategy of phages for better adaptation to the host organism and to avoid the defense systems [118; 119; 120]. [PERSON] et al. experimentally documented how mosaicism might have developed by the isolation of recombinant mycobacteriophages formed between a Butters's prophage (cluster N) and an infecting Island3 genome (subcluster I1) [120]. Unfortunately, the same strategy is used by bacteria to adapt to changing environmental conditions and to ensure survival in the presence of biocidal agents, including phages. It is estimated that up to 10% of the _M. tuberculosis_ genome may have been acquired by an evolutionarily recent horizontal gene transfer. What is more, phage-mediated gene transfer seems to play a significant role in the rearrangement of mycobacterial genomes, and mycobacteriajhages have become the genetic tools to engineer mycobacteria [119; 121]. The complete sequencing of mycobacteriajhages' genomes, understanding the function of individual genes and developing tools that enable genetic modification, have opened the way to phage engineering. One possibility is to delete fragments of the genome of wild-type phages to change their properties. For example, removing part of the repressor gene can change the replication mode of the phages from temperate, with the capacity to integrate into bacterial genomes, to lytic, which is necessary for therapeutic phages. [PERSON] et al. [122], using the BRED (Bacteriophage Recombineering of Electrorated DNA) methodology, described by [PERSON] et al. [123] constructed the ZoeJA45 mutant with the deletion of repressor gene 45. Due to that, the ZoeJA45, infecting a broad spectrum of mycobacterial hosts (_M. smegmatis, M. tuberculosis_, _M. avium_, _M. bovis BCG_, and _M. interjectum_) as parental phage ZoeJ, could not develop the lysogenic cycle [122]. Similar engineered mycobacteriophages BPs\(\Delta\)33 HTH_HRM10 and D29_HRMGD40 combined with antibiotics were used in vivo to treat a _M. abscess_ multidrug-resistant lung infection in a patient with cystic fibrosis (CF) [124]. BPs\(\Delta\)33 HTH_HRM10 is a host range mutant (HRM) of an engineered lytic derivative of phage BPs developed by removing part of the repressor gene, rendering it lytic [61; 124]. D29_HRMGD40 is an HRM of the D29 lytic mycobacteriophage isolated on an _M. abscess_ strain GD40, which, unlike the D29 parent, infects several _M. abscess_ clinical isolates [122; 124].