Antiviral activity of Bacillus clausii probiotic strain and bacterial supernatant against herpes simplex virus type 1: in vitro study
Antiviral activity of Bacillus clausii probiotic strain and bacterial supernatant against herpes simplex virus type 1: in vitro study
Mehdi Gholami Barzoki,1,*Somayeh Shatizadeh Malekshahi,2Mohammad Shayestehpour ,3
1. Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran 2. Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran 3. Department of Microbiology and Immunology, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran
Introduction: Herpes simplex virus-1 (HSV-1) is an enveloped double stranded DNA virus belonging to herpesviridae family. It can produce a wide spectrum of clinical disorders in skin, eye, central nervous system (CNS) and genital tract with multiple sequelae. There are several commercially available antivirals to treat lesions caused by HSV-1 including acyclovir, valacyclovir, penciclovir, and famciclovir. However, long-term treatment may lead to drug resistance ranging from 3.5 to 10% in the immunosuppressed patients and with a lower frequency in the immunocompetent individuals. Probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. Currently, the use of probiotic and their metabolic products have attracted research attention and represents a promising approach for the prevention and treatment of viral infections. Bacillus clausii (B. clausii) is a spore-forming, aerobic probiotic bacterium which its antimicrobial and immunomodulatory properties have been described previously. The aim of the present study was to investigate for the first time the potential antiviral activity of B. clausii probiotic strain and bacterial supernatant against HSV-1 in a series of in vitro experiments.
Methods: To determine the amount of infectious HSV-1 particle we performed 50% tissue culture infective dose (TCID50) assay. To evaluate the possible cytotoxicity of the B. clausii and bacterial supernatant the survival ability of the vero cells was determined by dimethylthiazolyl-diphenyl tetrazolium bromide (MTT) assay. The experimental conditions designed as pre-treatment, pre-incubation, competition and post-treatment assays which was performed by B. clausii, bacterial supernatant and HSV-1 on Vero cells, separately. For antiviral experiments, the HSV-1 was used with a multiplicity of infection (MOI) of 0.1. Bacterial with 106 CFU/mL and bacterial supernatant at 1/64 dilution was used for all the experiments. Vero cells treated with HSV-1 served as control. DNA was extracted by using the AmpliSens® RIBO-prep nucleic acid extraction kit (Moscow, Russia) according to the manufacturer’s instruction. Real-time PCR analysis was performed using a FirePol® EvaGreen® qPCR Mix (Solis BioDyne, Estonia) with ABI StepOnePlus™ instrument. Data related to real time PCR and TCID50 were analyzed by t-student test using GraphPad Prism version 6.0 and comparing each treatment with control. P value <0.05 was considered as statistically significant.
Results: The results of MTT assay indicated that by incubation of Vero cells with B. clausii at a concentration of 104, 105, and 106 CFU/mL more than 80% of cells were viable. Moreover, incubation of Vero cells with bacterial supernatant at a concentration of 1/32, 1/64, and 1/128 more than 80% of cells were viable. Then, the concentrations used to evaluate the antiviral activity was 106 CFU/mL of bacteria and 1/64 for bacterial supernatant in all subsequent assays. The HSV-1 titer in the absence of B. clausii (control) was 7.66 Log10 TCID50/mL. After incubation of B. clausii and HSV-1 into Vero cells under pre-treatment, pre-incubation, competition, and post-treatment assay, the titer of HSV-1 was estimated 4.08, 4.83, 5.33, and 7.08 Log10 TCID50/mL, respectively. HSV-1 titers in pre-treatment, pre-incubation, competition, and post-treatment assays decreased by about 3.6, 2.8, 2.3, and 0.6 Log10 TCID50/mL in comparison with control, respectively. After incubation of B. clausii supernatant and HSV-1 into Vero cells under pre-treatment, pre-incubation, competition, and post-treatment assay, the titer of HSV-1 was estimated 5.5, 6.08, 6.41 and 6.91 Log10 TCID50/mL, respectively. HSV-1 titers in pre-treatment, pre-incubation, competition, and post-treatment assays decreased by about 2.2, 1.6, 1.2, and 0.7 Log10 TCID50/mL in comparison with control, respectively. Using 2−∆∆C method, the amount of HSV-1 genomic DNA in different experimental assays was compared with the control and the results were presented as fold change. The greatest reduction in viral DNA occurs when either B. clausii or bacterial supernatant was added to the cells before adding the virus (pre-treatment).
Conclusion: These results collectively suggest that probiotic strain B. clausii and its supernatant have promising inhibitory activity towards HSV-1 in vitro. The possible mechanisms of action are aggregation of B. clausii on the cell surface and prevention of viral entry in early infection steps and production of antiviral compounds. Hence, B. clausii may be considered as a novel inhibitor of HSV-1 infection with potential therapeutic or prophylactic benefits. More research is needed to explore its potential activity in animal model.