Relationship Between CRISPR/Cas System And Antibiotic Resistance Of Shigella

Shigella is a kind of gram negative enteric pathogen which are highly infectious and pathogenic. Shigellosis that induced by shigella has been an important public health problem throughout the world, especially in developing countries. At present, the increasingly serious problem of drug resistance blocks effective treatment for Shigellosis. And horizontal gene transfer (HGT) as an important way of acquired resistance of bacteria has attracted enough attention. CRISPR/Cas system, mainly composed of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), is a heritable and acquired novel defense system in prokaryotes that deals with HGT through defensing foreign genetic materials in the basis of various spacers and versatile Cas. This mechanism may provide some help for resolving drug resistance spread. However, there is no a final conclusion about the relationship between CRISPR/Cas system and bacterial resistance, and the exact mechanism remains unclear either.ObjectTo explore relationship between antibiotic resistance and CRISPR/Cas system in Shigella based on the previous research.Methods1. Agar dilution method was used to determine the minimum inhibitory concentration (MIC) of Shigella.2. PCR amplification and nucleic acid sequence analysis were used to find loci of CRISPR/Cas system in Shigella.3. Clustal X2.1, Seqman, CRISPR Finder were used to find change of CRISPR/Cas system in different Shigella which were electroporated with plasmid PHSG398, induced by chloramphenicol in vitro with 1/2 X MIC method and concentration increase method, consecutive transferred without antibiotics, respectively.4. The combination of CRISPR Target and BLAST was applied for searching homologous plasmids or phages of spacers, then according to their accession numbers to lookup their information related to drug resistance. Drug resistance information of Shigella strains in GenBank and preserved in our laboratory which were isolated from clinic was detected by Bioedit and PCR method previously, respectively.Results1. No change of CRISPR/Cas system was found in transformants mel-sf1998024/PHSG398 and mel-sf2005127/PHSG398 except the insertion of IS600 in cse2 locus of mel-sf2005127/PHSG398.2. The sensitivity of three Shigella strains to different antibiotics was decreased in different degrees after the induction of drug resistance, and loci of CRISPR/Cas system were found no change.3. The sensitivity to certain antibiotics of four Shigella strains with different drug resistant spectrums after consecutive transfer 90 generations increased. The spacer and its upstream repeat in 3’end of CRJSPR3 were lost in mel-sfl998024/C and mel-sf2013004/C.4. Seven of 52 spacers were homologous to 4 plasmids which carry information related to drug resistance. Drug resistance gene blaTEM, aadA2 and dfrA12 were detected in both Shigella strains (mel-ss1998011/zz, mel-ss1998024/zz, mel-sf2005082/sx, mel-sf2013004/bj) and their spacer’s (CRISPR2-mel-3) homologous plasmid pUMNF18_IncFV. Similarly, drug resistance gene ampC and tetB were detected in both Shigella strain (sd1012) and its spacers’(CRISPR-Q1-S1, CRISPR-Q4-S1) homologous plasmid pM5A24P. In addition, drug resistance gene ampH, mrdA and were detected in both Shigella strain (mel-sf2000102/zz) and its spacer’(CRISPR3.-mel-33) homologous plasmid:5. Intriguingly, both of them carried virulence gene yafO, yaJN, virK, idrB, ychO. What’s more, we found type I-F CRISPR/Cas system in this plasmid.Conclusions1. In the case that plasmid was transformaed into Shigella by electroporation, the CRISPR/Cas system may not play the function.2. High concentrations of chloramphenicol may activate drug resistance efflux pump. Chloramphenicol pressure may not impact on CRISPR/Cas system.3. Shigella can reduce or lose their resistance to some antibiotics after consecutive transferred without antibiotics. CRISPR3 locus had dynamic spacers in Shigella and CRISPR3 locus and cas genes may co-evolved.4. Shigella strain and its spacer’s homologous plasmids have some similarity in distribution of resistance information. We speculate that in the process of acquiring new spacer, the other gene fragments of exogenous genetic material that carries protospacer may be integrated into the genome of Shigella which may provide clues for relationship between CRISPR/Cas system and drug resistance or virulence of bacteria. CRISPR may co-regulate drug resistance and virulence. The CRISPR/Cas system also exists on plasmids.