Preliminary Study Of Antibacterial Activity Of Antibacterial Peptides PJF11 And PLC13

BackgroundDental implant was supposed to be with a better stability and higher success rate according to a great amount of reports.Whereas accompany with the ascending of amounts of patients that accept implant surgery,the odds of peri-implantitis was increasing too.The consensus report from the 6th European Workshop on Periodontology has defined peri-implantitis as the presence of inflammation of the peri-implant mucosa and simultaneously loss of supporting bone.In addition,it has also been described as a site-specific infection that exhibits features comparable to those of chronic adult periodontitis.Therefore,the progression of infection around implants and natural teeth is also divergent.Moreover,tissues around implants are more prone to plaque-associated infections that spread into the alveolar bone.The best method for prevention of peri-implantitis is considered to be suitable infection control,along with strict aseptic techniche during the operation and teach the patients with right measures of oral hygiene protection,more implantly to consider is to apply local antimicrobial medicine before the inflammation taking place or slight inflammation being occured.This is especially important in the patients with a hitory of choronic periodontitis.According to research before,the occurrance rate was obviously higher in the patients with a hitory of choronic periodontitis in comparison to patients without such history.Fusobacterium nucleatum is a kind of gram negative bacilli which grows in a environment with a optimum temperature of 37 ? and is belong to obligate anaerobe.Fusobacterium nucleatum is commonly found in different oral choronic infecion and it is important pathogenic bacteria in both chronic periodontitis and peri-implantitis.Fusobacterium nucleatum is able to act on the metronidazole which is a kind of antibiotic that mainly kill anaerobe.After the interaction process between the bacteria and metronidazole m acetamide was produced and made metronidazole losing its efficacy,which is able to provide protection for another important pathogenic bacteria Porphyromonas gingivalis.Staphylococcus aureus is a kind of gram positive coccus which grows in a environment with a optimum temperature of 37 ? and is belong to facultative anaerobe.Staphylococcus aureus is commonly seen in different oral acute infecion such as postoperation of exodontia and periodontal surgery,and is also usually found in the postoperation of implant surgery.Although the relavant research of direct correlation with the bacteria to occurrance with peri-implantitis was relatively less,Staphylococcus aureus was often found in the tissues of patients with peri-implantitis.More recently,Staphylococcus aureus has been demonstrated to have the ability to adhere on titanium surfaces.This may be significant in the colonisation of dental implants and subsequent infections.Staphylococcus aureus is frequently responsible for infections associate with metallic biomaterials and indwelling medical infections in general.According to a recent research,Staphylococcus aureus has the ability to injure the osteoblast and interrupt the osteogenetic process.Furthermore,a case report had shown that a huge amount of Staphylococcus aureus was found in a patient with peri-implantitis that caused osteomyelitis.Therefore,selecting Fusobacterium nucleatum and Staphylococcus aureus as the research objects of the experiment was representative and was able to inflect the practical situation of clinic.At present,the commonly used local medicine in prevention and treatment of peri-implantitis in domestic Stomatological Department are Milo ring bases and Chlorhexidine gargle fluid.Among them Milo ring bases can be coated in the local red and swollen soft tissues or periodontal pockets,playing a long anti-inflammation effect.Chlorhexidine gargle fluid can swash bacteria that adhered on the teeth face and oral mucosa to some extent,meanwhile playing a bacteiral inhibited effect.These medicine were proved to be effective,owed strong bacteriacidal effect,and were widely and longly used in Stomatological Department,and till now they are still the common medication in postoperation infection prevention in implant surgery.However,since Antibiotic resistance occured in 1980s last century,the species of drug-resistant bacteria were increased year by year,researchers had to develop medicine with more strong effect.Beta lactam class of antibiotics(cephalosporin drugs)had been developed from the earlier first generation to the third generation with a continuously strengthen antimicrobial effect and broader antibacterial spectrum,even so the reports of bacteria tolerance to the third generarion cephalosporin were not rare.Ten years at least were needed in a new antimicrobial drug development by medical rearchers,while bacteria tolerance take place only within two years,which making the new medicine developments being far behind the occurance of bacteria tolerance.The reason that cause these problems was the abuse of antibiotics along with the restrict of the antibiotics themselves.It was elaborated by several domestic experts that China has become the country with most severe antibiotic abusement.In this overall background,the increase species of tolerant bacteria year by year brought a warm to the antibiotics use in imlpant surgery.Based on this,study focused on antimicrobial peptides have been widely spreaded since the last three decades.Antimicrobial peptides(AMPs)is a kind of polypeptide active substance with thousands molecular weight and composed of 12 to 50 amino acid residues.Through a great amount of studies by global rearchers,AMPs were supposed to have good antimicrobial effect and wide antibacterial spectrum along with a very rare occurrence rate of bacteria tolerance,and by reason of these merits,the study of AMPs have been widely spreaded.At preset research of several kinds of medicine such as Enfuvirtide and PG-1 had entered the third period of clinical trial,and Daptomycin had already been used in clinic.However,after several decades of rearch of AMPs,the development was relatively low,it was because that there were many important