| Part Ⅰ.Inducing the formation of persister cells on the surface of biomaterialsObjective:Finding out the method of inducing the formation of persister cells on the surface of biomaterials.Constructing of persister cells in the biomaterials polymicrobial biofilms model.Observing the morphological characteristics of persister cells and its influence on the structure of polymicrobial biofilms.Methods:1.The polymicrobial biofilm of Candida albicans and Staphylococcus epidermidis was induced by tetracycline,gentamycin,rifampicin,5-fluorocytosine(5-FC),caspofungin,fluconazole and CCCP at concentration of 10ug/mL、50ug/mL、100ug/mL for 0.5h,1h and 3h,respectively,and then ciprofloxacin and amphotericin B for 10ug/mL were used to kill the non-persister cells.Elute the polymicrobial biofilm with ultrasonic and plate colony count were used to compare the number of persister cells induced by various schemes,to find the best method to induce the formation of persister cells;drug sensitivity test was carried out to verify the formation of persister cells.2.The best inducing method was used to induce the polymicrobial biofilm on the surface of biomaterials,to construct the model of persister cells in the biomaterials polymicrobial biofilms in vitro.3.The model of persister cells in the biomaterials polymicrobial biofilms in vitro was used.The uninduced polymicrobial biofilm was used as the blank control.After 24 hours of culture,the morphological characteristics of the bacteria were observed by scanning electron microscope(SEM).The untreated polymicrobial biofilms was used as the blank control,and the uninduced but cured polymicrobial biofilms was used as the negative control.The laser confocal microscope was used(CLSM)to observe the effect of retention bacteria on the structure of mixed biofilmResults:1.According to the results of colony count,tetracycline and gentamicin can induce the persister of S.epidermidis,5-FC,caspofungin and fluconazole can induce the persister of C.albicans,all the drugs above at concentration of 100ug/mL and induce for 1h can get best effect.Rifampicin at concentration of 100ug/mL induce for 3 hours could persist S.epidermidis,but the effect was significantly lower than that of the above drugs.After the induction of each drug,S.epidermidis and C.albicans had no drug resistance,which proved that the living microorganisms were all persister cells.2.The best inducing method was used to induce the polymicrobial biofilm on the surface of biomaterials,to construct the model of persister cells in the biomaterials polymicrobial biofilms in vitro.3.SEM observation showed that there was no significant difference in the surface structure between the induced and the uninduced polymicrobial biofilms,both of which were based on C.albicans skeleton,and S.epidermidis adhered to the surface to form a complex polymicrobial biofilms structure;the surface of the induced C.albicans could show a bulge like morphological change,and the induced S.epidermidis cell division was reduced,and could find small cells;living/dead bacteria staining with CLSM observation,it can be seen that the uninduced polymicrobial biofilm was dissolved under the cure of antibiotics,but the polymicrobial biofilm which was induced with high proportion of persister cells was not dissolved;no matter the thickness of biofilm,persister cells can be seen,persister cells in the thinner part of biofilm are mixed with dead bacteria,and in the thicker part of biofilm could find mass of persister cells.Conclusions:1.5-FC and caspofungin can induce the persister of C.albicans,tetracycline and gentamicin can induce the persister of S.epidermidis,the induction effect appears time-dose-effect relationship,and the best induction effect can be obtained at the concentration of 100ug/mL and induced for 1h.2.Using tetracycline,gentamicin,5-FC,and caspofungin to induce the fungal-bacterial polymicrobial biofilms on biomaterials can successfully construct the model of persister cells in the biomaterials polymicrobial biofilms in vitro,which solves the technical problems that the small number of persister cells and the difficulty to obtain it.3.In the induced C.albicans,the surface of the cell was swelled,and the S.epidermidis cells showed decreased division and volume;a large number of persister cells could avoid the destruction of the polymicrobial biofilms by antibiotics;the complex polymicrobial biofilms structure was not the necessary condition for the formation of the bacteria retaining cells,but a higher level of persister cells could be produced in the complex mixed biofilm.Part Ⅱ.Study on the persister mechanism of the persister cells in the polymicrobial biofilms of bacteria