The mechanism of CRISPR-mediated drug resistance and the relationship between the characteristics of CRISPR and isolation site in Klebsiella pneumoniae
-
摘要:
目的 了解呼吸道标本肺炎克雷伯菌中成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats，CRISPR）对耐药的调控机制，并分析其CRISPR分布特征与分离地点的关系。 方法 收集并提取120株肺炎克雷伯菌的基因组DNA，通过扩增CRISPR/Cas（clustered regularly interspaced short palindromic repeats/CRISPR-associated）系统相关基因CRISPR 1、CRISPR 2来确定CRISPR阳性菌株。CRISPR阳性菌株的耐药表型用BD Phoenix-100细菌鉴定仪进行检测，利用CRISPR Target寻找间隔序列同源噬菌体或质粒，并在Center for Genomic Epidemiology上查找同源质粒或噬菌体的耐药信息并检测间隔序列所在菌株的耐药基因，分析两者携带耐药基因的关系。采用CRISPR Finder分析CRISPR并运用多序列比对分析间隔序列的一致性。 结果 CRISPR1、CRISPR2阳性率分别为12.50%和13.33%；间隔序列同源质粒与其所在菌株均携带共同的耐药基因，且菌株的耐药表型与其携带的耐药基因高度符合；相同地点菌株的CRISPR分布具有极高相似性。 结论 肺炎克雷伯菌通过将外来质粒的耐药基因片段整合到菌株的基因组中实现对菌株耐药性的调控；CRISPR中间隔序列的分布与菌株分离地点密切相关，为临床治疗和感染控制工作提供理论依据。 Abstract:Objective To understand the regulatory mechanism of clustered regularly interspaced short palindromic repeats (CRISPR) to drug resistance of Klebsiella pneumoniae in respiratory specimens, and to analyze the relationship between the characteristics of CRISPR and the location of isolation. Methods 120 strains of Klebsiella pneumoniae were collected and genomic DNA was extracted. CRISPR-positive strains were identified by amplifying CRISPR/CRISPR-associated(Cas) related genes CRISPR 1 and CRISPR 2. The resistance phenotype of CRISPR-positive strains was detected by the BD Phoenix-100 bacterial identification instrument. CRISPR Target was used to look for homologous bacteriophages or plasmids with spacer sequence, find the drug resistance information of homologous bacteriophages or plasmids in Center for Genomic Epidemiology and detect the drug resistance genes of the strain where the spacer sequence is located, and analyze the relationship between the drug resistance genes of the two. CRISPR Finder was used to analyze CRISPR and multi-sequence alignment was used to analyze the consistency of spacer sequences. Results The positive rates of CRISPR 1 and CRISPR 2 were 12.50% and 13.33%; The homologous plasmids of the spacer sequence and the strains in which they were both carried common drug resistance genes, and the resistance phenotype of the strain was highly consistent with the drug resistance genes they carry. At the same location, the CRISPR distribution of the strains was extremely similar. Conclusions Klebsiella pneumoniae regulates drug resistance by integrating foreign plasmid resistance gene fragments into the strain's genome. The distribution of spacer sequence in CRISPR is closely related to the location of the strain isolation, which provides a theoretical basis for clinical treatment and infection control work. -
