Detection Of Gene LuxS In Subgingival Plaque Under Different Periodontal Conditions

Objective:P.gingivalis and F.nucleatum,the representative bacteria in the red complex and orange complex of periodontal pathogens,were selected to construct a biofilm model to study the interaction of two kinds of bacteria under co-culture conditions and the difference of AI-2 signals produced by biofilm at different time points(12 h,24 h,48 h,72 h)of biofilm formation.At the same time,to further explore the distribution of gene LuxS content of F.nucleatum and P.gingivalis in subgingival plaque and its relationship with the number of bacteria in different severity of chronic periodontitis.Methods:1.P.gingivalis and F.nucleatum were cultured alone and co-cultured for 12 h,24 h,48 h and 72 h respectively,and the ultrastructure of biofilm was observed by scanning electron microscope.At the same time,the supernatants of three groups of bacteria cultured alone and co-cultured with two kinds of bacteria were collected and added to the suspension of reporter strain V.harveyi respectively.The growth was induced by AI-2 in the supernatant of bacteria.The signal value of V.harveyi was detected by enzyme labeling instrument,and the signal intensity of AI-2 produced in the process of biofilm formation was indirectly evaluated.2.Examinee,who from Huocheng County,Yining City,Xinjiang Province,were took part in the research project ’Xinjiang multi-ethnic cohort study’ from February to March,2019.According to the American CDC diagnostic criteria of chronic periodontitis,the examinee were divided into four groups:periodontal health group,mild periodontitis group,moderate and severe periodontitis group.The total DNA,of absorbent paper points from subgingival plaque was extracted and the specific primers of gene LuxS and 16s rRNA of F.nucleatum,P.gingivalis were designed.The distribution of two genes were detected by qRT-PCR.Results:1.Construction of biofilm in vitro and detection of AI-2 signal:1)Under 10000 times scanning electron microscope,the two kinds of bacteria showed growth in the ultrastructure of biofilm.2)There was a significant difference in the signal intensity of AI-2 produced by P.gingivalis and F.nucleatum when cultured separately.Among the six tests,the signal value of AI-2 produced by P.gingivalis cultured alone for 12 h and 24 h was higher than that of F.nucleatum,but after 48 h,the signal value of F.nucleatum was higher than that of the former.3)In the first three tests of 12 h and 24 h culture,the AI-2 signal intensity was P.gingivalis group>co-culture group>F.nucleatum group,while in the first three tests of 48 h and 72 h culture,the AI-2 signal intensity was F.nucleatum group>co-culture group>P.gingivalis group.With the increase of detection times,the AI-2 signal molecules weakened,and the difference between groups was not obvious.2.The distribution of gene LuxS content of F.nucleatum and P.gingivalis in subgingival plaque and its relationship with the number of bacteria in different severity of chronic periodontitis:1)The contents of P.gingivalis LuxS and F.nucleatum 16s rRNA were different among groups,and the difference was statistically significant.There was no significant difference in the distribution of LuxS of F.nucleatum and 16s rRNA of P.gingivalis between groups.2)The relationship between the concentration of LuxS and 16s rRNA of P.gingivalis and F.nucleatum is not monotonous.The concentration of LuxS increases first and then decreases with the concentration of 16s rRNA.The peak concentration of P.gingivalis is 0.0172 ng/μL,while that of F.nucleatum is 0.25 ng/μL.Conclusions:1.Independent of the early colonization bacteria,the middle colonization bacteria-F.nucleatum can co-aggregate with the late colonization bacteria-P.gingivalis.2.the AI-2 signal produced by F.nucleatum may promote the growth of P.gingivalis in co-aggregation culture.3.The severity of chronic periodontitis in patients is not related to the number of P.gingivalis bacteria,but may be related to LuxS gene.4.The severity of chronic periodontitis in patients is not related to the concentration of gene LuxS of F.nucleatum,but may be related to the number of bacteria.