N-Acetyl-l-Cysteine and Cysteamine as New Strategies against Mixed Biofilms of Nonencapsulated Streptococcus pneumoniae and Nontypeable Haemophilus influenzae

Abstract

Acute otitis media, a polymicrobial disease of the middle ear cavity of children, is a significant public health problem worldwide. It is most frequently caused by encapsulated Streptococcus pneumoniae and nontypeable Haemophilus influenzae, although the widespread use of pneumococcal conjugate vaccines is apparently producing an increase in the carriage of nonencapsulated S. pneumoniae Frequently, pneumococci and H. influenzae live together in the human nasopharynx, forming a self-produced biofilm. Biofilms present a global medical challenge since the inherent antibiotic resistance of their producers demands the use of large doses of antibiotics over prolonged periods. Frequently, these therapeutic measures fail, contributing to bacterial persistence. Here, we describe the development of an in vitro nonencapsulated S. pneumoniae-nontypeable H. influenzae biofilm system with polystyrene or glass-bottom plates. Confocal laser scanning microscopy and specific fluorescent labeling of pneumococcal cells with Helix pomatia agglutinin revealed an even distribution of both species within the biofilm. This simple and robust protocol of mixed biofilms was used to test the antimicrobial properties of two well-known antioxidants that are widely used in the clinical setting, i.e., N-acetyl-l-cysteine and cysteamine. This repurposing approach showed the high potency of N-acetyl-l-cysteine and cysteamine against mixed biofilms of nonencapsulated S. pneumoniae and nontypeable H. influenzae Decades of clinical use mean that these compounds are safe to use, which may accelerate their evaluation in humans.

Keywords: Haemophilus influenzae; Streptococcus pneumoniae; antioxidants; biofilms.

FIG 1

Influence of bacterial proportions on NE S. pneumoniae-NT H. influenzae mixed biofilm formation in vitro. Exponentially growing cultures of NE S. pneumoniae P233 and NT H. influenzae 54997 in s(C+Y) medium were diluted to ∼5 × 10⁶ CFU/ml and mixed in different proportions (NE S. pneumoniae-to-NT H. influenzae [NESp:NTHi] ratios of 1:40, 1:15, 1:3, 1:2, 1:1, 3:1, and 10:1). Two hundred microliters of the mixture was then distributed into the wells of a polystyrene microtiter plate and incubated for 6 h at 37°C under 5% CO₂. (A) The viability of NE S. pneumoniae (squares) and NT H. influenzae (circles) was determined by plate counting. (B) Gray bars indicate mixed biofilm formation determined by CV staining. The initial NE S. pneumoniae-to-NT H. influenzae proportions are shown inside the bars. Data represent the average of three experiments. Standard error bars are shown.

FIG 2

Time course of NE S. pneumoniae-NT H. influenzae mixed biofilm formation. Exponentially growing cultures of NE S. pneumoniae (NESp) P233 and NT H. influenzae (NTHi) 54997 in s(C+Y) medium were diluted in the same medium to ∼10⁶ CFU/ml and mixed in a 1:1 proportion. Two hundred microliters was then distributed into the wells of a polystyrene microtiter plate and incubated at 37°C in a 5% CO₂ atmosphere. At the times indicated, plate counting was performed. (A) Biofilm formation by NE S. pneumoniae. (B) Biofilm formation by NT H. influenzae. (C, D) Mixed biofilms. Open, black, and gray bars correspond, respectively, to the nonadherent cells of the biofilm and to biofilm-grown cells of NE S. pneumoniae and NT H. influenzae. Crosshatched and hatched bars indicate bacterial growth and biofilm formation in mixed cultures, respectively. The data represent the average of six experiments.

FIG 3

S. pneumoniae labeling in mixed biofilms with HPA. Biofilms formed by NE S. pneumoniae (NESp) P233 (A to C), NT H. influenzae (NTHi) 54997 (D to F), or both (G to I) were stained with a combination of SYTO9 (green) and HPA conjugated to Alexa Fluor 488 (pink). Projections were obtained in the x-y (individual scans at 0.5-μm intervals) and x-z (images at 6-μm intervals) planes. Scale bars, 25 μm.

FIG 4

Killing of S. pneumoniae P233 and NT H. influenzae 54997 in mixed biofilms with antioxidants. Mixed biofilms were washed with sterile distilled H₂O and incubated with different concentrations of NAC (A) or Cys (B) for 1 h at 37°C in a 5% CO₂ atmosphere. Black and gray bars correspond to NE S. pneumoniae (NESp) and NT H. influenzae (NTHi) viability, respectively. Data represent the average of five experiments.

FIG 5

CLSM of a mixed biofilm after exposure to NAC. An NE S. pneumoniae-NT H. influenzae biofilm was left untreated (A) or treated with 0.5 mg/ml NAC for 1 h at 37°C under 5% CO₂ (B). The cells in the biofilms were then stained with the BacLight LIVE/DEAD kit to reveal viable (green fluorescence) and nonviable (red fluorescence) bacteria. Scale bars, 25 μm.