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.

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.

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.

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