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.

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.

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.

N-Acetylcysteine Protects Bladder Epithelial Cells from Bacterial Invasion and Displays Antibiofilm Activity against Urinary Tract Bacterial Pathogens.

Introduction: Urinary tract infections (UTIs) affect more than 150 million individuals annually. A strong correlation exists between bladder epithelia invasion by uropathogenic bacteria and patients with recurrent UTIs. Intracellular bacteria often recolonise epithelial cells post-antibiotic treatment. We investigated whether N-acetylcysteine (NAC) could prevent uropathogenic E. coli and E. faecalis bladder cell invasion, in addition to its effect on uropathogens when used alone or in combination with ciprofloxacin. Methods: An invasion assay was performed in which bacteria were added to bladder epithelial cells (BECs) in presence of NAC and invasion was allowed to occur. Cells were washed with gentamicin, lysed, and plated for enumeration of the intracellular bacterial load. Cytotoxicity was evaluated by exposing BECs to various concentrations of NAC and quantifying the metabolic activity using resazurin at different exposure times. The effect of NAC on the preformed biofilms was also investigated by treating 48 h biofilms for 24 h and enumerating colony counts. Bacteria were stained with propidium iodide (PI) to measure membrane damage. Results: NAC completely inhibited BEC invasion by multiple E. coli and E. faecalis clinical strains in a dose-dependent manner (p < 0.01). This was also evident when bacterial invasion was visualised using GFP-tagged E. coli. NAC displayed no cytotoxicity against BECs despite its intrinsic acidity (pH ~2.6), with >90% cellular viability 48 h post-exposure. NAC also prevented biofilm formation by E. coli and E. faecalis and significantly reduced bacterial loads in 48 h biofilms when combined with ciprofloxacin. NAC visibly damaged E. coli and E. faecalis bacterial membranes, with a threefold increase in propidium iodide-stained cells following treatment (p < 0.05). Conclusions: NAC is a non-toxic, antibiofilm agent in vitro and can prevent cell invasion and IBC formation by uropathogens, thus providing a potentially novel and efficacious treatment for UTIs. When combined with an antibiotic, it may disrupt bacterial biofilms and eliminate residual bacteria.

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.

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 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.

Conditions Under Which Glutathione Disrupts the Biofilms and Improves Antibiotic Efficacy of Both ESKAPE and Non-ESKAPE Species.

Bacterial antibiotic resistance has increased in recent decades, raising concerns in hospital and community settings. Novel, innovative strategies are needed to eradicate bacteria, particularly within biofilms, and diminish the likelihood of recurrence. In this study, we investigated whether glutathione (GSH) can act as a biofilm disruptor, and enhance antibiotic effectiveness against various bacterial pathogens. Biological levels (10 mM) of GSH did not have a significant effect in inhibiting growth or disrupting the biofilm in four out of six species tested. However, exposure to 30 mM GSH showed >50% decrease in growth for all bacterial species, with almost 100% inhibition of Streptococcus pyogenes and an average of 94-52% inhibition for Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive S. aureus (MSSA) and multi-drug resistant Acinetobacter baumannii (MRAB) isolates, respectively. Klebsiella pneumoniae and Enterobacter sp. isolates were however, highly resistant to 30 mM GSH. With respect to biofilm viability, all species exhibited a >50% decrease in viability with 30 mM GSH, with confocal imaging showing considerable change in the biofilm architecture of MRAB isolates. The mechanism of GSH-mediated biofilm disruption is possibly due to a concentration-dependent increase in GSH acidity that triggers cleaving of the matrix components. Enzymatic treatment of MRAB revealed that eDNA and polysaccharides are essential for biofilm stability and eDNA removal enhanced amikacin efficiency. Combination of GSH, amikacin and DNase-I showed the greatest reduction in MRAB biofilm viability. Additionally, GSH alone and in combination with amikacin fostered human fibroblast cell (HFF-1) growth and confluence while inhibiting MRAB adhesion and colonization.

Application of the Morita-Baylis-Hillman reaction in the synthesis of 3-[(N-cycloalkylbenzamido)methyl]-2-quinolones as potential HIV-1 integrase inhibitors.

A practicable six-step synthetic pathway has been developed to access a library of novel 3-[(N-cycloalkylbenzamido)methyl]-2-quinolones using Morita-Baylis-Hillman methodology. These compounds and their 3-[(N-cycloalkylamino)methyl]-2-quinolone precursors have been screened as potential HIV-1 integrase (IN) inhibitors. A concomitant survey of their activity against HIV-1 protease and reverse-transcriptase reveals selective inhibition of HIV-1 IN.

Directed Growth of Orthorhombic Crystals in a Micropillar Array.

