满江红内生细菌的培养及其抗生素抗性基因的检测

陈坚; 郑益平; 陈彬; 郑斯平; 郑伟文

doi:10.19303/j.issn.1008-0384.2021.06.013

满江红内生细菌的培养及其抗生素抗性基因的检测

doi: 10.19303/j.issn.1008-0384.2021.06.013

福建省农业科学院生物技术研究所，福建　福州　350003

基金项目: 福建省农业科学院项目（A2017-2）；福建省科技计划引导性项目（2017N1001）

详细信息

作者简介:
陈坚（1965−）男，博士，研究员，研究方向：植物与微生物生化与分子生物学（E-mail：chenjian3@hotmail.com）

通讯作者:
郑伟文（1942−），男，研究员，研究方向：满江红与蓝细菌共生的细胞与分子生物学研究（E-mail：bcfaas01@hotmail.com）

中图分类号: S 184；S 188
计量
- 文章访问数: 713
- HTML全文浏览量: 185
- PDF下载量: 32
- 被引次数: 0
出版历程
- 收稿日期: 2021-03-09
- 修回日期: 2021-04-27
- 网络出版日期: 2021-06-06
- 刊出日期: 2021-06-28

Culture and Antibiotic Resistance Genes of Endophytic Bacteria in Azolla

Biotechnology Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350003, China

摘要

摘要: 目的水产养殖中广泛使用抗生素会导致抗生素抗性基因在水环境中的传播。水生植物满江红含有丰富的内生菌，建立满江红内生细菌群落及所含有的抗生素抗性基因的检测方法，可以为水环境中抗性基因的监测提供参考。方法用3种不同的培养基对小叶满江红的内生细菌进行培养并对培养物进行宏基因组测序及生物信息学分析。结果共得到高质量的序列数据量48.66 G，预测出54 180个基因（ORFs）。通过分类学信息数据库NR共注释出1712种微生物物种，在总门水平上属38个门，其中3种培养物中变形菌门（Proteobacteria）的物种丰度均占全部门类的99.14%以上。在总属水平，草螺菌属（Herbaspirillum）的丰度为91%左右，伯克霍尔德菌属（Burkholderia）可达1.2%左右。比对抗生素抗性基因数据库ARDB，3个样品共注释到10个与抗生素有关基因ARG，主要针对杆菌肽（bacitracin）和氯霉素（chloramphenicol）产生抗性。与综合抗生素抗性基因数据库 CARD进行比对，获得212抗生素相关基因ARO。按照抗性机制分类，其中对抗生素起直接作用的抗性类基因AR的丰度占88%以上；对抗生素有敏感作用的抗性基因AS的丰度均在7%左右；抗生素靶标类抗性基因AT占4%～6%。结论通过3种不同的培养基获得的满江红内生细菌的群落结构，抗生素抗性基因类型基本一致。
- 满江红 /
- 内生菌 /
- 抗生素抗性基因 /
- 宏基因组
Abstract: Objective Culture methods for and detection of the antibiotic resistance genes (ARGs) in the endophytic bacteria of Azolla were established to monitor and find means to curtail the drug pollution in aquatic environment. Method Three selected media were used to culture the endophytic bacteria in Azolla microphylla for metagenome sequencing and bioinformatical analysis on ARGs. Result A total of 48.66G clean data was obtained to yield 54 180 predicted ORFs. Through the taxonomic information database NR, 1 712 microbial species were annotated belonging to 38 phyla. On species abundance of the bacteria from the 3 cultures, Proteobacteria accounted for more than 99.14% of all at the phyla level, while at genus level, Herbaspirillum approximately 91% and Burkholderia 1.2%. Blasted with the ARDB database, 3 samples were annotated with 10 genes associated with the resistance to bacitracin and chloramphenicol. Compared to CARD, there were 212 antibiotic related genes (AROs) found in the samples. The genes resistant to antibiotics (AR) accounted for more than 88% of total AROs, while those sensitive to antibiotics (AS) approximately 7% and those resistant to target antibiotics (AT) 4-6%. Conclusion The community structure and ARG types of the endophytic bacteria in Azolla cultured in the 3 different media were basically same.
- Azolla /
- endophytic bacteria /
- antibiotic resistance genes /
- metagenome

