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Quantitative Microbial Population Characterizationto Reveal Sources of Bacteria in Indoor Air

D Hospodsky 1, J Qian 1, N Yamamoto 1,2, W NAZAROFF 3, J Peccia 11 Yale University, New Haven, CT USA; 2 Japan Society for thePromotion of Sciences, Tokyo, Japan; 3 University of California,

Berkeley, CA USA [Funding: Alfred P. Sloan Foundation]

Healthy Buildings 2012Brisbane, Australia

9 July 2012

Denina Hospodsky Jing Qian Naomichi Yamamoto WW Nazaroff Jordan Peccia

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Humans may affect the indoor microbiome

• Issue — To what extent and in what ways do occupantsinfluence airborne bacteria in indoor environments?

• Approach — Study the indoor microbiome in a universityclassroom.

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Study site: University classroom

HVAC intake MERV 8 filters (upstream) Filters (downstream) Coils

Volume = 90 m3; Room AER = 6 per hour

Outside air: 25-100%

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Methods

• Sampling: 2 × 2 (indoor, outdoor × occupied, unoccupied)- Sampled 4 d occupied (104 person-h); 4 d unoccupied- Continuous size-resolved particles (OPC, 0.3-10 µm)- Time-integrated filter samples (cascade impactor, PM10, PM2.5)- Floor dust (sieved, resuspended, and sampled as PM37 & PM10)- Carbon dioxide (air-exchange rate determination)

• Analytical methods- Gravimetric (PM mass)- DNA quantification: real-time PCR with universal bacterial primers

targeting 16S rRNA encoding genes- DNA sequencing: pyrosequencing on a GS-FLX 454 platform- Analysis via QIIME (“quantitiative insights into microbial ecology”)

5Ref.: J Qian et al., Indoor Air (in press, 2012).

Occupancy strongly influences particle levels

Unoccupied (19 Sep 09) Occupied (3 Nov 09)

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Each 1 person caseinvolved walking; the 30persons were seated

Ref.: D Hospodsky et al., PLoS One 7(4), e34867, 2012

Resuspension from carpet is a strong contributor

One person walking on carpet contributes more than 30 people sitting on plastic.

7Refs.: J Qian et al., Indoor Air (in press, 2012); D Hospodsky et al., PLoS One 7(4), e34867, 2012

Indoor PM mass strongly enhanced w/ occupancy

The “occupied” to “vacant” PM10 mass concentration ratio indoors was 15 to 1.

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Bacterial genomes even more strongly enhanced!

The “occupied” to “vacant” PM10 bacterial genome concentration ratio indoors was 66 to 1.

Refs.: J Qian et al., Indoor Air (in press, 2012); D Hospodsky et al., PLoS One 7(4), e34867, 2012

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Floor dust is especially enriched in bacterial DNA

Ref.: D Hospodsky et al., PLoS One 7(4), e34867, 2012; Bacterial mass estimated as 655 fgper bacterium with an average of four 16S rDNA gene copies per bacterium.

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Emission rates quantified by material balance

Ref.: J Qian et al., Indoor Air (in press, 2012).

Peak ~ 3-5 µmPeak > 9 µm

C = indoor concentration; f = indoor proportion of outdoorlevel; Cout = outdoor concentration; N = number ofoccupants; E = per person emission rate; Q = ventilationrate; k = deposition rate constant; V = volume

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Significant contributor: Human microbiome

Refs.: D Hospodsky et al., PLoS One 7(4), e34867, 2012; EK Costello et al., Science 326, 1694, 2009. In all, 16 samples wereanalyzed by 454 pyrosequencing. The effort yielded 10.7k partial 16S rDNA gene sequences with average length of 500 bp.

Samples show clear representation from the dominant bacteria of the human microbiome. Five taxa— Propionibacterineae, Staphyloccus, Streptococcus, Enterobacteriaceae, and Corynebacterineae— comprise 17% and 20% of all bacteria in indoor air and in floor dust, respectively.

Prominent bacterial taxa in the human microbiome (Costello et al., 2009)

12Refs.: D Hospodsky et al., PLoS One 7(4), e34867, 2012; EK Costello et al., Science 326, 1694, 2009.

Indoor air differs from HVAC filter cake

Bacterial communities in indoor air during human occupancy were significantly differentfrom the communities associated with the HVAC filter dust (p < 0.001).

Prominent bacterial taxa in the human microbiome (Costello et al., 2009)

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Summary and conclusions for this study site

• Human occupancy contributes strongly to the concentration of indoorairborne bacterial genomes.

• DNA sequences point to important contributions of human microflora,either by direct shedding, or resuspension from surfaces (e.g., carpet).

• Current or previous occupants of a room can contribute substantially tohuman inhalation exposure to bacterial DNA.

We live in this microbial soup,and a big ingredient is our ownmicroorganisms. Mostly,people are resuspendingwhat’s been deposited before.The floor dust turns out to bethe major source of thebacteria we breathe.

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Ongoing work: Additional classroom sites