Screening,identification And Characterization Of Clostridioides Difficile Antibiotic Resistance Genes

In recent years,Clostridioides difficile（C.difficile）has become a common pathogen in nosocomial infections.Due to the extensive use and abuse of broad-spectrum antibacterial drugs,the incidence of Clostridioides difficile antibiotic-associated pseudomembranous colitis and diarrhea is increasing worldwide.,CDI)are the main antibiotics.With the continuous emergence of drug-resistant mutant strains of Clostridioides difficile,the treatment of CDI is becoming more and more difficult.The discovery of drug resistant strains often comes from the isolation and culture of bacteria during clinical treatment.Most of the information on drug resistant mutation sites is obtained through whole-genome sequencing.At the same time,due to the lack of high throughput genetic engineering methods,it is difficult to obtain site-directed mutation drug resistant strains,and the research on antibiotic resistance genes and drug resistance mechanisms is slow.The Mariner transposon random insertion system was used to induce random insertion of transposons into the whole genome to cause single point mutations through tetracycline induction,and a library of single gene mutants with random insertions of transposons was constructed.Antibiotic resistant strains were screened from the library by applying antibiotic pressure,and two vancomycin resistant mutant strains and one fidaxomicin resistant mutant strain were successfully obtained.Based on the principle of Tail-PCR to identify transposons random Ly inserted into single gene mutation sites,three rounds of PCR degenerate primers and specific amplification primers were designed.By optimizing the number of PCR amplification cycles,reaction temperature,and template processing methods,a difficult The identification method of Clostridium Mariner transposon insertion site and the construction of Inverse PCR detection method proved the reliability and accuracy of the Tail-PCR Clostridioides difficile transposon mutation identification method.The three new drug resistant mutant strains of Clostridioides difficile obtained by the above method were detected,and the transposon insertion sites were successfully identified,which were the rel A/spot domain protein（CD630DERM＿17080）gene sequence and the K⁺channel regulatory protein（CD630DERM＿06970）gene sequence,Car D family transcriptional regulator（CD630DERM＿30370）gene sequence,no related drug resistance gene reported in the literature.The new drug resistant mutant strains and wild strains were detected by antibiotic MIC₅₀,and the MIC₅₀ of two vancomycin resistant mutant strains increased from 0.5-1μg/m L to 2-4μg/m L and 1-2μg/m L respectively,and one non vancomycin resistant mutant strain The MIC₅₀ of the daxamycin resistant mutant strain increased from 100 ng/m L to 300 ng/m L;through growth curve detection,after antibiotic pressure was applied,the growth of the wild strain was significantly inhibited,and the mutant strain still maintained a good growth trend,but the logarith MIC growth The plateau phase peak value decreased after the period was delayed;through the detection of Tcd B toxin expression,the expression level of toxin in the wild strain and the mutant strain was the same under no antibiotic pressure,and the expression level of Tcd B toxin in the mutant strain increased by 7 times under antibiotic pressure.In this work,the Mariner transposon random insertion system was used to successfully induce and obtain a single gene mutation library of Clostridioides difficile.Screening of antibiotic resistant mutants commonly used in clinical CDI treatment was carried out,and the transposon insertion site was established based on the principle of Tail-PCR The identification method successfully realized the identification of the mutation sites of the three mutant strains,and completed the performance characterization and difference analysis between the mutant strains and the wild strain.The results showed that the drug resistance of the mutant strains and the level of toxin expression under antibiotic pressure were higher than those of the wild strain,the genes CD630DERM＿17080,CD630DERM＿06970 and CD630DERM＿30370 are C.difficile related resistance genes to vancomycin and fidaxomicin,providing new methods and new directions for further research on C.difficile antibiotic resistance genes.