The Relationship Between CRISPR/Cas System And Drug Resistance In Shigella

Shigella is the main pathogen of infectious diarrhea, causing bacillary dysentery, its treatment mainly depends on antibiotics, however, variation took place frequently with antibiotics abuse, and many resistance Shigella strains emerged. Horizontal gene transfer(HGT) is the main way that bacteria acquire resistance genes. CRISPR exist widely in bacteria and archaebacteria, composed of repeat and spacer, together with cas, limit HGT.ObjectiveWe analyzed the relationship between CRISPR/Cas system and antibiotic resistance through detecting the CRISPR/Cas, antibiotic resistance and resistance genes. Moreover, we analyzed changes of CRISPR/Cas system in multi-drug resistance Shigella strains structured by drug induced and conjugation, so that we can clarify the regulation of CRISPR/Cas system to drug resistance in Shigella.MethodsRecovery and identification 59 Shigella strains, Kirby-Bauer was used to detect the drug resistance, PCR was used to detect resistance genes and cas; PCR amplified and sequenced CRISPR, CRISPR target and BLAST were used to analyze the homology between spacer and plasmids; Clustal X 2.1 and Bio Edit were used to analyze changes of CRISPR/Cas system in multi-drug resistance Shigella strains structured by drug induced and conjugation.Results1. The resistance rates of the 59 Shigella isolates to AM, CF, TE, C, SXT and NOR were respectively, 59.32%, 27.12%, 72.88%, 50.85%, 67.80% and 16.95%. The multi-drug resistance rate was 57.63%. The positive rates of resistance genes were respectively, tem 100%, oxa 59.32%, tet A 86.44%, tet B 84.75%, sul1 89.83%, sul2 76.27%, cat 83.05%, qnrA 77.97%.2. The positive rates of CRISPR1, CRISPR2 and CRISPR3 were respectively 79.66%, 84.75%, 83.05%. At least one CRISPR locus existed in each Shigella strain, forming 4 CRISPR spectral patterns(A-D), type A and B contained cas2-cas1-cas6e-cas5-cas7-cse2-cse1-cas3.3. The difference between multi-drug resistance and spacers on CRISPR1 was significant, also spacers on CRISPR2, and the spacer number was fewer in multi-drug resistance strains, and IS were found in CRISPR/Cas system in most of the multi-drug resistance strains.4. The difference between CRISPR1 and oxa was significant, also tet A, and all the strains without CRISPR1 carried oxa, but fewer carried tet A; The difference between CRISPR2 and oxa was significant, also tet A, and all the strains without CRISPR2 carried oxa, but fewer carried tet A, IS were found in CRISPR/Cas in most of strains with the corresponding resistance gene. The difference between spacers on CRISPR1 and oxa was significant, also tet A, sul2, cat; The difference between spacers on CRISPR2 and oxa was significant, also tet A, sul2, cat, and spacer number was fewer in strains with the corresponding resistance gene.5. 10 spacers matched with 25 plasmids, and resistance gene aph, aadA2, dfrA12, apr R, hph and tem were found in plamid p UMNF18_Inc FV which matched with spacer3. Spacer3 were found in 4 strains, and all the 4 strains carried tem.6. Only cas1, cas2 and isolated CRISPR3 were detected in both sensitive strain and multi-drug resistance strain structured by conjugation, but no changes of cas1, cas2 and CRISPR3 were found. CRISPR1, CRISPR2 and cas gene clusters were all detected in sensitive strain and multi-drug resistance strain structured by drug induced, only 4 bases changed at the end of cse1-cas3.Conclusion1. The multi-drug resistance rate and the positive rate of resistance genes were high in 59 Shigella strains.2. In multi-drug resistance Shigella strains, the spacers on CRISPR1 and CRISPR2 were fewer, and IS were found in CRISPR/Cas system. Fewer spacers were found in Shigella strains carried more resistance genes.3. No change of CRISPR3 was found in multi-drug resistance strain structured by conjugation. Only 4 bases changed at the end of cse1-cas3 in multi-drug resistance strain structured by drug induced.