areas. An earlier study using a GFP-expressing Escherichia coli (GFP-E. coli) as a model 23 organism demonstrated dispersal from drain biofilm in contaminated sinks. The present study 24 further characterizes the dispersal of microorganisms from contaminated sinks. Replicate hand-25 washing sinks were inoculated with GFP-E. coli, and dispersion was measured using qualitative 26 (settle plates) and quantitative (air sampling) methods. Dispersal caused by faucet water was 27 captured with settle plates and air sampling methods when bacteria were present on the drain. In 28 contrast, no dispersal was captured without or in between faucet events amending earlier theory 29 that bacteria aerosolize from P-trap and disperse. Numbers of dispersed GFP-E. coli diminished 30 substantially within 30 minutes after faucet usage, suggesting that the organisms were associated 31 with larger droplet-sized particles that are not suspended in the air for long periods. Retrospective and prospective surveillance investigations affirm that hospital sinks provide 48 habitats for several opportunistic pathogens, raising serious concerns (8, 24, 26, 28) . It is not the 49 mere presence of these drug-resistant pathogens in the hospital wastewater that is of concern, but 50 the ability of these organisms to colonize biofilms on the luminal surfaces of wastewater 51 plumbing and thereby withstand routine cleaning practices. While several gammaproteobacteria 52 detected from the sinks in hospitals have been linked to healthcare-associated infections, 53 opportunistic pathogens like Pseudomonas aeruginosa, Acinetobacter baumannii and 54 Stenotrophomonas maltophilia are typically known to be found in water environments (29) (30) (31) . In 55 contrast, emerging pathogens such as the carbapenemase-producing Enterobacteriaceae, many 56 having fecal origin, may survive within the biofilm formed on sink surfaces and wastewater 57 premise plumbing(32, 33). Often by acquiring mobile resistance elements through horizontal 58 gene transfer, carbapenemase-producing Enterobacteriaceae (CPE) infections are especially 59 threatening because they are more frequent causes of highly antibiotic resistant infections with 60 reduced treatment options.
In outbreak investigations species and strain matching between patient and sink isolates is 62 often attributed to sink source contamination, however direction (sink to patient versus patient to 63 sink) and precise mode of transmission remains inconsistent and elusive (29, 30) . Even with 64 4 increased recognition of transmission, a knowledge gap exists with regards to the precise 65 mechanism of transmission from sink reservoirs to the patient. Using a model of sink 66 colonization with green fluorescent protein (GFP)-expressing Escherichia coli (GFP-E.coli), we 67 recently demonstrated the source and the degree of dispersion from sink wastewater to the 68 surrounding environment(34). Factors effecting the rate and extent of droplet-mediated 69 dispersion were investigated, but particle size involved in dispersion was not measured in this 70 study. Studies that claim aerosols as the primary dispersion mechanism from sinks are based on 71 rudimentary findings (2, 23, 35, 36) or assumptions drawn based on these unsubstantiated 72 findings (3, 6, 10, 13, 21, 37 Advisory Committee (HICPAC) guidelines use a particle diameter of 5 μm to delineate between 75 bioaerosol (≤5 μm) and droplet (>5 μm) transmission (38, 39).
