Antiviral activity of Lactobacillus reuteri Protectis against Coxsackievirus A and Enterovirus 71 infection in human skeletal muscle and colon cell lines

L. reuteri Protectis (Deutsche Sammlung von Mikroorganismen 17938) [36] was re-activated from freeze-dried BioGaia ProTectis tablet. L. casei Shirota is a kind gift from A/Prof Lee Yuan Kun (Department of Microbiology and Immunology, National University of Singapore). Both L. reuteri Protectis and L. casei Shirota were grown in MRS broth or on MRS agar (Oxoid, United Kingdom) at 37 °C. All LAB cultures were incubated under microaerobic condition (closed cap without agitation) to stationary phase, between 16 and 18 h, to achieve a bacterial concentration of 10¹² colony-forming unit per mL (CFU/mL). Bacteria cultures were passaged twice from frozen stock ( −80 °C). Formaldehyde-inactivated L. reuteri Protectis was obtained by resuspending live bacteria pellet in phosphate buffered saline (PBS) containing 4 % v/v formaldehyde (Sigma-Aldrich, United States) overnight. Prior to infection assays bacteria were washed extensively with PBS to remove traces of MRS broth or formaldehyde, and serially diluted in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2 % (v/v) heat-inactivated fetal bovine serum (FBS).

Human rhabdomyosarcoma (RD) cells (ATCC® CCL-136™) were maintained in DMEM supplemented with 10 % v/v heat-inactivated FBS. Human Caco-2 cells (ATCC® HTB-37™) were cultured in DMEM containing 20 % v/v heat-inactivated FBS, 1 % v/v non-essential amino acids (100X), 1 % v/v GlutaMAX supplement (100X), 1.5 % v/v sodium bicarbonate (7.5 %) and 1 % v/v sodium pyruvate (100 mM). Both cell lines were culture in 5 % CO₂ atm at 37 °C, and were sub-cultured every 2–3 days. RD cells and Caco-2 cells were used between passages 13 and 40. All the virus strains, CA6 (NUH0026/SIN/08, Accession No. GU198758.1), CA16 (CA16-G-10, Accession No. U05876) [37], CB2 (KOR 04-279, Accession No. EF174469) and EV71 strain 41 (5865/SIN/00009, Accession No. AF316321) [38], used for this study were propagated in RD cells. All the reagents used to maintain cell cultures were purchased from Thermo Fisher Scientific (Gibco).

L. reuteri Protectis bacteria (10¹¹ CFU) were co-incubated with CA16 (10⁵ PFU) in 2 % DMEM at 37 °C for one hour. The mixture was then added to RD cells (10⁵ cells) for another hour to allow viral entry. CA16-infected RD cells served as control. After one hour incubation, the cells were washed thrice with PBS to remove unbound bacteria and viruses, and immediately fixed with ice cold methanol. The cells were then stained with mouse anti-CA16 antibody (MAB979, Merck, 1:1000 dilution) and rabbit anti-beta actin antibody (ab8227, Abcam, 1:1,000 dilution) followed by incubation with anti-mouse AF488-conjugated (A-11001, Invitrogen, 1:500 dilution) and anti-rabbit AF594-conjugated (R37117, Invitrogen, 1:500 dilution) secondary antibodies, respectively. Cell nuclei were stained with 4',6’-diamidino-2-phenylindole (DAPI) (Invitrogen) (1:100,000 dilution) at room temperature for 30 min in the dark. Images were captured using Olympus IX81 microscope. Cell fluorescence (viral signals, green) was measured using ImageJ software and the corrected total cell fluorescence (CTCF) was calculated using the equation: CTCF = Raw integrated Density – (Area of selected cell × Mean fluorescence of background readings).

RD cells (2.5 × 10⁴ cells/well) and Caco–2 cells (5 × 10³ cells/well) were seeded onto 96 well plates (Nunc, United States) and incubated overnight and for 6 days, respectively. The culture medium was changed every 2- 3 days. Monolayers were incubated with various concentrations of live bacteria for an hour and washed thrice with PBS before fresh 2 % DMEM supplemented with 50 μg/mL gentamicin (Sigma-Aldrich, United States) was added. After 24 h incubation at 37 °C and CO₂, 2 % v/v alamarBlue reagent (Invitrogen, United States) diluted in 2 % DMEM was added to each well. Fluorescence intensity was then measured at excitation wavelength of 570 nm and emission wavelength of 585 nm using a microplate reader (Infinite 200, Tecan). The relative percentage (%) of cell survival with respect to control wells containing untreated cells was calculated. Values were corrected for background fluorescence obtained with media only.

