## 5 New Technologies to Support the Development of Phage Therapy
abscessus_ are usually less susceptible to phage infection because of high glycopidolipid (GPL) content on the cell surface, whereas rough strains are more phage sensitive [131]. Therefore, we will probably never find appropriate phages for all _M. tuberculosis_ strains. Still, in light of the crisis of antibiotic therapy, we must try to expand the therapeutic repertoire to combat bacterial infections, in which the application of phage cocktails or a combination of phages and drugs targeting the bacterial cell surface may prove to be very valuable. Engineered mycobacteria analogs can also be used for TB diagnostics, e.g., to determine the presence of viable tubercle bacilli, drug susceptibility, or treatment efficacy. In 2009, [PERSON] et al. described the construction of fluoromycobacteriophages based on TM4, able to infect a broad range of mycobacterial hosts, including fast-growing and slow-growing strains such as _M. tuberculosis_[132]. Fluorophage derivatives of TM4 were developed by gfp or ZsYellow gene fusion with the constitutive _M. bovis_ BCG Hsp60 promoter in plasmid derivatives of pYUB854, transferred then into the phAE87 shuttle plasmid to generate phAE87:hsp60-EGFP and phAE87::hsp60-ZsYellow, respectively. Both engineered fluoromycobacteriophages could infect _M. smegmatis_ mc2155 and mark these bacteria fluorescently, allowing the detection by fluorescence microscopy or flow cytometry in as few as 100 bacterial cells. The effect was specific for mycobacteria, and _M. smegmatis_ was only labeled in a mixed culture containing _M. smegmatis_ and _E. coli_. However, since the TM4 phage infects a broad range of mycobacterial hosts, the constructed fluoromycobacteriophages detect both _M. smegmatis_ and _M. tuberculosis_ strains, which was described as a limitation for TB diagnostics [132]. Therefore, an addition of p-nitrobenzoic acid (PNB) was next proposed during the diagnostic assay with fluoromycobacteriophages for discrimination between the _M. tuberculosis_ complex and atypical or non-tuberculous mycobacteria strains [133]. [PERSON] et al. used a second-generation fluoromycobacteriophage, mCherrybomb\(\varphi\), a derivative of TM4 with the optimized expression of an mCherrybombgene in mycobacteria, with an improved fluorescent signal and shorter time to detection of _M. tuberculosis_ using fluorimetry (fluorescence microscopy; flow cytometry) [133]. The results obtained were very promising. Compared to the reference culture method for detecting _M. tuberculosis_ in sputum samples, the sensitivity and specificity of mCherrybomb\(\varphi\) after recovering the sample for 96 h reached 91.98 and 98.96%, respectively. Additionally, _M. tuberculosis_ cultures preincubated with PNB (500 ug/mL) before overnight infection with mCherrybomb\(\varphi\) had quenched fluorescence. In contrast, phage infection and expression of mCherrybomb were not affected in the non-tuberculous mycobacteria strains tested [133]. [PERSON] et al. constructed TM4 expressing the highly sensitive BRET-nanoluciferase-based reporter, GeNL (TM4::GeNL), obtaining LRP for drug screening. It was demonstrated that the _M. leprae_ luminescence output decreased dose dependence when exposed to rifampicin, dapsone, and Q203 for 24 and 48 h, followed by TM4::GeNL infection [134]. _M. leprae_ transformedby the CoIE1-integration proficient plasmid expressing GeNL was also detected in mice using an in vivo imaging system [134]. To provide a clear overview of the evolution of mycobacteriophage research, Figure 5 presents a chronological timeline highlighting major discoveries and advancements.