In-vitro Research Of Lonicera Japonica Decoction Reverse Metallo-?-lactamase Producing Pseudomonas Aeruginosa Resistance

Objective 1.To investigate the epidemic of Imipenem resistance caused by metallo-?-lactamses in Imipenem resistant Pseudomonas aeruginosa collected from Henan Hospital of TCM in 2015 and provide evidence for nosocomial infection control.2.To assess the horizontal transfer risk of metallo-?-lactamase gene through R plasmid and enrich the theory of metallo-?-lactamase gene horizontal transfer.3.To explore the difference between different PA iolates and whether inhibit effects exist upon MBL producing isolates by AST of PA on Lonicera japonica.4.To appraise the R plasmid curing effect of Lonicera japonica decoction,analyse the resistance reverse mechanism and enrich the theory of Lonicera Japonica decoction reverse the resistance of Pseudomonas aeruginosa clinical isolates by inspecting the in-vitro R plasmid curing experiments of Lonicera Japonica decoction on Pseudomonas aeruginosa clinical isolates.5.To analyse the resistance reverse capacity of Lonicera Japonica decoction by comparison with presennt results,and provide theory basis for anti-infection treatment of Chinese herb medicine and resistance horizontal transfer prevention and control.Method 1.WHONET 5.6 were applied to analyse antibiotic sensitivity test of IRPA collected from Henan Hospital of TCM in 2015 and resistance condition was preliminary estimated.2.IPM-EDTA double-disc synergy test(DDST)were used to screen Metallo-?-lactamase phenotype of IRPA.3.Four frequent Metallo-?-lactamase genotype,IPM,VIM,SPM and NDM screening test to IRPA which Metallo-?-lactamase phenotype were positive,completed by gradient PCR and agarose gel electrophoresis.4.Conjugative plasmid transfer expermient were applied to determine whether the plasmid containing Metallo-?-lactamase genet can be transferred to other species or not.5.R plasmid curing experiment were applied to analyse and appraise the plasmid curing capacity of Lonicera Japonica decoction and 5%SDS at 24 hours,48hours and 72 hours.Result 1.248 PA were collected in Henan Provincial Hospital of TCM in 2015,and 35.89%(89/248)of isolates were IRPA.The resistance rate of Ticarcillin,Ticarcillin/clavulnic acid,Piperacillin,Piperacillin/tazobactam,Ceftazidime,Cefepime,Cefoperazone,Cefopera zone/sulbactam,Imipenem,Meropenem,Gentamicin,Amikacin and Aztreonam are 75% ?48.79%?66.53%?19.76%?32.66%?21.77%?30.24%?15.73%?35.89%?31.85%?23.79%?17.34% and 39.52% respectively.2.23 of 89 IRPA were positive MBL phenotype isolates and positive rate reached 25.84%(23/89),including 23 positive genotype isolates which consist of 16 IPM strains and 7 VIM strains,but no SPM and NDM-1 positive strains were detected.7 of 23 positive MBL isolates which resistance transferred by conjugative plasmids were all IMP types PA isolates confirmed by PCR.3.The MIC of Imipenem sensitive PA,non-MBL producing IRPA MBL producing IRPA to Lonicera Japonica decoction were 120.96±1.42mg/ml,267.47±1.46mg/ml and 298.86±1.36mg/ml respectively,measured by crude herbal dose;The One-Way ANOVA results showed that significant difference existed betweeb Imipenem sensitive PA and non-MBL producing IRPA,MBL producing IRPA(p=0.000?0.05),but no significant difference existed between non-MBL producing IRPA and MBL producing IRPA(p=0.312?0.05).4.The average curing rate of 5%SDS and Lonicera Japonica decoction to MBL producing IRPA plasmid at 24 h,48h and 72 h were 0.24%?1.33%?1.09%? 0?0.71%?0.62% respectively.Conclusion 1.The resistance rate of IRPA to Penicillins,third generation cephalosporins,Ceftazidime and Carbapenems was very relatively high,but to Aminoglycosides Cefoperazone/sulbactam and Cefepime were relatively low;Compared with the data of CARSS,we found the feature of resistance was of geographical regions,and may be associated with prescribing custom and epidemic isolates.2.IPM resistance caused by MBL was one of important cause which account for IRPA in Henan hospital of TCM in 2015;Majority of MBL genotype was IPM and VIM,and highly consistent with prevalence MBL gene in our country,and IPM gene resistance can be transferred to other species through plasmid conjugation.3.Lonicera Japonica decoction can inhibit Imipenem sensitive PA,MBL producing IRPA and non-producing IRPA to some extent,but the MIC were different with presented results.Significant difference existed between Imipenem sensitive PA and MBL producing IRPA,non-producing IRPA,but no significant difference existed between MBL producing IRPA and non producing IRPA.4.Lonicera Japonica decoction was capable of curing R plasmids carried by MBL producing IRPA and the curing rate was lower than 5%SDS.But SDS was full of kinds of side effects and can't be used to food,Lonicera Japonica was good medicine which can clear away heat carambola and eliminate pathogens.