Isolation And Identification Of Chicken-derived Mcr-1 Positive Escherichia Coli And Screening For Its Traditional Chinese Medicine Inhibitors

Colistin,as the last defense for the Gram-negative bacterial infections,has gradually lost its efficacy due to the non-standard use.However,due to the irregular use of antibiotics in recent years,the effect has gradually lost.With the discovery of the plasmid-mediated colistin resistance gene mcr-1,the mystery of the spread of colistin resistance has been revealed.Therefore,it is urgent to find a new drug to target inhibit mcr-1 positive Escherichia coli is a reasonable strategy to solve the problem of drug resistance.In this study,185 chicken cloacal swab samples from a chicken farm in Qingdao,Shandong Province were collected as the research object,and these strains were isolated through selective medium and 16 S rRNA identification.The epidemic characteristics were analyzed by drug sensitivity test and drug resistance gene detection.Resistance inhibitors were screened from 102 natural compounds(flavonoids,alkaloids,organic acids,lignin,terpenoids,etc.)by the trace broth checkerboard method.The growth curve and bactericidal curve were used to evaluate the in vitro antibacterial activity of inhibitors,and the CCK8 cytotoxicity test was used as a preliminary method to evaluate their safety,scanning electron microscopy and lactate dehydrogenase(LDH)tests were used to explore the mechenisms underlying the action of the drug.173 Escherichia coli isolates were isolated from 185 samples,and the susceptibility test of 11 commonly used antibacterial drugs(including Amoxicillin,Cephalexin,and Tetracycline hydrochloride,etc.)was 0.58%～100.00%.The Colistin resistance rate was 75.72%(131/173),the mcr-1 gene detection rate was 38.15%(66/173).All strains are multi-drug resistant,none of the other subtypes of mcr gene were found.In the initial research,Dehydrovioline,Dihydrocapsaicin,Peimisine,Brazylinand and other 12 natural compounds had potential inhibitory effect on the tested bacteria,among which Brazylin had the most stable effect.The results showed that when 64 μg/mL Brazilian and 1 μg/mL Colistin were used in combination,they showed a synergistic effect(FICI=0.25～0.38)to the tested strains,and the MIC value was correspondingly reduced by 4～8 times(MIC decreased from 8 μg/mL and 0.25 μg/mL to 1 μg/mL and0.06 μg/mL).The growth curve verified that Brazilian has a short-term growth inhibitory effect on mcr-1positive bacteria at the used concentration;the time sterilization curve test verified that Brazilian can significantly restore the bactericidal activity of colistin.At the same time,the cytotoxicity showed that Brazilian caused certain damages to Vero,AML12,and HK2 cells,and it also had a good effect on SW480 colon cancer cells.The extracellular lactate dehydrogenase test and scanning electron microscopy test further confirmed the inhibitory mechanism of Brazilian on mcr-1.Under the combined drug co-cultivation,the release of intracellular LDH increased gradually within 6 h,and the bacterial cells were observed under the electron microscope.It shows that the combination of brazilian and colistin has a damaging effect on the cell membrane and cell wall of the strain.In summary,brazilian can be used as an mcr-1 drug-resistant inhibitor to improve the antibacterial activity of colistin,and provide a scientific basis for the treatment of colistin-resistant infections.It also provides data support and scientific ideas for the treatment of colistin resistant bacteria infection and the development of Chinese and Western antibacterial reagents.