Antimicrobial Activity Of Bactenecin And Its Derivatives Against Cariogenic Bacteria

1. Antimicrobial and antibiofilm activity of bactenecin and its derivative againstcariogenic microorganismsBactenecin, extracted from cattle neutrophils, is the smallest natural cationicpolypeptide, which contains12amino acids. The two cysteine residues form a disulfidebond, which makes the natural bactenecin a cyclic peptide. Previous studies have shownthat bacternecin has strong antibacterial activity against Escherichia coli andStaphylococcus aureus, possibly through an increase of the permeability of the bacterialcell membrane and the inhibition of bacterial DNA synthesis. By linear modification, thederivatives would enhance the antibacterial activity of Bactenecin, and decrease itscytotoxicity.In the first part of present study, we investigated whether bactenecin and itsderivative is an effective antibiotic peptide against common cariogenic microorganisms and performed a preliminary study of the antimicrobial mechanism. We assayed minimalinhibitory concentration (MIC) and minimal bactericide concentration (MBC). Theresults suggested that bac8c showed the stronger antimicrobial activity compared to otherbactenecin derivatives; therefore, we chose bac8c to perform further investigation ofcytotoxic and bactericidal dynamics. At the same time, we also studied the effect ofsaliva on the of bac8c antimicrobial activity.Our results of MIC, MBC and bactericidal dynamics test suggested that bac8c hasgood antibacterial activity against common cariogenic bacteria, and is able to completelykill S.mutans in a short period of time. Cytotoxicity results suggested that the cytotoxicityof bac8c is sort of time-dependent, and even a higher concentration of bac8c will not becytotoxic in a short time. Although components of saliva could affect antimicrobialactivity, bac8c dissolved in saliva still showed antimicrobial effects against oralmicroorganisms. Our findings proved that bac8c has the potential to kill dental biofilmsand to be finally used in the clinic.2. Preliminary study of the antibacterial mechanism of bac8c against commoncariogenic bacteria.The first part of our experiments have confirmed antibacterial ability of bac8cagainst cariogenic bacteria and smaller cell toxicity. Therefore, we further studiedantibacterial mechanism of bac8c. Microscopes are most commonly used in the study ofthe antibacterial mechanism, as microscope can directly observe the effect ofantimicrobial peptides on the microbial surface and internal ultrastructure, to determinethe area of antimicrobial peptide works.Observation of SEM showed that: surface of S.mutans changed after, and its roughsurfaces contained debris and irregular shape; membrane of Lactobacillus fermentumtreated with bac8c had slightly swelling and apertures formation. The results of TEMshowed that: Mutans treated with bac8c had damaged membrane and concentratednuclear; The Lactobacillus fermentum treated with bac8c had more serious injuries,including breaking down of the cell walls, outflow of the contents and nuclear material decomposition. In addition, some of Streptococcus mutans treated with bac8c retainednormal shape after death.Our findings suggested that effect of bac8c on Gram-positive bacteria such asS.mutans and Lactobacillus fermentum may function in two ways: one way is throughbacterial cell membrane, i.e. through pore formation and rupture of the cell membraneleading ultimately to the lysis of the bacterial cells; another way is to act directly on theintracellular targets, such as the synthesis of macromolecules. This way is similar to theway Bac8c function on the Gram-negative bacteria.3. Preliminary study of the antibacterial mechanism of bac8c against Streptococcusmutans biofilm.The third part of present study focused on the effect of bac8c on the S.mutansbiofilm formed under static conditions and dynamic bioflux systems, and studied themechanism of bac8c antimicrobial activity.The results of our study indicated that, bactericidal effect of bac8c against staticbiofilm and dynamic biofilm was concentration-dependent, and a high concentration canpenetrate into the internal biofilm to kill bacteria. The presence of the bac8c significantlydecreased the biofilm formation related gene expression of Streptococcus mutans, whichto some extent explained the reasons for the suppressed biofilm.For the prevention of dental caries, the most effective method is to prevent the earlyformation of a biofilm, or inhibite the growth of the existed biofilm. Our present dataindicated that the antimicrobial peptide bac8c can effectively inhibit the growth ofcariogenic bacteria and the formation of biofilms. This peptide with low cytotoxicity andhigh antibacterial activity provides a new method to be potencially used in the anti-cariesclinics.