Study On The Effect Of TiO₂ Nanotube Materials Coated With Gallium Nitrate On Staphylococcus Aureus And Escherichia Coli Mixed Biofilm

The development of medical biomaterials has become a crucial field of modern medicine.Biomaterials with high utility value can improve life quality and expand lifespan,but due to the enormous use of biomaterials,a series of serious complications caused by implant infection have been an urgent clinical problem to be solved.A large number of studies have reported that the reason why the internal plant infection is intractable,severe and drug resistant is the formation of complex bacterial biofilms on the surface of materials,which are regulated by several genes during the process of formation.Titanium(Ti)is the most widely used metallic material in clinic,especially in orthopedics.As the implantation of a variety of Ti with steel plates and screws causes an increasing number of infections,the mixed infection of Staphylococcus aureus-Escherichia coli in chronic osteomyelitis and other chronic infection is common.Because of its serious infection,it is a vital clinical problem that the lesion is protracted and the treatment is difficult.The research and development of antibacterial drugs and new biomaterials are the two main methods to treat the surface infection of the materials at present.Staphylococcus aureus-Escherichia coli co-infection in orthopaedics such as chronic osteomyelitis and other chronic infections are not uncommon.Because of its serious infection,focus delay,and difficult to treat,it becomes a tough clinical problem.Antimicrobial agents and new biomaterials are two main methods to treat the surface infection of materials.As a new antimicrobial agent,gallium nitrate interferes with the synthesis of DNA and protein for the competitive substitution of Fe3+ by Ca3+.TiO2 nanotubes have good biocompatibility,high drug loading and antibacterial properties.Therefore,the mixed infection of Staphylococcus aureus and Escherichia coli on Ti surface was studied from the perspective of mixed bacterial biofilm,and a new type of TiO2 nanotube coated with gallium nitrate was prepared by modifying Ti material to explore its effect on Staphylococcus aureus-Escherichia coli mixed infection.The above studies will provide theoretical basis for clinical refractory mixed infection,and have a significant impact on clinical prevention and treatment.Objectives of this study:(1)to investigate whether Staphylococcus aureus and Escherichia coli can form mixed biofilm on Ti material surface and its formation regularity.(2)To explore the role of Staphylococcus aureus icaA,icaD,SarA genes and Escherichia coli flhC;flhD,qseC genes in the formation of mixed biofilm(3)The effects of TiO2 nanotube materials coated with gallium nitrate on Staphylococcus aureus and Escherichia coli biofilm were investigated.Section ?:Study on the formation of mixed biofilm between Staphylococcus aureus and Escherichia coli on Ti SurfaceObjectivesTo observe whether Staphylococcus aureus-Escherichia coli can form a mixed biofiln on Ti surface,to discover its formation regularity,and to make a control research on the formation of single Staphylococcus aureus and single Escherichia coli biofilm on Ti surface.MethodCo-culture of Ti slice and TSB medium(Blank group),Staphylococcus aureus TSB liquid(S.aureus group),Escherichia coli TSB solution(E.coli group),Staphylococcus aureus-Escherichia coli TSB mixture(Mixed group)was adopted.Semi-quantitative detection was adopted by crystal violet staining after 6h,12h,24h,48h and 72h culture(Detection of bacterial adhesion,bacterial biofilm formation).The dynamic of bacterial biofilm formation was detected by XTT method,the thickness of bacterial biofilm was observed by laser confocal microscope,the ratio of living dead bacteria was measured,and the ultrastructure of biofilm was observed by scanning electron microscope.Results1.Number of bacterial adhesion:After 6h,12h,24h,48h and 72h of culture,6h Mixed group>E.coli group(P<0.05);12h Mixed group>E.coli group,Blank group respectively(P<0.05)and S.aureus group>Blank group(P<0.05).24h-48h Mixed group>E.coli group,S.aureus group,Blank group respectively(P<0.05);24h E.coli group,S.aureus group>Blank group respectively(P<0.05);48h S.aureus group>E.coli group,Blank group respectively(P<0.05),E.coli group>Blank group(P<0.05).72h Mixed group>E.coli group,Blank group respectively(P<0.05)and S.aureus group>E.coli group,Blank group respectively(P<0.05).The number of bacteria adhesion in Mixed group was the highest at 24-48h.2.Bacterial biofilm forming ability:No biofilm was formed in Blank group,E.coli group,S.aureus group and Mixed group for 6 h.The biofilm of weak bacteria was formed in S.aureus group and Mixed group at 12h,and there was no difference between the two groups(P>0.05);After 24 hours weak biofilm was formed in E.coli group,S.aureus and Mixed group(P<0.05);After 48 hours,weak biofilm was formed in E.coli group and S.aureus group,and mature biofilm was formed in Mixed group(P<0.05);After 72 hours,mature bacterial biofilm was formed in all three groups,and E.coli group>S.aureus group,E.coli group>Mixed group(P<0.05).3.Growth dynamics of bacterial biofilm:There was no difference in the growth dynamics of 4 group at 6h.After 12 hours,Mixed group>blank group,S.aureus group E.coli group respectively(P<0.05);There was no significantly difference in growth dynamics of bacterial biofilm of S.aureus group,E.coli group and Mixed group between 24 h and 48 h(P>0.05),which were stronger than the blank group(P<0.05);After 72h,S.aureus group,E.coli