WANG Lei,ZHAO Xia,WANG Wenjing,et al.Detection of CRISPR and cas expression analysis in induced drug resistance of Salmonella[J].Journal of Nanjing Agricultural University,2018,41(2):370-376.[doi:10.7685/jnau.201701021]





Detection of CRISPR and cas expression analysis in induced drug resistance of Salmonella
王磊 赵霞 王文静 张瑞良 代兴杨 曾明华 李琳
安徽农业大学动物科技学院, 安徽 合肥 230036
WANG Lei ZHAO Xia WANG Wenjing ZHANG Ruiliang DAI Xingyang ZENG Minghua LI Lin
College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
沙门菌诱导耐药成簇间隔短回文重复序列cas mRNA表达量
Salmonellainduced drug-resistanceclustered regularly interspaced short palindromic repeats sequenceexpression of cas mRNA
[目的]通过对诱导耐药沙门菌成簇间隔的短回文重复序列(CRISPR)比对分析,以及cas(CRISPR associated)mRNA表达水平的变化,探讨CRISPR/Cas与沙门菌耐药性的关系。[方法]对鼠伤寒沙门菌ATCC13311进行耐药诱导分别获得3株体外诱导耐药(环丙沙星、庆大霉素、氨苄西林)菌和1株体内诱导耐药(环丙沙星)菌,设计引物对CRISPR,进行PCR扩增并检测,应用CRISPR Finder分析CRISPR序列,对5株沙门菌的CRISPR序列进行比对;利用RT-qPCR检测耐药前后沙门菌cas1、cas6、casA mRNA表达水平的变化。[结果]成功扩增出2段CRISPR序列(CRISPR1、CRISPR2),耐药菌碱基的突变均发生在前导区和序列末端;CRISPR1序列的突变发生在前导区的1~4 bp和序列末端的1 136~1 144 bp区域内;CRISPR2序列的突变发生在前导区的1~14 bp和序列末端的780~796 bp区域内;重复间隔序列保守且发现有重复序列的退化现象(degeneration repeats,DRs),同时耐药菌cas1、cas6、casA mRNA的表达水平下降。[结论]提示:沙门菌CRISPR序列突变和cas mRNA表达水平下降与其耐药性有关。
[Objectives]By comparing and analyzing the clustered regularly interspaced short palindromic repeats sequence(CRISPR),and detecting the expression of mRNA in drug resistant Salmonella,the relationship between CRISPR/Cas and drug resistance was explored. [Methods]Salmonella typhimurium ATCC13311 strains were induced by in vitro methods to obtain ciprofloxacin-resistant strain,gentamicin-resistant strain,and ampicillin-resistant strain,and ciprofloxacin-resistant strain was induced in vivo. The CRISPR were obtained by PCR amplification method,and the CRISPR sequences were analyzed by CRISPR Finder. The CRISPR sequences of five Salmonella strains were compared and the mRNA expression of cas1,cas6 and casA of them were detected before and after drug resistance by RT-qPCR. [Results]Two CRISPR sequences(CRISPR1 and CRISPR2) were amplified successfully and the base mutation mainly happened at the leader and sequence ends. The mutation of the CRISPR1 sequence occurred in the area of 1-4 bp and 1 136-1 144 bp. The mutation of the CRISPR2 sequence occurred in the area of 1-14 bp and 780-796 bp. Repeat and spacer sequences were relatively conservative,the degeneration of repeats was found. Simultaneously,the expression level of cas1,cas6 and casA mRNA decreased. [Conclusions]The results suggested that the mutation of CRISPR sequences and the decrease of cas mRNA expression are related to drug resistance in Salmonella.


