| Objective:The relationship between class Ⅰ integron and drug resistance was analyzed by detecting class Ⅰ integron in clinical isolates of Pseudomonas aeruginosa(PA);Analysis of the antibacterial effect of Lonicera hypoglauca extract,a traditional Chinese medicine antibiotic,on class Ⅰ integron positive PA;Furthermore,the effect of Lonicera hypoglauca extract with sub-inhibitory concentration on the expression of virulence factors and quorum sensing(QS)genes was studied;To explore the effect and mechanism of Lonicera hypoglauca extract on QS system under sub-inhibitory concentration,and to provide theoretical basis for developing new Lonicera hypoglauca bacteriostatic agent.Methods:1.Use VITEK-2 compact bacterial identification instrument for strain identification and drug sensitivity test;the antibacterial activity of Lonicera hypoglauca extract was detected by agar dilution method.2.Polymerase chain reaction(PCR)was used to detect class Ⅰ integron and its variable region genes of PA clinical isolates,and the variable region genes were sequenced and compared.3.The inhibitory effect of Lonicera hypoglauca extract on quorum sensing was judged by report plate method and quantitative test of violacein;the minimum inhibitory concentration(MIC)of Lonicera hypoglauca extract on standard wild-type Pseudomonas aeruginosa 01(PA01)was determined by multiple dilution method,and then 1/4 MIC,1/2MIC and MIC were selected as experimental groups;Optical density method was used to detect the effect of Lonicera hypoglauca extract with sub-inhibitory concentration on virulence factors(pyocyanin,elastase,rhamnolipid);4.Quantitative Real-time PCR was used to detect the effect of Lonicera hypoglauca extract with sub-inhibitory concentration on the expression of PA01 quorum sensing system-related genes(las I,las R,rhl I,rhl R).Results:1.There are 8 gene cassettes in the variable region of class Ⅰ integron,and the resistance gene cassettes are consistent with PA resistance;2.The resistance rate of class Ⅰ integron positive strains to all kinds of antibiotics was higher than that of class Ⅰ integron negative strains,and the difference was statistically significant except for chloramphenicol(P<0.01);3.Lonicera hypoglauca extract with sub-inhibitory concentration(4 mg/ml)had a significant inhibitory effect on the expression of pyocyanin and elastase(P<0.05);and at the minimum inhibitory concentration had a significant inhibitory effect on rhamnolipid(P<0.01);4.The results of Quantitative Real-time PCR showed that Lonicera hypoglauca extract significantly inhibited the expression of rhl R gene at the minimum inhibitory concentration(P<0.01);At the sub-inhibitory concentration(4 mg/ml),the expression of rhl Ⅰ and las Ⅰ genes was significantly lower than that of the blank control group(P<0.01).Conclusion:1.In this hospital,Class Ⅰ integron-gene cassette system is one of the most important factors for drug resistance of Pseudomonas aeruginosa;2.It was confirmed for the first time that the Lonicera hypoglauca extract has stronger bacteriostatic effect on Class Ⅰ integron positive strains than Class Ⅰ integron negative strains;3.The Lonicera hypoglauca extract from Long hui County,Hunan Province.(Lonicera macrathoides Hand-Mazz)can inhibit the expression of virulence factors and quorum sensing genes,and its inhibition mechanism is related to the regulation of quorum sensing system. |
