Enviro-Biotics® inhibition of SARS-CoV2 virus infectivity.

  Francesco Piacente1, Laura Sturla1, Mirko Magnone1, Katia Cortese2, Cristina Gagliani2, Moti Rebhun3, Hila Weissman3, Rotem Link3, Martin H. Bluth3,4,5

1. ME.S.section of Biochemistry, University of Genova, 16132 Genova; Italy
2. ME.S. section of Human Anatomy, University of Genova, 16132 Genova, Italy
3. Department of Pathology, Maimonides Medical Center, Brooklyn, NY, USA
4. Wayne State University School of Medicine, Detroit, MI, USA

*Correspondence to: Martin H. Bluth, MD, PhD Professor of Pathology

Wayne State University School of Medicine toxdocs@gmail.com

Abstract

The covid-19 pandemic has affected the world in a variety of negative ways. While vaccination, therapeutics, and social distancing are critical for controlling viral spread, anti-viral disinfectant approaches remain less effective due to caustic skin responses of some chemical agents, repetitive application requirements, in addition to ineffective surface coverage realities and consumables costs, storage, and handling.

Recent effectiveness of Enviro-Biotics® demonstrates a paradigm shift in controlling infectious organisms in the environment. Application of Enviro-Biotics® to covid-19 infection may prove efficacious in reducing viral infectivity. In order to test the antiviral efficiency of Enviro-Biotics®, two Covid-19 virus constructs were generated, expressing the Spike SARS-CoV2 S protein and, in parallel green fluorescent protein (EGFP) or luciferase enzyme (LUC). Spike S protein presence on Covid-19 produced in HEK293T was confirmed by Western blot analysis; moreover, transmission electron microscopy analysis on the generated viruses confirmed that the size of the viral particles (131 nm) was similar to that of the natural virus SARS-CoV2.

Caco-2 cells were infected with viruses and incubated in the presence or absence of Enviro- Biotics® containing Bacillus spores and assessed for viral infectivity at different time points. Incubation of Bacillus species contained in Enviro-Biotics® decreased infectivity of both GFP and LUC Covid-19 constructs (>99% after three hours, p<0.001). Similar reductions in viral infectivity were observed after Enviro-Biotics® treatment via (1) confocal microscopy analysis of fluorescent Caco-2 cells incubated with EGFP-Covid-19 and (2) crystal violet quantitation after puromycin-induced colony formation.

In conclusion, Bacillus species contained in Enviro-Biotics® decreased viral infectivity of a Covid-19 virus model. This approach provides a unique, low-cost, effective, sustainable modality for the reduction of covid-19 infection and disease transmission.

Introduction

Coronavirus disease 2019 (COVID-19), caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the cause of a pandemic that has infected over 180 million people worldwide with a mortality of over 4 million people [https://www.worldometers.info/coronavirus/]. Despite improvements in public health conduct, diagnosis, and treatment, there is a dire need to optimize all resources to minimize the spread of the virus. While social distancing, wearing masks, and hand washing remain a staple of antiviral conduct, viral spread continues around the globe. Although effective, the use of face masks has limitations, including the type of masks worn (1), maintenance of a proper oropharyngeal seal (2), exacerbating chronic breathing difficulties (3,4), and questionable effective use in select populations such as children (5,6).

Other than vaccination strategies, additional approaches to stave viral transmission include antiseptic applications, including alcohol, detergents, iodine, sodium hypochlorite (bleach), hydrogen peroxide, chlorine dioxide, glutaraldehyde, ultraviolet irradiation, and air purification (7,8). However, decontamination approaches using detergents require continuous cycles of applications fostering irritation or disease manifestation of select agents to sensitive individuals (9,10) and resultant increased costs related to hazardous materials, shipping, storage, and frequent reapplication requirements. Furthermore, constant hand washing, a staple of anti-viral transmission reduction practice, has been associated with increased incidence of contact dermatitis and other dermatologic conditions (11,12). Clearly, there remains a need for alternative interventions.

