Bioaerosol mass spectrometry for rapid detection of individual airborne Mycobacterium tuberculosis H37Ra particles

Abstract

Single-particle laser desorption/ionization time-of-flight mass spectrometry, in the form of bioaerosol mass spectrometry (BAMS), was evaluated as a rapid detector for individual airborne, micron-sized, Mycobacterium tuberculosis H37Ra particles, comprised of a single cell or a small number of clumped cells. The BAMS mass spectral signatures for aerosolized M. tuberculosis H37Ra particles were found to be distinct from M. smegmatis, Bacillus atrophaeus, and B. cereus particles, using a distinct biomarker. This is the first time a potentially unique biomarker was measured in M. tuberculosis H37Ra on a single-cell level. In addition, M. tuberculosis H37Ra and M. smegmatis were aerosolized into a bioaerosol chamber and were sampled and analyzed using BAMS, an aerodynamic particle sizer, a viable Anderson six-stage sampler, and filter cassette samplers that permitted direct counts of cells. In a background-free environment, BAMS was able to sample and detect M. tuberculosis H37Ra at airborne concentrations of >1 M. tuberculosis H37Ra-containing particles/liter of air in 20 min as determined by direct counts of filter cassette-sampled particles, and concentrations of >40 M. tuberculosis H37Ra CFU/liter of air in 1 min as determined by using viable Andersen six-stage samplers. This is a first step toward the development of a rapid, stand-alone airborne M. tuberculosis particle detector for the direct detection of M. tuberculosis bioaerosols generated by an infectious patient. Additional instrumental development is currently under way to make BAMS useful in realistic environmental and respiratory particle backgrounds expected in tuberculosis diagnostic scenarios.

FIG. 1.

Schematic of aerosol generation, sampling, and detection experimental setups for BAMS mass spectral signature development using BAMS without its virtual impactor (VI) stage (A), BAMS' virtual impactor (VI) stage efficiency measurements (B), BAMS particle sampling and tracking efficiency measurements using BAMS without its VI stage (C), and the CDC-NIOSH bioaerosol chamber experiments (D).

FIG. 2.

Experimentally measured particle preconcentration, sampling, and detection efficiencies for the various stages of the BAMS. (A) Fractional efficiency with which particles are concentrated in the virtual impactor (VI) stage exit minor flow as a function of particle diameter [VI_eff(d_p)]. This is based on a theoretical concentration factor of 120. (B) Fraction of particles successfully tracked and sized out of all particles that are sampled by the BAMS after the VI stage as a function of particle diameter [TK_eff(d_p)]. (C) Fractional hit rate, defined as the fraction of particles whose molecules are desorbed, ionized, and produced a mass spectrum out of all particles successfully tracked and sized by the BAMS as a function of particle diameter [H_eff(d_p)]. This was measured with 1,000 M. tuberculosis H37Ra- and 1,000 M. smegmatis-containing particles.

FIG. 3.

Average of ∼1,000 single particle BAMS mass spectra of airborne M. tuberculosis H37Ra (A)-, M. smegmatis (B)-, B. atrophaeus (C)-, and B. cereus (D)-containing particles. Many of the individual spectra of M. tuberculosis H37Ra-containing particles (∼66%) can be differentiated from the three other organisms based on the presence of a m/z −421 peak.

FIG. 4.

Series of single-particle mass spectra from M. tuberculosis H37Ra-containing particles randomly selected to depict the typical wide variation in the m/z −421 signal intensity. It should also be noted that a subset of single-particle mass spectra from M. tuberculosis H37Ra-containing particles (∼34%) contain no significant signal at m/z −421. Within panel A is the structure of deprotonated trehalose-2-sulfate, the tentative identification of the m/z −421 peak.

FIG. 5.

Raw particle counts and calculated particle concentrations, regardless of their identity, as a function of particle diameter during the aerosolization of the high-concentration suspension (10⁸ M. smegmatis particles/ml) into the bioaerosol chamber measured by the APS (A) and BAMS (B) in the first 60 s of aerosolization.

FIG. 6.

Raw M. tuberculosis H37Ra CFU and particle counts and calculated M. tuberculosis H37Ra CFU and particle concentrations as a function of particle diameter during the aerosolization of the high-concentration suspension (2 × 10⁸ M. tuberculosis H37Ra particles/ml) into the bioaerosol chamber as measured by BAMS (A) and the Andersen six-stage sampler (B) in the first 60 s of aerosolization.