Antibacterial and anti-adherence effects of a plant extract mixture (PEM) and its individual constituent extracts (, ., and .) on single- and dual-species biofilms.

Background: Plant extracts mixture (PEM) and its individual constituent plant extracts( sp., sp., sp.) are known to have an anti-adhering effect towards oral bacteria in the single-species biofilm. To date, the adhering ability of the early and late plaque colonisers ( and ) to PEM-treated experimental pellicle have not been investigated in dual-species biofilms.

Methods: Fresh leaves of these plants were used in the preparation of the respective aqueous extract decoctions. The minimum inhibitory concentration (MIC) of the extracts towards ATCC BAA-1455 and ATCC 25175 was determined using a two-fold serial microdilution method. The sum of fractional inhibitory concentration (ΣFIC) index of PEM and its constituent plant extracts was calculated using the MIC values of the plants. The minimum bactericidal concentration (MBC) of the plant extracts was also determined. The anti-adherence effect of the plant extracts (individually and mixed) was carried out by developing simulated and respectively in single- and dual-species of biofilms in the Nordini's Artificial Mouth (NAM) model system in which the experimental pellicle was pretreated with the plant extract before bacterial inoculation. The bacterial population in the respective biofilms was quantified using ten-fold serial dilutions method and expressed as colony forming unit per ml (CFU/ml). The bacterial population was also viewed using Scanning Electron Microscope (SEM). All experiments were done in triplicate.

Results: The PEM compared with its respective constituent plants showed the lowest MIC towards (3.81 mg/ml) and (1.91 mg/ml) and exhibited a synergistic effect. The sp. (15.24 mg/ml) and, PEM and sp. (30.48 mg/ml) showed the lowest MBC towards and respectively. The anti-adherence effect of the PEM and its respective constituent plants (except sp.) was different for the two bacteria in the single-species biofilm. In the dual-species biofilms, PEM demonstrated similar anti-adherence effect towards and . The proportions of the bacterial population viewed under SEM appeared to be in agreement with the quantified population.

Discussion: The combination of the active constituents of the individual plant extracts in PEM may contribute to its low MIC giving rise to the synergistic effect. The different anti-adherence effect towards and in both single- and dual-species biofilms could be due to the different proportion of the active constituents of the extracts and the interaction between different bacteria. The better adhering ability of towards the PEM-treated pellicle when present together with in the dual-species biofilms may suggest the potential of PEM in controlling the balance between the early and late colonisers in biofilms.