Aerosol-mediated transmission and droplet-mediated transmission in the healthcare 77 environment will require conceptually different infection control strategies. Clarity regarding 78 aerosol versus droplet mediated dispersion in the context of sinks is critical. In the present study, 79 we aim to further define several outstanding knowledge gaps: i) dispersion mechanism of 80 bacteria, aerosol sized particles or droplets, from biofilms in handwashing sinks, ii) factors 81 triggering dispersion from a colonized sink drain and iii) role of biologically active aerosols 82 spontaneously dispersing from drain or P-trap without a triggering event. GFP-E. coli as the 83 surrogate organism for Enterobacteriaceae was used in this model study to investigate these 84 questions. E.coli (10 9 CFU/ml) was added into the P-trap water (~150ml) through the lower-most sampling 109 port on the tailpiece using a 60ml syringe attached to silicone tubing (Cole-Parmer, Vernon Hills, 110 6 IL). The inoculum was mixed with the P-trap water by repeated withdrawal and injection of the 111 inoculum, with precautions taken to avoid unintentional inoculation of drain (strainer) or sink 112 bowl (bowl). For the drain inoculation, a 10ml mid-log phase culture of GFP-E.coli (10 9 113 CFU/ml) was evenly applied on the surface of the drain using a sterile pipet. For establishment 114 of GFP-E.coli P-trap biofilm, a 10 ml mid-log phase culture of GFP-E.coli (10 9 CFU/ml) was 115 added into an unused P-trap and following inoculation, 25ml TSB and 25ml (x2) 0.85% saline 116 was added on a daily basis through the drain for 7 days to facilitate biofilm growth on the bowl (set at 6L/min for 50min) to collect a 300L air sample (Fig 2b) . In a biological safety 172 cabinet, the liquid from the impinger was transferred to a sterile tube, vortexed, filtered through plates were processed and counted at University of Virginia. Paired with air sampling particles in 179 size range 0.3, 0.5, 0.7, 1.0, 2.0 and 5.0 µm were measured using particle counter (GT-526, Met 180 One Instruments, Inc. Grants Pass, OR) placed 12 inches from the sink bowl (Fig 2. ). With a 181 runtime of 660 seconds each, 3 successive runs of particle counter were performed, first run 182 coinciding with t=0. Particle counter also recorded relative humidity and air temperature. water, plugged, and shipped by overnight courier to CDC for analysis. Swab sampling from the 203 tailpipe and P-trap, and sponge wipes were processed as already described to recover and 204 quantify biofilm organisms. Samples were plated on TSA and R2A media, and counted, as (Table 1) . So were the water and air temperatures ( Table 2 ). In comparison, the relative humidity (Table 2) . Dispersion detected by settle plates across the three sinks ranged from 35-107 CFU/plate. GFP-220 E.coli levels were higher on the counter space surrounding the sink bowl compared to lower 221 counts near the faucets (Fig 4b) . Dispersion was not detected on side-splatter shields. With the 222 exception of the detection of 1 CFU/m 3 at t=60 min using the impaction, dispersion of GFP-E. The objective of the present study was to characterize the mechanism of bacterial dispersion 264 from handwashing sinks, using a GFP plasmid-containing E. coli strain as a surrogate for assessed using a gravity method, and bioaerosol production was not evaluated.
The air sampling methods chosen and tested in the present study were three of the most widely 283 used methods previously reported (23, 35, 36, 40, 42, 43) and were selected to assess bioaerosol 284 production during sink usage. In the present study, GFP-E. coli dispersion was detected during a surfaces due to gravity rather than aerosol sized particles which remain in the air(44). Dispersion 292 of GFP-E. coli from sinks does not appear to be associated with the production of bioaerosols, 293 that is, particles smaller than 5 µm (35, 40, 45, 46) . Studies that measured air sampling lacked 294 the resolution between aerosols and droplets (35, 40, 41) . Air was sampled significantly closer 295 (~4 inch) to the sink drain or impact point of faucet water on the sink and therefore, might have 296 picked droplets rather than aerosols.
A consistent result from this work which is worth reemphasizing is the finding that for dispersion 298 to occur the presence of bacteria on drain and/or bowl surface is necessary(34). When GFP-E.
coli was inoculated into a new P-trap, dispersion was not detected using settle plates or air 300 sampling methods. This underscores the fact that as long as the sink drain and bowl remain free 301 of the target organisms (e.g., CPE or other antibiotic resistant Gammaproteobacteria), dispersion 302 can be controlled. However, under favorable conditions bacteria can grow or mobilize from the 303 P-trap into the drain piping (tailpiece) and colonize the sink drain surfaces, with the potential for 304 a dispersion event to occur. This further underlines the importance of sanitary hygiene practices, 305 strategic surveillance paired with hand washing only use of hand-washing sinks in the patient 306 care environment to reduce the risk of hand-washing sink contamination by the multi-drug-307 resistant microorganisms that can colonize ICU patients(8). This also emphasizes the necessity to 308 implement stricter measures to prohibit disposal of nutrients, body fluids and anything into the 309 sinks that could be a nutrient source for maintenance of microorganism biofilms in drains(23).
