Impact of relative humidity and collection media on mycobacteriophage D29 aerosol

Abstract

This study was conducted to evaluate the effect of aerosol generation, methods of sampling, storage conditions, and relative humidity on the culturability of the mycobacteriophage D29. The lytic phage D29 can kill Mycobacterium tuberculosis, and the phage aerosol can be treated as a potential tool for tuberculosis treatment. The culturability of D29 was tested using a test chamber designed for the bioaerosols research against three spray liquids (deionized water, phosphate-buffered saline [PBS], and normal saline), four collection media (suspension medium [SM], nutrient broth, PBS, and deionized water), two sampling systems (the all-glass impinger AGI-30 and the Biosampler) and across a range of humidities (20 to 90%). The effect of storage conditions on the culturability of collected sample was also evaluated for the AGI-30 impinger. The results proved that viable phage D29 particles generated by deionized water were approximately 30- and 300-fold higher than PBS and normal saline, respectively. As collection media, SM buffer and nutrient broth were observed to yield a higher number of plaques compared to PBS and deionized water. No difference was observed in collection efficiency between AGI-30 and Biosampler with two detection methods (culture-based technique and real-time PCR). The culturability of collected D29 in SM buffer or nutrient broth can be maintained up to 12 h irrespective of storage temperature. Relative humidity was found to strongly influence airborne D29 culturability which is 2- to 20-fold higher in low humidity (25%) than medium (55%) or high (85%) humidity. This research will help identify the optimal means for the application of D29 aerosol in animal inhalation experiments.

Fig 1

Schematic diagram of the generation and sampling system.

Fig 2

The results of plaque assays were used to calculate the total recovery rates. (a) Total recovery rates for D29 aerosols as a function of spray liquids; (b) total recovery rates of four sample liquids for D29 aerosols; (c) effect of relative humidity on the recovery rates of the D29 aerosols in the test chamber after it decayed for 15 min (■) and 30 min (●); (d) effect of storage temperature and storage time on phage D29 culturability (■, 4°C, the sample medium is nutrient broth; ●, 25°C, the sample medium is nutrient broth; ▲, 4°C, the sample medium is SM buffer; ▼, 25°C, the sample medium is SM buffer). Each column represents the mean value of at least three experiments, and the error bars represent the standard deviation.