Molecular and microscopic analysis of bacteria and viruses in exhaled breath collected using a simple impaction and condensing method

Abstract

Exhaled breath condensate (EBC) is increasingly being used as a non-invasive method for disease diagnosis and environmental exposure assessment. By using hydrophobic surface, ice, and droplet scavenging, a simple impaction and condensing based collection method is reported here. Human subjects were recruited to exhale toward the device for 1, 2, 3, and 4 min. The exhaled breath quickly formed into tiny droplets on the hydrophobic surface, which were subsequently scavenged into a 10 µL rolling deionized water droplet. The collected EBC was further analyzed using culturing, DNA stain, Scanning Electron Microscope (SEM), polymerase chain reaction (PCR) and colorimetry (VITEK 2) for bacteria and viruses.Experimental data revealed that bacteria and viruses in EBC can be rapidly collected using the method developed here, with an observed efficiency of 100 µL EBC within 1 min. Culturing, DNA stain, SEM, and qPCR methods all detected high bacterial concentrations up to 7000 CFU/m(3) in exhaled breath, including both viable and dead cells of various types. Sphingomonas paucimobilis and Kocuria variants were found dominant in EBC samples using VITEK 2 system. SEM images revealed that most bacteria in exhaled breath are detected in the size range of 0.5-1.0 µm, which is able to enable them to remain airborne for a longer time, thus presenting a risk for airborne transmission of potential diseases. Using qPCR, influenza A H3N2 viruses were also detected in one EBC sample. Different from other devices restricted solely to condensation, the developed method can be easily achieved both by impaction and condensation in a laboratory and could impact current practice of EBC collection. Nonetheless, the reported work is a proof-of-concept demonstration, and its performance in non-invasive disease diagnosis such as bacterimia and virus infections needs to be further validated including effects of its influencing matrix.

Figure 1. Exhaled breath condensate collection device (A) and method developed in this study: B) the EBC collection device cover, C) the collection device base with a layer of ice and hydrophobic film on the top, D) the exhaled breath condensate collection method: 5–10 µl DI water pipetted and scrolled over the hydrophobic film to scavenge EBC droplets.

Figure 2. Sketch of experimental setup for collecting exhaled breath samples.

Figure 3. Physical collection efficiency of the exhaled breath condensate collection device developed in this study under different collection time; control indicates the total volume of sample collected without breathing toward the device; data points represent averages and standard deviations of six EBC collection volume data by different subjects.

Figure 4. Particle size distributions in exhaled breath by mouth breathing using an Optical Particle Counter(OPC); x-axis shows the average diameters of 16 channel sizes of the OPC; data points represent averages and standard deviations of 20 min measurements by the OPC.

Figure 5. Culturable bacterial aerosol concentrations detected in exhaled breath condensate samples collected using the device from seven human subjects with symptoms listed in Table S2; F and M indicate Female and Male, respectively, 1–7 indicate the subject ID corresponding to those listed in Table S2; EBC collection time was 3 min; data points represent averages and standard deviations from at least three replicates.

Figure 6. Determination of total bacterial aerosols in EBC by qPCR; DNA standards (STD) used were 3.15, 3.15×10¹, 3.15×10², 3.15×10³ ng/µl Bacillus subtilis DNA; Sample 1–7 represent EBC samples collected from seven human subjects with their medical conditions listed in Table S2; DI water was used as the negative control.

Figure 7. Dissociation curve of bacterial aerosols in EBC samples amplified by qPCR; Samples 1–7 were those collected from seven human subjects with their medical conditions listed in Table S2; Bacillus subtilis species was used as the positive control and DI water was used as the negative control; the curves shown here include two duplicates for each EBC sample.

Figure 8. SEM images (different resolutions) of bacteria in EBC samples and images of colony forming units after culturing; the EBC samples were collected from human subjects and cultured using liquid broth overnight; different colored arrows point to likely different bacteria (different morphologies); Sphingomonas paucimobilis, Kocuria rosea, Bacillus lentus, Aerococcus viridians, Bacillus firmus, Kocuria kristinae, Staph.

Xylosus were identified in EBC samples from patients with respiratory symptoms using VITEK 2 system.