group and Mixed group>blank group,Mixed group<S.aureus group(P<0.05).4.Laser confocal detection:After 12-72 hours of culture,the structure of Mixed group biofilm was the most complex and the proportion of dead bacteria was the highest.The density and complexity of biofilm structure are:Mixed group>S.aureus group>E.coli group>Blank group.The increase of bacteria quantity and structure complexity in each group was along with the culture time,but the proportion of living bacteria decreases and the proportion of dead bacteria increases.5.Scanning electron microscopy:After 12-72h culture,the number of bacteria increased and the structure complexity of biofilm increased in the groups of Mixed group,S.aureus group,E.coli group.At each time point,the mixed biofilm of Staphylococcus aureus and Escherichia coli could be formed.The structure of mixed biofilm was more complicated than that of single staphylococcus aureus and Escherichia coli biofilm.The main growth of mixed biofilm was Escherichia coli.Conclusion1.Staphylococcus aureus-Escherichia coli could form mixed bacterial biofilm on the surface of pure titanium material,which was mixed with Staphylococcus aureus Escherichia coli and complex and compact structure.Escherichia coli was the main strain of the mixed biofilm.2.Compared with a single Staphylococcus aureus biofilms and single Escherichia coli,Complex and compact structure of mixed biofilm was increased with the extension of incubation time.The total number of bacteria was increased,but Rates of the living bacterium was down,Rates of dead bacteria was increased.This may be related to the competitive relationship between Staphylococcus aureus and Escherichia coli predominate.Escherichia coli is predominant.Section II:The role of icaA,icaD,SarA genes and Escherichia coli flhC,flhD,QseC genes in the formation of mixed biofilmObjectiveTo investigate the expression of Staphylococcus aureus icaA,icaD,SarA genes and Escherichia coli flhC,flhD,qseC genes in mixed biofilm formation.MethodThe relative expression of Staphylococcus aureus icaA,icaD,sarA Genes and Escherichia coli flhC,flhD,qseC genes in Staphylococcus aureus-Escherichia coli mixed biofilm were detected by real-time fluorescence quantitative PCR.ResultsThe relative expression of icaD,sarA genes of Staphylococcus aureus in Staphylococcus aureus-Escherichia coli mixed biofilm was up-regulated 0.62±0.01 and 5.03±0.75 fold respectively(P<0.05)and icaA gene was down-regulated 0.62±0.01 fold(P<0.05).The relative expression of flhC,flhD and qseC genes of Escherichia coli were up-regulated 383.78±88.01,468.69±107.65 and 561.11±121.48 fold respectively(P<0.05)in Staphylococcus aureus-Escherichia coli mixed biofilm fold respectively.ConclusionDuring the formation of mixed biofilm between Staphylococcus aureus and Escherichia coli,the expression of icaD,sarA,flhD,flhC and qseC genes were up-regulated and the expression of icaA was down-regulated,which may be related to the complex structure of mixed biofilm and competitive advantage of Escherichia coli.Section ?:Effect of TiO2 nanotubes coated with gallium nitrate on biofilm formation of Staphylococcus aureus and Escherichia coli mixed bacteriaObjectiveTo investigate the effects of TiO2 nanotube materials coated with gallium nitrate on the formation of Staphylococcus aureus and Escherichia coli biofilm.Methods1.TiO2 nanotubes were prepared by anodic oxidation method,and their morphologies were observed by SEM.2.Using solvent casting technology to prepare gallium nitrate coated Ti,TiO2 nanotubes,scanning electron microscopy to observe the morphology of the characteristics.3.Mixed bacterias was cultured on TiO2 nanotubes for 24h and SEM was used to observe the ultrastructure of bacterial biofilm.4.Four groups of Ti,TiO2 nanotubes,Ti and TiO2 nanotubes coated with gallium nitrate were implanted into SD rats and cultured in vivo with mixed bacteria solution.After feeding for 3 days,7 days and 14 days,the general state and activity of animals were detected.After the animals were killed,the incision and infection in vivo were detected,the formation of bacterial biofilm in vivo was observed by laser confocal microscope,and the ultrastructure of bacterial biofilm was observed by scanning electron microscope.Results1.Material Appearance:TiO2 nanotube was dark yellow,had certain roughness,SEM observation was porous dense tubular structure,upper opening,low-end seal and titanium base connected,arranged neatly,aperture size uniformity,average tube length 7.12±0.13?m,diameter was 146.72±13.14?m.Gallium nitrate coated Ti and TiO2 nanotubes could be seen by a uniform thin layer of material covering the surface of the material with varying sizes of massive material.2.After 24h in vitro culture,the bacterial biofilm was formed on the surface of TiO2 nanotube and the main bacteria is Escherichia coli.3.There was no difference in activity and body weight status among the four groups on the 3rd,7th and 14th day after operation.The TiO2 nanotubes coated with gallium nitrate had the least number of wound infection,the lightest tissue infection in vivo,and the strongest tissue repair ability.The formation of mature bacterial biofilm was not observed on the surface of Ti and TiO2 nanotubes coated with gallium nitrate by SEM and CLSMConclusion1.The TiO2 nanotube with gallium nitrate coating had the effect of inhibiting the formation of staphylococcus aureus-Escherichia coli mixed biofilm.2-Gallium nitrate coating on the surface of TiO2 nanotubes is a new method to prevent mixed infection of Staphylococcus aureus and Escherichia coli.