key: cord-0256565-oln1ujii
authors: Kalaiselvan, Parthasarathi; Yasir, Muhammad; Willcox, Mark; Vijay, Ajay Kumar
title: Efficacy of anti-microbial gel vapours against aerosolised coronavirus, bacteria, and fungi
date: 2021-10-29
journal: bioRxiv
DOI: 10.1101/2021.10.27.466182
sha: 9923cf9380d940422f4bc9260eb12b055c0dc432
doc_id: 256565
cord_uid: oln1ujii

Background The urban population spends up to 90% of their time indoors. The indoor environment harbours a diverse microbial population including viruses, bacteria, and fungi. Pathogens present in the indoor environment can be transmitted to humans through aerosols. Aim This study evaluated the efficacy of an antimicrobial gel containing a mix of essential oils against aerosols of bacteria, fungi, and coronavirus. Methods The antimicrobial gel was allowed to vapourize inside a glass chamber for 10 or 20 minutes. Microbial aerosols of Escerichia coli, Aspergillus flavus spores or murine hepatitis virus MHV 1, a surrogate of SARS CoV-2 was passed through the gel vapours and then collected on a 6-stage Andersen sampler. The number of viable microbes present in the aerosols collected in the different stages were enumerated and compared to number of viable microbes in control microbial aerosols that were not exposed to the gel vapours. Results Vaporizing the antimicrobial gel for 10 and 20 minutes resulted in a 48% (p = 0.002 Vs. control) and 53% (p = 0.001 Vs. control) reduction in the number of MHV-1 in the aerosols, respectively. The antimicrobial gel vaporised for 10 minutes, reduced the number of viable E. coli by 51% (p = 0.032 Vs. control) and Aspergillus flavus spores by 72% (p=0.008 Vs. control) in the aerosols. Conclusions The antimicrobial gel may be able to reduce aerosol transmission of microbes.

The majority of the urban population spend up to 90% of their time indoors [1, 2] . The 1 indoor environment harbours a diverse microbial population including viruses, bacteria, 2 fungi and protozoa [3] [4] [5] [6] that is referred to as the indoor microbiome. A major 3 component of indoor microbiome are endogenous microbes shed by human and 4 animal occupants with a minor constituent being the transient microbiota of external 5 9 filtration efficiency (BFE) test rig (CH Technologies, Westwood, NJ, USA) was used to 82 produce viral aerosols (Figure 1 ). The antimicrobial gel (10 g) was removed from its 83 container and allowed to vaporise into the glass aerosol chamber for 10 minutes or 20 84 minutes prior to the introduction of the virus. The viral inoculum (50 µL; 1.0 × 10 6 85 PFU/mL) was aerosolized using a continuous drive syringe pump through a nebulizer 86 with an airflow of 28.3 L/min for one minute and allowed to interact with vapours of the 87 antimicrobial gel as they passed through the glass tube. The size of the aerosols 88 produced was approximately 3.0 ± 0.3 µm and these travelled through the glass 89 aerosol chamber into an Anderson sieve sampler and were collected by flowing past 90 2% (w/v) agar plates. The largest (7 µm) sized aerosols were captured on the agar 91 plate at the top of the Anderson sieve and the smallest (0.65 µm) on the agar plate at 92 the bottom of the device. After one minute, the airflow was stopped to cease aerosol 93 generation, and the vacuum pump was run for further one minute to collect any 94 residual aerosols from the glass chamber. Following this, agar plates were flooded with 95 1.5 ml of either 20% BSA in DMEM (neutralised samples) or DMEM alone (non-96 neutralised samples), and viruses were carefully removed using a sterile cell scrapper. 97

Aliquots (100 µL) from each plate were placed in duplicate on A9 cells in 12-well cell 98 culture plates to culture any infectious viruses. The culture conditions were as 99 10 described above. Control runs were performed at the beginning of each experiment 100

prior to the addition of the gel in the glass aerosol chamber to collect infectious viruses 101 so that any reduction in the number of infectious viruses could be calculated as a 102 percentage of this control. Test and control runs were conducted in duplicate and 103 repeated twice. 104

The anti-bacterial activity of the gel vapourised for 10 minutes against E. coli and its 106 sporicidal activity against A. flavus spores was determined using a similar method as 107 described for MHV-1, except using 50 µL of E. coli or A. flavus spores (1 × 10 4 108 CFU/mL). Bacteria were collected on agar plates composed of tryptic soy agar (TSA; 109 BD, Macquarie Park, NSW, Australia) alone or containing TSA and the neutralizers 110

Tween® 80 (5 g/L) and lecithin (7 g/L). Fungal spores were collected on SDA plates 111 alone or containing the same neutralizers. The numbers of viable cells from each of the 112 6 plates in the Anderson sieve collector were enumerated following incubation at 37 ºC 113 for 24 hours for bacteria and at 25 ºC for 72 hours for fungal spores. Control runs were 114 conducted prior to the addition of the gel in the glass aerosol chamber to collect viable 115 bacteria, and fungal spores. Test and control runs were performed in duplicate and 116 repeated twice. The percentage of cells remaining viable after passage through the gel 117 11 vapours was calculated by comparing numbers in the absence (control) and presence 118 (test) of the gel vapours. Table I ). The smaller quantity (25 mg) of the gel reduced the numbers of 134 coronavirus by 98.6% after 30 minutes of incubation compared to control (p < 0.001). 135

The antimicrobial gel vapours were active against MHV-1 aerosols. The majority of the 137 viral particles travelled in aerosols of 3.30 to 0.65 µm in the absence of the gel ( Figure  138 2A) with most viral particles travelling in the 2.10 and 1.10 µm aerosols ( Figure 2A ). 139

After allowing the gel to vaporize in the chamber for 10 minutes, the numbers of viral 140 particles that were able to infect the mouse cells were reduced for most aerosol sizes, 141 with a significant reduction of 67% in the 1.10 µm aerosol (p = 0.011; Figure 2A) . A 142 slightly greater reduction of 78% was produced in the 2.10 µm aerosols compared to 143 the controls when the gel was allowed to vaporize for 20 minutes (p = 0.011; Figure  144 13 2B). Overall, exposure of MHV-1 aerosols to the antimicrobial gel vapours (vaporized 145 for 10 minutes) resulted a significant 48% reduction of all the aerosols sizes compared 146 to untreated control (p = 0.002; Table II ). Allowing the antimicrobial gel to vaporize for 147 20 minutes, resulted in a 53% reduction in the number of viable aerosolized viral 148 particles of all sizes compared to control (p = 0.001; Table II ). Following neutralization 149 with 20% BSA, the activity of the antimicrobial gel was slightly but not significantly (p = 150 0.078; Table II ) reduced, resulting in a 33% reduction in the viability of viral aerosols 151 compared to control (p = 0.001; Table II) . 152

The antimicrobial gel in vaporized form was active against aerosols of E. coli. In the 154 absence of antimicrobial gel, this bacterium mostly travelled in aerosol particle sizes 155 between 3.30 to 1.10 µm ( Figure 3 ). Overall, the antimicrobial gel produced a reduction 156 in bacterial viability of 29% (p = 0.018) when neutralised during bacterial growth and 157 51% (p = 0.032) when not neutralised during bacterial growth (Table III) 

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