and fungi on the surface of biomaterialsObjective:Analyzing the differential expression genes between the persister cells and non-persister cells in the polymicrobial biofilms of S.epidermidis and C.albicans on the surface of biomaterials.To finding the possible persister mechanism and persister related genes.Methods:1.Tetracycline and gentamicin were used to induce the persister of S.epidermidis in the polymicrobial biofilms on the surface of biomaterials,5-FC and caspofungin were used to induce the persister of C.albicans,to construct the model of persister cells in the biomaterials polymicrobial biofilms in vitro.The uninduced polymicrobial biofilms was used as blank control.The total RNA of each group of was extracted by Trizol method,and OD260/280 and agarose gel electrophoresis was used to test the purity and integrity of total RNA2.The transcriptome sequence of NCBI genomic database was used as the reference sequence for comparison.The total RNA was detected by RNA-Seq.The co-differential genes which were differentially expressed between the two drug-induced group and the blank control were screened,so as to exclude the gene differential expression caused by each drug and unrelated to persister,and analyze the function of these co-differential genes.3.GO enrichment analysis and KEGG pathway analysis were carried out for co-differential genes to find the main metabolic pathway involved in co-differential genes.Results:1.The total RNA of bacteria and fungi was extracted successfully.The OD260/280 of total RNA in each group was more than 1.8.The 28s and 18S rRNA bands of C.albicans and 23S and 16S rRNA bands of S.epidermidis were complete,which proved that the purity and integrity of total RNA were good.2.There are 684 genes co-differentially expressed by C.albicans and 102 genes co-differentially expressed by S.epidermidis.The functions of genes differentially expressed by C.albicans are mainly to enhance ribosome function,decrease cell division,weaken virulence and enhance tolerance to various stresses.Extracellular matrix 1(ECM1)and other adhesion related genes were significantly up-regulated.The up-regulation of DnaK and sced genes in S.epidermidis can increase the resistance of bacteria to antibiotics and adverse environment,but there is no other co-differential expression gene.3.The results of GO enrichment analysis showed that the up-regulated genes of C.albicans were mainly enriched in molecular functions:methyltransferase activity,transferase activity transferring one-carbon groups,nucleoside-triphosphatase activity,pyrophosphatase activity,ATPase activity,coupled;biological processes:methylation,rRNA processing,rRNA metabolism process,ribosome biogenesis,ncRNA metabolism process,etc There was no significant enrichment of down-regulated genes of C.albicans and differential expression genes of S.epidermidis.The results of KEGG enrichment analysis showed that the up regulated genes of C.albicans were mainly enriched in the following pathways:Ribosome biogenesis in eukaryotes,RNA polymerase,pyrimidine metabolism,purine metabolism,ABC transporters;the down regulated genes of C.albicans were mainly enriched in the pentose phosphate pathway,starch and sucrose metabolism,glutathione metabolism,inositol phosphate metabolism,RNA degradation.The up-regulated genes of S.epidermidis were enriched in RNA degradation pathway,but the down regulated genes were not enriched.4.RT-qPCR showed that the ECM1 of C.albicans was up-regulated in each experimental group,and the PLC1,PRR2,SAC6 and TORI were down-regulated in each experimental group.The DnaK of S.epidermidis was up-regulated in each experimental groups.Conclusions:1.The persister in the C.albicans-S.epidermidis polymicrobial biofilms on the surface of biomaterials is a complex process caused by multiple factors and genes to increase the tolerance and viability of microorganisms to external pressure.This process is accompanied by redistribution of intracellular resources,and may lead to the decline of metabolism,division of pathogenicity.2.After the adhesion stage of C.albicans adheres on the surface of biomaterials,it can reduce PRR2,SAC6,PLC1 and other genes to form the persister cells that are resistant to a variety of environmental pressures but with cell growth inhibition and low function.ECM1 can promote the adhesion of C.albicans,which may be the key gene of C.albicans persister cells.3.Tetracycline and gentamicin may promote the persister cells formation of S.epidermidis through relatively independent genes and pathways.After the formation of S.epidermidis persister cells,the ability to withstand multiple environmental pressures can be obtained by up-regulating DnaK. |
