Halogenated Dihydropyrrol-2-One Molecules Inhibit Pyocyanin Biosynthesis by Blocking the Pseudomonas Quinolone Signaling System.

Quorum-sensing (QS) systems of Pseudomonas aeruginosa are involved in the control of biofilm formation and virulence factor production. The current study evaluated the ability of halogenated dihydropyrrol-2-ones (DHP) (Br (4a), Cl (4b), and F (4c)) and a non-halogenated version (4d) to inhibit the QS receptor proteins LasR and PqsR. The DHP molecules exhibited concentration-dependent inhibition of LasR and PqsR receptor proteins. For LasR, all compounds showed similar inhibition levels. However, compound 4a (Br) showed the highest decrease (two-fold) for PqsR, even at the lowest concentration (12.5 µg/mL). Inhibition of QS decreased pyocyanin production amongst P. aeruginosa PAO1, MH602, ATCC 25619, and two clinical isolates (DFU-53 and 364707). In the presence of DHP, P. aeruginosa ATCC 25619 showed the highest decrease in pyocyanin production, whereas clinical isolate DFU-53 showed the lowest decrease. All three halogenated DHPs also reduced biofilm formation by between 31 and 34%. The non-halogenated compound 4d exhibited complete inhibition of LasR and had some inhibition of PqsR, pyocyanin, and biofilm formation, but comparatively less than halogenated DHPs.

The effect of N-acetylcysteine in a combined antibiofilm treatment against antibiotic-resistant Staphylococcus aureus.

BACKGROUND: The WHO declared Staphylococcus aureus as a 'pathogen of high importance' in 2017. One-fifth of all bloodstream-related infections in Australia and 12 000 cases of bacteraemia in the UK (2017-18) were caused by the MRSA variant. To address the need for novel therapies, we investigated several permutations of an innovative combination therapy containing N-acetylcysteine (NAC), an antibiotic and an enzyme of choice in eradicating MRSA and MSSA biofilms. METHODS: Biofilm viability (resazurin assay) and colony count methods were used to investigate the effect of NAC, antibiotics and enzymes on S. aureus biofilm disruption and killing. The effects of NAC and enzymes on the polysaccharide content of biofilm matrices were analysed using the phenol/sulphuric acid method and the effect of NAC on DNA cleavage was determined using the Qubit fluorometer technique. Changes in biofilm architecture when subjected to NAC and enzymes were visualized using confocal laser scanning microscopy (CLSM). RESULTS: NAC alone displayed bacteriostatic effects when tested on planktonic bacterial growth. Combination treatments containing 30 mM NAC resulted in ≥90% disruption of biofilms across all MRSA and MSSA strains with a 2-3 log10 decrease in cfu/mL in treated biofilms. CLSM showed that NAC treatment drastically disrupted S. aureus biofilm architecture. There was also reduced polysaccharide production in MRSA biofilms in the presence of NAC. CONCLUSIONS: Our results indicate that inclusion of NAC in a combination treatment is a promising strategy for S. aureus biofilm eradication. The intrinsic acidity of NAC was identified as key to maximum biofilm disruption and degradation of matrix components.

Covalent Immobilization of N-Acetylcysteine on a Polyvinyl Chloride Substrate Prevents Bacterial Adhesion and Biofilm Formation.

Biofilm formation and antimicrobial resistance at surgical implant sites result in high morbidity and mortality. Identifying novel molecules that inhibit biofilm formation to coat surgical biomaterials is essential. One such compound is N-acetylcysteine (NAC), a potent antioxidant precursor for glutathione, necessary in mammalian cells and known to disrupt/prevent biofilms. In this study, NAC was covalently immobilized onto functionalized polyvinyl chloride surfaces using plasma immersion ion implantation (PIII) treatment that achieves covalent binding without the need for linker groups. NAC immobilization was characterized using water contact angles, Fourier-transform infrared, and X-ray photoelectron spectroscopy techniques. Bacterial viability and biofilm formation on NAC surfaces were assessed using resazurin assays, phase contrast microscopy, and colony counting experiments. Effect of NAC on bacterial polysaccharide production and DNA cleaving was investigated using the phenol-sulfuric acid method and the Qubit fluorometer. Surface thermodynamics between the NAC coating and bacterial cells were measured using the Lewis acid-base method. Surface characterization techniques demonstrated superficial changes after PIII treatment and subsequent covalent NAC immobilization. NAC-coated surfaces significantly reduced biofilm viability and the presence of Gram-negative and Gram-positive bacteria. NAC also decreased polysaccharide production and degraded DNA. This led to unfavorable conditions for biofilm formation on NAC-coated surfaces, as demonstrated by surface thermodynamic analysis. NAC-coated surfaces showed no cytotoxicity to human fibroblast cells. This study has successfully utilized NAC as an antibiofilm coating, which may pave the way for improved prophylactic coatings on medical implant devices in the future.

