Cephalosporin-NO-donor prodrug PYRRO-C3D shows β-lactam-mediated activity against Streptococcus pneumoniae biofilms

Bacterial biofilms show high tolerance towards antibiotics and are a significant problem in clinical settings where they are a primary cause of chronic infections. Novel therapeutic strategies are needed to improve anti-biofilm efficacy and support reduction in antibiotic use. Treatment with exogenous nitric oxide (NO) has been shown to modulate bacterial signaling and metabolic processes that render biofilms more susceptible to antibiotics. We previously reported on cephalosporin-3\u27-diazeniumdiolates (C3Ds) as NO-donor prodrugs designed to selectively deliver NO to bacterial infection sites following reaction with β-lactamases. With structures based on cephalosporins, C3Ds could, in principal, also be triggered to release NO following β-lactam cleavage mediated by transpeptidases/penicillin-binding proteins (PBPs), the antibacterial target of cephalosporin antibiotics. Transpeptidase-reactive C3Ds could potentially show both NO-mediated anti-biofilm properties and intrinsic (β-lactam-mediated) antibacterial effects. This dual-activity concept was explored using Streptococcus pneumoniae, a species that lacks β-lactamases but relies on transpeptidases for cell-wall synthesis. Treatment with PYRRO-C3D (a representative C3D containing the diazeniumdiolate NO donor PYRRO-NO) was found to significantly reduce viability of planktonic and biofilm pneumococci, demonstrating that C3Ds can elicit direct, cephalosporin-like antibacterial activity in the absence of β-lactamases. While NO release from PYRRO-C3D in the presence of pneumococci was confirmed, the anti-pneumococcal action of the compound was shown to arise exclusively from the β-lactam component and not through NO-mediated effects. The compound showed similar potency to amoxicillin against S. pneumoniae biofilms and greater efficacy than azithromycin, highlighting the potential of C3Ds as new agents for treating pneumococcal infections

Low concentrations of nitric oxide modulate Streptococcus pneumoniae biofilm metabolism and antibiotic tolerance

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 hypothesised that addition of low concentrations of NO to pneumococcal biofilms would improve antibiotic efficacy and 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

Nasal mucosal immunoexpression of the mast cell chemoattractants TGF-β, eotaxin, and stem cell factor and their receptors in allergic rhinitis

BackgroundAllergic rhinitis is characterized by the epithelial accumulation of cells, particularly mast cells and eosinophils. There is little information relating to the chemotaxins responsible for mast cell epithelial accumulation in this disease.ObjectiveExpression of the mast cell chemoattractants TGF-β, eotaxin, and stem cell factor and their receptors was investigated in tissue sections from biopsy specimens obtained from patients with naturally occurring allergic rhinitis.MethodsSpecific immunohistochemical staining was performed on thin sections of inferior turbinate biopsy specimens from patients with perennial and seasonal allergic rhinitis and, for comparison, from nonatopic and, where relevant, atopic healthy volunteers without rhinitis. Sequential staining of adjacent 2-μm sections was undertaken to colocalize TGF-β receptors to mast cells.ResultsEvidence was found of significantly increased epithelial immunoreactivity for TGF-β1, TGF-β2, TGF-β3, TGF-β receptor I, TGF-β receptor II, and TGF-β receptor III in patients with perennial and seasonal allergic rhinitis compared with that seen in healthy control subjects. TGF-β receptors I and II were found to colocalize to mast cells. Eotaxin epithelial immunoreactivity was significantly increased in the perennial group, although there were no corresponding disease-related differences found in relation to CCR-3 immunoreactivity at this site. There was no increase in stem cell factor immunoreactivity within the epithelium in naturally occurring disease. Significant correlations were found between epithelial immunoreactivity for TGF-β1, TGF-β2, TGF-β receptor I, TGF-β receptor II, and the number of epithelial mast cells.ConclusionThese findings of enhanced epithelial TGF-β immunoreactivity in patients with rhinitis, the correlation with intraepithelial mast cell numbers, and the colocalization of TGF-β receptors to mast cells suggest that the epithelial expression of TGF-β might represent an important biologic process involved in either the recruitment or retention of mast cells within the epithelium in naturally occurring allergic rhinitis