Key words:
- Klebsiella pneumoniae /
- CRISPR /
- Isolation location /
- Drug resistance
-
表 1 耐药基因引物信息
Table 1. Primers of drug-resistant genes
耐药基因 引物序列 长度
(bp)退火
温度(℃)aac(6')-Ib ATGACTGAGCATGACCTTGC
TTAGGCATCACTGCGTGTTC519 53 aac(6')-Ib-cr ATGAGCAACGCAAAAACAAAGTTAGGC
GTGAAACCCAACATACCCCTGA600 53 blaOXA ATGAAAAACACAATACATATCAACTTCG
TTATAAATTTAGTGTGTTTAGAATGGTGATCG831 46 blaSHV TCTCCCTGTTAGCCACCCTG
CCACTGCAGCAGCTGC529 56 catB3 ATGACCAACTACTTTGATAGCCCCT
TTAGACGGCAAACTCGAGCCA633 53 aadA2 ATGAGGGAAGCGGTGA
TCATTTACCAACTGACTTGATGATCTC792 50 sul1 CTAGGCATGATCTAACCCTCGGT
CTAGGCATGATCTAACCCTCGGT927 53 dfrA12 ATGAACTCGGAATCAGTACGCATTTAT
TTAGCCGTTTCGACGCGC498 50 aac(3')-IIa ATGCATACGCGGAAGGCAAT
CTAACCGGAAGGCTCGCAAG861 53 qnrB1 ATGACGCCATTACTGTATAAAAAAACA
CTAACCAATCACCGCGATG681 47 tetA GTGAAACCCAACATACCCCTGA
TCAGCGATCGGCTCGTTG750 53 catA1 ATGGAGAAAAAAATCACTGGATATACCACCGTTGAT
TTACGCCCCGCCCTGCC630 55 mph(A) ATGACCGTAGTCACGACCGC
CTATATCGACGTTCGCTCATTCCG921 52 
下载: 导出CSV
表 2 CRISPR1、CRISPR2在120株肺炎克雷伯菌中的检出率和间隔序列数目
Table 2. Detection rate and number of spacer sequences of CRISPR1 and CRISPR2 in 120 strains of Klebsiella pneumoniae
CRISPR 间隔序列数目 菌株数 阳性率(%) CRISPR 1 7 1 12.50 9 13 12 1 CRISPR 2 6 1 13.33 11 13 14 2
下载: 导出CSV
表 3 间隔序列同源质粒携带耐药基因情况
Table 3. Spacer sequence homologous plasmids carrying resistant genes
间隔序列编号 所在菌株 间隔序列 同源质粒登记号 同源质粒上的耐药基因 C1/(A1-A12)-CR2-24 C1 CCGCCGTTTAATCGCGGTGATGATATCCGGCA plasmid pUCLAOXA232-6(NZ_CP012567) aac(6')-Ib A1-A12 aac(6')-Ib-cr blaOXA, blaSHV catB3 B1-CR1-11 B1 AGTTAAAGCGCCACCAGCTAAGCCTGTGCCGGT plasmid unnamed1(NZ_CP027613) aadA2，bla-SHV sul1，dfrA12 B1-CR1-14 B1 TGAAGCCCAGCGGAATGGCCGGGAAAAATTTAT plasmid pCN1_1(NZ_CP015383) aac(6')-Ib aac(6')-Ib-cr aac(3')-IIa qnrB1, catB3 blaOXA, dfrA12 tetA B2-CR1-25 B2 TGAAGCGTAGAAAAGCAGGCAGCTTTTACCCTGG plasmid pKPN-498(NZ_CP008829) aac(3')-IIa，aadA2, catA1, sul1, dfrA12, mph(A) 注：C1/(A1-A12)-CR2-24表示菌株C1或菌株A1-A12的CRISPR2位点中第24个间隔序列；B1-CR1-11表示菌株B1的CRISPR1位点中第11个间隔序列；B1-CR1-14表示菌株B1的CRISPR1位点中第14个间隔序列；B2-CR1-25表示菌株B2的CRISPR1位点中第25个间隔序列。
下载: 导出CSV
表 4 间隔序列所在肺炎克雷伯菌携带耐药基因情况
Table 4. Drug-resistant genes carried by Klebsiella pneumoniae which spacer sequence located
间隔序列编号所在菌株 耐药基因 C1/(A1-A12)-CR2-24 C1 bla-SHV, aac(6')-Ib, aac(6')-Ib-cr, catB3 A1 bla-SHV, catB3 A2 bla-SHV, catB3 A3 bla-SHV, catB3 A4 bla-SHV, catB3 A5 bla-SHV, catB3 A6 bla-SHV, catB3 A7 bla-SHV, catB3 A8 bla-SHV, catB3 A9 bla-SHV, catB3 A10 bla-SHV, catB3 A11 bla-SHV, catB3 A12 B1-CR1-11 B1 bla-SHV, aac(3')-IIa, tetA B1-CR1-14 B1 bla-SHV, aac(3')-IIa, tetA B2-CR1-25 B2 aac(3')-IIa, catA1, mph(A)
下载: 导出CSV
表 5 16株CRISPR阳性菌株耐药表型
Table 5. Drug resistance phenotypes of 16 CRISPR positive strains