Dispersion from a contaminated sink reservoir can result in transmission to patients either 311 directly or indirectly mediated through numerous contact surfaces. Herruzo and colleagues 312 demonstrated the potential for microbial transfer from contaminated hands, which continued to 313 disperse microorganisms after more than 10 successive contacts with surfaces (25).
The droplet dispersion load observed on settle plates was similar and consistent with our 315 previous work(34). Total dispersion measured in corresponding experiments in the previous 316 study was higher, which may be attributed to one or more of the following factors: i) fewer settle 317 plates were used in the present study (22 vs 90), ii) a higher water flow rate was used in the 318 present study (8 vs 1.8-3.0 L/min) and iii) air sampling methods performed in conjunction with 319 the settle plate method may have captured a portion of the dispersed droplets. Settle plates were 320 found to be a reliable method to assess the large-droplet dispersion from sinks. In this study 22 321 settle plates (=11.24 m 2 ) were used which accounted for a defined surface area and locations on 322 the sink counter. Dispersion could have been higher in locations of the sink counter other than 323 those chosen in the present study, and the dispersion load recorded in this study may not be the 324 absolute value. Of the three methods investigated for air sampling, impaction and filtration were 325 found to be reliable and consistent. In the same amount of air sampled using impaction and 326 filtration, comparable counts were recorded; however, air sampled using the impinger method 327 was unable to capture the dispersion of GFP-E.coli under similar testing conditions.
Mannequin hands functioned as obstruction to direct impact of faucet water on the sink drain, 329 and therefore no dispersion was detected. This rationale behind testing mannequin hands was to 330 simulate hand washing, but in reality the water would be flowing before, after and during a hand 331 washing event. In other words, an actual handwashing event is more dynamic than static 332 mannequin hands and there is likely direct impact of water on the sink drain at least for brief 333 periods when the water is running. There is also the scenario where the sinks and faucets may be 334 used outside of hand washing (e.g. dumping liquid wastes) (5, 8, 23) . This finding we think 335 further defines and supports another important dynamic that may minimize dispersion in 336 healthcare settings (i.e., avoid faucet water flow directly onto drains to minimize dispersion). All 337 of these findings must be taken in the context of an experimental water stream which directly hits 338 the drain which is outside FGI guidance but thought to be frequently found in health care sink 339 design.
This study has several limitations. First, the dispersion experiments were not performed in a 341 controlled environment. Each dispersion experiment lasted at least 12h, therefore it was not 342 possible to maintain precisely the same conditions with regards to air flow velocity, air 343 temperature, relative humidity, and bacterial and/or fungal burden in the laboratory space 344 harboring the sinks. These parameters may have direct or indirect influence on the dispersion 345 pattern and load recorded across experiments(47). To address this issue, we monitored the 346 heterotrophic plate counts, relative humidity and particle concentration in the air. Particle counts 347 recorded in the absence of faucet event (control) were higher or equal to that in the presence of 348 faucet event (test). This observation implies that particle concentrations in the air were driven by 349 relative humidity and/or temperature of the air. This trend was observed in all the experimental 350 methods (Drain, P-trap inoculation and Drain colonization) (Supplemental Figure S1 ). In other 351 words, particle counts were largely consistent across the day for a given experiment (control 352 preceding test). Further particle counter used in the study could not resolve or measure particles 353 >5μm, which defined droplet particles. Another limitation was that air samples were collected at 354 only one location relative to the sink bowl, so it is not possible for this data set to define a 355 "splash zone" pattern without additional measurements collected from various positions and 356 distance from the source of dispersed organisms. 357 We have provided data to support the position that microorganisms will disperse from 358 contaminated sink bowl and drain surfaces primarily as large droplets that are generated during 359 faucet usage. These droplet-associated organisms remain viable with the potential to contaminate 