This study aimed to detect aerosolized viable bacteria during operation of an acoustic-assisted flow cytometer and to compare the detected bacteria with those cultured from air samples collected in the same laboratory space when the cytometer was not in use, and in a diagnostic clinical microbiology laboratory.

Flow cytometry techniques have been used to analyze bacteria for several decades 1 , 2 , and for assessing the effects of antimicrobial agents since the 1980s 3 – 5 . The bacteria analyzed then included species that can be safely handled on an open bench in a suitably equipped microbiology laboratory, providing a series of standard biosafety procedures are adhered to 6 . Concerns about laboratory biosafety and containment increased after 2001 7 , 8 and led to higher physical containment levels for select biothreat agents and some bacterial species such as Neisseria meningitidis, prone to transmission by aerosols generated during laboratory procedures 9 , 10 . Given these concerns about bioaerosol transmission risks, it is not surprising that standards for bioaerosol risk assessment and mitigation have been recommended for fluorescence-activated cell sorting protocols 11 , 12 . Our use of flow cytometry was not for cell sorting, but for a less aerosol-prone cellular analysis. We commenced use of our analytic flow cytometer in a physical containment level two laboratory while developing a flow cytometry-assisted antimicrobial susceptibility test (FAST) assay method with Klebsiella pneumoniae 13 . Though the cytometer we used had no cell sorting function and therefore would not generally produce aerosols, 14 , 15 we decided to conduct an assessment to confirm that viable bacteria are not aerosolized during use before progressing with any analysis of potentially more hazardous aerosol-transmitted species such as Neisseria meningitidis, Mycobacterium tuberculosis and Burkholderia pseudomallei .

Methods

Laboratory locations. Two laboratory locations in adjacent buildings were used. One was a university research laboratory approximately 54.7 m2 and 145.5 m3 equipped with two acoustic-assisted flow cytometers, and two class two biosafety cabinets, peripheral benches and one central bench. One of the flow cytometers is housed and used in a biosafety cabinet while the other one, the focus of this study, is on the open bench. The other location was a large, open plan clinical laboratory microbiology laboratory approximately 400.5 m2 and 1081.4 m3 operating a range of high throughput bacteriology procedures serving an on-campus 700 bed teaching hospital and an extensive regional hospital network. Notably, the clinical laboratory does not have an acoustic-assisted flow cytometer. Both laboratories were air-conditioned and equipped with high efficiency particulate air filtration on external air outlets. Detailed information about the number of complete air exchanges per hour was not available for either location.

The clinical laboratory was included for comparative purposes. While the study aimed to determine if aerosolized viable target bacteria (Burkholderiathailandensis,K. pneumoniae andStreptococcus pneumoniae) could be detected in air in the research laboratory, the likelihood was that other microorganisms, commonly present in indoor air, would be detected. Published data regarding the range and concentration of bacteria present in air in microbiology laboratories, either research or clinical, are scarce. Without data for comparison, data on the viable bacteria detected in air samples from the research laboratory would be entirely without context. Work practices in the clinical laboratory are designed to maintain a safe working environment and the background level of viable bacteria detected in clinical laboratory air could therefore serve as a proxy indicator of the acceptable level of viable bacteria in air in the research laboratory.

Since background microorganisms would be detected in the air and, in ana priori effort to put them in some context, we sampled a non-flow cytometer site within the same research laboratory (preparation bench) and a non-flow cytometry laboratory (clinical laboratory) that handles numerous human bacterial pathogens using standard diagnostic microbiological techniques and biosafety risk management procedures.

Flow cytometer equipment and reagents. An Attune NxT (ThermoFisher Scientific, Eugene, OR) acoustic-assisted flow cytometer was the focus of these air sampling investigations. The instrument uses acoustic radiation pressure to align particles in the center of a sample stream. This pre-focused stream is then injected into the sheath stream, which supplies an additional conventional hydrodynamic pressure to the sample. The instrument uses a sheath fluid branded “focusing fluid” that hydrodynamically focuses samples just prior to analysis. The focusing fluid is a proprietary mix of reagents including an unspecified broad spectrum antimicrobial agent (personal communication M. Ward, ThermoFisher, Eugene OR, USA).

Workflow. The air sampling study was conducted over a one month period in which the research laboratory was intensively used by up to nine people at one time during office hours, often with both flow cytometers in use at once. Each flow cytometer procedure was staffed by one cytometer operator and another scientist preparing bacterial suspensions for FAST and other cytometer assays, plus at least one of the above authors engaged in the air sampling procedure. The majority of flow cytometer experiments analyzed theK. pneumoniae isolates as previously reported13. The other two species analyzed wereS. pneumoniae andB. thailandensis. The clinical laboratory was staffed between 7.30am and 9pm by up to 20 people, with 1–3 people per side of each laboratory bench, conducting predominantly manual procedures with liquid and solid bacterial cultures. While clinical specimens were opened and blood cultures were subcultured in a class two biosafety cabinet, the majority of bacteriologic procedures were performed on the open bench in accordance with clinical laboratory safety policy6.

