Browsing by Author "Faust, S. N."

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    Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa
    (Oxford University Press, 2019-09-17) Soren, O.; Rineh, A.; Silva, D. G.; Cai, Y.; Howlin, R. P.; Allan, Raymond N.; Feelisch, M.; Davies, J. C.; Connett, G. J.; Faust, S. N.; Kelso, M. J.; Webb, J. S.
    Objectives: The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (DiEthylAmin-Cephalosporin-3’- Diazeniumdiolate) has been showed to initiate the dispersal of biofilms formed by Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of P. aeruginosa and its effect in combination with two anti-pseudomonal antibiotics, tobramycin and colistin, in vitro. Methods: -lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs against P. aeruginosa clinical isolates were measured using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results: DEA-C3D was confirmed to selectively release NO in response to contact with bacterial -lactamase. Despite lacking direct, cephalosporin/-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions: DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.
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    Cephalosporin-3’-diazeniumdiolate NO-donor prodrug PYRRO-C3D enhances azithromycin susceptibility of non-typeable Haemophilus influenzae biofilms
    (American Society for Microbiology, 2016-12-05) Collins, S. A.; Kelso, M. J.; Rineh, A.; Yepuri, N. R.; Coles, J.; Jackson, C. L.; Halladay, G. D.; Walker, W. T.; Webb, J. S.; Hall-Stoodley, L.; Connett, G.; Feelisch, M.; Faust, S. N.; Lucas, J. S. A.; Allan, Raymond N.
    Objectives: PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO)-donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against non-typeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. Methods: The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free LC/MS proteomic analyses were performed to identify differentially expressed proteins following NO treatment. Results: PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking β-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log-fold reduction in viability (p<0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log reduction was observed (p<0.01). Label-free proteomics showed that NO increased expression of sixteen proteins involved in metabolic and transcriptional/translational functions. Conclusions: NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm associated antibiotic tolerance.
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    D-methionine interferes with non-typeable Haemophilus influenzae peptidoglycan synthesis during growth and biofilm formation
    (Microbiology Society, 2017-07-12) Dawe, H.; Berger, E.; Sihlbom, C.; Angus, E. M.; Howlin, R. P.; Laver, J. R.; Tebruegge, M.; Hall-Stoodley, L.; Stoodley, P.; Faust, S. N.; Allan, Raymond N.
    Non-typeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that plays a major role in a number of respiratory tract infections, including otitis media, cystic fibrosis and chronic obstructive pulmonary disease. Biofilm formation has been implicated in both NTHi colonization and disease, and is responsible for the increased tolerance of this pathogen towards antibiotic treatment. Targeting metabolic pathways that are important in NTHi biofilm formation represents a potential strategy to combat this antibiotic recalcitrance. A previous investigation demonstrated increased expression of a putative D-methionine uptake protein following exposure of NTHi biofilms to the ubiquitous signalling molecule, nitric oxide. We therefore hypothesized that treatment with exogenous D-methionine would impact on NTHi biofilm formation and increase antibiotic sensitivity. Treatment of NTHi during the process of biofilm formation resulted in a reduction in biofilm viability, increased biomass, changes in the overall biofilm architecture and the adoption of an amorphous cellular morphology. Quantitative proteomic analyses identified 124 proteins that were differentially expressed following D-methionine treatment, of which 51 (41 %) were involved in metabolic and transport processes. Nine proteins involved in peptidoglycan synthesis and cell division showed significantly increased expression. Furthermore, D-methionine treatment augmented the efficacy of azithromycin treatment and highlighted the potential of D-methionine as an adjunctive therapeutic approach for NTHi biofilm-associated infections.
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    Low concentrations of nitric oxide modulate Streptococcus pneumoniae biofilm metabolism and antibiotic tolerance
    (American Society for Microbiology, 2016-03-25) Allan, Raymond N.; Morgan, S.; Brito-Mutunayagam, S.; Skipp, P.; Feelisch, M.; Hayes, S. M.; Hellier, W.; Clarke, S. C.; Stoodley, P.; Burgess, A.; Ismail-Koch, H.; Salib, R. J.; Webb, J. S.; Faust, S. N.; Hall-Stoodley, L.
    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.
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    Multi-excitation Raman spectroscopy for label-free, strain-level characterisation of bacterial pathogens in artificial sputum media
    (ACS, 2022-01-03) Lister, A. P.; Highmore, C. J.; Hanrahan, N.; Read, J.; Munro, A. P. S.; Tan, S.; Allan, Raymond N.; Faust, S. N.; Webb, J. S.; Mahajan, S.
