| The beef cattle breeding industry in China is in a period of rapid development,and Tongliao City,Inner Mongolia Autonomous Region,as the core beef cattle production area,has a stock of over 4 million heads.Due to insufficient intensification and modernization in beef cattle farming,various diseases occur frequently.Among them,calf diarrhea is one of the main obstacles affecting the healthy development of beef cattle breeding at this stage,and the direct or secondary infection of pathogenic Escherichia coli is the main reason for calf diarrhea.Type Ⅰ quorum sensing system is mediated by AHL(N-acylhomoserine lactone)molecule,which mainly exists in Ellan-negative bacteria,and has been proved to be involved in regulating the drug resistance and acid resistance of Escherichia coli.In this study,bacterial isolation,Gram staining,16 S r RNA sequencing and other methods were used to isolate and identify calf diarrhea Escherichia coli in Tongliao area.K-B method was used to detect the sensitivity of isolated strains to 15 kinds of antibiotics,and PCR method was used to detect common drug resistance genes.A severe multidrug-resistant strain TL-13 was screened for complete genome sequencing.Adding200μM of type Ⅰ quorum-sensing molecule AHL(C8)in the medium to detect the regulatory effect on the resistance of strain TL-13.After the drug ofloxacin with obvious regulatory effect was screened out,transcriptomic sequencing was performed on the substatin concentration treatment group,substatin concentration plus C8 group and control group.The results showed that all the 20 strains of E.coli with calf diarrhea had multiple drug resistance.Strain 4(TL-13)had the most severe resistance,and was only sensitive to amoxicillin clavulanate potassium,moderately sensitive to doxycycline,and resistant to the other 13 antibiotics.Among the drug-resistant genes examined,the detection rates of aph A1,str B,TEM-1 and qnr S were 100%.Whole genome sequencing of strain TL-13 showed that its genome size was 4 897 185 bp,GC content was 50.68%,and it carried two plasmids with the size of 108 288bp(p TL13-1)and 64 018bp(p TL13-2),respectively.The plasmid carries a total of 18 mobile drug resistance genes.After adding the AHL signaling molecule C8,the regulatory effect on ofloxacin resistance is significant.The minimum inhibitory concentration(MIC)of ofloxacin in TL-13 strain can be adjusted from 32 μg/m L increase to 128 μg/m L.The results of transcriptome sequencing showed that there were 415 differentially expressed genes between the control group and the treatment group,of which201 were up-regulated and 214 were down-regulated.There were 125 significantly differentially expressed genes between the sub inhibitory concentration treatment group and the sub inhibitory concentration added C8 group,of which 102 were upregulated and23 were downregulated.It mainly involves bacterial metabolism 、 ribosome 、 quorum sensing、two-component system and other pathways.The above experimental results show that the drug resistance level of calf diarrhea Escherichia coli in Tongliao area is high,and there is a risk of horizontal transmission.Therefore,the drug regimen should be optimized to avoid the continued increase and spread of drug resistance.The addition of AHL signaling molecule C8 can significantly increase the resistance of the tested bacteria to ofloxacin.A large number of differentially expressed gene data were obtained by transcriptomic sequencing,which could provide experimental basis for the subsequent study on the mechanism of regulation of ofloxacin resistance in calf diarrhea Escherichia coli by type Ⅰ quorum sensing system. |
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