Fluoromycobacteriophages Can Detect Viable Mycobacterium tuberculosis and Determine Phenotypic Rifampicin Resistance in 3–5 Days From Sputum Collection

Abstract

The World Health Organization (WHO) estimates that 40% of tuberculosis (TB) cases are not diagnosed and treated correctly. Even though there are several diagnostic tests available in the market, rapid, easy, inexpensive detection, and drug susceptibility testing (DST) of Mycobacterium tuberculosis is still of critical importance specially in low and middle-income countries with high incidence of the disease. In this work, we have developed a microscopy-based methodology using the reporter mycobacteriophage mCherrybombϕ for detection of Mycobacterium spp. and phenotypic determination of rifampicin resistance within just days from sputum sample collection. Fluoromycobacteriophage methodology is compatible with regularly used protocols in clinical laboratories for TB diagnosis and paraformaldehyde fixation after infection reduces biohazard risks with sample analysis by fluorescence microscopy. We have also set up conditions for discrimination between M. tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) strains by addition of p-nitrobenzoic acid (PNB) during the assay. Using clinical isolates of pre-XDR and XDR-TB strains from this study, we tested mCherrybombΦ for extended DST and we compared the antibiotic resistance profile with those predicted by whole genome sequencing. Our results emphasize the utility of a phenotypic test for M. tuberculosis extended DST. The many attributes of mCherrybombΦ suggests this could be a useful component of clinical microbiological laboratories for TB diagnosis and since only viable cells are detected this could be a useful tool for monitoring patient response to treatment.

Nanoluciferase Reporter Mycobacteriophage for Sensitive and Rapid Detection of Mycobacterium tuberculosis Drug Susceptibility

“…The performance of this system was subsequently improved through methods development (26), and phage modifications (27,28), enabling studies using phage for MTB detection and DST from isolates and clinical specimens (29,30). Additional phage modifications including introduction of a stronger constitutive promoter and use of reporter cassettes encoding one or more fluorescent proteins (fluorophages) enabled differentiation of antibiotic tolerance versus resistance in a mixed bacterial population, measurement of bactericidal activity of antibiotics against MTB in vitro and in ex vivo sputum samples from TB patients, and DST assays directly on sputum samples from TB patients identifying rifampicin heteroresistance (31)(32)(33)(34)(35). The combined conclusions of these efforts indicate that it is possible to use the TM4 mycobacteriophage encoding different reporter gene cassettes for DST and to measure bactericidal activity of anti-MTB agents.…”

Engineered Bacteriophages for Practical Applications

“…Thus, faster, simpler and cheaper methods are needed. With the development of luciferase reporters, M. tuberculosis-infecting phages able to deliver these reporters were developed, 35) and after some iterations 36,37) they have evolved into powerful tools (Fig. 3).…”

“…For example, they can detect M. tuberculosis in sputum samples with 91.98% sensitivity and 98.96% specificity compared to the reference method, with a sample recovery time of only 96 h (compared to 3-6 weeks), and with an estimated cost of 2 USD, using infrastructure already available in most laboratories working with M. tuberculosis. 38) Since this method detects only metabolically active bacteria, it can detect resistance phenotypes that might not be detected by sequencing, which is used in most of the current fast-diagnostic techniques. 38) Phage-based detection of pathogenic bacteria in the food industry has followed a similar path.…”

“…38) Since this method detects only metabolically active bacteria, it can detect resistance phenotypes that might not be detected by sequencing, which is used in most of the current fast-diagnostic techniques. 38) Phage-based detection of pathogenic bacteria in the food industry has followed a similar path. For example, in the case of Listeria, luciferase-based reporters were also created, 39,40) and these reporters, coupled with the high affinity of phage lysins for Listeria cell wall carbohydrates, 41,42) were used to construct a robust assay, enabling detection limits of 0.1-1.0 cfu/g Listeria in less than 24 h, versus 96 h by the standard method.…”

Rapid ultrasensitive diagnosis of pneumonia caused by Acinetobacter baumannii using a combination of enrichment and phage-based qPCR assay

“…Bacteriophages have been developed for various assays, such as the detection of Mycobacterium tuberculosis in sputum. [13][14][15][16][17] Conventional methodologies based on phage for the recognition of bacteria are focused on counting the formed plaques by the double-layer plate culture after the infection of phage to the bacteria, where it takes two to three days to get the results. [18] In recent decades, increasing methods relying on the high affinity of the bacteriophages to the host bacteria have been developed for the detection of pathogenic bacteria.…”