Exerimental Study On The Effect Of Human Beta-defensin3on Biofilms Of Staphylococcus Aureus

Staphylococcus aureus (Staphylococcus aureus) is a common pathogenic bacteria, andalso is a creature of many bacteria infections. Data from the traumatic intensive care unitshows that Staphylococcus aureus is the second common cause of ICU implant infectionssepticemia (15.4%). The Staphylococcus aureus biofilm has a high resistance through therecognition of the collective immune system. The infection is not easy to be controlledcollectively. Hence, it is valuable to study on inhibition of the Staphylococcus aureusbiofilm.Human beta-defensin3(HBD-3) is a kind of novel antimicrobial peptides found fromhuman bone cells.HBD-3has strong lethal effects on S. aureus. Alfa-defensin has thesimilar structure with HBD-3and can inhibit biofilms.But there is no report about the effectof HBD-3on biofilms. We hypothesized that HBD-3may also have an effect on theStaphylococcus aureus biofilm. Therefore, we studied the effect of HBD-3on theStaphylococcus aureus biofilm.The process of Staphylococcus aureus biofilm formation can be divided into clustering(0-3hours), adhesive (3-6hours), preliminary form (6-12h), mature (18-24h) and agingspread (72h) stage.we studied the effects of HBD-3in the various stages of Staphylococcusaureus biofilm according to the process.At present, the formation mechanism of Staphylococcus aureus biofilms is still unclear.Studies have shown that bacterial cells attached quickly to the polymer surface atfirst.Thereafter multilayer growth-dependent accumulation of bacterial cell clusters areenclosed in the synthesis of a polysaccharide matrix adhesion induced slime (PIA)，whichis catalyzed by the ica operon encoding product.This process involves many genes. Thelaboratory study of specific strain S. aureus knockout icaA gene showed that icaA deletionstrain can form biofilms under certain conditions. In addition, dltABCD operon is alsoconsidered one of the key factors in the regulation. RNA interference can effectively resist the targeted gene, and is very similar with the"knockout", which is a phenomenon that genes can not express after siRNA transcription. Ithas been reported that it can replace the gene targeting. We tried to construct siRNAplasmid of dltB gene for RNA interference, so as to provide a new idea to investigate themechanism.Materials and methods:Strain: the standard strains of Staphylococcus aureus ATCC25923. Animal：Wister SDrats. Reagent: HBD-3, vancomycin, clindamycin, white fluorescent and LIVE/DEADBacLightTM Bacterial Viability Kits.Sample: Ni Cr alloy, aluminum alloy and ultrahighmolecular polyethylene. Instrument: laser scanning confocal microscopy.1. Effect of HBD-3on Staphylococcus aureus biofilms: Experimental groups weredivided according to the categories of reagent and the concentration gradient of Minimuminhibitory concentration（MIC）：1/2MIC、1MIC、2MIC、4MIC,HBD-3concentrationof4,8,16,32ug/ml,vancomycin concentration of0.25,0.5,2,8ug/ml,clindamycinconcentration of0.125,0.25,0.5,1.0ug/ml.Respectively,after S. aureus were cultured3,6,12,18,24h, reagents were added in each group. Each group was dyed and observe after6h. White Fluorescent agent dyeing biofilms was used to assess the biofilm change;LIVE/DEAD kits were used for differential staining of live bacteria and died bacteria toassess the viable rate within the biofilms. Laser scanning confocal microscope was used toobserve the HBD-3, vancomycin, clindamycin on the s. aureus biofilm and the survivalrate of viable organism within biofilms.2. Effect of HBD-3on the mechanism of staphylococcus aureus biofilms. S. aureuswere cultured by adding HBD-3, vancomycin, clindamycin. Total RNA was extracted aftercultivating6h,24h. Reverse transcription was used for cDNA synthesis. PCR primerswere designed based on the experimental demand, and the resulting strains cDNA was takenas a template for PCR amplification test.3. According to bioinformatic, three target sequence were screened and three pairs of64nt oligonucleotides were synthesized.They