We report directed growth of orthorhombic crystals of potassium permanganate in spatial confinement of a micropillar array. The solution is introduced by spontaneous wicking to give a well-defined film (thickness 10-15 µm; volume ∼600 nL) and is connected to a reservoir (several microliters) that continuously "feeds" the evaporating film. When the film is supersaturated, crystals nucleate and preferentially grow in specific directions guided by one of several possible linear paths through the pillar lattice. Crystals that do not initially conform are stopped at an obstructing pillar, branch into another permitted direction, or spontaneously rotate to align with a path and continue to grow. Microspectroscopy is able to track the concentration of solute in a small region of interest (70 × 100 µm2) near to growing crystals, revealing that the solute concentration initially increases linearly beyond the solubility limit. Crystal growth near the region of interest resulted in a sharp decrease in the local solute concentration (which rapidly returns the concentration to the solubility limit), consistent with estimated diffusion time scales (<1 s for a 50 µm length scale). The ability to simultaneously track solute concentration and control crystal orientation in nanoliter samples will provide new insight into microscale dynamics of microscale crystallization.

Influence of Sample Volume and Solvent Evaporation on Absorbance Spectroscopy in a Microfluidic "Pillar-Cuvette".

Spectroscopic analysis of solutions containing samples at high concentrations or molar absorptivity can present practical challenges. In absorbance spectroscopy, short optical path lengths or multiple dilution is required to bring the measured absorbance into the range of the Beer's Law calibration. We have previously reported an open "pillar-cuvette" with a micropillar array that is spontaneously filled with a precise (nL or µL) volume to create the well-defined optical path of, for example, 10 to 20 µm. Evaporation should not be ignored for open cuvettes and, herein, the volume of loaded sample and the rate of evaporation from the cuvette are studied. It was found that the volume of loaded sample (between 1 and 10 µL) had no effect on the Beer's Law calibration for methyl orange solutions (molar absorptivity of (2.42 ± 0.02)× 10(4) L mol(-1) cm(-1)) for cuvettes with a 14.2 ± 0.2 µm path length. Evaporation rates of water from methyl orange solutions were between 2 and 5 nL s(-1) (30 - 40% relative humidity; 23°C), depending on the sample concentration and ambient conditions. Evaporation could be reduced by placing a cover slip several millimeters above the cuvette. Importantly, the results show that a "drop-and-measure" method (measurement within ∼3 s of cuvette loading) eliminates the need for extrapolation of the absorbance-time data for accurate analysis of samples.

Heteroditopic P,N ligands in gold(I) complexes: synthesis, structure and cytotoxicity.

New heteroditopic, bi- and multidentate imino- and aminophosphine ligands were synthesised and complexed to [AuCl(THT)] (THT=tetrahydrothiophene). X-ray crystallography confirmed Schiff base formation in three products, the successful reduction of the imino-group to the sp(3)-hybridised amine in several instances, and confirmed the formation of mono-gold(I) imino- and aminophosphine complexes for four Au-complexes. Cytotoxicity studies in cancerous and non-cancerous cell lines showed a marked increase in cytotoxicity upon ligand complexation to gold(I). These findings were supported by results from the 60-cell line fingerprint screen of the Developmental Therapeutics Programme of the National Institutes of Health for two promising compounds. The cytotoxicity of some of these ligands and gold(I)complexes is due to the induction of apoptosis. The ligands and gold(I)complexes demonstrated selective toxicity towards specific cell lines, with Jurkat T cells being more sensitive to the cytotoxic effects of these compounds, while the non-cancerous human cell line KMST6 proved more resistant when compared to the cancerous cell lines. Results from the NIH DTP 60 cell-line fingerprint screen support the observed enhancement of cytotoxicity upon gold(I) complexation. One gold(I)complex induced high levels of apoptosis at concentrations of 50 µM in all the cell lines screened in this study, while some of the other compounds selectively induced apoptosis in the cell lines. These results point towards the potential for selective toxicity to cancerous cells through the induction of apoptosis.

The evaluation of statins as potential inhibitors of the LEDGF/p75-HIV-1 integrase interaction.

Lovastatin was identified through virtual screening as a potential inhibitor of the LEDGF/p75-HIV-1 integrase interaction. In an AlphaScreen assay, lovastatin inhibited the purified recombinant protein-protein interaction (IC50 = 1.97 ± 0.45 µm) more effectively than seven other tested statins. None of the eight statins, however, yielded antiviral activity in vitro, while only pravastatin lactone yielded detectable inhibition of HIV-1 integrase strand transfer activity (31.65% at 100 µm). A correlation between lipophilicity and increased cellular toxicity of the statins was observed.

4-(3-Azaniumylpropyl)morpholin-4-ium chloride hydrogen oxalate: an unusual example of a dication with different counter-anions.

