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-Lcysteine 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, biofilmsA cute otitis media (AOM) is defined as the presence of inflammation in the middle ear accompanied by the rapid onset of signs and symptoms of an ear infection. Frequent suppurative complications of AOM are mastoiditis, hearing loss, and meningitis. AOM is a significant public health problem worldwide (incidence rate, ϳ11%) (1). Indeed, Ͼ80% of children suffer at least one episode of AOM by their third birthday and 40% will suffer six or more recurrences by the age of 7 years (2). Pediatric AOM, which is often treated with antibiotics (56 to 95% of cases, depending on the country) (3), represents a significant health care burden, annually accounting for some $2.88 billion in medical costs in the United States (4). A polymicrobial disease, AOM is typically caused by colonization of the middle ear space by opportunistic bacteria that normally form part of the nasopharyngeal microbiota. The most frequently involved bacterial pathogens are Streptococcus pneumoniae, nontypeable (nonencapsulated) Haemophilus influenzae (NT H. influenzae), and, to a lesser extent, Moraxella catarrhalis and other species (5). Several respiratory viruses may also cause AOM (6).

Widening the antimicrobial spectrum of esters of bicyclic amines: In vitro effect on gram-positive Streptococcus pneumoniae and gram-negative non-typeable Haemophilus influenzae biofilms

“…In fact, S. pneumoniae and NTHi are the predominant othopathogens associated with otitis media as identified through bacterial culture [10] and frequently form mixed biofilms. Taking advantage of the in vitro mixed NTPn-NTHi biofilm development procedure recently developed [27], we tested the antimicrobial effect of EBA 31 and showed that this compound also demonstrated inhibiting and disintegrating properties in this system (Fig. 6).…”

“…The bacterial strains used for mixed biofilms were NTHi 54997 and the novobiocin-resistant (Nov r ) NTPn P233 (Table 2). Biofilm formation was assessed with crystal violet [27]. NTHi 54997 was grown at 37C in sBHI medium as described above to an OD550 of 0.5, sedimented by centrifugation, resuspended in an equal volume of C+Y (individual scans at 0.5 µm intervals) and x-z (images at 6 µm intervals) planes.…”

“…6C). Figure 6D shows the distribution of the populations that form the biofilm using SYTO 59 together with the Helix pomatia agglutinin HPA, which recognizes the GlcNAc residues of the cell wall teichoic and lipoteichoic acids of S. pneumoniae [27,33]…”

Demolishing the great wall of biofilms in Gram‐negative bacteria: To disrupt or disperse?

“…The FDA‐approved N ‐acetyl cysteine (NAC), whose mucolytic ability to dissociate disulfide bonds in extracellular mucin proteins and reduce their viscosity, was subsequently expounded into a potential anti‐biofilm agent. NAC was employed in modern applications, such as combinatorial treatment with cysteamine, an antioxidant against mixed biofilms of Streptococcus pneumoniae and Haemophilus influenzae . Another garlic‐derived compound, raffinose, could inhibit biofilms by binding to lectin‐A, which is an exopolysaccharide‐binding protein in P. aeruginosa …”

Monitoring quinolone resistance due to Haemophilus influenzae mutations (2012–17)

“…Haemophilus influenzae is the second most common causative bacterium of community-acquired pneumonia, following Streptococcus pneumoniae , and has taken the leading position after pneumococcal vaccines became popular (1). β-Lactam antibiotics have previously been used for the treatment of community-acquired pneumonia.…”