Phage MS2

Spiked stool samples, clinical stool samples, and swabs were extracted with the QIAamp Stool DNA Mini kit (Qiagen, Valencia, CA), after a bead beating step and 95°C incubation. For stool specimens, 200 mg of raw stool was first lysed with QIAamp ASL buffer, beaten for 2 min with 212 to 300-μm glass beads (Sigma, St. Louis, MO), and incubated at 95°C for 5 min. The samples were centrifuged at full speed for 1 min to pellet stool particles, then 400 μl of ASL lysate were extracted and eluted in 200 μl of elution buffer following the manufacturer’s instructions. For swabs, the dry swab was mixed with the lysis buffer and glass beads, then subjected to bead beating directly and extracted following the same procedure as that for stool. Two extrinsic controls, Phocine Herpesvirus (PhHV) and bacteriophage MS2, were spiked into lysis buffer to monitor extraction and amplification efficiency. For comparison of extraction methods, one aliquot of each sample was extracted with QIAamp Stool DNA Mini kit and another aliquot was extracted with the QIAamp Viral RNA mini kit (Qiagen) or QuickGene RNA Tissue kit (FujiFilm, Tokyo, Japan). During all extractions an extraction blank was incorporated to monitor for lab contamination.

Bacteriophage MS2 (ATCC 15597-B1) was used as a bioaerosol test challenge for this work. Fresh cultures of MS2 were prepared from frozen bacteriophage stocks and propagated in Escherichia coli using nutrient broth (Oxoid) without supplements. Briefly, the preliminary E. coli culture (E. coli ATCC^® 15597) was prepared by adding 1 mL E. coli stock to 100 mL nutrient broth (1.3% w/v) and maintained at 37°C for 3–4 h in a shaking incubator at 70 rpm. A 100 μL volume of MS2 stock was added to the E. coli host culture and maintained overnight at 35°C in a shaking incubator at 70 rpm.
After incubation, the MS2 suspension was centrifuged at 3000 rpm (1690 g) for 10 min to remove host cell debris. The supernatant was filtered consecutively through 0.45 and 0.2 μm filters (Millipore, United Kingdom). To quantify the stock MS2 as plaque forming units per mL (PFU/mL), the freshly filtered stock was diluted using a 10-fold serial dilution in phosphate-buffered saline (PBS). One hundred microliters of each dilution was mixed with 300 μL E. coli (3–6 h culture). The MS2:E. coli mix was then added to 3 mL nutrient agar overlay and poured onto 1.5% Oxoid nutrient agar plates without supplements and incubated overnight at 35°C. PFU/mL of the MS2 stock was then back calculated. Forty milliliters volumes of MS2 were added to the Collison nebulizer before each test run.
A bioaerosol of MS2 was generated in the exposure chamber using a Collison 6-jet nebulizer,²⁰ operated from breathing quality compressed air delivered at 25 psi for 30 min. This provides a nebulizer flow delivery rate of 12 L/min. The liquid bacteriophage suspension was prepared so as to sit at a starting level exactly 1 cm above the lower tip of the Collison inlet tube, as recommended by the supplier. An assessment of particle sizes generated by the Collison nebulizer, based on Optical Particle Sizer (OPS) measurement, confirmed that the majority of nebulized particles fell within 0.1–1.4 μm size range (data not shown).
This is consistent with the findings of others^{21 (link),22} and is within the respirable particle size fraction, that is, if inhaled, a particle of this size may penetrate deep into the lungs, rather than depositing in the upper airways. It is known that the diameter of droplets generated by sneezing, coughing or speech may vary from <1 to 100 μm, but also recognized that those particles in the medium to large size range may either fall out of the airborne state, or else reduce in diameter due to rapid drying effects while in the airborne state.^{23–25 (link)} Based on these earlier reports, the particle sizes produced by the Collison nebulizer were deemed appropriate for this air cleaning challenge study.