Low Concentrations of Nitric Oxide Modulate Streptococcus pneumoniae Biofilm Metabolism and Antibiotic Tolerance

Abstract

g Streptococcus pneumoniae is one of the key pathogens responsible for otitis media (OM), the most common infection in children and the largest cause of childhood antibiotic prescription. Novel therapeutic strategies that reduce the overall antibiotic consumption due to OM are required because, although widespread pneumococcal conjugate immunization has controlled invasive pneumococcal disease, overall OM incidence has not decreased. Biofilm formation represents an important phenotype contributing to the antibiotic tolerance and persistence of S. pneumoniae in chronic or recurrent OM. We investigated the treatment of pneumococcal biofilms with nitric oxide (NO), an endogenous signaling molecule and therapeutic agent that has been demonstrated to trigger biofilm dispersal in other bacterial species. We hypothesized that addition of low concentrations of NO to pneumococcal biofilms would improve antibiotic efficacy and that higher concentrations exert direct antibacterial effects. Unlike in many other bacterial species, low concentrations of NO did not result in S. pneumoniae biofilm dispersal. Instead, treatment of both in vitro biofilms and ex vivo adenoid tissue samples (a reservoir for S. pneumoniae biofilms) with low concentrations of NO enhanced pneumococcal killing when combined with amoxicillin-clavulanic acid, an antibiotic commonly used to treat chronic OM. Quantitative proteomic analysis using iTRAQ (isobaric tag for relative and absolute quantitation) identified 13 proteins that were differentially expressed following low-concentration NO treatment, 85% of which function in metabolism or translation. Treatment with low-concentration NO, therefore, appears to modulate pneumococcal metabolism and may represent a novel therapeutic approach to reduce antibiotic tolerance in pneumococcal biofilms.

Cephalosporin-3′-Diazeniumdiolate NO Donor Prodrug PYRRO-C3D Enhances Azithromycin Susceptibility of Nontypeable Haemophilus influenzae Biofilms

“…This was confirmed by KEGG pathway analysis of all differentially expressed proteins, which showed overrepresentation of ribosome and glycolysis/ gluconeogenesis pathways. A similar response was recently observed in Streptococcus pneumoniae biofilms, where low-dose NO modulated both translation and metabolism (26). This is particularly interesting given that S. pneumoniae also lacks the GGDEF, EAL, and HD-GYP domain-containing proteins associated with the c-di-GMP pathway.…”

“…Previous research has shown that NO treatment of biofilms formed by several bacterial species reduces their tolerance toward antibiotics (26,27). Treatment of established 72-h NTHi biofilms with 4 mg/ml azithromycin produced a slight reduction in viable NTHi within the biofilm (P ϭ 0.0019; Fig.…”

“…NO treatment of E. coli has also been shown to inhibit a global regulator (Fur) that uses iron as a cofactor, affecting a wide range of metabolic processes, such as the stress response and iron metabolism (39), and has been implicated in NTHi virulence (40). While several studies have shown that NO-mediated dispersal of biofilms reduces antibiotic tolerance (19)(20)(21), the regulation of pneumococcal biofilm metabolism was recently shown to provide an alternative mechanism for reducing tolerance (26). As PYRRO-C3D increases the susceptibility of NTHi biofilms to treatment with azithromycin, it is possible that a mechanism similar to that observed in pneumococcus is also responsible for reduction in tolerance observed here.…”

Study of crystal formation and nitric oxide (NO) release mechanism from S-nitroso-N-acetylpenicillamine (SNAP)-doped CarboSil polymer composites for potential antimicrobial applications

“…In this regard, NO has been reported to kill both gram-positive and gram-negative bacteria [27–32]. It has also been reported that NO can modulate biofilm metabolism and make bacteria within biofilm more susceptible to antibiotics [33,34]. …”

Biofilms 2015: Multidisciplinary Approaches Shed Light into Microbial Life on Surfaces

“…Dr. Hall-Stoodley showed that low doses of nitric oxide affect metabolism and decrease antibiotic tolerance. This suggests that adjunctive treatment with low doses of NO, which do not trigger biofilm dispersal, could reduce antibiotic tolerance in pneumococcal biofilms and improve antibiotic efficacy (60). Another treatment strategy was championed by Bob Hancock (University of British Columbia, Vancouver, British Columbia, Canada), who made a strong case for the use of broad-spectrum cationic antibiofilm peptides against biofilm infections.…”