| 【Objective】Pathogens which were separated from udder of cows in Lanzhou and nearbyareas used for drug resistance analysis; The screened Chinese medicines with betterinhibitory effect on Staphylococcus aureus and Escherichia coli were used as eliminationagents to explore the drug resistance and elimination mechanisms of the resistant bacteria andto provide experimental basis for the cure of diseases caused by drug resistant bacteria, bydetecting the effect of medicine extraction solution on drug resistant plasmids, expression andcontent of drug resistant enzymes as well as drug resistance of genes of the drug resistantbacteria.【Methods】Pathogens were collected, isolated and identified from dairies of Lanzhou andnearby areas to obtain drug resistant staphylococcus and E. coli through drug sensitive tests,the minimum and sub-minimum inhibitory concentrations were measured using broth dilutionmethod; Plasmids were screened and extracted with replica planting and alkaline lysismethod, respectively, and then been conducted the electrophoresis with agarose gel method,to analyze and compare plasmid bands change and difference of drug resistant bacteria afterbeing affected by the minimum inhibitory concentrations o f different Chinese medicines;Using paper acid quantitative method to screen ultra-broad-spectrum β-lactamase enzyme,and using ultrasonication to extract crude enzyme solution, and sub-inhibitory concentrationsextraction of Chinese medicines were used to treat drug resistant bacteria, to analyze theeffect of Chinese medicines on ultra-broad-spectrum β-lactamase enzyme activity and content;the target fragments in mecA gene were sequenced with PCR method according to publishedgene sequences in GenBank, and the deactivation test of Chinese medicine on drug resistantgene mecA were conducted to compare the effect of Chinese medicine on mecA gene beforeand after deactivation test.【Results】Pathogens causing mastitis were highly detected, rifampicin, chloramphenicol,vancomycin, gentamicin, ciprofloxacin had stronger anti-bacterial activity, and were suitablefor clinical use; But there were resistant to penicillin, amoxicillin, erythromycin, kanamycin,gentamicin, sulfa drugs, tetracycline, cefazolin and oxacillin, and were not suitable forclinical use; MIC extract solutions of Chinese medicines were used to treat drug-resistantStaphylococcus aureus for24h, and then been conducted the plasmid elimination tests,elimination rate:1.4%in gall, skullcap was1.3%, compound2(berberine, baicalin, plum)was1.8%, gall and skullcap can eliminate two plasmid bands, compound2can eliminatethree, with better elimination effect compared with single herb; to gall, Schisandra, leaves,rhubarb, berberine, baicalin, three needles and ebony, using sub-inhibitory concentration water and alcohol extractions of the9Chinese herb to affect Staphylococcus aureus andEscherichia coli, and got the result that all the9herbs could decrease the activity andexpression of ESBLs, and was significantly different from the negative controlStaphylococcus aureus that producing extended-spectrum β-lactamase (P<0.05), and nochanges were found in sequence after the herbal mecA inactivation test treated by herbextract solutions.【Conclusions】In this study, pathogens were highly detected with relatively severeresistance and broader spectrum, and showed multiple drug resistance; herbal extractsolutions had no effect on the drug resistance of mecA gene, but can eliminate plasmids.Compound were found better than single herbs extract solutions in elimination effect, gall,Schisandra, leaves, rhubarb, berberine, baicalin, three needles, ebony, the9sub-inhibitoryconcentration Chinese herb’s water and alcohol extracting solutions could inhibit theexpression and activity of ESBLs, and alcohol extract was found with better inhibitory effect,indicating that traditional Chinese medicine extract can reduce resistance of mastitispathogens, and can provide theoretical basis for the cure of clinical mastitis infected byStaphylococcus aureus and Escherichia coli. |
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