Enviro-Biotics® represents a newly emerging field of environmental probiotics where select naturally occurring innocuous microorganisms such as Bacillus species (i.e., B. subtilis, B. pumilus, and B. megaterium) provide a microbiome for the environment (13). Enviro-Biotics® are able to colonize surfaces to which they are applied and competitively exclude other harmful organisms by counteracting the proliferation and survival of such infectious agents, thereby providing a balanced, hygienically stable environment (14). Furthermore, Enviro-Biotics® are dispensable through aerosolization and are able to survive on and colonize non-biological surfaces. This provides better “coverage” against infectious agents compared with a conventional towel or pad-based distribution of detergents that can be excluded in cracks and uneven surfaces. Such administration can offer effect longevity with minimal product consumption and less cost. Enviro-Biotics® have been shown to be effective in sanitizing hospital environments and providing a “green” alternative to common chemical disinfectants. Previous studies have shown the beneficial effects of Enviro- Biotics® against S. aureus, P. aeruginosa, K. pneumoniae, E. faecalis, C. Difficile, A. baumannii and

1. Albicans, as representative organisms responsible for hospital-acquired infections (HAI) (14,15).

Those studies also showed that Enviro-Biotics® interventions were up to 80% more effective than conventional chemical disinfectants in reducing potentially pathogenic microbial loads. Furthermore, despite the continuous potential for healthcare-associated infections (HAI) recontamination (from visitors, patients, and staff), the observed reduction in microbial load was stably maintained at low levels post-application (14).

The role of Enviro-Biotics® in viral diseases has also demonstrated beneficial effects in a murine model of respiratory syncytial virus (16) by increasing anti-viral effector molecules and that specific peptidoglycans (PG) from Bacillus subtilis reduced viral infection greater than 10,000-fold compared with PG from other bacterial species (17).

Here, we assessed the ability of Enviro-Biotics® on a Covid-19 analogue to reduce viral infectivity as a potential novel intervention against Covid-19.

METHODS

 Cell culture

 HEK293T cells (American Type Culture Collection, Rockville, MD, USA) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Merck, Milan, Italy) supplemented with 10% fetal bovine serum (Merck), 2mM L-glutamine and 1% penicillin/streptomycin solution (Merck) at 5% CO2 and 37 °C.

Caco-2 cells (Cell factory, San Martino Hospital, Genova, Italy) were cultured in Eagle’s Minimum Essential Medium (Merck) supplemented with 20% fetal bovine serum (Merck), 2mM L-glutamine and 1% penicillin/streptomycin solution (Merck) at 5% CO2 and 37 °C.

Lentiviral Covid-19 production

Two different typed lentivirus constructs packaging the Spike SARS-CoV2 protein was generated via co-transfecting HEK293T with the following vectors: for the production of typed lentivirus expressing Spike S protein and EGFP protein (EGFP-Covid-19) was used incorporating the vectors pRP-CMV-SARS-CoV2 S protein, pLV-Puro-CMV-EGFP (Vector Builder, Chicago, IL, USA) and the packaging plasmid pCMV Delta R8.2 encoding Gag-Pol, Tat, and Rev (Addgene, MA,.USA). For the production of typed lentivirus expressing Spike S protein and luciferase enzyme (LUC-Covid-19), all the vectors were the same as the EGFP- Covid except for the vector pLV-Puro-CMV-EGFP which was replaced by the pLV-Puro- EF1A-Luciferase vector (Vector Builder). Briefly, HEK293T cells (300,000 cells on 6-cm Petri) were seeded. After 24 h, a mix of lentiviral vectors (1 µg of pRP-CMV-SARS-CoV2 S, 0.5 µg of pLV- Puro-CMV-EGFP or pLV-Puro-EF1A-Luciferase, 0,9 µg of pCMV Delta R8.2) was incubated for 20 min in 400 µl of DMEM containing 8 µl of Transit-293 Transfection Reagent (Mirus, Tema Ricerca, Milan, Italy) and added to HEK293T cells in the presence of DMEM without penicillin-streptomycin to promote cells transfection. 24 h after transfection, the HEK293T medium was replaced by complete DMEM to boost the viral production. The supernatant containing lentiviral particles was harvested at 48 h (first cycle) and 72 h (second cycle) after transfection, filtered with a 0.45-μm-diameter filter and purified following the protocol described in Kutner, R.H. et al. (18). Briefly 12 ml of supernatant was deposited on a cushion containing 10% sucrose in 50 mM Tris-HCl pH 7.4, 100 mM NaCl and 0.5 mM EDTA and centrifuged for 2 h at 100,000xg at 4°C. After centrifugation supernatant was discharged and the pellet containing the virus was washed once with saline phosphate buffer (PBS) and resuspended in 100 µl of PBS.

