Novel insights into the antibiofilm activity of borneol, citral and their combination

The problem of increased antibiotic resistance and improved environmental adaptability from bacteria has become a challenge for food safe and our public health, and this is of particular concern when bacteria form biofilms. Phytochemicals have shown great advantages and application prospects for the prevention of complex biofilms due to their excellent antibacterial properties, hard to develop resistance, and multi-target antibacterial strategies. Therefore, it is particularly important to understand their antibiofilm activity and mode of action against biofilm bacteria in different biofilm models, which is particularly important for realizing the control of pathogenic bacteria biofilm. Many different types of biofilms occur in food industry and clinical patients, but two of the most common ones are surface-attached biofilms and chronic biofilm aggregates that are found in pulmonary infections in cystic fibrosis patients and wound infections. Hence, the overall aim of this dissertation is to explore borneol and citral-based approaches for the prevention and treatment of different biofilms. The primary contents and results are as follows: (1) Bacterial isolates from 480 food raw material samples were collected and investigated. Tthe prevalence of antibiotic resistance, biofilm forming ability and incidence of class 1 integron of foodborne pathogenic isolates was assessed. Antimicrobial resistance was widespread amongst the obtained pathogenic isolates. 61.5% isolates were resistant to at least one antibiotic and 17.7% were multi-resistant to three or more antibiotics. Resistant strains isolated from fresh/frozen pork and fresh chicken, including 12 isolates of E. coli and 18 isolates of Salmonella spp., carry class I integron genes which was most likely related to the abuse of antibiotics in animal husbandry. 6 isolates of Salmonella spp. carrying the positive gene cassette had weak or moderate biofilm formation ability, and of which 3 isolates had multidrug resistance; all E. coli isolated had multidrug resistance, and of which 7 isolates carrying the positive gene cassette had weak or moderate biofilm-forming ability. Correlation analysis showed that there was no significant difference between the biofilm formation ability of Salmonella spp. carrying class I integron and bacterial drug resistance, indicating that the horizontal transfer of drug resistance genes is universal. (2) The synergistic antimicrobial and antibiofilm activity of citral and borneol against common pathogenic bacteria in several in vitro surface-attached models were evaluated. Both citral and borneol have broad-spectrum antibacterial activity, and the synergistic antibacterial effect is significant. These phytochemicals inhibited biofilm formation by preventing bacterial adhesion and a dose-dependent manner was observed. They can effectively eradicate 90.73-100% of the preformed biofilm grown on the surface of the polycarbonate membrane. Leakage of cell contents and changes of biofilm morphology indicated that these two phytochemicals disrupted the cell membranes of planktpnic bacteria and bacteria within biofilms. Citral and borneol exert anti-biofilm activity mainly by inhibiting bacterial adhesion and disrupting the integrity of cell membranes. (3) Pickering emulsions with capsulized phytochemicals including citral or borneol and citral (C-Cap/BC-Cap) stabilized by hydrophilic amine-functionalized silica nanoparticles (SiO2-NH2 NPs) were fabricated first. The optimum preparation conditions of Pickering emulsion were determined, and this emulsion could be stored stably for more than 20 days at 40℃. Then Pickering emulsions as novel antibacterial agents to combat E. coli and S. aureus infections as well as their mode of action were assesed. Compared to citral or BC, the antibacterial effect of C-Cap/BC-Cap increased by 2-8 folds. Indeed, both Pickering emulsions exhibited effective antibiofilm activities against E. coli and S. aureus in in vitro biofilm models. They can effectively eradicate 99.95-100% of the preformed biofilms grown on the surface of the polycarbonate membranes. Pickering emulsion can effectively improve the water solubility and chemical stability of these two phytochemicals, penetrate biofilm matrix aand effectively deliver them to the bacterial surface; and then these two phytochemicals disrupted the integrity of the cell membrane, kill the cells, and cause the collapse of the biofilm structure. (4) The antibiofilm activity of borneol, citral and BC as well as their Pickering emulsions against S. aureus and P. aeruginosa was examined in an in vivo-like SCFM2 model, an in vivo-like wound model and an in vivo Galleria mellonella model, respectively. The Pickering emulsions demonstrated an enhanced biofilm-inhibitory activity as compared to both citral and BC, reducing the MBIC values up to 2-4 times against P. aeruginosa PAO1 and 2-8 times against S. aureus P8-AE1 in SCMF2 medium. Specially, citral, the combination borneol/citral and their Pickering emulsions can completely eliminate the established biofilm of S. aureus P8-AE1. Interestingly, effectiveness of Pickering emulsions was also demonstrated in the wound model with a reduction of up to 4.8-log units in biofilm formation by S. aureus Mu50. Furthermore, citral and Pickering emulsions exhibited a significant degree of protection against S. aureus infection in the G. mellonella model. Combination of phytochemicals and encapsulation in Pickering emulsion are promising strategies to improve the efficacy of citral, especially against biofilms under challenging host-mimicking conditions. In conclusion, this dissertation shows that combination of phytochemicals and encapsulation in Pickering emulsion are promising strategies to improve the efficacy of citral against biofilms in both in vitro and in vivo-like conditions. These capsules have potential applications as a general surface disinfectant as well as an antibiofilm agents for the treatment of lung infections in cystic fibrosis and wound infections