Sampling sites and sample types
Sampling of bioaerosols and surface swabs was performed at workplaces in five different wastewater treatment plants (A, B, C, D, and E – see Tab. 1). All investigated plants had a minimum capacity of 60,000 m3 of wastewater per day and treated municipal and hospital sewage using mechanical-biological technology. All tested workplaces were located indoors and at all of them the treatment processes were open or only partially hermetically sealed. Additionally in this study, the influent and effluent samples were also microbiologically examined. All samples were taken during normal operating hours after obtaining the appropriate permits from the authorities of investigated WWTPs.
Bioaerosol sampling
In this study, two different instruments were used to collect air samples: Coriolis®μ impinger (Bertin Technologies, St-Quentin-en-Yvelines, France) and MAS-100NT impactor (MBV AG, Stäfa, Switzerland) [38-39]. During the measurements, both samplers were placed at a height of 1–1.5 m above the floor level to simulate aspiration from the human breathing zone [40] and at least 1 m apart to avoid any interference between them. In total, 52 bioaerosol samples (26 Coriolis®μ and 26 MAS-100NT, respectively) were collected at the following sampling sites: wastewater pumping section (4 and 4), screens section (10 and 10), grit chamber (4 and 4), and dewatering and thickening sludge section (8 and 8) (Table 1).
A cyclone-based Coriolis®μ impinger samples airborne particles into a liquid medium. Each time, the air samples were collected for 10 min at a flow rate of 200 L/min using sterile sampling cones filled with 15 mL of universal viral transport medium (VTM) (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) [41-42]. After each sampling session, the external and internal surfaces of both the impinger inlet and aspiration tube were cleaned and disinfected with isopropyl alcohol, the cone removed from the sampler and the sample stored in 0–4 °C until further analysis.
A single-stage MAS-100NT impactor operates by aspirating the air through a 400-hole perforated inlet plate onto a Petri dish containing biological collection media. Each time, the air samples were collected for 20 min at a flow rate of 100 L/min on standard Petri dishes filled with the bi-phase medium consisting of solid phase mycoplasma base agar (MBA, Oxoid Ltd., Basingstoke, UK) covered with thin layer liquid-phase VTM to maximize the potential of viral particle recovery [39, 43]. After each sampling session, an impactor inlet was cleaned and disinfected with isopropyl alcohol. The air samples were collected on standard Petri dishes filled with the bi-phase medium consisting of solid phase mycoplasma base agar (MBA, Oxoid Ltd., Basingstoke, UK) covered with thin layer liquid-phase VTM to maximize the potential of viral particle recovery [39, 43]. After collection, the samples were transported to laboratory within 12 h where they were stored in −80 °C until further analysis [44].
Surface swab sampling
In total, 54 swab samples were collected from stainless steel and plastic surfaces (machine valves, machine handles, hatch handles, machine controllers, handrails) with sterile polyester fiber-tipped swabs (Deltaswab PurFlock Ultra ViCUM, Deltalab, Barcelona, Spain) pwetted in 0.9% saline solution, which ensures the most effective recovery of viruses from nonporous fomites [45-46] (Table 1).
Table 1. Description of studied wastewater treatment plant (WWTP) sites and number and characteristics of surface swab samples.
WWTP
|
Site
|
|
Performed tasks
|
A, D
|
Wastewater pumping section
|
|
Pumping wastewater into treatment system
|
A, B, C, D, E
|
Screens section
|
|
Removal of big objects, screens storage
|
B, C
|
Grit chamber section
|
|
Removal of heavier solid particles with aeration, grease traps
|
B, C, D, E
|
Dewatering/thickening sludge section
|
|
Dewatering and thickening of sludge intended for incineration
|
|
|
|
|
Workplace
|
Number of samples
|
ST
|
P
|
Wastewater pumping section
|
8
|
6
|
Screens section
|
10
|
7
|
Grit chamber section
|
5
|
4
|
Dewatering/thickening sludge section
|
8
|
6
|
Total
|
31
|
23
|
Notes: ST – steel, P – plastic.
Wastewater samples
Fifteen wastewater influent and the same number of effluent samples were collected (each of them into a sterile 1000 mL glass container) and kept in 4 °C for less than 24 h until further analysis.
Laboratory analysis
Aerosol, surface swab and wastewater samples
All liquid media with air samples were concentrated by ultrafiltration using Amicon® Ultra-15 (molecular weight cut-off 30 kDa) centrifugal filter device (Merck Millipore Ltd., Livingston, UK) at 3200 × g for 20 min in 4 °C [41, 47]. Centrifugal concentration step was repeated until the entire volume of the sample passed through the filter. The concentrated samples (400 µL) were intended for further analysis. In turn, the swab shafts of swab samples were cut off, then placed into 400 µL of 1 × PBS (pH = 7.2) and vortexed thoroughly using a programmable rotator-mixer (Multi RS-60, Biosan, Riga, Latvia) at 800 rpm for 15 min. Influent and effluent wastewater samples were centrifuged at 4500 × g for 5 min in 4 °C and each obtained supernatant was concentrated as described above [47].