HTML全文

表 1 满江红内生菌的培养基

Table 1. Culture media for endophytic bacteria of Azolla

样本编号 Sample	培养基 Medium	培养基配方 Formulation/（mg·L⁻¹）
N	Nickell	蔗糖 20000; KNO₃ 202; KCl 150;KH₂PO₄ 136; Cu（NO₃）₂·4H₂O 708; CaCl₂ 167;MgSO₄·7H₂O 246; MgCl₂ 96; H₃BO₃ 0.1; MnSO₄·4H₂O 0.1; ZnSO₄·4H₂O 0.3; CuSO₄·5H₂O 0.1; Na₂MoO₄·2H₂O 0.1; EDTA-Na₂ 370; FeSO₄·7H₂O 280；pH 6.5。
R	R2A	酵母膏 500；蛋白胨 500；酪蛋白水解物 500；葡萄糖 500；可溶性淀粉 500；丙酮酸钠 300；K₂HPO₄300；MgSO₄ · 7H₂O 50。pH 6.5。
T	TRN	胰蛋白胨 10000；NaCl 5000；pH 7.0。

下载: 导出CSV

表 2 宏基因组测序数据的统计

Table 2. Summary of metagenomic sequencing data

样本 Sample	原始读长数 Raw reads	原始碱基数 Raw bases	质控读长数 Clean reads	质控碱基数 Clean bases	质控覆盖度 Coverage of clean data/%
N	110062874	16619493974	108759958	16410855235	98.74
T	114872990	17345821490	113816354	17176889285	99.03
R	101353338	15304354038	99910034	15078111760	98.52

下载: 导出CSV

表 3 拼接片段与预测的功能基因数据的统计

Table 3. Summary of contigs and predicted genes

样本 Sample	拼接片段数 Contigs	拼接片段碱基数 Contigs’ bases/bp	预测基因 ORFs	预测基因总长度 Total length of ORFs/bp	预测基因平均长度 Average length of ORFs/bp
N	13482	19400680	27163	16927256	623.17
T	1711	15510096	15204	13741823	903.83
R	3762	10455606	11813	9163870	775.74

下载: 导出CSV

表 4 不同样本的微生物在门与属分类水平上的物种丰度

Table 4. Microbial community abundance of cultured samples at phylum and genus levels　　　　　　（单位：%）

分类水平 Taxonomic level	物种名称 Specific name	R	T	N
门 Phylum	变形菌门 Proteobacteria	99.36	99.33	99.14
门 Phylum	其余37门	0.64	0.67	0.86
属 Genus	草螺菌属 Herbaspirillum	92.15	91.11	91.25
	伯克霍尔德菌属 Burkholderia	1.23	1.19	1.24
	其余710属 The other 710 genera	6.62	7.70	7.51

下载: 导出CSV

表 5 样本中抗生素抗性基因的类型与序列读长数

Table 5. Types and read amounts of ARGs in cultured samples

基因类型 Gene type	抗生素类型 Antibiotic	R	T	N
baca	杆菌肽	36648	41236	35060
bcra	杆菌肽	2	0	0
bl2a_iii	青霉素	2	0	0
ceob	氯霉素	28166	30174	26246

下载: 导出CSV

表 6 不同样本中检测的ARO抗生素抗性基因的数量及在抗性机制类别水平的比例

Table 6. Amount and classified percentage of detected AROs in cultured samples　　　　　　　　　　（单位：%）

样本 Sample	ARO抗性基因数量 No. of AROs	在抗性机制类别水平的比例 Percentage at the level of ARO Class
样本 Sample	ARO抗性基因数量 No. of AROs	直接抗性类 Antibiotic resistance	敏感抗性类 Antibiotic sensitive	靶向抗性类 Antibiotic target
R	154	88.29	7.33	4.37
T	162	88.11	7.50	4.37
N	201	88.71	7.12	4.15

下载: 导出CSV

表 7 不同样本中前10位ARO抗生素抗性基因的序列读长数

Table 7. Read amounts of top 10 AROs in cultured samples

抗性基因 AROs	R	T	N
macB	194258	224126	208220
mdtB	127482	142556	139274
sav1866	124974	142704	133410
PmrB	114658	128638	122986
mexT	114430	124156	120530
PmrA	94842	105144	102598
adeH	84666	95750	91498
evgS	59998	76478	79418
mdtD	70826	84288	77766
rosB	56552	67874	61222

下载: 导出CSV

参考文献(29)

