The Distribution And Characteristic Of CRISPR In Shigella

Shigella is the main pathogen causing acute infective diarrhea. The incidence ofbacillary dysentery caused by Shigella is highest in infectious diarrhea. Owing to thewide use of antibiotics, the emergence of resistant strains of Shigella spp. is of greatinterest both in developing and industrialised countries. The drug resistance can beimproved mainly by getting resistance genes caused by horizontal gene transfer(HGT).Recent studies show that clustered regularly interspaced short palindromicrepeat(CRISPR) closely related to HGT in bacteria. It can resist the invasion offoreign genetic material such as phage and plasmid to avoid HGT in bacteria. Theantibiotic resistant strains in clinic are mainly caused by HGT, so exploring thedistribution and characteristics of CRISPR can help us know more about antibioticresistant spectrum and the HGT of antibiotic resistance genes. And the distribution ofCRISPR in Shigella is barely reported.The aim of this study was to describe the distribution and characterizations ofCRISPR in Shigella. CRISPR,cas1and cas2sequences were detected and sequencedin Shigella. To provide basic data in order to reveal the rules of HGT and theassociation with drug resistance.MethodsSelecting bacteria from the suspicious shigella system by microbial culture andbiochemical identification.196clinical Shigella strains were isolated from differentplaces in China,including Beijing, Henan and Jiangxi province;Susceptibility test wasperformed by Kirby-Bauer. PCR amplification was used to detect cas1, cas2andCRISPR.Sequencing cas1, cas2and CRISPR, and analyzing the gene sequences.Digoxin labelled random primers was used to fabricate DNA probe of CRISPRassociated genes in Shigella.Using digoxin labeled DNA probe to detect CRISPRassociated genes in Shigella by dot blot hybridization. Results1. All the196Shigella strains carry cas1(a), cas1(b) and cas2genes. Sequencedstrains were divided into two groups by isolated time. And the distribution ofCRISPR-S1and CRISPR-S4were different before and after2004.2.260spacers were detected in90Shigella.Among them,207spacers exited in theCRISPR DB of Shigella and other53spacers were new found.3. The repeat sequences in the same CRISPR were homologous in different strains,but were different in different CRISPRs. Deletion of nucleotides existed in the5’endor3’ end in some repeats.4. Cas1(a), cas1(b) and cas2genes by BLASTN in GenBank shows: Shigella werehomologous to Escherichia coli. In addition, repeat and spacers sequences whichwere homologous to Escherichia coli, Salmonella enteritidis and even some distantlygenetic related bacteria.were found in CRISPR DB.5. Cas1(a), cas1(b) and cas2genes were sequenced. By comparing them withcorresponding gene sequences of Shigella sonnei53G published in GeneBank,different levels of mutations appeared in all the sequenced cas1(a), cas1(b) and cas2genes. Some of these mutations may be related to antibiotic resistance.6. Probes of cas2and repeats were designed successfully. The Shigella strainsdetected were positive by Dot blot hybridization.Conclusion1. The CRISPR system widely exist in shigella. The distribution of CRISPR S1andS2is different in Shigella strains isolated in different time.2. The gene mutations of cas2genes may lead to different antibiotic resistance inShigella.3. The CRISPR of Shigella and Escherichia coli are high homologous.4. Completed the establishment of probe and hybridization of Shigella CRISPRsystem.