Oral mitis group streptococci reduce infectivity of influenza A virus via acidification and H2O2 production

Abstract

Members of the mitis group streptococci are the most abundant inhabitants of the oral cavity and dental plaque. Influenza A virus (IAV), the causative agent of influenza, infects the upper respiratory tract, and co-infection with Streptococcus pneumoniae is a major cause of morbidity during influenza epidemics. S. pneumoniae is a member of mitis group streptococci and shares many features with oral mitis group streptococci. In this study, we investigated the effect of viable Streptococcus oralis, a representative member of oral mitis group, on the infectivity of H1N1 IAV. The infectivity of IAV was measured by a plaque assay using Madin-Darby canine kidney cells. When IAV was incubated in growing culture of S. oralis, the IAV titer decreased in a time- and dose-dependent manner and became less than 100-fold, whereas heat-inactivated S. oralis had no effect. Other oral streptococci such as Streptococcus mutans and Streptococcus salivarius also reduced the viral infectivity to a lesser extent compared to S. oralis and Streptococcus gordonii, another member of the oral mitis group. S. oralis produces hydrogen peroxide (H2O2) at a concentration of 1-2 mM, and its mutant deficient in H2O2 production showed a weaker effect on the inactivation of IAV, suggesting that H2O2 contributes to viral inactivation. The contribution of H2O2 was confirmed by an inhibition assay using catalase, an H2O2-decomposing enzyme. These oral streptococci produce short chain fatty acids (SCFA) such as acetic acid as a by-product of sugar metabolism, and we also found that the inactivation of IAV was dependent on the mildly acidic pH (around pH 5.0) of these streptococcal cultures. Although inactivation of IAV in buffers of pH 5.0 was limited, incubation in the same buffer containing 2 mM H2O2 resulted in marked inactivation of IAV, which was similar to the effect of growing S. oralis culture. Taken together, these results reveal that viable S. oralis can inactivate IAV via the production of SCFAs and H2O2. This finding also suggests that the combination of mildly acidic pH and H2O2 at low concentrations could be an effective method to inactivate IAV.

Fig 1. Viable S. oralis reducs the infectivity of influenza A virus (IAV).

(A) IAV in brain heart infusion (BHI) broth was incubated with growing S. oralis wild type (WT) (1 × 10⁸ to 2 × 10⁹ cfu) at 37°C in a 5% CO₂ atmosphere. After incubation for 3 h, the bacterial growth was stopped by adding antibiotics, and the IAV-bacteria mixture was centrifuged to precipitate the bacteria. The IAV titer in the supernatants was determined using a plaque assay. The IAV titer was expressed as the pfu/ml (A left) and % of the untreated control IAV (A right). (B) S. oralis WT was heat-inactivated at 60°C for 30 min in phosphate buffered saline (PBS). The heat-inactivated S. oralis (equivalent to 2 × 10⁹ to 2 × 10¹⁰ cfu) was incubated with IAV for 3 h, and centrifuged to precipitate the bacteria. The titer in the supernatants was determined using a plaque assay. (C) IAV in BHI broth was incubated with or without growing S. oralis WT (2 × 10⁹ cfu) at 37°C for 0, 1, 2, and 3 h. The bacterial growth was stopped by adding antibiotics, and the IAV-bacteria mixture was centrifuged to precipitate the bacteria. The titer was determined using a plaque assay. : with S. oralis; : without S. oralis. The data are shown as mean ± SD values of triplicate samples. *p < 0.05 as compared with the untreated control (no bacteria, or no S. oralis).

Fig 2. Effect of viable oral streptococci on the infectivity of IAV.

IAV in BHI broth was incubated with growing S. oralis WT (WT), S. oralis spxB KO (KO), S. gordonii (gor), S. salivarius (sal), S. mutans (mut), or S. sobrinus (sor) (2 × 10⁹ cfu) at 37°C in a 5% CO₂ atmosphere. After incubation for 3 h, the bacterial growth was stopped by adding antibiotics, and the bacteria were removed by centrifugation. The IAV titer in the supernatants was determined using a plaque assay. The data are shown as mean ± SD values of triplicate samples. *p < 0.05 as compared with the untreated control (no bacteria; None).

Fig 3. Effect of buffer and catalase on the S. oralis-induced inactivation of IAV.