Covid-19 titration

 Titration of Covid-19 was performed in order to ascertain the number of viral particles per volume employed in each sample of those utilized in assays. Briefly, the number of transduction units per milliliter (TU/ml) present in Covid-19 preparation was calculated as the number of fluorescent Caco-2 cells in the wells of each dilution done and detected by FACS analysis (Calibur, Beckman Coulter, U.S.A.). The TU/ml was calculated in the wells where the number of fluorescent cells was less than the 30% of the total number of cells. The values of TU/ml of each dilution were averaged to result the final Covid19 concentration which was 6.9 x 105 TU/ml in each batch produced.

Western blot analysis.

 Expression of the Sars-CoV2 spike protein on the generated Covid-19 construct, after its production in transfected HEK293T cells, was evaluated by Western blot analysis Briefly, 30 ml of the produced and purified virus (with or without Spike S protein expression) was added with 10 ul of the loading buffer solution as previously described (19) and boiled 10 min. Then the samples were subjected to 8% SDS-PAGE followed by Western blot on a nitrocellulose membrane (Bio-Rad, Milan, Italy). The Spike S protein transferred onto the membrane after Western blot was detected by a specific primary antibody diluted 1:1000 (code #PA5112048, Thermo Scientific, U.S.A). Goat anti- Rabbit IgG HRP conjugate diluted 1:5000 was used to detect the primary antibody. The chemiluminescence signal of the secondary antibody was acquired by Chemi-Doc System (Bio-Rad) after membrane incubation with ECL™ Western blotting detection reagent (GE-Healthcare Life Sciences, Milan, Italy).

Transmission Electron Microscopic (TEM) analysis on Covid-19

TEM analysis on isolated lentiviral Covid-19 preparations was performed as follows. The viral preparations were resuspended in 20 μL PBS (pH 7.4) and fixed by adding an equal

volume of 2% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4).  viruses were then adsorbed for 10 minutes to formvar-carbon coated copper grids by floating the grids on five μl drops of parafilm. Subsequently, grids with adhered viruses were rinsed in PBS and negatively stained with 2% uranyl acetate for 5 minutes at room temperature. Stained grids were embedded in 2.5% methylcellulose for improved preservation and air-dried before the examination. Electron micrographs were taken at Hitachi TEM microscope (HT7800 series, Tokyo, Japan) equipped with Megaview 3 digital camera and Radius software (EMSIS, Germany).

Covid-19 residual infectivity test in the presence of Enviro-Biotics®

 The interaction between the Enviro-Biotics® (BetterAir, Israel), containing Bacillus spores, and Covid-19 was tested using a sterile plastic plate cover containing 96 wells. The experiments were carried out in a switched-off, hermetically sealed biological hood with an inner volume of 0.42 m3 at a temperature of 25 °C and at 80% RH.

Briefly, the Enviro-Biotics® Bacillus spores, contained in the commercial Betterair’s spray can, were sprayed over the well covers until reaching a volume of 30 µl/well, followed by adding 10 µl of saline buffer to each well. As a negative control, 40 µl of saline buffer was added in dedicated wells in the absence of spores. The plates were pre-incubated in a negative flow hood for 4 h.

In the first experiment, 30 µl of EGFP-Covid-19 (containing approximately 20.7 x 103 Covid-19 particles according to the titration performed) were added to each well, yielding a final volume of 70 µl/well. Five different bacteria-virus incubation times were tested: 0 min, 15 min, 30 min, one h, and three h in triplicate.

At the end of each time point incubation, samples were collected from the plastic well surface, transferred to a 0.2 ml tube, centrifuged 3,500xɡ for 8 minutes, and the recovered supernatant was filtered (0.2µm absolute filter) to eliminate bacterial debris.

Immediately after the filtration, the recovered supernatant of each sample was transferred to 96-well plates where Caco-2 cells were seeded (15,000 cells/well, seeded 24 hours prior to the stimulation). The Caco-2 cells were incubated with the recovered supernatant for 72 h; at the end of such incubation, the Caco-2 cells (deriving from each different experimental time point) were measured for their fluorescence by spectrofluorimeter (BMG Labtech, Clario star plus) to quantify the residual infectivity of EGFP-Covid-19 after its incubation or not with Enviro-Biotics®. After this fluorimetric detection, a qualitative analysis acquisition by confocal fluorescence microscopy was performed on the same cells.

