Shuanghuanglian Preparation Against Klebsiella Pneumoniae In Vitro Antibacterial Effect And Its Effect On Biofilm Formation

Klebsiella Pneumoniae(KP)is one of the serious multidrug-resistant(MDR)bacteria,often causing bacterial pneumonia,cow mastitis,etc.Biofilm(Bacterial biofilm,BF)is produced by KP important mechanisms of drug resistance.BF is a highly tolerant and complex microbial community composed of bacteria and extracellular polymeric substance(EPS)produced by bacteria to adapt to maladaptive environments,which can adhere to biotic or abiotic surfaces and cause persistent and chronic infection.Shuanghuanglian(SHL)is often used to treat respiratory diseases.In the production of SHL oral liquid preparations,the volatile oil is often not recycled.This study investigated the effects of SHL oral liquid containing volatile oil,SHL oral liquid without volatile oil and recovered pure SHL volatile oil on multidrug-resistant Klebsiella pneumoniae(MDR KP)and their effects on the transcription level of genes related to BF formation,so as to provide a basis for the clinical application of SHL in the treatment of respiratory diseases.In this study,the antibacterial effects of SHL oral liquid with volatile oil,SHL oral liquid without volatile oil and pure SHL volatile oil alone or in combination with antibacterial drugs were determined on MDR KP.Crystal violet staining method was used to determine the BF formation ability of MDR KP and the inhibitory effect of three SHL preparations on BF formation of MDR KP.The distribution of BF formation-related genes of MDR KP was detected by PCR.The effects of three SHL preparations on the transcription levels of BF formation-related genes of MDR KP were determined by RT-PCR.The main results are as follows:1.The MIC of SHL oral liquid without volatile oil to KP1 and KP2 is 375mg/m L,and the antibacterial effect of SHL oral liquid with volatile oil is 4 times higher than that of SHL oral liquid without volatile oil,which is 93.75mg/m L.The antibacterial effect of pure SHL volatile oil is the best at 0.05 mg/m L.After being used in combination with clindamycin,oxacillin,doxycycline,amikacin,meropenem and ciprofloxacin,the volatile oil of SHL shows a synergistic effect in combination with ciprofloxacin and clindamycin,respectively.The volatile oil of SHL combined with meropenem and oxacillin,the combination of SHL oral liquid containing volatile oil and doxycycline,clindamycin,and oxacillin respectively,and the combination of SHL oral liquid without volatile oil and meropenem show additive effect.It can effectively reduce the concentration of antibacterial drugs.2.The MDR KP strain has a very strong ability to form BF(P<0.01).At the concentrations of 1/8MIC,1/4MIC,1/2MIC and MIC,the three SHL preparations has extremely significant inhibitory effects on the formation of BF in a concentration-dependent manner(P<0.01).At 1/16 MIC concentration,only pure SHL volatile oil has a very significant inhibitory effect on the formation of BF in KP1 strain(P<0.01).All three SHL preparations could significantly reduce the transcription level of mrk D gene(P<0.05).However,only SHL oral liquid containing volatile oil and pure SHL volatile oil could significantly reduce the transcription levels of wbbm and wzm genes(P<0.01).The above results show that the three SHL preparations combined with antibiotics can additively or synergistically inhibit MDR KP.The three SHL preparations can significantly inhibit the formation of MDR KP BF(P<0.01)in a dose-dependent manner.SHL preparations can significantly reduce the transcription level of mrk D gene(P<0.05).SHL oral liquid containing volatile oil and pure SHL volatile oil can significantly reduce the transcription levels of wbbm and wzm genes(P<0.01).