"Dormancy Technique" Induces The Formation And Structure Of Retained Bacteria In Mixed Bacterial-fungal Biofilms

To explore a reliable method for inducing the formation of retention bacteria in the mixed bacterial-fungal biofilm,and to provide a reliable experimental model and research method for the study of the retention mechanism in the mixed biofilm of bacteria and fungi on the surface of biological materials.Observe the growth and characteristics of retained bacteria in mixed biofilms,explore the effects of retained bacteria on the formation and structure of mixed biofilms,and explore the relationship between the formation of retained bacteria and the structure of mature biofilms.[Methods]1.Cultivate the mixed biofilm of Staphylococcus epidermidis-Candida albicans on the surface of biological materials,check the relevant literature and verify the optimal induction concentration and cultivation time.Use 100ug/mL of tetracycline,gentamicin,rifampicin,5-fluorocytosine,caspofungin,fluconazole to induce culture of mixed biofilm for 1h to promote the formation of retained bacteria,and then use 10ug/mL Profloxacin and amphotericin B kill non-retentive bacteria.The mixed biofilms were eluted by ultrasound and cultured on MH agar plates and Shakespeare's agar plates for 24 hours.The plate colony counting method was used to count the number of retained bacteria to find the best drug-inducing drugs.At the same time,drug sensitivity testing was performed to rule out drug resistance.2.Use Scanning Electron Microscope(SEM)to observe the morphological characteristics of the retained bacteria in the mixed bacterial-fungal biofilm and the changes of the mixed biofilm at 24h.3.Use Confocal laser scanning microscope(CLSM)to observe the proportion of retained bacteria in the mixed biofilm structure and the change of mixed biofilm at 24h.[Results]1.The standard strains of Candida albicans atcc10231 and Staphylococcus epidermidis atcc35984 were inoculated and cultured to the logarithmic growth period,and the concentration of the bacterial solution was adjusted to 2 ×106cfu/ml respectively.The mixed bacterial solution with the concentration of 1×106cfu/ml was obtained by mixing the two groups of bacterial solutions according to the volume of 1:1.The mixed bacterial solution was cultured on the surface of titanium sheet biomaterials to form a mixed biofilm on which Candida albicans and Staphylococcus epidermidis were attached.2.After being induced by tetracycline and gentamicin,the level of staphylococcus epidermidis cells in the mixed biofilm was significantly higher than that of the negative control group(P<0.001).The level of staphylococcus cells in the tetracycline group was higher than that of the gentamicin group.(63.02%vs 53.60%,P<0.001).After induction with 5-fluorocytosine and caspofungin,the level of retention cells of Candida albicans in the mixed biofilm was significantly higher than that of the negative control group(P<0.001).The levels of retention cells of 5-fluorocytosine group were higher In the Caspofungin group(52.09%vs 34.99%,P<0.001).The results of drug sensitivity tests showed that there was no significant change in the sensitivity of Staphylococcus epidermidis and Candida albicans to ciprofloxacin,amphotericin B,erythromycin,penicillin,levofloxacin and other antibiotics.2.Observed under SEM,it can be seen that the induced Candida albicans appeared bulge-like changes on the surface of the cell,the induced Staphylococcus epidermidis cell division decreased,and the cell volume decreased.Mixed biofilms with a higher percentage of retained bacteria had more clumpy colonies on the surface of the negative control group,the three-dimensional structure of the mixed biofilms was more prominent,and the complexity of the mixed biofilms was higher.3.After the live/dead bacteria staining,CLSM observation shows that the mixed biofilm with a high retention rate will not be dissolved under the action of antibacterial drugs,and the mixed biofilm with a low retention rate is dissolved by a large piece.Mixed biofilm thickness blank control group>induction treatment group>negative control group(37.1um vs 15.7um vs 6.3um).Retaining bacteria and dead bacteria are mixed in the thinner part of the biofilm,and large pieces of retaining bacteria can be seen in the thicker part of the biofilm.[Conclusions]1."Dormant technology" helps the formation of retained bacteria in the mixed bacterial-fungal biofilm.Among them,5-FC and caspofungin can effectively induce the formation of Candida albicans retained bacteria.The effect of 5-FC is better,tetracycline,Gentamicin can effectively induce the formation of Staphylococcus epidermidis retention bacteria,and the effect of tetracycline is better.2.The retained bacteria in the mixed biofilm will undergo morphological changes.A large number of retained bacteria cells can prevent the mixed biofilm from being destroyed by antibacterial drugs.The generation of retainer cells does not depend on the formation of complex mixed biofilm structures,but the induction of mixed biofilms to produce retainer cells will make the structure of mixed biofilms more stable and complex,and will be more resistant to antibiotics.