A second inactivation test on Covid-19 infectivity in the presence of the Enviro-Biotics® was performed by using LUC-Covid-19 (instead of EGFP-Covid-19) in order to validate the Bacillus virucidal efficiency with a different system of detection. All the experimental conditions were identical to the EGFP-Covid-19 experiment except for (1) the incubation time on Caco- 2 cells with the recovered supernatants that lasted 120 h and (2) the evaluation of the residual infectivity on the cells which was quantified by chemiluminescence acquisition as luciferase activity (BMG Labtech, Clario starplus).

Qualitative fluorescence confocal microscopy of infected Caco-2 cells

After 72 h of incubation of Caco-2 cells (15,000 cells/well seeded in a 96-well plate) with the supernatants deriving from the time 0 and time three h of the residual infectivity test with EGFP- Covid-19 in the presence or not of Enviro-Biotics®, qualitative images were obtained with a fluorescence confocal microscope.

Colony forming assay

 Colony forming assay was performed on the same Caco-2 cells incubated for 72 h with the supernatants recovered by the EGFP-Covid-19 inactivation experiment after the fluorimetric acquisition of the residual viral infectivity. Briefly, 2 mg/ml puromycin (Merck, U.S.A.) were added every 48 h for 12 days to Caco-2 cells. After 12 days, colonies were stained with a 0.5% crystal violet

solution. The difference in colonies amount between samples was detected by dissolving the dye with 2% SDS solution and quantified by spectrophotometric absorbance at 570 nm.

Covid-19 inactivation.

 The number of the active bacteria deriving from the Enviro-Biotics spores used in the experiments of Covid-19 inactivation was calculated by performing a colony bacterial growth on solid Agar medium (agar 15g/L, two g/L yeast extract, two g/L skim milk powder, 16 g/L glucose, five g/L sodium chloride). Briefly, the time zero incubation point of the experiment to evaluate the Covid-19 inactivation was centrifuged at 3,500xg for 8 min, and the pellet, containing the bacteria, was resuspended in 200 ul of PBS (bacteria stock suspension). Serial dilutions were made from the bacteria stock suspension (1:100, 1:10,000, and 1:1,000,000), and 250 mL of each dilution were spread in duplicate on a petri dish containing solid agar medium (composition: agar 15g/L + 2 g/L yeast extract, two g/L skim milk powder, 16 g/L glucose, five g/L sodium chloride). A petri dish with the agar medium alone was used as a control. After overnight incubation at 37°C, the colonies were counted. The count of the colonies was possible only at the dilution 1:1,000,000, where each colony was well separated from another in the whole petri surface. The final average amount of bacteria was calculated as CFU/mL.

RESULTS

Confirmation of Covid-19 S-protein expression and viral characterization.

The expression of SARS-CoV2 S-protein on the generated viruses after HEK293T cell transfection was confirmed by Western blot analysis on produced and purified virus lysates. As shown in Fig. 1, the produced Covid-19 expressed the S protein typical of the natural SARS-

CoV2, which was fully recognized with the specific anti-Spike protein antibody. Transmission electron microscopy (TEM) analysis on the generated viruses, both expressing EGFP and LUC, confirmed that the size of the viral particles was similar to that of the natural virus SARS-CoV2 (20) and that the Covid-19 obtained from HEK293T cell transfection was intact (Fig. 2).

Titration results

 A virus titration was performed to quantify the concentration of viral particles in the preparations used in all the experiments. The concentration of viral particles was expressed as transduction units per milliliter (TU/ml). FACS analyzed caCo-2 cells transduced with two-fold dilutions of EGFP-Covid-19 to detect the number of fluorescent cells in the wells of each dilution. The TU/ml was calculated in the wells where the number of fluorescent cells was less than 30% of the total (Fig. 3). The TU/ml resulted in 6.9 x 105, thus in each experimental point, the number of virus particles was 20.7 x 103.

Covid-19 inactivation by Enviro-Biotics® Bacillus cells

Enviro-Biotics® Bacillus spore suspension was applied to the tested surface four h prior to viral inoculation. The Bacillus cell concentration sprayed on the tested surface, after 4 hours of pre-incubation and prior to the Covid-19 addition, were counted resulting with an average value of

1.42 x 108 CFU/ml for each time point incubation (data not shown).