were connected to plasmid pRNAT-U6.1/NEOdigested by BamHI and HindIII Then,they were transformed into Escherichia coli DH5a.. Theplasmids were amplified and tested.4. Effect of HBD-3on implant material to form staphylococcus aureus biofilm in vitro and in vivo. The experiment was grouped according to the material of Nickel chromium alloy,aluminum alloy and polyethylene (uhmwpe) and were cultured with staphylococcus aureus.HBD-3, vancomycin, clindamycin and HBD-3+vancomycin or HBD-3+clindamycinwere added, then were observed by confocal laser scanning microscopy after dyeing. In vivotests are divided into HBD-3group, vancomycin group and clindamycin group. Ratssubcutaneous bursa model of the medical plant infection were set up. Ultra-high molecularpolyethylene samples were inserted.Before the insertion, the samples were soaked into physiological saline, HBD–3group with a concentration of8ug/ml, vancomycin group with a concentration of0.5ug/mland chlorine group with a concentration of0.25ug/ml, respectively for20minutes.Staphylococcus aureus bacteria liquid were injected in subcutaneous bursa. After operation,we observed vital signs change of rats and the survival rate of different time points, andobserved staphylococcus aureus biofilms on the sample surface and the survival rate ofviable organism.Results:1. There are three Staphylococcus aureus for biofilm formation, of which theStaphylococcus aureus ATCC25923was the most obvious. In the initial adhesion stage,HBD-3, vancomycin, clindamycin promoted the increase of Staphylococcus aureus ATCC25923secreting polysaccharide matrix, and fastened the bacteria to secrete polysaccharidematrix. When drugs were acting on Staphylococcus aureus biofilm formation, HBD-3andclindamycin can reduce Staphylococcus aureus biofilm, vancomycin had no effect on thematured biofilm. For the Staphylococcus aureus which has formed biofilm, the concentrationof HBD-3greater than2MIC can reduce the rate of live bacteria in biofilm.2.HBD-3had no effect on the levels of expression of dltB and icaA of S.aureus25923atthe adhesion phase of bacterial culture and formed biofilms. But clindamycin couldup-regulate the dltB and icaA gene expression. Vancomycin had no effect on the levels ofexpression of dltB at the adhesion phase，up-regulated the icaA gene expression only at thephase of biofilms formation.3. Three SiRNA plasmid vector of dltB gene was constructed successfully, named aspRNAT-U6.1/NEO-shRNA-1, pRNAT-U6.1/NEO-shRNA-2, pRNAT-U6.1/NEO-shRNA-3,respectively. The insertion element was conformed to be accurate. 4. In vitro, after joining HBD-3, the biofilm formation on the surface of groups of NiCr alloy, aluminum alloy and ultrahigh molecular were decreased. And groups of HBD-3+Vancomycin or Hbd-3+Clindamycin can significantly reduce the formation ofStaphylococcus aureus biofilm. Rats in groups of vancomycin were all survival. One rat ingroups of clindamycin died at4thday.Two rats in HBD-3groups dead at3rdand4thdays.Four rats in control groups dead at2nd,3rd,4thand5thdays. But biofilms were notsignificantly reduced in groups of HBD-3in the in-vivo test.Conclusions:1.HBD-3could significantly inhibit the Staphylococcus aureus biofilm formation ability,reduce the number of live bacteria in biofilm formation, have the same inhibitory effect on themature biofilms. The antibacterial peptide is effective for treating Staphylococcus aureusinfection and for implant infection in surgery.2.Unlike the Clindamycin and Vancomycin, HBD-3could not up-regulate the expressionof the gene dltB and icaA, which were the key factors in biofilm formation. So HBD-3wouldn’t induce the biofilm forming.3.HBD-3can reduce staphylococcus aureus biofilm on the surface of implant materialand had co-application effect with vancomycin and clindamycin in vitro.But its ability ofanti-biofilm was not confirmed in the in-vivo test.These results may lead to a better understanding on the inhibition effect of HBD-3onStaphylococcus aureus biofilms and provide a new pathway to solve the infection ofStaphylococcus aureus biofilms.