problems hadn't been solved.Among these,the biggest ones were complex synthetic technology and expensive price,and a quite amount of AMPs haven't been controled in a range of safe cytotoxicity.As the length of the amino acid sequence increased,these points above were simultaneouly ascending.The widely studied AMPs such as SMAP29,LL-37,HBD2,HBD3,Cecropin B were proved to be with strong antimicrobial effect,but these AMPs also owned long length of amino acid sequence and incresed cytotoxicity.A survey showed that AMP can only be used widely in clinic when the synthetic price bring down to 10 dollars per gram.Therefore in the view of future application in clinic,we not only have to find effective AMPs,but to develop AMP s with better cost performance.Based on this,the experiment had chosen a nonapeptide which was proved to be with good effect in our pre-experiment,then we took this peptide as a model to synthesize two new AMPs PJF11 and PLC13,after that we took them as our research objects.We are looking forward to establish a litte basic for the future use of AMPs in stomatological department.Chapter one:Determination of antimicrobial activity and cytotoxicity of PJF11 and PLC13Objective1.To determine antimicrobial activity of antimicrobial peptides(AMPs)PJF11 and PLC13 on common bacteria(Fusobacterium nucleatur,Staphylococcus aureus)from peri-implantitis2.To determine the cytotoxicity of PJF11 and PLC13.3.To compare the antimicrobial ability for AMPs and Chlorhexidine gargle fluid.Method1.Bacteria were grown to a mid-log phase and diluted to 106CFU/ml.Experimental group,positive control group,negative control group were set simultaneously.The MIC was defined as the lowest peptides concentration that resulted in no bacterial growth.After that 100 ul of inoculum/peptide solution were spirally spreadered on the plate medium.The MBC was defined as the lowest dilution at which bacteria colonies were nolonger viable on subculture.2.Hemolysis was measured as the absorbance at 540 nm using a microplate reader.Human blood erythrocyte suspensions were diluted in 5%Physiological saline(vol/vol).The experiment group was determined plus a various contrations of PJF11/PLC13.The positive control group and the negative control group were set simultanneously.Hemolysis was measured as the absorbance at 540 nm using a microplate reader.The percentage of hemolysis was calculated afterwards.3.Bacteria were grown to a mid-log phase and then were coated on the plate medium,after that disks with a various dose of PJF11/PLC13 were placed on the plate.After that experimental plates were taken out,and the result was captured by camera.Result1.The MIC was ranged from 12.5ug/ml to 25ug/ml,the MBC was ranged from 12.5ug/ml to 50ug/ml.2.The hemolytic activity assay showed that PJF11 had a hemolysis of 2.8%and 1.2%at the concentration of 100ug/ml and 50ug/ml,while PLC13 had a hemolysis of 6.8%and 1.8%at the concentration of 100ug/ml and 50ug/ml.Below 25ug/ml,both peptides showed no hemolysis on the human erythrocyte.3.In both group of Fusobacterium nucleatum and Staphylococcus aureus,obvious inhibition zone were seen m as the concentrations got lower,the inhibition zone also become smaller.In comparison with the antibiotic group,with less medicine,showed bigger zones.Chapter two:Preliminary Study of antimicrobial mechanism of AMPs PJF11 and PLC13ObjectiveTo explore the antimicrobial mechanism of PJF11 and PLC13 from the aspect of membrane-active mechanism.Method1.Fusobacterium nucleaturm and Staphylococcus aureus were grown in a mid-logarithmic phase and then adjusted to profit concentration,after that PJF11/PLC13 were mixed with the suspension culture with the final AMPs concentration at MBC.Bacteria without AMPs was took as control group.After 1 hour,bacteria were pelleted and through a process include rinse,fixation,dehydration,replacement,desiccation,etc,and finally observed under SEM.2.Fusobacterium nucleaturn and Staphylococcus aureus were grown in a mid-logarithmic phase and then adjusted to profit concentration,500ul of PJF11/PLC13 were mixed with the suspension culture with the final AMPs concentration at MBC,MIC,0.5MIC.Control cells were not treated with the peptide.After rinsed and mixed with Propidium iodide,the samples were finally observed under a fluorescence microscope.3.Bacteria genomic DNA was extracted using a TIANamp Bacteria DNA Kit.Bacteria Total RNA was extracted using a RNAprep Bacteria Kit.Briefly,gel retardation experiments were performed by mixing the bacteria genomic DNA or the bacteria total RNA with varying concentrations of peptides.And DNA or RNA without AMPs were set for control group.The reaction mixtures were incubated at room temperature for 30 min.Subsequently,for DNA/RNA-peptides mixtures or pure DNA/RNA solution,4ul of native loading buffer was added and then the reaction solution was applied to a 1%agarose gel electrophoresis with 0.5ug/ml ethidium bromide(final concentration)in 0.5× Tris borate-EDTA buffer.Result1.Observed under SEM,it was found that in the absence of peptides,bacteria had intact and smooth surfaces.While after the exposure of the bacteria to a MBC of PJF11/PLC13 resulted in a little bit change in the appearance of the cells include membrane blebbing and roughening,and the regular shape were lost a little.2.As the results showed,when both bacteria cells were incubated with PI without pretreatment with PJF11/PLC13,no appreciable fluorescent signal was observed.In contrast,in experimental group,the cells emitted a red fluorescence after exposure to different concentrations of PJF11/PLC13 as the result of PI uptake.3.Our results demonstrate that PJF11/PLC13 could not inhibit the migration of bacterial genomic DNA below concentrations of 100ug/ml,when at 100ug/ml PJF11/PLC13 seemed to partly cause retardation of DNA mobility of both bacteria.Nevertheless,our results demonstrate that PJF11/PLC13 could not inhibit the migration of bacterial Total RNA,even at concentrations up to 100ug/ml.Conclusion of the full textAMPs PJF11 and PLC13 are able to inhibit Fusobacterium nucleatum and Staphylococcus aureus from peri-implantitis with a low cytotoxity.AMPs PJF11 and PLC13 are inhibiting the bacteria mainly through a membrane-active mechanism.