In the initial test, the EGFP-Covid-19 construct was used. Enviro-Biotics® Bacillus spores were able to induce a rapid inactivation of the viral particles: 67% of the EGFP viral particles were already neutralized after 15 min incubation with the Enviro-Biotics® when compared to the control (virus incubated with only saline buffer), and this percentage of inactivation reached 97.7% after three h incubation (Fig. 4; Table 1A). The Log reduction value, calculated for each incubation point as the difference between the Log10 of the control’s fluorescence (virus alone) and the

Log10 of the fluorescence measured in the sample where the virus was in the presence of the spores was 0.5 and 1.6 in the presence of EGFP-Covid-19 at 15 min and three h, respectively (Table 1A). A qualitative fluorescence confocal microscopy acquisition confirmed the almost complete virus inactivation in 3 h (Fig. 5). Moreover, after spectrofluorimetric detection, the addition of puromycin to each well of the Caco-2 host cells with regard to colony formation assessed an evaluation of the Bacillus cell-mediated virucidal efficiency. As shown in Fig. 6, the cells treated with the supernatants derived from the incubations where Covid-19 was incubated in the presence of Bacillus cells strongly decreased Covid-19 infected colony formation and survival. Indeed, the Crystal violet quantitation by spectrophotometric absorbance at 570 nm of the dissolved crystals, corresponding to the colonies formed in the colony-forming assay (Fig. 7), was in line with the fluorescence values, representing virus inactivation reported in Figure 4.

Additional confirmation of Bacillus containing Enviro-Biotics® antiviral efficiency was obtained by challenging Caco-2 cells with a different Covid-19 construct where a LUC replaced EGFP. Similar to the experiment that demonstrated the inactivation of EGFP-Covid-19, the LUC-Covid-19 infected cells were also found susceptible to the Enviro-Biotics® Bacillus-containing cells. The % of inactivation mediated by Enviro-Biotics® Bacillus spores, when detected as chemiluminescence acquisition, was 70% after 15 min incubation and approximately 99% after three h (Fig. 8; Table 1B), practically the same results obtained with the EGFP-virus construct studies. Further, the calculated Log reduction at 15 min and three h was 0.8 and 1.85, respectively (Table 1B). The results obtained in these alternate LUC virus construct studies confirmed the efficacy of Enviro-Biotics® inactivation effects observed in the EGFP virus construct studies, providing additional support for the use of Enviro-Biotics as an antiviral modality by two uniquely different approaches.

Discussion

The present studies demonstrate that Enviro-Biotics® may provide protection against covid-19.

This is important because current ways to decrease covid-19 viral transmission and infectivity, including social distancing, masking, and hand washing, are not completely effective or universally used. To this end, incorporating Enviro-Biotics® may provide an additional and necessary protection way in the arsenal to reduce viral transmission.

The following important Enviro-Biotics® characteristics stand out in that they (1) are stable, spore-forming bacilli that can be maintained in a variety of containers without attention to special temperature and/or hazardous material shipping and handling conditions, making them universally available compared with many chemical detergent compositions that require permits, personal protective equipment or special facilities, (2) can be dispensed via aerosolization to the majority of surfaces. This can be expanded by spraying in select environments that may be prone to infection propagation through enclosed spaces (automobiles, hotel rooms), or metered dispensation via programable units (concert venues, home heating/cooling vents), (3) can be dispensed via aerosolization which will provide more effective surface coverage than conventional foams and liquids when considering uneven surfaces such as those containing pores, cracks or spaces, (4) can actively compete with infectious organisms when present and remain dormant when infectious agents are no longer a threat, only to repopulate when a new infectious threat presents itself.

Such a way would be cost-effective, sustainable, re-populatable, environmentally friendly, and foster the longevity of protective effects providing a unique advantage to the healthcare marketplace compared with conventional interventions.