    The current methods for diagnosis of acute and chronic infections are complex and skill-intensive. For complex clinical biofilm infections, it can take days from collecting and processing a patient’s sample to achieving a result. These aspects place a significant burden on healthcare providers, delay treatment, and can lead to adverse patient outcomes. We report the development and application of a novel multi-excitation Raman spectroscopy-based methodology for the label-free and non-invasive detection of microbial pathogens that can be used with unprocessed clinical samples directly and provide rapid data to inform diagnosis by a medical professional. The method relies on the differential excitation of non-resonant and resonant molecular components in bacterial cells to enhance the molecular finger-printing capability to obtain strain-level distinction in bacterial species. Here, we use this strategy to detect and characterize the respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus as typical infectious agents associated with cystic fibrosis. Planktonic specimens were analyzed both in isolation and in artificial sputum media. The resonance Raman components, excited at different wavelengths, were characterized as carotenoids and porphyrins. By combining the more informative multi-excitation Raman spectra with multivariate analysis (support vector machine) the accuracy was found to be 99.75% for both species (across all strains), including 100% accuracy for drug-sensitive and drug-resistant S. aureus. The results demonstrate that our methodology based on multi-excitation Raman spectroscopy can underpin the development of a powerful platform for the rapid and reagentless detection of clinical pathogens to support diagnosis by a medical expert, in this case relevant to cystic fibrosis. Such a platform could provide translatable diagnostic solutions in a variety of disease areas and also be utilized for the rapid detection of anti-microbial resistance.
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    Primary ciliary dyskinesia ciliated airway cells show increased susceptibility to Haemophilus influenzae biofilm formation
    (European Respiratory Society, 2017-09-10) Walker, W. T.; Jackson, C.; Allan, Raymond N.; Collins, S. A.; Kelso, M. J.; Rineh, A.; Yepuri, N. R.; Nicholls, B.; Lau, L.; Johnstone, D.; Lackie, P.; Faust, S. N.; Lucas, J. S. A.; Hall-Stoodley, L.
    Non-typeable Haemophilus influenzae (NTHi) is the most common pathogen in primary ciliary dyskinesia (PCD) patients. We hypothesised that abnormal ciliary motility and low airway nitric oxide (NO) levels on airway epithelial cells from PCD patients might be permissive for NTHi colonisation and biofilm development. We used a primary epithelial cell co-culture model to investigate NTHi infection. Primary airway epithelial cells from PCD and non-PCD patients were differentiated to ciliation using an air–liquid interface culture and then co-cultured with NTHi. NTHi adherence was greater on PCD epithelial cells compared to non-PCD cells ( p<0.05) and the distribution of NTHi on PCD epithelium showed more aggregated NTHi in biofilms (p<0.001). Apart from defective ciliary motility, PCD cells did not significantly differ from non-PCD epithelial cells in the degree of ciliation and epithelial integrity or in cytokine, LL-37 and NO production. Treatment of PCD epithelia using exogenous NO and antibiotic significantly reduced NTHi viability in biofilms compared with antibiotic treatment alone. Impaired ciliary function was the primary defect in PCD airway epithelium underlying susceptibility to NTHi biofilm development compared with non-PCD epithelium. Although NO responses were similar, use of targeted NO with antibiotics enhanced killing of NTHi in biofilms, suggesting a novel therapeutic approach.
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    Pronounced metabolic changes in adaptation to biofilm growth by Streptococcus pneumoniae
    (PLOS, 2014-09-04) Allan, Raymond N.; Skipp, P.; Jefferies, J.; Clarke, S. C.; Faust, S. N.; Hall-Stoodley, L.; Webb, J.
    Streptococcus pneumoniae accounts for a significant global burden of morbidity and mortality and biofilm development is increasingly recognised as important for colonization and infection. Analysis of protein expression patterns during biofilm development may therefore provide valuable insights to the understanding of pneumococcal persistence strategies and to improve vaccines. iTRAQ (isobaric tagging for relative and absolute quantification), a high-throughput gel-free proteomic approach which allows high resolution quantitative comparisons of protein profiles between multiple phenotypes, was used to interrogate planktonic and biofilm growth in a clinical serotype 14 strain. Comparative analyses of protein expression between log-phase planktonic and 1-day and 7-day biofilm cultures representing nascent and late phase biofilm growth were carried out. Overall, 244 proteins were identified, of which .80% were differentially expressed during biofilm development. Quantitatively and qualitatively, metabolic regulation appeared to play a central role in the adaptation from the planktonic to biofilm phenotype. Pneumococci adapted to biofilm growth by decreasing enzymes involved in the glycolytic pathway, as well as proteins involved in translation, transcription, and virulence. In contrast, proteins with a role in pyruvate, carbohydrate, and arginine metabolism were significantly increased during biofilm development. Downregulation of glycolytic and translational proteins suggests that pneumococcus adopts a covert phenotype whilst adapting to an adherent lifestyle, while utilization of alternative metabolic pathways highlights the resourcefulness of pneumococcus to facilitate survival in diverse environmental conditions. These metabolic proteins, conserved across both the planktonic and biofilm phenotypes, may also represent target candidates for future vaccine development and treatment strategies. Data are available via ProteomeXchange with identifier PXD001182.