Reporter mycobacteriophages: versatile tools for TB-diagnosis and antitubercular drugs screening

PAYASLIAN FLORENCIA; SEIJO AGUSTÍN; RONCONI LISANDRO; PIURI *, MARIANA

Congreso; Viruses of Microbes 2023; 2023

About 40% of tuberculosis cases go undiagnosed and consequently not treated. WHO recommends the use of rapid molecular tests to speed up tuberculosis drug susceptibility testing (DST). Nevertheless, due to the cost, Ziehl Neelsen staining of Mycobacterium spp. in sputum, with subsequent culture and DST is often the method of choice. Fluoromycobacteriophages are reporter mycobacteriophages containing a fluorescent gene. These phages are a simple and rapid mean of revealing the metabolic state of M.tuberculosis cells, and their response to antibiotics.We constructed a Fluoromycobacteriophage, mCherrybomb, and developed a simple microscopy-based methodology for detection of viable Mycobacterium spp. and phenotypic determination of RIFR in 3-5 days from sputum sample collection. Paraformaldehyde fixation after infection reduces biohazard risks. mCherrybomb can also discriminate between pre-XDR and XDR-TB and between MTBC and NTM using PNB (para-nitro benzoic acid). mCherrybomb could also be used for HTS (high throughput screening) of drugs in an automated format but we further developed a new reporter mycobacteriophage expressing the NLuc enzyme. This new phage enabled us to decrease the number of cells and MOI (multiplicity of infection) for this assay. Complete DST could be done in 96 h and while the background:signal ratio using mCherrybomb was 1:5, with the new Nluc was 1:52, highly increasing the activity window of the assay. Reporter mycobacteriophages could facilitate therapy and prevent the spread of drug-resistant strains in low resource settings and be used for a rapid, easy and highly sensitive automated whole cell assay for HTS of new anti TB drugs.Keywords: Reporter Mycobacteriophages-Tuberculosis diagnosis -HTS