Flushing significantly increased the number of particles 3 μm and less in diameter and bioaerosol concentrations. However, we did not observe a statistically significant difference in bioaerosol concentrations across time (i.e., 30 min sampling period) or distance (i.e., .15, .5 and 1.0 m from the toilet). The results of our study are significant, as no previous study has measured bioaerosol concentrations in normal hospital bathrooms without flushing, or bioaerosols generated from flushing unmanipulated loose human waste in a hospital setting during active patient care.
Johnson et al.,  reported similar findings and stated their study found the majority of particles produced from toilet flushes were less than 2 μm. Unlike Johnson et al.,  the composition of the particle concentrations measured in our study are unknown. Particles sampled may result from water droplets or microorganisms produced from the agitation of toilet water resulting from the flush.
Airborne particle concentrations remained constant in the bathroom when the toilet was not flushed (Fig. 3). Toilet flushing without waste had a greater particle concentration increase than flushing with waste. The presence of toilet paper, consistency of waste, and amount of loose fecal waste may lessen the movement of toilet water during the flush preventing the generation of particles. While particles can be generated from a multitude of sources (e.g., people moving in a room), the increase of particle concentrations immediately following a flushing event provides evidence that flushing (both with and without waste) increases the airborne concentration of particles in hospital bathrooms. Additionally, particles less than 3 μm significantly increased (p-values< 0.05). Johnson et al.  also found that various toilet designs produce an aerosol where the largest proportion of particles are smaller than 2 μm. Therefore, particles less than 3 μm in diameter may be a significant source of human exposure and environmental contamination as particles of this size can remain airborne for hours .
Pure water droplets evaporate in unsaturated conditions where relative humidity is less than 100%, however soluble nuclei (i.e., microorganisms like bacteria) can grow in unsaturated atmospheric conditions by a phenomenon called heterogeneous nucleation . Studies have confirmed that both gram positive and gram negative bacteria may act as a source of soluble nuclei, which have been found to grow to a diameter of 20 μm [33, 34]. These large particles have a much greater settling velocity in comparison to small particles . Faster settling rates may prevent large particle concentrations (i.e., 20 μm or greater) from being observed. Johnson et al.,  reported particles larger than 5 μm to reach max concentration within 30 s after a flush. Our room conditions where toilets were flushed had observable changes in particle concentrations immediately following the flush only in bin sizes 3, 1, 0.5, and 0.3 μm (Figs. 4 and 5). The faster settling rates of particles within the 5 and 10 μm bins may have caused us to not detect differences within comparisons of the particle concentration of those size bins. Furthermore this phenomenon may contribute to surface contamination near the toilet (i.e., the floor).
Bioaerosols generated from toilets containing fecal waste resulted in the highest average concentration of 278 CFU/m3 (SD ± 149), compared to the average concentration of 210 CFU /m3 (SD ± 138) near toilets that were idle with no waste present. The no waste no flush and the fecal waste with flush conditions were significantly different from one another (p-value = 0.005). The results of our study are significant, as no previous study has measured bioaerosol concentrations in normal hospital bathrooms without flushing, or bioaerosols generated from flushing unmanipulated loose human waste in a hospital setting during active patient care. While differences in bioaerosol concentrations across our experimental condition do not differ substantially, the amount of microorganism required to develop a HAI is often unknown and some healthcare acquired infections (e.g., Escherichia coli 0157–10 CFU) occur at low doses . Earlier studies have found that flushing toilets seeded with bacteria, increases the bioaerosol concentration of a bathroom; however these results are not applicable of the bioaerosols produced from flushing manipulated waste [22, 23, 26,27,28,29]. The increased concentration of bioaerosols from flushing loose fecal waste supports previous research that identifies a positive correlation between the amount of bacteria used to seed a toilet and measured bioaerosol concentration [22, 23, 26].
Our study detected bioaerosols produced from toilets that were flushed containing no waste, suggesting bacteria remain in the toilet from previous toilet use. We initially hypothesized that differences would be detected between conditions where toilets were flushed containing fecal waste and no waste. However, no differences were observed in the two flushing conditions of waste and no waste, suggesting bacterial residues from previous patient use remained in the water and along the sides of the bowl. When flushed, the agitation of the water could loosen bacteria attached to the wall and be released into the air. Other studies have suggested this phenomenon after seeding toilets with bacteria in the water and along the walls of the toilet [22, 23, 26, 27, 29]. After several flushes, residual bacteria have still been detected in the water or swabbed from toilet surfaces [22, 23, 26, 29].
The study did not observe statistically significant differences in bioaerosol concentration across horizontal distance of the samplers from the toilet or time after the flush (p-values = 0.925, 0.977). Prior to this study, there was little information available on the variance in bioaerosol concentrations across horizontal distance and time after a flush. Since our study detected greatest particle concentrations immediately after the flush, the distance and time closest to the toilet flush were hypothesized to have the greatest bioaerosol concentration, which our study results do not support. The plume produced from toilets is unknown due to many toilet varieties and other variables such as toilet height, water usage, and flush energy. The results do suggest that bioaerosol plumes produced during a toilet flush may extend beyond the distances sampled in this study (i.e., 1 m). The magnitude of the bioaerosol plume may be the reason why we did not observed a statistically significant difference across time (i.e., 30 min sampling period) or distance (i.e., .15, .5 and 1.0 m from the toilet). These results are concerning as a large bioaerosol plume may result in the contamination of surfaces bioaerosol exposures for healthcare staff and patients. Toilets with greater energy flushes have been shown to generate higher concentrations of bacteria and particles into the air than toilets with less energy [22, 25, 28]. In hospitals and other public places, toilets with high energy flushes are installed to comply with standards and to reduce the amount of water used and costs . Future research should include longer sampling times at greater distances from the toilet to understand how time or distance from the flush impact bioaerosol concentrations.
During the study, particle and bioaerosol concentrations were collected for 30 sampling events, therefore due to the variability observed in bioaerosol concentrations; our study may have been underpowered which may have resulted in an inability to detect small differences between experimental conditions (i.e., time and distance). The study procedures were conducted in one hospital ward, therefore the observations of this study may not be generalizable to other hospital wards. We chose to perform our study in a single hospital ward with the same toilet design to reduce variability between bathrooms such as make, model and height of toilets. The position of the toilet seat may have affected particle and bioaerosol concentrations. In our study, for the majority of the sampling trials, the toilet seat was left in the “up” position. The bioaerosol concentrations were measured with TSA plates under aerobic conditions. Therefore, our observed concentrations are likely an underestimate due to culture bias. Also, we did not collect data on stool volume added to the toilet prior to flushing. These data may help explain variability in bioaerosol concentrations measured in the field. Our bioaerosol concentration is limited to viable, culturable, fast growing microorganisms (i.e., 24 h) on TSA. Fecal wastes are composed of various species of bacteria with differing oxygen requirements, bioaerosol concentrations being underestimated. In the future studies, samples should be collected with selective growth media or using non-culture and non-target based analysis (e.g., metagenomic shotgun sequencing) to determine the characteristics of bioaerosols generated from toilets [36, 37]. Additionally, this study design allowed a better understanding of how bioaerosol samples could be collected in patient rooms where bathrooms were frequently used by patients and staff. While our design limits the generalizability to other hospitals, our field-based study provides direction to further investigate the role of toilets in aerosolizing fecal waste in a patient-care setting.