Mycobacteriophage D29-mediated lysis improves recovery of mycobacterial genomic DNA from low-biomass samples

<jats:p>Background: Detection of rare mycobacterial genotypes, including those associated with antibiotic resistance or population heterogeneity is important for diagnostic, therapeutic and research applications. This depends on efficient recovery of genomic DNA (gDNA) from sampled populations, a challenging requirement in paucibacillary clinical materials. Mycobacteria have uniquely lipid-rich, structurally robust cell envelopes which resists cell lysis by conventional methods. Here, we characterize mycobacteriophage D29-mediated lysis at the single-cell level, evaluating its utility as a biological lysis strategy for mycobacterial DNA isolation, benchmarked against the standard cetyltrimethylammonium bromide (CTAB) extraction method. Methods: Conditions for mycobacteriophage D29 infection of Mycobacterium smegmatis (Msm) were established, and single-cell phage adsorption and phage-mediated lysis visualized through live-cell time-lapse fluorescence microscopy (FM). A mycobacteriophage D29-based lysis method was applied to both Msm and M. tuberculosis (Mtb), and extraction efficiencies compared with the standard CTAB method. Cell lysis efficiency was quantified by colony forming units (CFU), flow cytometry (FC) and FM; DNA yield was determined by quantitative polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR). Results: Mycobacteriophage D29 adsorption was observed at the poles and septa of individual mycobacterial cells. Phage infection was associated with loss of cytoplasmic green fluorescence protein (GFP) reporter protein, with uptake of a cell death marker propidium iodide (PI). Mycobacteriophage D29 infection resulted in a marked loss of cell viability, with &gt;6log10 reduction in CFU, and cell lysis efficiencies calculated as 93.3% (FC) and 96.8% (FM). Molecular quantification (qPCR and ddPCR) indicated that the mycobacteriophage-based lysis achieved between 4- to 7-fold greater gDNA yields in Msm and between 3- to 12-fold greater gDNA yields in Mtb H37Ra compared with the CTAB method. Notably, gDNA extraction efficiencies in both mycobacterial species exceeded 92% in low-biomass samples containing approximately 100, 175 and 320 bacilli. Conclusion: These results demonstrate the utility of the mycobacteriophage D29-based method for improved DNA extraction yields from mycobacteria through direct lysis of individual bacilli, with performance suited to low-biomass samples.</jats:p>