Various studies have demonstrated the beneficial effects of Enviro-Biotics®. LaFauci and colleagues (14) have employed Enviro-Biotics® containing various Bacillus species (i.e., B. subtilis, B. pumilus, B. megaterium) in their Probiotic Cleaning Hygiene System (PCHS). In those studies, surfaces (washbasin, floor, and desk) were seeded with infectious organisms, subsequently sanitized with Enviro-Biotics®, and assessed for recontamination. The addition of Enviro-Biotics® reduced HIA-related organisms (S. aureus, P. aeruginosa, K. pneumoniae, A. baumannii, E. faecalis and C. albicans) over 92% after 24 h. Additionally, field trials, to evaluate the efficacy of probiotics

to contain pathogens in a standard hospital environment (staff, patients, family), also demonstrated elimination of pathogenic bacteria after only six hours even when re-contaminated. This further demonstrates the durability of Enviro-Biotics® application over time, which is thought to be due to the stabilization of biofilms which continues to contain and reduce proliferation of pathogenic microorganisms (21). Studies from Vandini and colleagues (22) showed that application of a Bacillus containing Enviro-Biotics® solution in a hospital setting reduced the microbial load of S. aureus, Coliforms, Pseudomonas spp. and Candida spp., over 80% and maintained the antimicrobial effect over five days.

Despite the reported beneficial effects of Enviro-Biotics®, and the fact that Bacillus species are generally recognized as safe (GRAS) by the FDA (23), the question as to whether these applications are safe for humans in the clinical setting cannot be ignored. To this end, Caselli and colleagues

(15) investigated the impact of an Enviro-Biotics® Bacillus-based cleanser on pathogens in a healthcare setting. In addition, they assessed the ability of the Enviro-Biotics® cleanser-derived Bacilli to infect hospitalized patients. Not only did they report a decrease of antibiotic resistance genes in the contaminating microbial population, but the authors also found that HAI-positive patients in those studies were negative for Bacilli, suggesting that they can be safely used for pathogen reduction. Furthermore, their PCHS Enviro-Biotics® intervention showed a 33%-100% decrease of resistant strains to antibiotics, a 60% decrease in HAI antimicrobial drug consumption and a 75% reduction in the associated disease related costs (15). Tarricone and colleagues (13) have similarly reported that Enviro-Biotics® based use instead of conventional cleansers would avert 31,000 HAIs and 8500 antibiotic resistances over five years and reduce costs by 14 million euros ($16.8 million USD), where over 80% of those costs are for the treatment of resistant HAIs. Similarly, recent reports by Kim et al (24) have shown the beneficial effects of Enviro-Biotics® in animal asthma models by assessing safety, antimicrobial efficacy, and activity against airborne pathogens. In those studies, intranasal instillation and inhalation of Enviro-Biotics® (B. subtilis and

1. amyloliquefaciens) in an ovalbumin (OVA)-induced lung inflammation murine model reduced T

helper type 2 (Th2) cytokines at transcriptional and protein levels. Enviro-Biotics® decreased IL-4 and IL-5 mRNA expression and/or protein levels without altering IL-10 in OVA-induced lung inflammation in vivo emphasizing their immunomodulatory effects. Bacillus species metabolites including 2,3-butanediol, propylene glycol, malonic acid, 2-methylbutyric acid, lactic acid, subtilosin and aconitic acid were identified as possible anti-microbial anti-fungal agents, acting individually and/or via additive and synergistic effects to mediate the observed inhibitory processes. Application of Enviro-Biotics® is not limited to laboratory and healthcare-based institutions. Buildings that are subject to bacterial or mold growth and suffer from sick building syndrome (25) can cost hundreds of thousands of dollars to remedy by conventional contractors or be subject to demolition. In addition, office buildings and school systems can benefit from Enviro-Biotics® to maintain a hygienically stable work environment, decrease infectious disease transmission and reduce workdays or school days lost as well as litigious experiences as a result of workplace harms (26-28).

The application of Enviro-Biotics® in viral infection has also been reported. Hong and colleagues

• studied the effects of intranasal administration of Bacillus subtilis containing Enviro-Biotics® in a murine model of RSV. They found that Enviro-Biotics® administration induced expansion of alveolar macrophages in addition to activation and enhanced production of inflammatory cytokines, macrophage differentiation and a profound reduction of viral titers and RSV related lung injury, suggesting its use as a potential therapeutic approach against RSV.

