Streptococcus pneumoniae (Pneumococcus) infections affect millions of people worldwide, cause serious mortality and represent a major economic burden. Despite recent successes due to pneumococcal vaccination and antibiotic use, Pneumococcus remains a significant medical problem. Airway epithelial cells, the primary responders to pneumococcal infection, orchestrate an extracellular antimicrobial system consisting of lactoperoxidase (LPO), thiocyanate anion and hydrogen peroxide (H₂O₂). LPO oxidizes thiocyanate using H₂O₂ into the final product hypothiocyanite that has antimicrobial effects against a wide range of microorganisms. However, hypothiocyanite’s effect on Pneumococcus has never been studied. Our aim was to determine whether hypothiocyanite can kill S. pneumoniae. Bactericidal activity was measured in a cell-free in vitro system by determining the number of surviving pneumococci via colony forming units on agar plates, while bacteriostatic activity was assessed by measuring optical density of bacteria in liquid cultures. Our results indicate that hypothiocyanite generated by LPO exerted robust killing of both encapsulated and nonencapsulated pneumococcal strains. Killing of S. pneumoniae by a commercially available hypothiocyanite-generating product was even more pronounced than that achieved with laboratory reagents. Catalase, an H₂O₂ scavenger, inhibited killing of pneumococcal by hypothiocyanite under all circumstances. Furthermore, the presence of the bacterial capsule or lytA-dependent autolysis had no effect on hypothiocyanite-mediated killing of pneumococci. On the contrary, a pneumococcal mutant deficient in pyruvate oxidase (main bacterial H₂O₂ source) had enhanced susceptibility to hypothiocyanite compared o its wild-type strain. Overall, results shown here indicate that numerous pneumococcal strains are susceptible to LPO-generated hypothiocyanite.

Bacterial killing measured by colony counting

Components of the LPO-based antibacterial system were used as described previously (8). Briefly, the following concentrations were used: 6.5 µg/ml LPO, 400 µM SCN^-, 5 mM glucose and 0.1 U/mL glucose oxidase. The reaction volume was set to 120 µL. Catalase (700 U/mL) was also used when indicated to inhibit the system by scavenging H₂O₂. The components were assembled in a sterile 96-well microplate in triplicates with the bacteria being added last at a maximal concentration of 5x10⁵ CFU/ml. The plates were then placed in a 37°C incubator with 5% CO₂. After 6 hours of incubation, 40 µL was spread onto BAP in triplicate and incubated at 37°C with 5% CO₂. After 24 hours, the colonies were counted and CFU/mL was determined. Agar plates exposed to only the assay medium without Spn were always used to ensure that no potential contaminants were detected. A time 0 condition was also counted to make sure that bacterial death was due to OSCN^- and not related to an unknown variable, and thatno significant changes in bacterial numbers were observed in samples containing only bacteria during the duration of the experiments. All the reagents were ordered from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise.

Bacteriostatic activity measured by a microplate-based growth assay

The bacteriostatic activity of OSCN^- was measured by a microplate-based assay described previously (23). Briefly, the components (mentioned in the cell-free assay) were assembled in a sterile 96-well plate with the bacteria being added last. Bacterial growth was measured in a microplate spectrophotometer [Eon (BioTek Instruments Inc., Winooski, VT, USA) or Varioskan Flash (Thermo Scientific, Rochester, NY)] on the basis of following increases in OD as a measure of bacterial density. This method enables fast and very reproducible measurement of bacterial growth (23). Spn strains were grown at 37°C for 14 hours, and OD at 600 nm was measured every 3 minutes. Each sample was run in triplicate. The time required for the positive control (Spn alone) to reach an OD of 0.4 (exponential growth phase) was used as the reference point for all other conditions, and OD values of other samples were compared to this. All the Spn strains used in this work were tested individually for their suitability for this method.

The commercial 1^st line™ immune support product

1^st line™ is an over-the-counter product that is marketed as an immune supplement (distributed by Profound Products). This product uses a proprietary technology to keep OSCN^- stable for a longer period of time allowing for a better antimicrobial effect. To our knowledge, this product is the only commercially available product producing OSCN^-. We tested this product in conjunction with our previously described cell-free system. Briefly, 0.1 g of LPO was reconstituted in 25 mL of HBSS. 750 µL of H₂O₂ solution was added to a 15 mL conical tube followed by the addition of 700 U/mL of catalase. The solution was incubated for 10 minutes to allow catalase to scavenge all H₂O₂ present. 12.5 mL of LPO solution was then added to each tube and mixed. Following this step, 750 µL of SCN^- was also added to each sample and mixed thoroughly. Finally, 750 mL poly aluminum chloride was administered to each solution and mixed well. Samples were then incubated for 30 minutes at room temperature allowing the generation of OSCN^-. By the end of this incubation time, the solution separates into two distinct phases. The top, clear phase containing OSCN^- was used for experiments while the pelleted precipitate was discarded.

Bacterial H₂O₂ production

Generation of H₂O₂ in bacterial suspension was measured by the ROS-Glo^TM luminescence kit following the manufacturer’s instructions (Promega Corporation, Madison, WI, USA). This sensitive assay enables specific and direct detection of low amounts of H₂O₂. TIGR4 wild-type or DspxB bacteria (5x10⁶/ml) suspended in HBSS buffer were incubated at 37 ºC for 30 minutes, followed by centrifugation to collect supernatants for analysis of H₂O₂ production. Ros-GLo^TM reagent was added to bacterium-free supernatants and luminescence was read using a Varioskan Flash microplate luminometer (Thermo Scientific, Rochester, NY). The assay was run in triplicates. Results are expressed as relative luminescence units (RLU).

Quantitation of OSCN^- generation

          Production of OSCN^- was assessed using the photometric 5-thio-2-nitrobenzoic acid (TNB) oxidation assay (24). OSCN^- converts TNB that absorbs light at 412 nm, into a colorless disulfide (5,5’-dithio-bis-[2-nitrobenzoic acid]) (DNTB, Ellman’s reagent). OSCN^- production is measured as decrease in OD at 412 nm and is calculated based on the Lambert-Beer Law and the absorption coefficient e₄₁₂ = 14,100 M^-1 cm^{-1 (25)}. OSCN^- production is expressed as concentration of OSCN^- produced in the volume of the cell-free system under different conditions in 30 minutes.