The mixed organic-inorganic title salt, C7H18N2O(2+)·C2HO4(-)·Cl(-), forms an assembly of ionic components which are stabilized through a series of hydrogen bonds and charge-assisted intermolecular interactions. The title assembly crystallizes in the monoclinic C2/c space group with Z = 8. The asymmetric unit consists of a 4-(3-azaniumylpropyl)morpholin-4-ium dication, a hydrogen oxalate counter-anion and an inorganic chloride counter-anion. The organic cations and anions are connected through a network of N-H···O, O-H···O and C-H···O hydrogen bonds, forming several intermolecular rings that can be described by the graph-set notations R3(3)(13), R2(1)(5), R1(2)(5), R2(1)(6), R2(3)(6), R2(2)(8) and R3(3)(9). The 4-(3-azaniumylpropyl)morpholin-4-ium dications are interconnected through N-H···O hydrogen bonds, forming C(9) chains that run diagonally along the ab face. Furthermore, the hydrogen oxalate anions are interconnected via O-H···O hydrogen bonds, forming head-to-tail C(5) chains along the crystallographic b axis. The two types of chains are linked through additional N-H···O and O-H···O hydrogen bonds, and the hydrogen oxalate chains are sandwiched by the 4-(3-azaniumylpropyl)morpholin-4-ium chains, forming organic layers that are separated by the chloride anions. Finally, the layered three-dimensional structure is stabilized via intermolecular N-H···Cl and C-H···Cl interactions.

Gold-phosphine binding to de novo designed coiled coil peptides.

The coordination of the therapeutically interesting [AuCl(PEt(3))] to the de novo designed peptide, TRIL23C, under aqueous conditions, is reported here. TRIL23C represents an ideal model to investigate the binding of [AuCl(PEt(3))] to small proteins in an effort to develop novel gold(I) phosphine peptide adducts capable of mimicking biological recognition and targeting. This is due to the small size of TRIL23C (30 amino acids), yet stable secondary and tertiary fold, symmetric nature and the availability of only one thiol binding site. [AuCl(PEt(3))] was found to react readily with the Cys side chain in a 1:1 ratio as confirmed by UV-visible, (31)P NMR and mass spectrometry. Circular dichroism confirmed that the coiled coil structure was retained on coordination of the {Au(PEt(3))}(+) unit. Redesign of the exterior of TRIL23C based on a biologically relevant recognition sequence found in GCN4, did not alter the coordination chemistry of [AuCl(PEt(3))]. To the best of our knowledge, this represents the first report on the coordination of gold(I) phosphine compounds to de novo designed peptides, and could lead to the generation of novel gold(I) phosphine peptide therapeutics in the future.

{µ-1,2-Bis[bis-(4-meth-oxy-phen-yl)phosphan-yl]-1,2-diethyl-hydrazine-κP:P'}bis-[chloridogold(I)] tetra-hydro-furan disolvate.

The title compound, [Au(2)Cl(2)(C(32)H(38)N(2)O(4)P(2))]·2C(4)H(8)O, is formed from a bidentate phosphine ligand complexed to two linear gold(I) nuclei [P-Au-Cl = 175.98 (3)°]. The nuclei are 3.1414 (2) Šapart. The mol-ecule exhibits a twofold symmetry axis. Stacks of the compound are formed through inter-molecular C-H⋯Cl inter-actions, while the tetra-hydro-furan (THF) solvate is further attached to the stacks through weak C-H⋯O hydrogen bonding from the THF O atom to two separate H atoms on the complex.

{µ-1,2-Bis[bis(4-meth-oxy-phen-yl)phosphan-yl]-1,2-dimethyl-hydrazine-κP:P'}bis-[chloridogold(I)] tetra-hydro-furan disolvate.

The title compound, [Au(2)Cl(2)(C(30)H(34)N(2)O(4)P(2))]·2C(4)H(8)O, is formed from a bidentate phosphine ligand complexed to two almost linearly coordinated gold(I) atoms [P-Au-Cl = 175.68 (3) Å]. The nuclei are 3.122 (2) Šapart. The mol-ecule exhibits a twofold rotation axis.

[µ-1,2-Bis(diphenyl-phosphan-yl)-1,2-diethyl-hydrazine-κP:P']bis-[chlorido-gold(I)] tetra-hydro-furan disolvate.

The title compound, [Au(2)Cl(2)(C(28)H(30)N(2)P(2))]·2C(4)H(8)O, was synthesized from a bidentate phosphine ligand complexed to two linear gold(I) chloride moieties. The Au(I) atom is in an almost linear coordination with a P-Au-Cl angle of 179.22 (4)°. The complex molecules reside on a twofold rotation axis.

Dictyostatin flexibility bridges conformations in solution and in the ß-tubulin taxane binding site.

Dictyostatin (DCT, 1) is a complex, flexible polyketide macrolide that demonstrates potent microtubule-polymerization activity. Both a solution structure (2a) and a possible binding mode for DCT (Conf-1) have been proposed by earlier NMR experiments. In the present study, the conformational landscape of DCT in DMSO-d(6) and methanol-d(4) was explored using extensive force-field-based conformational searches combined with geometric parameters derived from solution NMR data. The results portray a diversity of conformations for dictyostatin that illustrates the molecule's flexibility and excludes the previously suggested dominant solution conformation 2a. One conformation present in DMSO-d(6) with a 7% population (Conf-2, 0.6 kcal/mol above the global minimum at 298°) also satisfies the TR-NOESY NMR parameters of Canales et al. that characterize the taxane binding-site interaction between DCT and assembled microtubules in water. Application of several docking methods (Glide, Autodock, and RosettaLigand) has identified a low-energy binding model of the DCT/ß-tubulin complex (Pose-2/Conf-2) that is gratifyingly compatible with the emerging DCT structure-activity data.