(A) IAV in BHI broth was incubated with growing S. oralis WT and S. oralis spxB KO (2 × 10⁹ cfu) at 37°C in a 5% CO₂ atmosphere. After incubation for 3 h, the bacterial growth was stopped by adding antibiotics, and the bacteria were removed by centrifugation. The IAV titer in the supernatants was determined using a plaque assay. (B) IAV in BHI broth containing HEPES buffer (0.1 M, pH 7.2; left) or phosphate buffer (0.1 M, pH 7.2; right) was incubated with growing S. oralis WT and S. oralis spxB KO (2 × 10⁹ cfu) at 37°C for 3 h. The bacterial growth was stopped by adding antibiotics, and the bacteria were removed by centrifugation. The titer was determined by the plaque assay. (C) IAV in BHI broth containing catalase (0–200 U/ml) was incubated with growing S. oralis WT (2 × 10⁹ cfu) at 37°C for 3 h. The bacterial growth was stopped by adding antibiotics, and the bacteria were removed by centrifugation. The titer was determined using a plaque assay. The data are shown as mean ± SD values of triplicate samples. *p < 0.05 as compared with the control (no bacteria; None).

Fig 4. Effect of pH on the infectivity of IAV.

(A) Effect of mildly acidic pH on IAV inactivation was studied using BHI broth containing NaOAc buffer (0.1 M; pH 4.0, 4.5, 5.0 and 5.5). IAV in these BHI broth was incubated at 37°C in a 5% CO₂ atmosphere for 3 h. The titer of the IAV was determined using a plaque assay. The data are shown as mean ± SD values of triplicate samples. *p < 0.05 as compared with the control (None; no NaOAc buffer). (B) Final pH of the streptococcal cultures was measured. S. oralis WT (WT), S. gordonii (gor), S. salivarius (sal), S. mutans (mut), and S. sobrinus (sor) were cultured in BHI broth at 37°C in a 5% CO₂ atmosphere for 3 h, as the same condition for the IAV inactivation study. Then, the pH of the cultures was directly measured using a pH meter (LAQUA F-71; HORIBA, Kyoto, Japan).

Fig 5. H₂O₂ reduces the infectivity of IAV.

(A) IAV in BHI broth was incubated with H₂O₂ (1, 2, 5, or 10 mM) at 37°C for 3 h. The IAV titer was determined using a plaque assay. (B) IAV in BHI broth with or without 0.1 M NaOAc (pH 5.0) was incubated with H₂O₂ (0, 1, and 2 mM) at 37°C for 3 h, and the titer of the IAV was determined using a plaque assay. The data are shown as mean ± SD values of triplicate samples. *p < 0.05 as compared with the untreated control (no H₂O₂).

Fig 6

Fluorescence staining of MDCK cells infected with IAV. IAV was incubated with viable S. oralis WT (2 × 10⁹ cfu) in BHI broth at 37°C for 3 h. Other samples of IAV were incubated in broths containing 2 mM H₂O₂, or NaOAc buffer (0.1 M; pH 5.0) with or without H₂O₂. These IAV preparations (50 μl) were inoculated to the MDCK cells in 24-well culture plates. The cells were fixed, and stained with FITC-anti IAV antibody (Green) and DAPI (Blue). The fluorescence of the cells was observed using a fluorescent microscope system. Bar = 10 μm.

Fig 7. Effect of acidic pH and H₂O₂ on neuraminidase and hemagglutinin activities of IAV.

(A) IAV in 0.1 M buffers (phosphate buffer, pH 7.2; NaOAc buffer, pH 5.0) with or without H₂O₂ (2 mM) was incubated at 37°C for 3 h. Neuraminidase activity of IAV was measured by using a neuraminidase assay kit. (B) IAV was incubated in buffers (phosphate buffer pH 7.2; NaOAc buffer pH 5.0) with or without H₂O₂ (2 mM) at 37°C for 3 h. Serial two fold dilutions of the virus suspensions (50 μl) were prepared in 96-well round bottom plates using PBS. After addition of 50 μl of a 5% (v/v in PBS) guinea pig blood to each well, the plates were incubated at 4°C for 1 h, and the visible aggregation of the red blood cells was observed.

Fig 8. Summary of this study.

Mitis group streptococci, such as S. oralis, produce SCFAs and H₂O₂. SCFAs decrease environmental pH, and the combination of mildly acidic pH (around pH 5.0) and H₂O₂ (around 2 mM) reduces the infectivity of IAV.