The Efficacy of an N-Acetylcysteine-Antibiotic Combination Therapy on Achromobacter xylosoxidans in a Cystic Fibrosis Sputum/Lung Cell Model.

Cystic fibrosis (CF) is a disorder causing dysfunctional ion transport resulting in the accumulation of viscous mucus. This environment fosters a chronic bacterial biofilm-associated infection in the airways. Achromobacter xylosoxidans, a gram-negative aerobic bacillus, has been increasingly associated with antibiotic resistance and chronic colonisation in CF. In this study, we aimed to create a reproducible model of CF infection using an artificial sputum medium (ASMDM-1) with bronchial (BEAS-2B) and macrophage (THP-1) cells to test A. xylosoxidans infection and treatment toxicity. This study was conducted in three distinct stages. First, the tolerance of BEAS-2B cell lines and two A. xylosoxidans strains against ASMDM-1 was optimised. Secondly, the cytotoxicity of combined therapy (CT) comprising N-acetylcysteine (NAC) and the antibiotics colistin or ciprofloxacin was tested on cells alone in the sputum model in both BEAS-2B and THP-1 cells. Third, the efficacy of CT was assessed in the context of a bacterial infection within the live cell/sputum model. We found that a model using 20% ASMDM-1 in both cell populations tolerated a colistin-NAC-based CT and could significantly reduce bacterial loads in vitro (~2 log10 CFU/mL compared to untreated controls). This pilot study provides the foundation to study other bacterial opportunists that infect the CF lung to observe infection and CT kinetics. This model also acts as a springboard for more complex co-culture models.

Disruption of biofilms and killing of Burkholderia cenocepacia from cystic fibrosis lung using an antioxidant-antibiotic combination therapy.

Cystic fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The resulting chloride and bicarbonate imbalance produces a thick, static lung mucus. This mucus is not easily expelled from the lung and can be colonised by bacteria, leading to biofilm formation. CF lung infection with Burkholderia cepacia complex (BCC), particularly the subspecies B. cenocepacia, results in higher morbidity and mortality. Patients infected with BCC can rapidly progress to "cepacia syndrome", a fatal necrotising pneumonia. The aim of this study was to identify whether a combination therapy (CT) of selected antioxidants and antibiotics significantly disrupts B. cenocepacia biofilms and to determine the optimum CT level for treatment. Using controlled in vitro spectrophotometry, colony-forming unit and microscopy assays, three antioxidants (N-acetylcysteine [NAC], glutathione and vitamin C) and three antibiotics (ciprofloxacin, ceftazidime and tobramycin) were screened and assessed for their ability to disrupt the early and mature biofilms of six B. cenocepacia CF isolates. A combination of NAC and ciprofloxacin produced a statistically significant biofilm disruption in all strains tested, with growth inhibition (>5-8 log10) observed when exposed to 4890 or 8150 µg/mL NAC in combination with 32 or 64 µg/mL ciprofloxacin. NAC-mediated biofilm disruption may be aided by the acidic pH of NAC at higher concentrations. This study showed that NAC is an effective disruptor that reduces the necessity for high concentrations of antibiotic. Further research will focus on the host toxicity and efficacy in ex vivo CF models.

Effect of N-Acetylcysteine in Combination with Antibiotics on the Biofilms of Three Cystic Fibrosis Pathogens of Emerging Importance.

Cystic fibrosis (CF) is a genetic disorder causing dysfunctional ion transport resulting in accumulation of viscous mucus that fosters chronic bacterial biofilm-associated infection in the airways. Achromobacter xylosoxidans and Stenotrophomonas maltophilia are increasingly prevalent CF pathogens and while Burkholderia cencocepacia is slowly decreasing; all are complicated by multidrug resistance that is enhanced by biofilm formation. This study investigates potential synergy between the antibiotics ciprofloxacin (0.5-128 µg/mL), colistin (0.5-128 µg/mL) and tobramycin (0.5-128 µg/mL) when combined with the neutral pH form of N-Acetylcysteine (NACneutral) (0.5-16.3 mg/mL) against 11 cystic fibrosis strains of Burkholderia, Stenotrophomonas and Achromobacter sp. in planktonic and biofilm cultures. We screened for potential synergism using checkerboard assays from which fraction inhibitory concentration indices (FICI) were calculated. Synergistic (FICI ≤ 0.5) and additive (0.5 > FICI ≥ 1) combinations were tested on irreversibly attached bacteria and 48 h mature biofilms via time-course and colony forming units (CFU/mL) assays. This study suggests that planktonic FICI analysis does not necessarily translate to reduction in bacterial loads in a biofilm model. Future directions include refining synergy testing and determining further mechanisms of action of NAC to understand how it may interact with antibiotics to better predict synergy.