The Genotyping Method Based On CRISPRs And The Relationship Between The CRISPR/Cas And The Virulence And Resistant In Escherichia Coli

Pathogenic Escherichia Coli(E.coli)strains are capable of causing a number of disease states in human and animals,including diarrhea,hemorrhagic colitis,hemolytic uremic syndrome,thrombotic thrombocytopenic purpura,sepsis,and meningitis,remaining a threat of public health around the world.The prophage or plasmid carried many genes associated with virulence and drug resistance move from one bacterial to another,which called horizontal gene transfer(HGT),leading to the emergence of the new high pathogenicity or multidrug-resistant bacteria,especially the new bacterial agent.CRISPR/Cas had been found in 90% of archaea and 50% of bacteria.The variable CRISPRs spacers could be used as a target for molecular typing and evolution of bacteria,and the CRISPR/Cas performed an adaptive immune system that provides protection from bacteriophages and plasmids.Objective1 To analyze the structural features of the CRISPR/Cas and its flanking sequence of the strains with complete genome sequences and explore whether the CRISPRs spacer can be used as a target for the classification of E.coli strains,then evaluating its effect.2 To establish the method based on the CRISPRs,and forecast its serotype according to the CRISPRs type,then evaluating its prediction effect.3 To describe the spatiotemporal distribution,serotype and CRISPRs type of Shiga toxin-producing E.coli strains with whole genome shotgun sequence,and elaborate whether the CRISPRs type have the significance being epidemiological study more.4 To discuss the distribution of phage,plasmid,the acquired virulence and antibiotic resistance genes,then analyze the relationships between the CRISPR/Cas and phage,plasmid,the acquired virulence and antibiotic resistance genes and provide the reference for the further studies on the immune mediated by CRISPR/Cas.Methods1 The CRISPR sequences were obtained through the BLAST with the repeat sequence against the publicly complete genome in Gen Bank related to the E.coli.The 4kb sequence(the repeat ± 2 kb)was extracted,and the repeat and spacer were analyzed by CRISPRs finder and BLAST.The Clustal X was used to perform multi-sequences alignment of spacers,cas and flanking sequences and the Mega5.1 was used to draw the unrooted tree.We obtained the serotype and ST by using the Sero Type Finder and MLST.2 PCR was used to amplify the CRISPRs,then sequencing.The sequences were analyzed by CRISPRs finder and BLAST to obtain CRISPRs type.The serotyping was identified by serum agglutination test.3 Recording the time and region of STEC strains,and the serotype and CRISPRs type were analyzed by Sero Type Finder and CRISPRs finder.4 The information of chromosome and plasmid of the strains with complete genome was retrieved from Gen Bank.The phage,the acquired virulence genes and drug-resistant genes were obtained by PHAST,Virulence Finder and Res Finder.The Chi-square test was used to compare and analyze the distribution of CRISPR/Cas types and plasmid,the acquired antibiotic resistance genes.Pearson correlation analysis was used to explore the association between CRISPR/Cas and phage,the acquired virulence gene.Results1 The distribution of CRISPR/Cas in E.coliA total of 203 strains of E.coli with complete genome sequences from NCBI were used.73.4% strains contained the I-E CRISPR/Cas while 8.4% strains carried the I-F CRISPR/Cas,and 17.2% strains only owned CRISPR3-4(non-CRISPR/Cas).There are two strains-B7 A and Co6114 both contained four CRISPR loci,and complete I-E cas and I-F cas.Only cas2 and cas1 presented in strain O177:H21 and the cas2 and cas3 existed in strain 1303.Moreover,the complete I-E cas genes were absent in 12 B strains.IS4,IS66 or IS 30 were detected in cas or CRISPR2.2 The method to identify the E.coli and ShigellaThe similarity is more than 99% for the CRISPR1 upstream flanking sequences only in E.coli and Shigella.The sensitivity and specificity were greater than 91% by PCR amplification the region to identify the E.coli and Shigella.3 The method based on CRISPRs for typing E.coliThe number of the spacers in CRISPR1,CRISPR2,CRISPR3,CRISPR4 