| Background: A wide variety of microorganisms colonize the inner wall of the root canal and the surface of periapical tissues in the form of biofilm,which is essential for the progression and persistence of periapical inflammation.The study of bacterial diversity and abundance in the infected root canal microbial community is of great significance to the infection control in the root canal,to improve the success rate of root canal treatment and to improve the quality of life of patients.In addition,the complex three-dimensional structure of oral bacterial biofilm is the basis of drug resistance and immune escape and is one of the main reasons for recurrent apical periodontitis.At present,there are many models about infected root canal biofilm,and there is no standard model.The research on the diversity of the infected root canal biofilm is helpful to establish the model of the infected root canal biofilm based on the dominant bacterial species.On this basis,on the one hand,the interaction between the dominant bacteria is studied,and on the other hand,it provides a standard tool for the research and evaluation of the control drugs of the bacterial biofilm.Objective:（1）To study the species and abundance of bacterial microbial communities in the primarily infected root canals,providing a theoretical basis for the construction of standard biofilm models of infected root canals.（2）A biofilm model of the infected root canal was preliminarily constructed based on the sequencing results of the microbial community,providing an effective tool for studying interactions between dominant bacteria and evaluating root canal flushing fluid effect.（3）The above-constructed biofilm model was used to evaluate the inhibitory effect and mechanism of the two small molecular antibacterial peptides buCa THL4 B and Im-4,which were effective against dental plaque biofilm screened out in the previous study,so as to provide a theoretical basis for the clinical application of antibacterial peptides in the control of root canal infection.Methods:（1）Primary apical periodontitis cases were collected,infection materials in root canals were collected,16 S rDNA sequencing was performed,bacterial diversity and abundance in samples were analyzed by bioinformatics methods,and potential biological functions of species were predicted.（2）Based on 16 S rDNA sequencing results and combined with each species’ detection rate and abundance values,two dominant species were screened and mixed cultured in a certain proportion under a simulated oral environment to form a two-species biofilm.In vitro infection root canal biofilm model was constructed,and the growth of the two species in the model was characterized and detected.（3）The biofilm model was constructed using the above method.The inhibition effect of two antimicrobial peptides from different sources alone and combined with chlorhexidine was detected by laser confocal microscopy.Results:（1）A total of 13 samples were included in the standard data analysis,and 359 strains were detected,which could be divided into 40 phylum,mainly distributed in Bacteroidetes（32.05%）,Firmicutes（31.23%）,and Proteobacteria（12.49%）.A total of 359 strains were detected from 13 samples,among which the top five strains were Fusobacterium nucleatus（100%,5.76%）,Lactobacillus plantarum（100%,1.12%）,Wolbachia endosymbiont（100%,1.00%）,Ralstonia pickettii（100%,0.23%）and Klebsiella oxytoca（100%,0.22%）.The top five bacteria were Porphyromonas gingivalis（7.02%,53.85%）,Fusobacterium nucleatum（5.76%,100%）,Dialister invisus（5.57%,84.62%）,Pyramidobacter piscolens（5.31%,61.54%）and Porphyromonas endodontalis（3.66%,84.62%）.The results of diversity analysis showed that there were significant individual differences among different samples.The prediction results of potential biological functions showed that the species in infected root canals were enriched in metabolism,environmental information processing,genetic information processing,and biological processes.（2）Based on 16 S rDNA sequencing results,combined with the detection rate and abundance values of each species,the detection rate of Fusobacterium nucleatum was the highest times the abundance value,and Enterococcus faecalis（16S rDNA detection rate 83.33%,abundance 1.19%）was the most used in root canal biofilm model research.Therefore,Fusobacterium nucleatum and Enterococcus faecalis were selected to construct biofilms.The two strains were mixed 1:1（colony number: 1.5×106 CFU/m L）and inoculated in a hydroxyapatite disk to construct a bacterial biofilm model of infection of the two strains in vitro.The growth and death rate of biofilm was calculated by confocal laser scanning microscope.From 24 h to 48 h,the volume of biofilm increased significantly,and after 48 h,the volume of biofilm increased gently.The rate of dead bacteria was lower than 10%,which was controlled at a good level.The colony numbers of Fusobacterium nucleatum and Enterococcus faecalis in the biofilm were calculated by real-time PCR.With the increase of time,the number of Fusobacterium nucleatum and Enterococcus faecalis in the biofilm increased significantly.The number of Fusobacterium nucleatum in 24 h biofilm was more than Enterococcus faecalis（P = 0.02）,there was no significant difference between them in 48 h biofilm（P = 0.169）,and the number of Fusobacterium nucleatum in 72 h biofilm was significantly more than Enterococcus faecalis（P < 0.01）.（3）The bactericidal rates of buCa THL4 B and Im-4 were（46.25 ± 3.58）% and（48.30 ± 2.80）%,respectively,using the biofilm model constructed above.The bactericidal rates of buCa THL4 B and Im-4 combined with chlorhexidine were（58.98 ± 6.64）% and（65.39 ± 4.70）%,respectively.The bactericidal effects of buCa THL4 B and Im-4 were better than those of chlorhexidine alone（P < 0.001）.The results showed that the total biofilm volume in the buCa THL4 B group was（9.60 ± 3.11）×106 μm3,（2.28 ± 0.30）×107 μm3,（3.52 ± 0.98）×107 μm3 at 24 h,48 h,and 72 h,respectively.The total biofilm volume of the Im-4 group was（4.93 ± 2.65）×106 μm3,（1.37 ± 0.55）×107 μm3 and（2.19 ± 0.32）×107 μm3 at 24 h,48 h,and 72 h,respectively,which were lower than that of the blank group.They were（2.99 ± 0.27）×107 μm3,（4.47 ± 0.23）×107 μm3,（4.62 ± 0.28）×107 μm3,respectively,with significant differences（P < 0.001）.The effect of Im-4 on biofilm inhibition was better than that of the buCa THL4 B group（P < 0.01）.The bactericidal rates of the buCa THL4 B group at 24 h,48 h,and 72 h were（54.06 ± 13.53）%,（62.97 ± 2.77）%,and（54.18 ± 4.29）%,respectively.Bactericidal rates of（33.60 ± 8.16）%,（37.89 ± 8.04）%,and（36.68 ± 4.16）% in the Im-4 group at 24 h,48 h,and 72 h were significantly higher than those in the blank group（7.50 ± 4.79）%,（6.56 ± 2.20）%,and（5.19 ± 3.27）%,respectively.The difference was significant（P <0.01）.The bactericidal effect of the buCa THL4 B group was better than that of the Im-4 group,the difference was significant（P < 0.001）.Conclusion: The primary infection of the periapical root canal has abundant bacterial composition,among which the detection rate of Fusobacterium nucleatum is the highest and the abundance value is also high,indicating that it plays an important role in the formation of biofilm.A two-species biofilm model was successfully constructed with Enterococcus faecalis,which is mostly used in root canal biofilm model.This model can effectively evaluate the effect of buCa THL4 B and Im-4 on inhibiting the infected root canal biofilm,and provide a laboratory basis for the clinical application of small molecular antibacterial peptides.In the later stage,biofilms of three species,four species,or five species will be constructed on this basis to further study the interaction between bacteria and the mechanism of action,and evaluate the effect of various root flushing drugs. |
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