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.

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 (NAC_neutral) (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.

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.

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.