and CRISPR3-4 were 1 to 25,1 to 27,4 to 17,2 to 22,and 1-2,respectively.According to the sequence identity by Clustal X,the total spacers were classified into 346,341,57,66 and 6 types of unique spacers in each CRISPR locus.We defined the I-E CRISPR/Cas as CT-I,I-F CRISPR/Cas as CT-II and only CRISPR3-4 as CT-III.We designated each unique arrangement spacer profile as a unique CRISPRs type.A total of 79 types were identified for CT-I with 64 types,CT-II with 9 types,CT-III with 6 types.Meanwhile,76 clear serotypes and 66 STs were identified.The CRISPRs type could divide the same Serotype(ST)into two subtypes,for example the O157:H7(ST11),O104:H4(ST678)and O26:H11(ST21)were divided into CT I 4 ?CT I 5,CT I 11 ? CT I 12,CT I 15 ? CT I 16.The detection rate of CRISPR1,CRISPR2 CRISPR3,CRISPR4 and CRISPR3-4 were 81.1%,94.5%,1.4%,1.4% and 4.6%;the accuracy was 95.0%,according to the distribution of spacers to forecast O157: H7.4 The typing method based on CRISPRs for STEC strainsWe collected 705 STEC strains including 13 serotypes,and the top three serotypes were O157:H7,O26:H11,O111:H8 and the proportion were 44.0%,16.3%,9.8%,respectively.The time distributions showed that the STEC strain presented a peak in 2010 and 2011.The top two countries of isolates were the United States and Netherlands,and the proportion were 50.9% and 7.2%,respectively.The STEC strains with the same serotype could be divided into several types based on the CRISPRs spacers.Type A of O157:H7,type B of 104:H4 and type A of O111:H8 accounted for 89.0%,87.9%,and 91.3% of their respective strains.Type A and type B of O145:H28 were widely spread in Europe whereas type C,D,E and G isolated from North America.Type H and Z of O26:H11 were distributed in Europe and United States,respectively.However,type B and type C of O26:H11 were both spread around North America and Europe.Type A and B of O69:H11 were separated in the United States whereas the type C isolated from Netherlands.All the strains belonged to the type C of O118:H16 were from the United States.Type A of O45:H2 was found in North America while type B isolated from China.5 The relationship between the CRISPR/Cas and phage,plasmid,the acquired virulence and resistance genesAll the strains contained the phage except K-12 MDS42,and the number of identified phage ranged from 1 to 22,we observed a clear inverse correlation between the I-E CRISPR/Cas spacer and phage(r=-0.450,P<0.001).The data showed that 56.2% strains carried plasmid.The distribution difference of plasmid between the strains with I-E CRISPR/Cas and non-CRISPR/Cas was statistical significance(?2=6.583,P=0.010).53 strains contained the plasmid with resistance genes.The distribution difference of plasmid with resistance genes between the strains with non-CRISPR/Cas and I-E CRISPR/Cas as well as I-F RISPR/Cas was statistical significance(?2=19.756,P<0.001 and P =0.017,respectively).All the chromosome of strains contained the acquired virulence genes except the strain ERS742059,and the number ranged from 2 to 28.We observed a clear inverse correlation between the I-E CRISPR/Cas spacers and the acquired virulence genes(r=-0.623,P<0.001).The data showed that 30.01% chromosome of strains contained the acquired resistance genes,and the number ranged from 1 to 21.The distribution difference of chromosome with resistance genes between the strains with I-E CRISPR/Cas and I-F CRISPR/Cas was statistical significance(?2=9.206,P=0.002).Conclusion1 The CRISPR/Cas was widely distributed in E.coli.The absence of partial or intact I-E cas and IS elements were detected in some strains.2 The upstream of CRISPR1 can achieve a preliminary identification of E.coli and Shigella.3 The method based on CRISPRs spacer can be used as a typing target for E.coli.4 A classification based on the CRISPRs for the same serotype of STEC strains can actually distinguish the highly pathogenic strain and track the source.5 The I-E CRISPR/Cas may inhibit the acquisition of phage and virulence genes whereas the I-F CRISPR/Cas may inhibit the acquisition of drug-resistant genes.The strains without the I-E CRISPR/Cas and I-F CRISPR/Cas may be more likely to gain the plasmids,especially the resistant plasmids.