Our studies investigated the effect of Bacillus species containing Enviro-Biotics® on coronavirus infectivity. We utilized a  coronavirus that expressed the wild-type coronavirus spike protein unique to CoV2. This virus was used to infect Caco-2 cells that contain the ACE2 receptor

• in the presence and absence of Enviro-Biotics®. We found that Enviro-Biotics® reduced covid infectivity >97% after three hours using both GFP and LUC reporter constructs and that puromycin induced colony formation was also reduced in the treatment group. These findings are in concert with other studies that assessed the effect of Enviro-Biotics® in other viral infections

including severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome CoV (MERS-CoV) systems (17). In those studies, peptidoglycan (PG) components from

1. subtilis reduced infection >10,000-fold, while PG from other bacterial species did not. The authors identified a cyclic lipopeptide – surfactin – that was responsible for the observed anti-viral effects and that surfactin treatment was efficacious against many enveloped viruses in vitro, including influenza A virus (strains H1N1 and H3N2), Zika, Dugbe, Nipah, Crimean-Congo hemorrhagic fever, Chikungunya, Una, Mayaro, and Ebola. Collectively, these results highlight remarkable anti-viral effects of Enviro-Biotics® and their potential to impact viral pathogenesis.

CONCLUSION

Enviro-Biotics® represent an emerging environmental probiotic application to the curtailment of infectious organisms. The results herein reported indicate the significant anti-viral activity of Bacillus species containing Enviro-Biotics® on a Covid-19 viral infection construct, having full homology with the natural SARS-CoV-2 virus, and the near eradication of infectivity in short time. These results, in conjunction with previously reported beneficial effects in various disease transmission systems, further edify the potential use of Enviro-Biotics® as a novel intervention to reduce SARS-CoV2 infection.

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Fig. 1

Fig. 1 A representative Western blot analysis, performed as described in Methods, on lysates of virus expressing or not the Spike S protein of Covid-19 used for all the tests. The Spike S was identified using a specific mAb (Thermo Scientific, U.S.A) protein on the lysate viruses samples.

Fig. 2

Fig. 2 A representative image of TEM acquisition of the produced Covid-19 (magnification 50000x; 100 Kv). Red arrows indicate the viral particles in the field of acquisition; the yellow box is a zoom image of a single  viral particle. The table summarizes the 2D dimension of Covid-19.

Fig.3

Fig. 3. FACS analysis of EGFP-Covid-19 titration. In panels A-C, a representative sample of non-transduced cells shows the cells totally negative for the presence of green fluorescence. In panels D-E, a representative sample of transduced cells with EGFP-Covid-19 shows a percentage of cells positive for green fluorescence.

Fig. 4

Fig. 4. Fluorimetric quantitation of EGFP-Covid-19 residual infectivity on Caco-2 cells detected as GFP fluorescence presented as Relative Fluorescence Units (RFU), as described in Methods. The background of auto-fluorescence deriving from untreated Caco-2 cells was subtracted for each experimental time point. Each bar is the mean±SD of a triplicate. P value was calculated by unpaired t-Test. ***p< 0.001

Fig. 5

Fig. 5. Fluorescence confocal microscope images (20X magnification) of Caco2 cells incubated for 72 h with the recovered supernatant of EGFP-Covid-19 test at time 0 (T0) and time three h (T3). Panels on the left represent the controls (supernatant of EGFP-Covid-19 alone); panels on the right represent the fluorescence of Caco-2 cells treated with the supernatants of EGFP-Covid-19 incubated with Enviro- Biotic® at T0 (Initiation of Bacillus–virus incubation) and at T3 of the residual infectivity test.

Fig. 6

Fig. 6. Colony forming assay, as described in Methods, of Caco-2 cells incubated for 12 days with the supernatants of the different time point incubations deriving from the EGFP-Covid-19 residual infectivity test. Upper lane: cells incubated with the supernatants of Covid-19 alone; lower lane: cells incubated with the supernatants of Covid-19 treated in the presence of Enviro-Biotics®.

Fig. 7

Fig. 7. Crystal violet quantitation by spectrophotometric absorbance at 570 nm of the dissolved crystals corresponding to the colonies formed in the colony forming assay. Black bars: control (EGFP-Covid- 19 alone); white bars: EGFP-Covid-19 incubated with Enviro-Biotics®

Fig. 8

Fig. 8. Chemiluminescence quantitation of the LUC-Covid-19 residual infectivity detected as luciferase activity, presented as Relative Luciferase Units (RLU). Each bar is the mean±SD of a triplicate. P value was calculated by unpaired t-Test; ***p< 0.001