RD cells (1.25x10⁵ cells/well) were seeded onto 24 well plates (Nunc) and infected with 200 μL of 10-fold serially diluted viral supernatant. After 1 h incubation at 37⁰C and CO₂, 1 % w/v sodium carboxymethyl cellulose (Sigma-Aldrich) in Minimum Essential Medium (MEM) (Invitrogen, United States) supplemented with 2 % v/v heat-inactivated FBS and 1.5 % v/v sodium bicarbonate (7.5 %) was added to the wells. After 3 days incubation, the cells were fixed with 4 % v/v formaldehyde and stained with 1 % w/v crystal violet (Sigma-Aldrich). Plaques were scored visually and the virus titers were expressed as plaque-forming units per mL (PFU/mL). Three technical replicates were performed for each dilution of a biological sample. The limit of detection for the plaque assay was set at 10 PFU/mL.

Four experimental set-ups were designed namely, pre-incubation, pre-treatment, co-treatment and post-treatment. Virus infection was carried out at a multiplicity of infection (MOI) of 1 (10⁵ PFU/mL) for all the set-ups. Cell monolayers were washed with PBS thrice and were maintained in 2 % DMEM supplemented with 50 μg/mL of gentamicin after contact time with bacteria. Culture supernatant was harvested at 12 (CB2-infection) or 24 (CA6, CA16, or EV71-infection) hours post-infection. Samples were stored in −80 °C and plaque assay was performed subsequently to determine the viral titer. The virus titers were compared with the titer obtained with cells exposed to virus only.

Live L. reuteri Protectis bacteria were co-incubated with CA6, CA16, EV71 or CB2 virus in 2 % DMEM at 37 °C and CO₂ for an hour. The bacteria-virus mixtures were then spun down at 4,000 x g for 5 min at 4 °C. The culture supernatant was filtered with 0.22 μm syringe filter unit (Millipore, United States) before viral titer determination in RD cells (section 2.4).

The results were expressed as mean ± standard deviation (SD) of three technical replicates. All the experiments were performed twice independently. Comparison between control and different treatment groups was statistically analyzed by one-way analysis of variance (ANOVA) with Dunnett’s post-test or by Mann–Whitney U test as indicated, using GraphPad Prism version 5.00 for Windows, GraphPad Software (San Diego California USA, www.​graphpad.​com). Probability values (p) of < 0.05 were considered statistically significant.

Prior to evaluating the antiviral activity of L. reuteri Protectis bacteria in human RD and intestinal Caco-2 cell lines, a cell viability assay was performed to assess the cytotoxicity of these probiotic bacteria. Results indicated that 1 h incubation of up to 10¹¹ CFU of live L. reuteri Protectis bacteria with RD and Caco-2 cell monolayers did not lead to significant cell viability loss (≥80 % viability) (Fig. 1). Next, various incubation conditions were performed to test the antiviral activity of L. reuteri Protectis (Fig. 2). In the pre-incubation set-up, bacteria and virus particles were incubated together for 1 h prior to infection of mammalian cells with the mixture. In the pre-treatment set-up, cell monolayers were incubated with bacteria for 1 h, and washed with PBS prior to virus infection. In the co-treatment set-up, cell monolayers were incubated for 1 h with virus and bacteria concomitantly. Finally, in the post-treatment set-up, cell monolayers were incubated with the virus for 1 h, and washed with PBS prior to incubation with bacteria for another hour. The culture supernatants were sampled at 12 or 24 h post-infection to determine the virus titers. CA6, CA16, CB2 and EV71 strains were tested. Virus alone, treated under the same experimental conditions, was used as a positive control (POS). The results indicated that L. reuteri Protectis bacteria displayed a significant antiviral activity against CA6, CA16 and EV71 but not against CB2 virus (Fig. 3a-d). Furthermore, the pre-incubation set-up where bacteria and virus are co-incubated prior to incubation with mammalian cells, led to the strongest reduction in virus titers compared to the positive control. This observation suggested a direct interaction between bacteria and virus particles, which may impair virus entry. The inhibitory effects observed were dose-dependent and virus-dependent whereby the greatest antiviral activity was observed against CA16 with more than 2–3 log reduction in virus titers when pre-mixed with 10¹¹ bacteria (Fig. 3b). In the same conditions, reduction in CA6 and EV71 virus titers was approximately 2 log and 1 log, respectively (Fig. 3a and c). A dose-dependent antiviral activity was also observed in the co-treatment set-up where virus and bacteria were added concomitantly to the cell monolayers. However, reduction in virus titers was less dramatic than those obtained in the pre-incubation set-up. In contrast, no dose-dependent antiviral activity was clearly observed in the pre-treatment and post-treatment set-ups with CA6 and CA16 (Fig. 3a and b), but was seen with EV71 (Fig. 3c). Furthermore, comparable observations were made with both cell lines, suggesting that the antiviral activity of L. reuteri Protectis is not cell type-dependent and likely affects an important step of the infection cycle.

