Zwittergent 3 14

The exopolysaccharide (EPS) of bacteria was extracted as previously described [47 (link)]. Briefly, a 500 μl overnight bacterial culture (OD₅₉₅ = 1) was mixed with 100 μl of 1% Zwittergent 3–14 (Sigma-Aldrich) in 100 mM citric acid (pH 2.0). After 20 min at 50 °C, the mixture was centrifuged at 17,700 × g for 10 min. A 250 μl aliquot of the supernatant was transferred to a clean tube and precipitated with 1 ml of ethanol at 4 °C for 20 min. After centrifugation, the pellet was dried and dissolved in ddH₂O. The sample was appropriately diluted, and then 1,200 μl of 12.5 mM sodium tetraborate (Sigma-Aldrich) in H₂SO₄ was added. The mixture was boiled for 5 min. After cooling, 20 μl of 0.15% 3-hydroxydiphenol (Sigma-Aldrich) was added, and the absorbance at 520 nm was measured. A standard curve ranging from 0.9375 to 30 μg of D-glucuronic acid (Sigma-Aldrich) dissolved in 200 μl of ddH₂O was used to calculate the concentration of the diluted EPS [48 (link)]. The extracted EPS from 3 ml of the tested strains ranged from ~ 70 to ~ 200 μg (different in each strain) in 100 μl of ddH₂O.

Agarose gel (1%) was prepared with agarose powder (SeaKem^® ME Agarose, Lonza, Basel, Switzerland) and 1× PBS with 0.05% NaN₃. The gel was mixed with the purified goat anti-E7HA serum. The ratio of serum to agarose was 1:75. The gel was placed in a circular mould and wells were generated by gel puncher. The purified viruses were treated with 1/10 V of 10% Zwittergent® 3-14 (Sigma-Aldrich, St. Louis, MO, USA) solution for 30 min and then diluted with 1× PBS with 0.05% NaN₃. The diluted antigens were added to the wells and left overnight. After that, the gel was washed and then dried at 37 °C. Finally, the gel was stained with Coomassie blue to show the precipitin rings [26 (link)].

The bacterial CPS was extracted by using a method described by Domenico (Domenico et al., 1989). Briefly, 500 μl of overnight‐cultured bacterium was mixed with 100 μl of 1% Zwittergent 3–14 (Sigma‐Aldrich, Milwaukee, WI, USA) in 100 mM citric acid (pH 2.0) and then incubated at 50°C for 20 min. After centrifugation, 250 μl of the supernatant was transferred to a new tube, and the CPS was precipitated with 1 ml of ethanol. The mixture was incubated at 4°C for 20 min. After centrifugation, the pellet was dried and dissolved in 100 μl of distilled water. The sample was appropriately diluted, and then, 1200 μl of 12.5 mM sodium tetraborate (Sigma‐Aldrich, St Louis, MO) in H₂SO₄ was added. The mixture was vigorously mixed and boiled for 5 min. After cooling, 20 μl of 0.15% 3‐hydroxydiphenol (Sigma‐Aldrich, Milwaukee, WI) was added. The tubes were shaken, and the absorbance at 520 nm was measured. Uronic acid content was determined from a standard curve of d‐glucuronic acid (Sigma‐Aldrich, Milwaukee, WI).

Quantification of capsule was performed using glucuronic acid assays, as previously described [56 (link)]. Briefly, bacteria were grown overnight in LB broth at 37°C under static growth conditions. Cultures were pelleted and resuspended in 1x PBS to an OD₆₀₀ of 1.0. A total of 500 μL of normalized culture was mixed with 100 μL of 1% Zwittergent 3–14 (Sigma) in triplicate in 100 mM citric acid and incubated at 50°C for 20 min. After centrifugation, supernatants from samples were precipitated with cold ethanol at 4°C for 20 min. Upon precipitation, samples were recentrifuged and the pellets were dissolved in 200 μL sterile water and 1200 μL of 12.5 mM tetraborate in concentrated H₂SO₄. Samples were vortexed, boiled at 95°C for 5 min, and mixed with 20 μL of 0.15% 3-hydroxydiphenol (Sigma) in 0.5% NaOH. Absorbance was measured at 520 nm using a microplate reader (Bio-Tek). The uronic acid concentration of each sample was determined using a standard curve of glucuronic acid (Sigma). The limit of detection (LOD) was previously defined [57 (link)] and reported here. Significance was determined using Mann-Whitney nonparametric tests with p<0.05. All graphs and statistics were generated using GraphPad Prism version 9.