Dual-Reporter Mycobacteriophages (Φ ² DRMs) Reveal Preexisting Mycobacterium tuberculosis Persistent Cells in Human Sputum

Abstract

Persisters are the minor subpopulation of bacterial cells that lack alleles conferring resistance to a specific bactericidal antibiotic but can survive otherwise lethal concentrations of that antibiotic. In infections with Mycobacterium tuberculosis, such persisters underlie the need for long-term antibiotic therapy and contribute to treatment failure in tuberculosis cases. Here, we demonstrate the value of dual-reporter mycobacteriophages (Φ2DRMs) for characterizing M. tuberculosis persisters. The addition of isoniazid (INH) to exponentially growing M. tuberculosis cells consistently resulted in a 2- to 3-log decrease in CFU within 4 days, and the remaining ≤1% of cells, which survived despite being INH sensitive, were INH-tolerant persisters with a distinct transcriptional profile. We fused the promoters of several genes upregulated in persisters to the red fluorescent protein tdTomato gene in Φ2GFP10, a mycobacteriophage constitutively expressing green fluorescent protein (GFP), thus generating Φ2DRMs. A population enriched in INH persisters exhibited strong red fluorescence, by microscopy and flow cytometry, using a Φ2DRM with tdTomato controlled from the dnaK promoter. Interestingly, we demonstrated that, prior to INH exposure, a population primed for persistence existed in M. tuberculosis cells from both cultures and human sputa and that this population was highly enriched following INH exposure. We conclude that Φ2DRMs provide a new tool to identify and quantitate M. tuberculosis persister cells.

Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture

“…Additionally, in the normal course of M. tuberculosis infection, some bacteria exist as viable non-culturable persister organisms that are hypothesised to cause the high relapse rate seen following treatment of insufficient duration. Although these organisms may be identified in sputum by techniques such as reporter phages or culture with resuscitation promoting factors(29, 30) they are likely to be missed by any sequencing method reliant on standard culture.…”

PerSort facilitates characterization and elimination of persister subpopulation in mycobacteria

“…However, the non-growing subpopulation was not isolated and characterized further, most likely due to low fluorescence levels of the reporter. In another study, Jain et al developed a dual-reporter mycobacteriophage (ϕ 2 DRM) system and used fluorescent activated cell sorting (FACS) to isolate drug tolerant MTB cells from in vitro cultures and human sputa (6). However, the necessity to re-infect daughter cells with ϕ 2 DRM limited the ability to follow isolated cells over generations and study their regrowth patterns.…”

RbpA and σB association regulates polyphosphate levels to modulate mycobacterial isoniazid‐tolerance

“…However, the number of living cells began to increase after 72 h probably due to the generation of INH‐resistant mutants (Fig. A), which is consistent with previous results (Jain et al ., ). Therefore, we determined the percentage of tolerant and resistant cells in the survivors after INH treatments over time by plating the cells on 7H10 plates with and without INH.…”

“…As one of the most successful pathogens, Mycobacterium tuberculosis ( Mtb ) employs distinct mechanisms to survive under drug exposure. One example of such a mechanism is by generating persisters that can survive at high concentrations of drugs without genetic modification (Zhang et al ., ; Cohen et al ., ; Jain et al ., ). Persisters of Escherichia coli are a marginal subpopulation of bacteria that evolve into a non‐growing state (Balaban et al ., ).…”