PMA dye ptreatment
All processed samples were divided into two equal aliquots (200 µL). The first one was intended for direct viral DNA/RNA isolation, the second one for PMA dye ptreatment allowing detection of potentially infectious viral particles. In this case, the samples were treated with PMAxx™ Dye (20 mM in H2O; Biotium, Inc., Hayward, USA) for a final concentration of 60 µM [48]. Tubes were gently mixed by inverting several times and then incubated in the dark for 15 min at room temperature with rotation at 200 rpm. The treated samples were exposed to 40 W LED light with a wavelength of 460 nm for 15 min using a photo-activation system (PMA-Lite™ LED Photolysis Device; Biotum Inc.).
Viral DNA/RNA extraction
The extraction of viral DNA/RNA from all samples was carried out with Kogene Power pp Viral DNA/RNA Extraction Kit CE-IVD (Kogene Biotech, South Korea) according to the manufacturer’s instructions to produce a final volume of 45 μL. Obtained RNA/DNA samples were stored in −20 °C until further analysis.
Quantitative PCR/Reverse-Transcription quantitative PCR (qPCR/RT-qPCR) and viability quantitative PCR/viability Reverse-Transcription quantitative PCR (v-qPCR/v-RT-qPCR) assays
Both qPCR/v-qPCR (for DNA viruses) and RT-qPCR/v-RT-qPCR (for RNA viruses) were performed using CFX96 real-time PCR thermocycler (Bio-Rad, Hercules, USA). The detection of AdVs, HBoV, RoVs, NoVs, IAV, and SARS-CoV-2 were carried out with Adenovirus, Bocavirus, Rotavirus, Norovirus (GI and GII), Influenza A, and SARS-CoV-2 VIASURE Real Time PCR Detection Kits (all: CerTest Biotec S.L., Zaragoza, Spain), respectively, according to procedures recommended by the manufacturer. The applied PCR kits have a detection limit of ≥ 10 RNA/DNA copies per reaction.
The target genes employed for PCR-based detection and identification of viruses repsent conserved regions with the hexon gene for AdVs, the NSP3 gene for RoVs, the ORF1-ORF2 junction for NoV genogroup I (GI) and NoV genogroup II (GII), the M1 gene for IAV, the ORF1ab and N genes for SARS-CoV-2.
The cycling conditions for DNA viruses were as follows: polymerase activation at 95 °C for 2 min, then 45 cycles of denaturation at 95 °C for 10 s, and annealing at 60 °C for 50 s. In case of RNA viruses, the reverse transcription at 45 °C for 15 min was followed by initial denaturation at 95 °C for 2 min, then 45 cycles of denaturation at 95 °C for 10 s, and annealing at 60 °C for 50 s. According to the manufacturer’s procedure, the fluorogenic data were collected through the FAM, ROX, and HEX channels. Both negative and positive controls, purchased from CerTest Biotec, were included in each run. All samples were tested in duplicates.
All qPCR/RT-qPCR and v-qPCR/v-RT-qPCR data were collected and quantification cycles (Cq) were calculated using CFX96 manager software (Bio-Rad). According to the manufacturer’s instruction, the samples with Cq ≤ 40 for AdVs, HBoV, NoV GI, NoV GII, RoVs, and IAV as well as with Cq ≤ 38 for SARS-CoV-2 were considered as positive. In case of SARS-CoV-2, if only N gene target was positive, the interptation was psumably positive for SARS-CoV-2 and the differentiation of SARS-CoV-2 from other coronaviruses, including animal ones, requires further analysis. The negative samples and the samples with Cq > 40 were reanalyzed after 10-fold dilution to evaluate the possible psence of inhibitors. Quantification analyses were performed based on standard curves, obtained by amplification of positive control 10-fold dilutions (standard from 1 × 101 to 1 × 107 gene copies/reaction), and log RNA/DNA copies were plotted against Cq value. All standard curves had efficiencies between 90% and 110% and r2 above 0.98.
To minimize the potential contamination, all analytical steps were performed in separate rooms, including RNA/DNA isolation, pparation of reagents, sample pparation, and amplification. All analyzes were carried out using the sterile RNase/DNase-free filter pipette tips only. The obtained results were expssed as the number of viral genome copies per 1 m3 of the air (gc/m3), per 100 cm2 of tested surfaces (gc/100 cm2), and per 1 L of influent and effluent wastewater (gc/L).
Temperature and relative humidity
During sampling, the temperature and relative humidity of the air were measured using portable thermo-hygrometer (Omniport 20; E+E Elektronik GmbH, Engerwitzdorf, Austria).
Statistical analysis
The obtained results were statistically analyzed with Shapiro-Wilk, Fisher Exact, Kruskal-Wallis and Mann-Whitney test as well as Spearman’s rank correlation coefficient using STATISTICA data analysis software system, version 7.1 (StatSoft Inc., Tulsa, USA). Probability values at p below 0.05 were considered statistically significant.