The data obtained indicated that the pre-incubation set-up led to the greatest reduction in virus titers, thus suggesting that L. reuteri Protectis bacteria interfere with the virus entry step. To further test this hypothesis, L. reuteri bacteria and CA16 virus were co-incubated for 1 h in a cell free environment prior to infection of RD cells as described for the pre-incubation experimental setup. After 1 h infection, the cell monolayer was washed thoroughly, fixed and processed for immunostaining using anti-CA16 and anti-actin antibodies, while nuclei were stained with DAPI. The corrected total cell fluorescence (CTCF) specific to the virus signal (green) was calculated and indicated significantly lower signal intensity with the (CA16 + L. reuteri)-infected cells compared to CA16-infected cells only (Fig. 4). These data therefore further supported that L. reuteri bacteria interfere with CA16 entry into mammalian cells.

We next asked whether L. reuteri bacteria physically interact with the virus particles during the pre-incubation phase, thereby compromising viral entry into the mammalian cells subsequently. To test this hypothesis, a dose range of live L. reuteri Protectis bacteria were co-incubated with a fixed amount of virus particles for one hour in a mammalian cell-free system. The mixtures were then centrifuged, the supernatants were collected and filtered to remove intact bacteria, and the amount of virus particles was determined by plaque assay. The results indicated a reduction in concentration of virus particles in the supernatant compared to the virus alone control (Fig. 5). This reduction was dependent on the amount of bacteria that were co-incubated with the virus and was seen with CA6, CA16 and EV71, but not with CB2 virus, thus correlating with the observation that L. reuteri Protectis bacteria do not impact CB2 infectivity (Fig. 3). Together, these data support that L. reuteri Protectis bacteria interact directly and physically with CA6, CA16 and EV71 virus particles and likely interfere with viral entry into the mammalian cells.

We next asked whether live L. reuteri Protectis bacteria were necessary to display a significant antiviral activity. To test this hypothesis, formaldehyde-treated L. reuteri Protectis bacteria were pre-incubated with CA6 or CA16 virus according to the pre-incubation set-up described above. The data showed that a bacteria dose-dependent reduction in virus titers was observed that was comparable to that observed with live L. reuteri Protectis bacteria (Fig. 6). Therefore, the data indicated that the antiviral activity of L. reuteri Protectis bacteria does not depend on bacteria replication and/or bacterial product secretion. They support that the antiviral mechanism relies on a physical interaction between bacteria and virus particles which likely interferes with the ability of the virus to bind to its mammalian receptor(s).

The potential antiviral activity of another widely consumed probiotic bacterium namely L. casei Shirota was explored. First, the cytotoxicity of L. casei Shirota was determined by incubating a dose range of bacteria with RD and Caco-2 cells. The results indicated that 10¹¹ CFU of live L. casei Shirota bacteria appears to be toxic (< 80 % viability) to both RD cells and Caco-2 cells (Fig. 7). This cytotoxicity is likely due to the high amounts of lactic acid produced by L. casei Shirota bacteria which results in acidification of the culture medium as evidenced by the colour shift of the pH indicator (data not shown). Therefore, the antiviral assays were conducted with concentrations of L. casei Shirota ranging from 10⁹ to 5 × 10¹⁰ CFU.

The antiviral activity against CA16 and EV71 of L. casei Shirota was assayed in the pre-incubation, pre-treatment, co-treatment and post-treatment set-ups. The results indicated significantly lower CA16 virus titers with L. casei Shirota concentrations of 10¹⁰ and/or 5 × 10¹⁰ CFU in all the experimental set-ups (Fig. 8a). However, significant cytotoxicity was clearly observed at these bacterial concentrations, with cells lifting off from the bottom of the wells (data not shown). Similar observations were made with EV71-infected cells (Fig. 8b). Therefore, these data indicate that L. casei Shirota does not appear to display a significant antiviral activity against CA16 and EV71.