Validation of a Sponge Processing Method for Characterizing Microbes in the Bullitt Center

Abstract

Background: The Bullitt Center is a six-story, 50,000 square-foot green building that emphasizes energy efficiency and environmental performance based on design and operation. Microorganisms found in indoor surface environments most often come from human sources or by transport from the outdoor environment. Quantity and location of surface microbes can change due to re-aerosolization with human activity and building air movement. Few studies have been performed on surface microbes in green buildings. The aims are 1) Validate a sponge processing method based on literature review and 2) Characterize microbial populations from surfaces in the Bullitt building. Methods: Three processing methods (shaker table, hand massage, and stomacher 400 circulator) were evaluated for microbial recovery from 3MTM sampling sponges with 10 ml neutralizing buffer. Sponges were spiked with 1 to 3E8 E. coli B cells and cells recovered by each method. DNA from recovered cells was extracted (MO BIO PowerSoilTM DNA Isolation Kit), quantified by nanodrop (ND-3300) using picogreen, and E. coli specific targets amplified by qPCR (with fluorescent probe). The best processing method was determined by examining both percent recoveries of CFU and C(t) values. The Bullitt surface samples were processed by the most efficient elution method followed by DNA extraction, nanodrop quantification of nucleic acid, and Illumina 16S metagenomic sequencing to characterize the microbes that were present. Results: Hand massage was determined to be the most effective elution method based on the highest and most consistent calculated percent recoveries of CFUs ranging on average from 32.2-69.3%. C(t) values gave inconsistent results using undiluted nucleic acid, but tighter results using 10-fold dilutions of the sponge extractions ranging from (16.7-35.8% for shaker table, 15.3-42.1% for hand massage, and 25.7-52.7% for stomacher) suggesting the presence of inhibitors. Throughout the study, nucleic acid results ranged from (0-5121.5 ng/ml). The highest nucleic acid values were observed in June ranging from (78.1-5121.5 ng/ml). Results from Illumina 16S (>10% OTUs) showed changes in microbial populations over time at each location. Results from all 16S data showed that the high bloom conditions for Streptophyta were observed in April 2014 with a percent abundance as high as 93.9%. The high bloom conditions for Sandaracinobacter were observed in August/November 2013 with a percent abundance as high as 40.1%, then decreases significantly for the rest of the timeline. Data on the top ten microbes based on the number of reads in each sample showed high abundance levels of environment associated microbes and distinct levels of human associated microbes in June. Conclusion: Validation of hand massage as a sponge processing method was the best compared to the shaker table and stomacher methods and can be used for future studies to characterize microbial populations. Nanodrop and Illumina 16S results provided information in the change of quantity of surface microbes at each location over time in the indoor environment. Seasonality, temperature, humidity, and human occupancy all relate to one another in creating the indoor microbial environment. Both environmental and human associated microbes are present in the indoor microbiome suggesting the environment and humans as major sources. Human occupancy contributes to the characterization of the indoor environment by resuspension of dust that results in microbial similarities at different locations within the same building. More surface sampling and analysis in green buildings needs to be investigated to understand the green indoor microbiome.