Abstract
Disinfection as a component of infection control is recognized as an essential consideration in healthcare particularly in light of the risk from emerging pathogens and the increasing percentage of vulnerable, at-risk individuals in populations. This thesis begins by reviewing the principles and guidelines concerning disinfection in healthcare settings, and the requirements and methods to evaluate the effectiveness of chemical biocides. It discusses disinfection requirements in the dental healthcare settings as well as in home environments, focusing on acrylic removable dental prostheses (RDPs). While highlighting the need for research into the non-toxic, low-cost, and ‘green’ chemical biocide, electrolysed oxidising water (EOW), it was found that the inter-relationship and significance of EOW physicochemical properties determine its type, efficacy, and shelf-life. Moreover, research investigating the molecular mechanism of action of neutral-pH EOW on microbial cells, especially the opportunistic fungal pathogen Candida albicans (that causes denture stomatitis), is minimal. There is a lack of clarity on the specific gaps in knowledge regarding EOW use in dental healthcare and so a detailed scoping literature review was undertaken to map the published research.
The scoping review revealed a paucity of data on ready-to-use (RTU), neutral-pH EOW pertaining to its shelf-life and effectiveness as a denture disinfectant as well as the effects of its repeated, long-term use on denture base resins. Most studies investigated ‘point-of-use’ produced EOW that requires installation and maintenance of generators, making it unsuitable for resource-challenged communities and home-disinfection needs of individuals.
This research aimed to investigate, in vitro, the storage-related stability and antimicrobial efficacy of RTU neutral-pH EOW and its suitability as a denture disinfectant by evaluating its effectiveness against C. albicans biofilms formed on denture acrylic resin, its molecular mechanism of action against C. albicans, and its long-term effects on denture base resins.
Sixty-four RTU neutral-pH EOW (Envirolyte, New Zealand) bottles (100 mL) that were procured in 13 batches were tested over five months from manufacture. Weekly evaluations of the RTU neutral-pH EOW properties (pH, redox, available chlorine content, hypochlorous acid (HOCl) concentration and antimicrobial efficacy against Staphylococcus aureus ATCC6538 and C. albicans ATCC10231) were performed over 28 days from bottle opening, for EOW samples that were stored at 4°C, room temperature (21°C) and 37°C. Minimum inhibitory concentrations (MIC90) of EOW-HOCl were determined against S. aureus ATCC6538 and C. albicans ATCC10231, SC5314 and recent human isolates. Antibiofilm efficacy against C. albicans ATCC10231 was determined by measuring the metabolic activity of biofilms formed on denture resin discs using a 2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. The EOW became moderately acidic with increasing storage duration. The [HOCl] decreased by 8% when stored at 21°C a reduction similar to storage at 4°C (p = 0.1); in contrast, [HOCl] decreased by 18% in samples stored at 37°C. A contact time of 1 min with EOW stored under any condition caused a >6 log10 reduction in S. aureus colony forming units (cfu) and >5 log10 reduction in C. albicans cfu, in line with British and European test standard requirements for chemical disinfectants and antiseptics. The mean MIC90 EOW-HOCl for a 5 min contact time was 37 ±10 µM against S. aureus and 54 ±13.7 µM against C. albicans. Furthermore, 5 min exposure to undiluted EOW caused an 87% reduction in the metabolic activity of C. albicans biofilms, a significantly greater reduction than that of a commercial denture cleaning tablet (DCT, 43%; p <0.05).
Mid-logarithmic phase C. albicans SC5314 cultures were incubated with sub-inhibitory concentrations of RTU neutral-pH EOW for 60 min. Cells were harvested, and total RNA extracted. RNA-sequencing and transcriptomic analyses revealed that EOW induced a concentration-dependent stress adaptive response in C. albicans, further validated by qRT-PCR assays. At a moderate sub-inhibitory concentration (15 µM; 0.125x of MIC90 for a cell density of 1 x 107 cells/mL) EOW induced a significant upregulation (log2 fold change >2) of genes that respond to oxidative stress (EBP2, GAP6), weak organic acid stress (PRN1), and heat-shock (HSP21). This response indicates an adaptive mechanism to HOCl-based biocides in yeast, and which could be crucial for yeast survival, antimicrobial-resistance or biocide-tolerance, and pathogenicity. At a higher sub-inhibitory concentration (60 µM; 0.5x of MIC90) EOW caused a significant downregulation of most genes (notably, 1.9 to 3 log2 fold changes in SUT1, HNM3, STP4), a cessation of growth, and an upregulation of genes involved in ammonia transport, carbohydrate metabolism, endoplasmic reticulum unfolded protein response and apoptotic response pathway mechanisms (ATO2, IRE1).
Five min daily disinfection with undiluted neutral-pH EOW of conventional heat-polymerized, 3D-printed, or CAD/CAM-milled denture resin samples performed over the long-term (up to 3.0 y) was simulated in a customized cycling device and effects on resin properties (colour, surface roughness, surface hardness, flexural strength) were measured. Effects were compared with those caused by tap water and a commercial DCT. Resin properties were affected both by the type of denture resin and the immersion agent. RTU neutral-pH EOW did not significantly change the colour (∆E00), the surface roughness (∆Ra) or the flexural strength (MPa) of denture resins. Results were comparable to treatment with the DCT (p >0.05), although the mean ∆E00 caused by the DCT for the heat-polymerized and the CAD/CAM-milled denture resins was above the perceptibility threshold (threshold where change in colour becomes perceivable). The mean surface hardness (Vickers; GPa) was not adversely impacted by either the EOW or the DCT treatments at 3.0 y, except for 3D-printed denture resins, where it decreased by 23% with both agents compared to the baseline (p <0.0001), and when compared to a 13% decrease caused by tap water (p<0.01).
In summary, the research described in this thesis demonstrates RTU neutral-pH EOW to be stable for up to five months from manufacture and longer shelf-life can be achieved under refrigerated conditions. The effectiveness of RTU neutral-pH EOW against C. albicans test strains, recent human isolates and biofilms formed on denture resin surfaces, and the overall minimal effects on denture resin properties from long-term exposure, supports its use as a denture cleaner. Furthermore, the investigation revealed novel and informative data on the molecular mechanism of action of EOW and C. albicans adaptive responses that, with further investigation, will help to understand and expand its application. The results indicate that neutral-pH EOW is a suitable agent for studying HOCl-stress adaptive responses and resistance mechanisms in other fungal and bacterial species.