菌株
编号氨苄
西林氨曲南 环丙
沙星左氧氟
沙星哌拉
西林头孢
噻肟头孢
唑林头孢
吡肟头孢
他啶亚胺
培南美罗
培南庆大
霉素阿莫西
林/克
拉维酸氨苄西
林/
舒巴坦哌拉西
林/他
唑巴坦阿米
卡星氯霉素 复方
磺胺四环素 C2 1 1 1 1 1 1 1 1 2 0 0 0 0 0 0 0 1 1 1 C1 1 1 0 0 1 1 1 1 0 0 0 0 2 1 0 0 1 1 1 B1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 B2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 A1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 A2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 A3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 A4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 A5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 A6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 A7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 A8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 A9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 A10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 A11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 A12 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 0 0 注：C1/(A1-A12)-CR2-24表示菌株C1或菌株A1-A12的CRISPR2位点中第24个间隔序列；B1-CR1-11表示菌株B1的CRISPR1位点中第11个间隔序列；B1-CR1-14表示菌株B1的CRISPR1位点中第14个间隔序列；B2-CR1-25表示菌株B2的CRISPR1位点中第25个间隔序列。
下载: 导出CSV
表 6 间隔序列一致性分析结果
Table 6. Results of spacer sequence consistency analysis
菌株编号 CR1 CR2 CRISPR 1 CRISPR2 C2 9 14 1-9 1-8 1-7 1-6 1-5 1-4 1-3 1-2 1-1 2-14 2-132-122-11 2-10 2-9 2-8 2-7 2-6 2-5 2-4 2-3 2-2 2-1 C1 0 11 2- -25 2-24 2-23 2-22 2-21 2-20 2-19 2-18 2-17 2-16 2-15 B1 7 14 1-16 1-15 1-14 1-13 1-12 1-11 1-10 2 -39 2-38 2-37 2-36 2-35 2- 34 2-33 2-32 2-31 2-302-292-282-27 2-26 B2 12 6 1-9 1-8 1-26 1-25 1-24 1-23 1-22 1-21 1-20 1-19 1-18 1-17 2-45 2-44 2-43 2-42 2-41 2-40 Al 9 11 1-9 1-8 1-7 1-6 1-30 1-29 1-5 1-28 1-27 2-25 2-24 2-23 2-22 2-21 2-20 2-19 2-18 2-17 2-16 2-15 A2 9 11 1-9 1-8 1-7 1-6 1-30 1-29 1-5 1-28 1-27 2-25 2-24 2-23 2-22 2-21 2-20 2-19 2-18 2-17 2-16 2-15 A3 9 11 1-9 1-8 1-7 1-6 1-30 1-29 1-5 1-28 1-27 2-25 2-24 2-23 2-22 2-21 2-20 2-19 2-18 2-17 2-16 2-15 A4 9 11 1-9 1-8 1-7 1-6 1-30 1-29 1-5 1-28 1-27 2-25 2-24 2-23 2-22 2-21 2-20 2-19 2-18 2-17 2-16 2-15 A5 9 11 1-9 1-8 1-7 1-6 1-30 1-29 1-5 1-28 1-27 2-25 2-24 2-23 2-22 2-21 2-20 2-19 2-18 2-17 2-16 2-15 A6 9 11 1-9 1-8 1-7 1-6 1-30 1-29 1-5 1-28 v 2- -25 2-24 2-23 2-22 2-21 2-20 2-19 2-18 2-17 2-16 2-15 A7 9 11 1-9 1-8 1-7 1-6 1-30 1-29 1-5 1-28v 2-25 2-24 2-23 2-22 2-21 2-20 2-19 2-18 2-17 2-16 2-15 A8 9 11 1-9 1-8 1-7 1-6 1-30 1-29 1-5 1-28 1-27 2-25 2-24 2-23 2-22 2-21 2-20 2-19 2-18 2-17 2-16 2-15 A9 9 11 1-9 1-8 1-7 1-6 1-30 1-29 1-5 1-28 1-27 2-25 2-24 2-23 2-22 2-21 2-20 2-19 2-18 2-17 2-16 2-15 A10 9 11 1-9 1-8 1-7 1-6 1-30 1-29 1-5 1-28 1-27 2-25 2-24 2-23 2-22 2-21 2-20 2-19 2-18 2-17 2-16 2-15 A11 9 11 1-9 1-8 1-7 1-6 1-30 1-29 1-5 1-28 1-27 2-25 2-24 2-23 2-22 2-21 2-20 2-19 2-18 2-17 2-16 2-15 A12 9 11 1-9 1-8 1-7 1-6 1-30 1-29 1-5 1-28 1-27 2-25 2-24 2-23 2-22 2-21 2-20 2-19 2-18 2-17 2-16 2-15 注：间隔序列用相应的编号代替：CRISPR 1中共30个完全不同的间隔序列，分别标记为1-1至1-30；CRISPR 2中共25个完全不同的间隔序列，分别标记为2-1至2-25。相同标记的均为完全相同的间隔序列。
