Inhibitory Effect Of Carboxylated Nanodiamond On Oral Pathogenic Bacteria Streptococcus Mutans

ObjectiveNano-diamonds have high biocompatibility and are also found to inhibit bacterial vitality or growth.With potential antibacterial properties,nano-diamonds could have applications in various dental materials.Currently,research concerning the putative antimicrobial properties of nano-diamonds is limited,and possible inhibitory effects on oral pathogenic bacteria are not established.The purpose of this study was to assess the putative inhibitory effects of carboxylated nano-diamonds(cND)on Streptococcus mutans and to analyze the relevant antibacterial mechanisms involved.Methods(1)Main materialscND samples were sourced from commercial manufacturers.The experimental oral pathogen Streptococcus mutans was purchased from American Type Culture Collection(ATCC).(2)Physical and chemical properties of cND surfaceFourier transform infrared spectroscopy(FTIR)was used to analyze the surface characteristics of cND,and confirm the existence of carboxyl groups on the surface of cND.(3)Determination of minimum inhibitory concentration(MIC)A suspension of bacterial cells with cND suspension was incubated in 96-well plates using the Anaero Pack System for 16 h,and optical density(OD)values at 600 nm wavelength were measured before and after incubation using a Microplate Reader.(4)Determination of minimum bactericidal concentration(MBC)100 μl of bacterial culture solution samples with different concentrations of cND were diluted 10 times continuously,cultured on BHI agar medium,and incubated at 37℃ for 48 h.The colonies were counted to determine bacterial viability at each concentration.(5)Analysis of time killing curvecND suspensions of 16 MIC,8 MIC,and 4 MIC groups were prepared,and bacterial suspension was added.Surviving bacterial quantities in the groups were measured after culture for 2 min,5 min,15 min,30 min,1 h,2 h,and 4 h,each.(6)Determination of antibacterial effects on biofilm by LIVE/DEAD stainingUsing a 12-well plate,cover slips were added to each well,and Streptococcus mutans biofilms were cultured.Four wells were selected and 50 μl of 16 μg/ml cND was added to treat the biofilm samples.The treated biofilm samples were transferred for LIVE/DEAD Staining using the Live/Dead Baclight Bacterial Viability Kit,and photographed using a laser confocal microscope.(7)Effects of cND on bacterial cell membrane surface observed by scanning electron microscopy(SEM)Streptococcus mutans was treated with cND at a concentration of 4 MIC and cultured in an anaerobic chamber at 37℃ for 4 h.Centrifugation was performed at 1800 g for 2 min at normal temperature to remove the supernatant.After rinsing twice with PBS buffer solution,re-centrifugation,and removal of excess supernatant,the bacterial solution was resuspended and images were obtained using SEM.(8)Effects of cND on bacterial cell internal structure observed by transmission electron microscope(TEM)Bacterial samples were prepared using the same procedure as for SEM.The OD600 value of the bacterial culture was adjusted to 0.8,the samples were centrifuged at 1800 g for 10 min,centrifuged again at 11000 g,fixative solution was added,and images were obtained using TEM.ResultsThe results of FTIR showed that the commercially purchased cND had homogeneous functionalized carboxyl groups.Obvious inhibition and killing effects on Streptococcus mutans,with an MIC of 4 μg/ml and MBC of 16 μg/ml were observed.The time-killing curve analysis showed that bacterial inhibition was time-dose dependent within a specific concentration range(16-64 g/ml).cND displayed definitive inhibitory effects on simulated Streptococcus mutans biofilms in static state.SEM observations demonstrated that cND induced antibacterial effects by destroying the cell membrane of Streptococcus mutans.TEM analysis showed that cND could penetrate the cell wall of Streptococcus mutans,resulting in rupture of the cell membrane,formation of transmembrane pores on bacterial surfaces,which eventually led to bacterial cell death.ConclusioncND induced inhibitory effects on Streptococcus mutans,where a time-and concentration-dependent bactericidal effect was evident within a specific concentration range.cND also inhibited Streptococcus mutans biofilms.The relevant antibacterial mechanisms included destruction of the bacterial cell wall and membrane.Thus,the results support cND as a candidate antibacterial agent for incorporation in dental materials,and further research is warranted for clinical translation.