[1] 侯雪娇,吴科敏,莫国东,等. 食源性沙门氏菌耐药表型与耐药基因的研究[J]. 食品科学,2016,37(19):166-170. Hou X J,Wu K M,Mo G D,et al. Evaluation of drug-resistant phenotypes and genes in foodborne Salmonella isolates[J]. Food Science,2016,37(19):166-170(in Chinese with English abstract).
[2] Lillesto P K,Redder P,Barrett A,et al. A putative viral defence mechanism in archaeal cells[J]. Archaea,2006,2(1):59-72.
[3] Mojica F J,Diez-Villaseonr C,Soria E,et al. Biological significance of a family of regularly spaced repeats in the genomes of Archaea,Bacteria and mitochondria[J]. Molecular Microbiology,2000,36(1):244-246.
[4] Touchon M,Charpentier S,Pognard D,et al. Antibiotic resistance plasmids spread among natural isolates of Escherichia coli in spite of CRISPR elements[J]. Microbiology,2012,158(12):2997-3004.
[5] Burley K M,Sedgley C M. CRISPR Cas,a prokaryotic adaptive immune system,in endodontic,oral and multidrug-resistant hospital-acquired Enterococcus faecalis[J]. Journal of Endodontics,2012,38(11):1511-1515.
[6] Horvath P,Barrangou R. CRISPR/Cas,the immune system of bacteria and archaea[J]. Science,2010,327(5926):167-170.
[7] Wiedenheft B,Sternberg S H,Doudna J A. RNA-guided genetic silencing systems in bacteria and archaea[J]. Nature,2012,482(7385):331-338.
[8] 孙磊,裘鹏,刘晓琳,等. 细菌耐药性产生对毒力的影响研究进展[J]. 畜牧与兽医,2016,48(11):120-123. Sun L,Qiu P,Liu X L,et al. Research progress onbacterial resistance influencing virulence[J]. Animal Husbandry and Veterinary Medicine,2016,48(11):120-123(in Chinese with English abstract).
[9] 李东,左其生,张亚妮,等. 基因组编辑技术的研究进展[J]. 畜牧与兽医,2015,47(7):124-128. Li D,Zuo Q S,Zhang Y N,et al. The research progress of genome editing techniques[J]. Animal Husbandry and Veterinary Medicine,2015,47(7):124-128(in Chinese with English abstract).
[10] Cain A K,Boinett C J. Genmone WATCH A CRISPR view of genome sequences[J]. Nature Reviews Microbiology,2013,11(4):226.
[11] Horvath P,Romero D A,Coûté-Monvoisin A C,et al. Diversity,activity,and evolution of CRISPR loci in Streptococcus thermophiles[J]. Journal of Bacteriology,2008,190(4):1401-1412.
[12] Comas I,Homolka S,Niemann S,et al. Genotyping of genetically monomorphic bacteria:DNA sequencing in Mycobacterium tuberculosis highlights the limitations of current methodologies[J]. PLoS ONE,2009,4(11):e7815.
[13] Sun C L,Tomas B C,Barrangou R,et al. Metagenomic reconstructions of bacterial CRISPR loci constrain population histories[J]. The ISME Journal,2016,10(4):858-870.
[14] Makarova K S,Haft D H,Barrangou R,et al. Evolution and classification of the CRISPR cas systems[J]. Nat Rev Microbiol,2011,9(6):467-477.
[15] Wright A V,Nunez J K,Doudna J A. Biology and applications of CRISPR systems:harnessing nature’s toolbox for genome engineering[J]. Cell,2016,164(1):29-44.
[16] Dixit B,Ghosh K K,Fernandes G,et al. Dual nuclease activity of a Cas2 protein in CRISPR Cas subtype I-B of Leptospira interrogans[J]. FEBS Letters,2016,509(7):1002-1016.
[17] 冯帅,汪莹,李琳,等. 沙门菌体内耐药性诱导模型的建立和体外诱导耐药基因差异分析[J]. 中国兽医学报,2014,34(12):1926-1930. Feng S,Wang Y,Li L,et al. The establishment of the drug-resistance inducing model in vivo of Salmonella enterica typhimurium in the Caenorhabditis elegans and dissimilarity analysis of drug-resistance inducing S.typhimurium in vivo and in vitro[J]. Chinese Journal of Veterinary Science,2014,34(12):1926-1930(in Chinese with English abstract).
[18] 贾坤,廖燕红,徐磊,等. 酰基-高丝氨酸内酯(AHL)对鼠伤寒沙门氏菌生物菌膜形成的影响[J]. 南京农业大学学报,2017,40(4):744-749. DOI:10.7685/jnau.201611023. Jia K,Liao Y H,Xu L,et al. Effect of acylated homoserine lactones(AHL)on the biofilm formation of Salmonella typhimurium[J]. Journal of Nanjing Agricultural University,2017,40(4):744-749(in Chinese with English abstract).
[19] 王琳琳,王颖芳,段广才,等. 志贺菌CRISPR的检测及其与耐药的关系[J]. 微生物学报,2015,55(4):476-483. Wang L L,Wang Y F,Duan G C,et al. Detection of CRISPR and its relationship to drug resistance in Shigella[J]. Atca Microbiologica Sinica,2015,55(4):476-483(in Chinese with English abstract).
[20] Kunin V,Sorek R,Hugenholtz P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats[J]. Genome Biology,2007,8(4):61.
[21] Grissa I,Vergnaud G,Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats[J]. BMC Bioinformatics,2007,8(1):172.
[22] Deveau H,Garneau J E,Moineau S. CRISPR/Cas system and its role in phage-bacteria interactions[J]. Annual Review of Microbiology,2010,64:475-493.
[23] Sampson T R,Napier B A,Schroeder M R,et al. A CRISPR cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion[J]. Proc Natl Acad Sci USA,2014,11l(30):11163-11168.
[24] 宋春花,黄学勇,郗园林,等. 志贺菌敏感株与基因转移多耐药株蛋白组学分析[J]. 中国公共卫生学,2008,24(1):42-45. Song C H,Huang X Y,Xi Y L,et al. Analysis on proteomics between sensitive strain and gene transferred multi-drug resistant strain of Shigella flexneri[J]. Chin J Public Health,2008,24(1):42-45(in Chinese with English abstract).
[25] Palmer K L,Gilmore M S. Multidrug-resistant enterococci lack CRISPR-cas[J]. mBio,2010,1(4):e00227-10.
[26] Bikard D,Hatoum-Aslan A,Mucida D,et al. CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection[J]. Cell Host & Microbe,2012,12(2):177-186.


更新日期/Last Update: 1900-01-01