下载: 导出CSV
-
[1] Ostria-Hernández ML, Sánchez-Vallejo CJ, Ibarra JA, et al. Survey of clustered regularly interspaced short palindromic repeats and their associated Cas proteins(CRISPR/Cas) systems in multiple sequenced strains of Klebsiella pneumoniae [J]. BMC Res Notes, 2015, 8:332. DOI: 10.1186/s13104-015-1285-7. [2] 徐鑫鑫, 陈立凌, 田健美, 等. 2011-2018年苏州市住院儿童肺炎的常见病原分布及流行特征[J].中华疾病控制杂志, 2020, 24(3):264-268. DOI: 10.16462/j.cnki.zhjbkz.2020.03.004.Xu XX, Chen LL, Tian JM, et al. The distribution and flow of common causes of hospitalized children' pneumonia in Suzhou from 2011 to 2018 [J]. Chin J Dis Control Prev, 2020, 24(3):264-268. DOI: 10.16462/j.cnki.zhjbkz.2020.03.004. [3] 穆玉姣, 王若琳, 张白帆, 等. 2013-2017年河南省婴幼儿志贺菌流行特征与耐药分析[J].中华疾病控制杂志, 2019, 23(7):835-839. DOI: 10.16462/j.cnki.zhjbkz.2019.07.018.Mu YJ, Wang RL, Zhang BF, et al. Epidemiological characteristics and drug resistance surveillance of Shigella in infants and young children in Henan Province from 2013 to 2017 [J]. Chin J Dis Control Prev, 2019, 23(7):835-839. DOI: 10.16462/j.cnki.zhjbkz.2019.07.018. [4] 胡付品, 郭燕, 朱德妹, 等. 2016年中国CHINET细菌耐药性监测[J].中国感染与化疗杂志, 2017, 17(5):481-491. DOI: 10.16718/j.1009-7708.2017.05.001.Hu FP, Guo Y, Zhu DM, et al. CHINET surveillance of bacterial resistance across China: report of the results in 2016 [J]. Chin J Infect Chemother, 2017, 17(5):481-491. DOI: 10.16718/j.1009-7708.2017.05.001. [5] Makarova KS, Haft DT, Barrangou R, et al. Evolution and classification of the CRISPR-Cas system [J]. Nat Rev Microbiol, 2011, 9(6):467-477. DOI: 10.1038/nrmicro2577. [6] 张冰.志贺菌中CRISPR/Cas系统与其耐药关系的探讨[D].郑州: 郑州大学, 2016.Zhang B. Study on the relationship between CRISPR/Cas system and drug resistance in Shigella [D]. Zhengzhou: Zhengzhou University, 2016. [7] Kunin V, Sorek R, Hugenholtz P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats [J]. Genome Biol, 2007, 8(4):R61. DOI: 10.1186/gb-2007-8-4-r61. [8] Shah SA, Hansen NR, Garrett RA. Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism [J]. Biochem Soc Trans, 2009, 37(Pt1):23-28. DOI: 10.1042/BST0370023. [9] Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea [J]. Science, 2010, 327(5962):167-170. DOI: 10.1126/science.1179555. [10] Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea [J]. Nature, 2012, 482(7385):331-338. DOI: 10.1038/nature10886. [11] Lin TL, Pan YJ, Hsieh PF, et al. Imipenem represses CRISPR-Cas interference of DNA acquisition through H-NS Stimulation in Klebsiella pneumoniae [J]. Sci Rep, 2016, 6:31644. DOI: 10.1038/srep31644. [12] Wang PF, Zhang B, Duan GC, et al. Bioinformatics analyses of Shigella CRISPR structure and spacer classification [J]. World J Microbiol Bioterchnol, 2016, 32(3):38. DOI: 10.1007/s11274-015-2002-3. [13] Davision J. Genetic exchange between bacteria in the environment [J]. Plasmid, 1999, 42(2):73-91. DOI: 10.1006/plas.1999.1421. [14] Dutta C, Pan A. Horizontal gene transfer and bacterial diversity [J]. J Biosci, 2002, 27(1 Suppl 1):27-33. DOI: 10.1007/bf02703681. [15] Garneau JE, Dupuis Mè, Villion M, et al. The CRISPR/Cas bacterial immune sysytem cleaves bacteriophage and plasmid DNA [J]. Nature, 2010, 468(7320):67-71. DOI: 10.1038/nature09523. [16] Jansen R, Embden JD, Gaastra W, et al. Identification of genes that are associated with DNA repeats in prokaryotes [J]. Mol Microbiol, 2002, 43(6):1565-1575. DOI: 10.1046/j.1365-2958.2002.02839.x. [17] Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA [J]. Science, 2008, 322(5909):1843-1845. DOI: 10.1126/science.1165771. [18] Shen J, Lv L, Wang X, et al. Comparative analysis of CRISPR-Cas systems in Klebsiella genomes [J]. J Basic Microbiol, 2017, 57(4):325-336. DOI: 10.1002/jobm.201600589. [19] 张冰, 王鹏飞, 段广才, 等. CRISPR/Cas系统间隔序列同源质粒耐药信息与志贺菌耐药的关系[J].中国病原生物学杂志, 2016, 11(10):881-887. DOI:10.13350/j.cjpb.161004.Zhang B, Wang PF, Duan GC, et al. Information on the drug resistance of plasmids that are homologous to spacers in a CRISPR/Cas system and its relationship to information on the drug resistance of Shigella [J]. Journal of Parasitic Biology. 2016, 11(10):881-887. DOI: 10.13350/j.cjpb.161004.j.cjpb.161004. [20] 王鹏飞, 王颖芳, 段广才, 等.志贺菌成簇的规律间隔的短回文重复序列系统结构特征的生物信息学分析[J].生物医学工程杂志, 2015, 32(2):343-349. DOI: 10.7507/1001-5515.20150063.Wang PF, Wang YF, Duan GC, et al. Bioinformatics analysis of clustered regularly interspaced short palindromic repeats in the genomes of Shigella [J]. Journal of Biomedical Engineering, 2015, 32(2):343-349. DOI: 10.7507/1001-5515.20150063. [21] Grissa I, Vergnaud G, Pourcel C. The CRISPR db database and tools to display CRISPRs and to generate dictionaries of spacers and repeats [J]. BMC Bioinformatics, 2007, 8:172. DOI: 10.1186/1471-2105-8-172. [22] Deveau H, Barrangou R, Garneau JE, et al. Phage response to CRISPR-encoded resisitance in Streptococcus thermophilus [J]. J Bacteriol, 2008, 190(4):1390-1400. DOI: 10.1128/JB.01412-07. [23] 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. DOI: 10.1016/j.chom.2012.06.003. -
