| Objective Acinetobacter is a large class of Gram-negative bacteria that are non-spore and incapable of movement.In recent years,the proportion of multidrug-resistant(MDR)strains has increased rapidly,which has attracted more and more public attention.Clustered regularly interspaced short palindromic repeats(CRISPR)are polymorphic loci widely found in many of the bacterial and archaeal genomes that provide adaptive immunity against invasive DNA elements.On the one hand,bacteria evolved mechanisms such as the CRISPR system for the stability of species to resist the interference of foreign genes.On the other hand,under the pressure of survival,new genes need to be obtained from the outside world to adapt to the new environment.Understanding how bacteria coordinate the conflicts requires a more in-depth study of their CRISPR systems.Methods CRISPR system of Acinetobacter was analyzed from two aspects,including the bioinformatics analysis of the CRISPR system in Acinetobacter spp.genome published in NCBI database and the study of CRISPR array and cas1 gene of the clinical isolates.CRISPR array were retrieved from the CRISPR database,genome sequences downloaded from Nucleotide at the NCBI.The alignment of the repeats was done by Meg Align.Then we used CRISPRmap software to analyze the resulted repeats and predicted RNA secondary structure and MFE.Sequence families exhibit varying patterns of repeat sequence conservation.All the spacers were searched by the BLAST platform in Gen Bank database to find homology sequences(identity≥85%,coverage≥85%).We selected 100 bp non-coding sequences located at the 5′ end of the first repeat as the putative leader sequences and aligned using Clustal W software.The conservation of leader sequences was represented by Web Logo.The presence of the promoter was predicted by promoter 2.0 Prediction Server.All cas loci were detected by the gene annotation of the Acinetobacter complete genome sequences downloaded by NCBI,and the extracted cas gene sequences were input into MEGA7.0 for Multiple sequence alignments and phylogenetic analyses.The CRISPR-Cas system structure diagram was drawn based on the information related to the CRISPR array and the cas gene locus.The complete genome sequence of 185 Acinetobacter strains was submitted to MLST database to search for the allele numbers and sequence types corresponding to different housekeeping genes.A total of 141 Acinetobacter strains with complete genome sequence and gene annotation published by Genomes database were used to find related drug resistance genes.105 clinical isolates of Acinetobacter spp.and their drug resistance phenotypes information were collected from the laboratory of three hospitals in Qingdao.The cas1 gene was amplified by PCR and divided into two subtypes,I-Fa and I-Fb.Statistical analysis of the relationship between the CRISPR system and drug-resistant genes or drug-resistant phenotypes was performed using SPSS 13.0 software.Results 1.In this study,397 identified CRISPR arrays were selected from 185 Acinetobacter strains to investigate their genomic structural characteristics and potential functions using bioinformatics tools.72 CRISPR repeats were identified and are typically28 nt in length,the repeat sequences referred to herein can form typical conserved RNA secondary structures.Additionally,only 9.7 % of spacers are homologous with known phage or plasmid nucleotide sequences.Interestingly,the 38.4% spacers matche the chromosome regions of other bacteria,while none of them are located in the CRISPR array regions of these bacteria.Interestingly,about 16.7% spacers matche to the sequences of the eukaryotic genome,most of which encode hypothetical proteins.We analyzed locus structure,gene organization and sequence of cas genes,most CRISPR-Cas systems in Acinetobacter belongs to type I-F(I-Fa 39% and I-Fb59%),the remaining strains are untyped or type III-A.We found a degree of evolutionary coherence among the Phylogenetic trees based on cas1,cas3,csy2,csy3 and csy4 nucleotide sequences,the strains located in corresponding clusters of these trees are mostly similar.We found that the cys1 gene is only found in strains with the I-Fb subtype.Additionally,many Acinetobacter strains with same CRISPR subtype shared the same MLST type.The analysis results of the drug resistance genes in the 141 Acinetobacter genomes are shown that the detection rates of some resistance genes in the absence of CRISPR system are significantly higher than those in the presence of CRISPR system(includes questionable structures)group.2.Analysis of the source of 105 Acinetobacter specimens revealed that it was mainly derived from the lower respiratory tract.The positive rate of PCR amplification of cas1 gene was 23.8%,which is basically consistent with the positive rate of cas1 in the Acinetobacter genome in the first part of the study(17.5%,20.6%).The presence of cas1 may affect the resistance of Acinetobacter to Cefoperazon+Sulb,Cefepim,Imipenem and Ciprofloxacin.Conclusions The results of this study showed that: 1.In the CRISPRdb database,the carrying rate of the CRISPR array in Acinetobacter genome was 21.3%,and the carrying rate of the cas gene was 17.5%,which was lower than that in most clinically common bacteria.The repeat of CRISPR system in the Acinetobacter is highly conserved within the species and can form a more stable RNA secondary structure.Partially spaced sequences are highly homologous to phage and plasmids.The CRISPR system in the Acinetobacter genome is almost I-F type and can be divided into two subtypes,I-Fa and I-Fb.It was found for the first time that the presence of the csy1 gene can be used for genotyping of the CRISPR array in Acinetobacter genome,in addition,gene sequences such as cas3,csy2,csy3 and csy4 can be used independently for genotyping.2.The results of drug resistance rate of clinical strains indicate that the resistance of Acinetobacter is very serious.The existence of CRISPR array or cas gene is closely related to certain resistance genes or drug resistance phenotypes of Acinetobacter. |
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