[1]	MARTÍNEZ J L. Antibiotics and antibiotic resistance genes in natural environments [J]. Science, 2008, 321(5887): 365−367. doi: 10.1126/science.1159483
[2]	RAZAVI M, MARATHE N P, GILLINGS M R, et al. Discovery of the fourth mobile sulfonamide resistance gene [J]. Microbiome, 2017, 5(1): 160. doi: 10.1186/s40168-017-0379-y
[3]	高盼盼, 罗义, 周启星, 等. 水产养殖环境中抗生素抗性基因(ARGs)的研究及进展 [J]. 生态毒理学报, 2009, 4(6):770−779. GAO P P, LUO Y, ZHOU Q X, et al. Research advances of antibiotic resistance genes (ARGs) in aquaculture environment [J]. Journal of Ecotoxicology, 2009, 4(6): 770−779.(in Chinese)
[4]	张骞月, 赵婉婉, 吴伟. 水产养殖环境中抗生素抗性基因污染及其研究进展 [J]. 中国农业科技导报, 2015, 17(6):125−134. ZHANG Q Y, ZHAO W W, WU W. Antibiotic resistance gene contamination in aquaculture environment and its research advances [J]. China Agricultural Science and Technology Review, 2015, 17(6): 125−134.(in Chinese)
[5]	胡莹莹, 王菊英, 马德毅. 近岸养殖区抗生素的海洋环境效应研究进展 [J]. 海洋环境科学, 2004, 23(4):76−80. doi: 10.3969/j.issn.1007-6336.2004.04.020 HU Y Y, WANG J Y, MA D Y. Advances on marine environmental effects of antibiotics in inshore aquaculture areas [J]. Marine Environmental Science, 2004, 23(4): 76−80.(in Chinese) doi: 10.3969/j.issn.1007-6336.2004.04.020
[6]	何基兵, 胡安谊, 陈猛, 等. 九龙江河口及厦门污水处理设施抗生素抗性基因污染分析 [J]. 微生物学通报, 2012, 39(5):683−695. HE J B, HU A Y, CHEN M, et al. Studies on the pollution levels of antibiotic resistance genes in Jiulong River estuary and wastewater treatment plants in Xiamen [J]. Microbial Bulletin, 2012, 39(5): 683−695.(in Chinese)
[7]	ZHU Y G, ZHAO Y, LI B, et al. Continental-scale pollution of estuaries with antibiotic resistance genes [J]. Nature Microbiology, 2017(2): 16270.
[8]	刘中柱, 郑伟文. 中国满江红[M]. 北京: 农业出版社, 1989.
[9]	BECKING J H. Endophyte transmission and activity in the Anabaena-Azolla association [J]. Plant and Soil, 1987, 100(1/2/3): 183−212.
[10]	PETERS G A, MEEKS J C. The Azolla-Anabaena symbiosis: Basic biology [J]. Annual Review Plant Physiology and Plant Molecular Biology, 1989, 40: 193−210. doi: 10.1146/annurev.pp.40.060189.001205
[11]	ITO O, WATANABE T. The relationship between combined nitrogen uptaken and nitrogen fixation in Azolla-Anabaena symbioses [J]. New Phytologist, 1983, 95: 647−654. doi: 10.1111/j.1469-8137.1983.tb03528.x
[12]	GATES J E, FISHER R W, CANDLER R A. The occurrence of coryneform bacteria in the leaf cavity of Azolla [J]. Archives of Microbiology, 1980, 127(2): 163−165. doi: 10.1007/BF00428020
[13]	SERRANO R, CARRAPICO F, VIDAL R. The presence of lectins in bacteria associated with Azolla-Anabaena symbiosis [J]. Symbiosis, 1999, 15: 169−178.
[14]	LECHNO Y S, NIERZWICKI-BAUER S A, RAI A N, et al. Cyanobacteria in symbiosis[M]. Dordrechr. The Netherlands: Kluwer Academic Publishers, 2002: 153-178.
[15]	ZHENG S P, CHEN B, GUAN X, et al. Diversity analysis of endophytic bacteria within Azolla microphyllausing PCRGGE and electron microscopy [J]. Chinese Journal of Agricultural Biotechnology, 2008, 5(3): 269−276. doi: 10.1017/S1479236208002441