Air sampling. Air sampling was performed with a compact impinger air sampler (MAS-100 Eco, EMD Millipore, Merck) that drew a defined volume of room air over an agar plate positioned in the air sampling unit under the air-permeable lid. Every air sampling in this study was performed at a rate of 100 L per minute for 10 minutes. This is the method used in clean room and operating theatre air quality assessment in government health settings in Western Australia. The device used an Anderson sampler principle to draw air at a constant rate pre-set by the operator onto a 90 mm diameter Petri dish containing agar culture medium, after an initial timed delay to allow the operator to withdraw from its vicinity. The lid surfaces were cleaned with 70% isopropyl alcohol before and after each use in accordance with the manufacturer’s instructions.

Two types of media were used; 5% horse blood agar (HBA) and MacConkey agar (MAC) (both supplied by PathWest Laboratory Medicine WA, Mt Claremont WA, Australia). HBA was included as a non-selective medium intended to allow the growth of any of the three target bacteria. MAC was included to minimize the growth of background non-target microorganisms (predicted to be mostly fungi or Gram positive bacteria) while still allowing the growth ofB. thailandensis orK. pneumoniae. Samples taken on HBA whileS. pneumoniae was being used in the research laboratory were incubated in the presence of 5% CO 2 . MAC plates were never incubated in CO 2 . All plates were incubated aerobically at 35°C. Colony forming units on both types of solid media were recorded after 24 hr incubation and expressed as CFU/1000 L air. Positive growth controls to confirm the ability of each of the three target bacteria to grow on the media under the chosen incubation conditions were performed.

Air was sampled for identical periods, volumes and locations, onto both agar media on each occasion. Air sampling was conducted before, during and at the end of a day to give a range of times reflecting different levels of research laboratory use and occupation. The same pattern of sampling was conducted for comparison in the clinical bacteriology laboratory. Two main sampling sites were used in the research laboratory (see sampling sites 1 and 2,Supplementary Figure 1). The second of these; sample site 2, was immediately adjacent to the sample introduction port of the flow cytometer between the cytometer and its operator. For comparison, sampling site 1 was on the preparation bench behind the operator approximately 2.5 m from the flow cytometer where 1 ml samples from bacterial cultures (generally 20–30 ml in trypticase soya broth or Mueller Hinton broth) were washed by centrifugation, mixed, diluted and further handled prior to analysis on the flow cytometer. In general, samples analyzed on the cytometer contained approximately 106 bacteria/ml or less. During data acquisition for FAST assays, up to 12 samples were analyzed per bacterium. Sample acquisition halted after 1–3 minutes, and each FAST sample was acquired in technical triplicate.

Air sampling was conducted at two sites in the clinical laboratory; the open bench where wound swabs were plated directly onto agar, and adjacent to the rotary plating device used to inoculate agar plates with bacterial suspensions for disk diffusion antimicrobial susceptibility tests (see sites 4 and 5,Supplementary Figure 1).

In the research laboratory, further sets of air samples were collected on multiple occasions after a detergent and ethyl alcohol interventional clean of all laboratory surfaces, and replacement of laboratory gowns. Interventional cleaning was performed late on a Friday and post-clean air samples were acquired beginning the following Monday morning. Cleaning practices in the clinical laboratory were not changed during the course of this study.

Equipment and bench surfaces. On completion of repeated air sampling over one month, surface swabs were collected from the external flow cytometer housing above, below and around the flow sample introduction port, the nearby open bench and the preparation bench opposite before any interventional cleaning had occurred. Swabs (peel pouch Dryswab™ catalogue number MW112; Medical Wire and Equipment Company, Corsham, Wiltshire, England) were immersed in sterile 0.9% normal saline before rubbing vigorously over a 2.5 cm diameter circular area for 1.5 minutes in a spiral motion beginning at the centre and rotating the swab continuously. Swabs were inoculated onto HBA first and then MAC using opposite sides of the swab for each plate. Swabs were collected at each site on at least two occasions. Incubation conditions were as previously described for air samples. Colony forming units on both types of solid media were recorded after 24 hr incubation at 35°C and expressed as CFU/swab.

Bacterial identification. All bacteria growing on MAC were identified using the clinical bacteriology laboratory’s identification protocol. In short, after macroscopic examination and discretionary Gram stain analysis, definitive identification was by matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF) from an extended mass spectrometer profile library applying thresholds of 1.80 and 2.00 as the acceptable lower limits for genus and species level identifications, respectively. Initial borderline identification was repeated on the same sample on the stainless steel target with repeated MALDI-TOF analysis. Potential clinically significant isolates known to be problematic by MALDI-TOF or showing borderline acceptable identification were then subject to supplementary methods such as substrate utilisation panels (e.g. API20E, BioMerieux, France). Microbial growth on HBA was frequently heavy making it difficult to isolate and identify all colonies. Consequently, only the six commonest colony types were identified plus any that resembled the three target bacteria being interrogated by flow cytometry. Due to the selective nature of MAC, fewer colonies occurred and attempts were made to identify all isolates growing on MAC.

Statistical analysis. Column statistics, Chi squared test and non-parametric tests (Mann-Whitney U test) were conducted with Prism statistical software (Prism 6.0, GraphPad, San Diego, CA).