Development of an oral biofilm model to investigate the combination of mechanical forces and novel antimicrobial chewing gums on oral bacteria.

Abstract

Oral biofilms are the massive-multispecies communities of bacteria which live in the oral cavity. Over time there can be shifts in this community, which lead to disease; this is called dysbiosis. Oral diseases can range from gum inflammation to periodontitis and can lead to systemic diseases. People have an average of around 250 bacterial species in their oral cavity at any single timepoint; however, the total oral microbiome comprises over 700 species. With 56% of the global population experiencing an oral disease during their lifetime, the discovery of novel methods for removing oral biofilms is an important unmet need. This thesis aimed to investigate the use of antimicrobials in conjunction with chewing gum, to reduce and prevent the formation of oral biofilms. A novel model for investigating oral biofilms, which includes aspects of the oscillatory mechanical forces experienced during chewing, was developed. A combination of literature searches and lab characterisation was used to create this novel model, building on the work completed by Wessel et al., observing bacteria being trapped in chewing gums. Eight antimicrobial products were tested against planktonic Streptococcus mutans. Of these, four, cinnamaldehyde, methyl salicylate, propolis and chlorhexidine, were seen to cause a significant reduction in bacterial culturability. These were developed into chewing gum prototypes, and their antimicrobial release concentrations were characterised as ranging from 0% to 19% of the total added. S. mutans biofilms were then exposed to the chewing gum diffusates, but no effect was observed due to the low release concentration. However, when biofilms were exposed to chewing in the presence of cinnamaldehyde, an additive effect was detected. Culturable counts and confocal laser scanning microscopy were used to provide insight into the effects that the antimicrobials and chewing forces have on ex vivo biofilms. When chewed, the culturable counts show a 3.5-log reduction in the number of culturable bacteria in both aerobic and aerotolerant species. However, a decrease was not observed when exposed to antimicrobials at 1X MIC or 4X MIC. When using confocal image analysis to investigate the changes in biofilm viability and structure, a high level of variation was observed between reactors. Finally, this project investigated the methods of improving the DNA concentration yielded from oral biofilm extraction without the use of PCR. Fifteen extraction protocols and optimisation steps were tested. Using a phenol/chloroform extraction method with a mechanical lysis step instead of using a Qiagen PowerBiofilm kit; and swapping from a hydroxyapatite coupon to a plastic coupon were the most promising steps for improving yield.

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Identifiers

ORCID for Katherine Emma Roe: orcid.org/0000-0002-6511-3087

ORCID for Jeremy Webb: orcid.org/0000-0003-2068-8589

ORCID for Paul Stoodley: orcid.org/0000-0001-6069-273X

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