[16]	郑斯平, 陈彬, 王瑾, 等. 小叶满江红(Azolla microphylla)内生细菌多样性的T-RFLP分析 [J]. 安徽农业科学, 2012, 40(29):14185−14187, 14270. doi: 10.3969/j.issn.0517-6611.2012.29.014 ZHENG S P, CHEN B, WANG J, et al. T-RFLP Analysis on diversity of endophytic bacteria in Azolla microphylla [J]. Journal of Anhui Agriculture Sciences, 2012, 40(29): 14185−14187, 14270.(in Chinese) doi: 10.3969/j.issn.0517-6611.2012.29.014
[17]	SU J Q, AN X L, LI B, et al. Metagenomics of urban sewage identifies an extensively shared antibiotic resistome in China [J]. Microbiome, 2017, 5(1): 1−15. doi: 10.1186/s40168-016-0209-7
[18]	彭司华, 吴智超, 孙丹, 等. 宏基因组学在抗生素抗性基因鉴定中的应用 [J]. 基因组学与应用生物学, 2019, 38(9):4102−4109. PENG S H, WU Z C, SUN D, et al. Application of Metagenomics in the Identification of Antibiotic Resistance Genes [J]. Genomics and Applied Biology, 2019, 38(9): 4102−4109.(in Chinese)
[19]	白克智, 于赛玲, 陈维纶, 等. 无藻满江红和满江红鱼腥藻的分离与培养 [J]. 科学通报, 1979, 14:664−666. BAI K Z, YU S L, CHEN W L, et al. Isolation and pure culture of algae-free Azollaand Anabaena azollae [J]. Chinese Sciences Bulletin, 1979, 14: 664−666.(in Chinese)
[20]	BÜNGER W, JIANG X, MÜLLER J, et al. Novel cultivated endophytic Verrucomicrobia reveal deep-rooting traits of bacteria to associate with plants [J]. Scientific Reports, 2020, 10(1): 8692. doi: 10.1038/s41598-020-65277-6
[21]	LIU B, POP M. ARDB: antibiotic resistance genes database [J]. Nucleic Acids Research, 2009, 37(Database issue): D443−D447.
[22]	JIA B F, RAPHENYA A R, ALCOCK B, et al. CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database [J]. Nucleic Acids Research, 2016: gkw1004.
[23]	曾秀丽, 王志, 罗利, 等. 茶树内生草螺菌ZXN111生长素合成及其对云抗-10号植物的促生功能 [J]. 微生物学报, 2020, 60(10):2198−2210. ZENG X L, WANG Z, LUO L, et al. Auxin synthesis in tea plant endophytic Herbaspirillum sp. and the plant growth promotion on Yunkang-10 [J]. Acta Microbiologica Sinica, 2020, 60(10): 2198−2210.(in Chinese)
[24]	沈应博, 史晓敏, 沈建忠, 等. 全基因组测序与生物信息学分析在细菌耐药性研究中的应用 [J]. 生物工程学报, 2019, 35(4):541−557. SHEN Y B, SHI X M, SHEN J Z, et al. Application of whole genome sequencing technology and bioinformatics analysis in antimicrobial resistance researches [J]. Chinese Journal of Biotechnology, 2019, 35(4): 541−557.(in Chinese)
[25]	杨兵, 梁晶, 刘林梦, 等. 耐药基因数据库概述 [J]. 生物工程学报, 2020, 36(12):2582−2597. YANG B, LIANG J, LIU L M, et al. Overview of antibiotic resistance genes database [J]. Chinese Journal of Biotechnology, 2020, 36(12): 2582−2597.(in Chinese)
[26]	MODI S R, LEE H H, SPINA C S, et al. Antibiotic treatment expands the resistance reservoir and ecological network of the phage metagenome [J]. Nature, 2013, 499(7457): 219−222. doi: 10.1038/nature12212
[27]	NESME J, CECILLON S, DELMONT T O, et al. Large-scale metagenomic-based study of antibiotic resistance in the environment [J]. Current Biology, 2014, 24(10): 1096−1100. doi: 10.1016/j.cub.2014.03.036
[28]	谭文彬. 细菌耐药的基因机理研究进展 [J]. 中国病原生物学杂志, 2009, 4(7):543−544, 555. TAN W B. Research advances in genetic mechanisms of bacterial antibiotic resistance [J]. Journal of Pathogen Biology, 2009, 4(7): 543−544, 555.(in Chinese)
[29]	朱阵, 曹明泽, 张吉丽, 等. 细菌耐药性研究进展 [J]. 中国畜牧兽医, 2015, 42(12):3371−3376. ZHU Z, CAO M Z, ZHANG J L, et al. Research progress on bacterial resistance [J]. China Animal Husbandry and Veterinary Medicine, 2015, 42(12): 3371−3376.(in Chinese)