| Salmonella is an important zoonotic pathogen,and its increasing virulence and drug resistance have attracted wide attention.As the main way of signal transmission between bacteria,bacterial quorum sensing system plays an important role in regulating bacterial virulence,drug resistance,acid resistance and other biological functions.The type I quorum sensing system mainly exists in Gram-negative bacteria and plays a role through AHLs signaling molecules.In this study,a clinical dead calf organ was used as the material,and the pathogen was isolated and identified by conventional methods such as bacterial isolation and purification and 16 S r RNA gene sequencing.The virulence of the isolated strains was detected by mouse challenge test.The minimum inhibitory concentration(MIC)values of the isolated strains against commonly used drugs were detected by broth microdilution method.The virulence and drug resistance genes of the isolated bacteria were detected by PCR.The whole genome of strain S-1-1 was sequenced by nanopore method.The serotype of strain S-1-1 was identified using the Seq Sero 1.2 database.The virulence genes,virulence islands,drug resistance genes and mobile elements of strain S-1-1 were analyzed by using virulence gene database and antibiotic resistance gene database.By adding AHL signal molecules C6 and C8 to detect its regulatory effect on the drug resistance of strain S-1-1,the drugs with obvious regulatory effect were screened.Crystal violet staining was used to detect the biofilm formation ability of the screened drugs at sub-inhibitory concentration and after sub-inhibitory concentration plus AHL molecules.The expression of biofilm formation related genes in different treatment groups was detected by fluorescence quantitative PCR.The results are as follows : The same pathogen was isolated from multiple organs of dead calves and identified as bovine Salmonella,named S-1-1.The mouse challenge experiment showed that the strain had strong virulence,and the median lethal dose was 2.8× 106 CFU / m L.Strain S-1-1 was a multidrug-resistant bacterium,which was sensitive to polymyxin B and thiamphenicol,moderately sensitive to doxycycline and enrofloxacin,and resistant to florfenicol,tetracycline,and sulfadiazine sodium.The analysis of the whole genome sequencing results of the strain S-1-1 showed that the strain was Salmonella Dublin.The genome size of the strain was 4 965 370 bp,and the GC content was 52.12 %.The strain carried two plasmids,which were 79 524 bp(p TLS-1)and 45 301 bp(p TLS-2).The strain S-1-1 contained 996 virulence genes and 24 virulence islands,carrying 42 drug resistance genes,of which 4 were horizontal transfer genes,and there were 9 mobile genetic elements such as insertion sequences and transposons.After the addition of AHLs,the drug resistance of doxycycline,polymyxin,florfenicol,tetracycline and thiamphenicol was regulated,and the MIC value of thiamphenicol was increased from 0.5 μg / m L to 4 μg/ m L.After adding 200 μmol / L molecule at sub-inhibitory concentration,the biofilm formation ability of strain S-1-1 increased significantly before 8 h and decreased after 8 h,and the expression trend of biofilm formation related genes was consistent.Through the above experimental contents,it was found that there was a risk of transmission of Salmonella Dublin from cattle in Tongliao area,and the virulence and drug resistance of the strains were strong.The addition of type I quorum sensing molecule AHL can regulate some drug resistance,and AHL can significantly affect the biofilm formation ability of isolated strains.This study can provide a reference for the mechanism of type I quorum sensing system regulating the resistance mechanism of bovine Salmonella Dublin,and provide a research basis for further study of the mechanism of type I quorum sensing system regulating biofilm formation. |
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