基于功能宏基因组学挖掘抗生素耐药基因研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2022-0537

摘要/Abstract

摘要：

随着各类抗生素被广泛用于治疗细菌感染以及抗生素在临床上的大量使用,驱动了各类抗生素耐药基因的不断进化,导致了耐药问题日趋严重。耐药基因与耐药细菌的广泛传播与普遍流行严重威胁公共卫生体系并引起了巨大的经济损失。值得注意的是,抗生素的广泛施用不仅造成了动物体内耐药细菌的产生,还提高了对环境中微生物的选择压力,间接推动了耐药基因的发展与进化,使环境微生物成为耐药基因新的储库。因此对耐药细菌的广泛监测与新型耐药基因的提前发掘具有重要的临床意义与研究价值。但是传统的耐药性调查手段过度依赖对耐药细菌的培养,难以全面的展现固定生态位中微生物耐药性的全貌。而功能宏基因组学技术利用其表型筛选和高通量测序相结合的优势,不依赖于对携带目标基因的特定细菌的培养,因此在发掘新型功能基因方面有着巨大的优势。本文综述了功能宏基因组在抗生素耐药方面的研究进展,讨论了功能宏基因组学方法在检测新型基因研究中的意义及存在的问题,为后续深入开展对抗生素耐药机制的探究提供了坚实的理论基础。

关键词: 功能宏基因组学, 环境微生物, 新型耐药基因, 抗生素

Abstract:

The widespread use of various antibiotics in the treatment of bacterial infections and the widespread clinical use of antibiotics drives the continuous evolution of various antibiotic resistance genes（ARGs）, leading to the increasing problem of drug resistance. The global spread of antibiotic-resistant genes and bacteria severely threatens the public health and results in tremendous economic losses in recent decades. Of note, the excessive usage of antibiotics not only led to the occurrence of antibiotics-resistant bacteria, but also constitutively elevated the selection pressure of environmental microbiome, indirectly accelerated the development and evolution of antibiotics-resistant genes, making environmental microbiome as the storage reservoir of antibiotics-resistant genes. Therefore, the extensive monitoring of antibiotics-resistant bacteria and the early exploration of new antibiotics-resistant genes are of great clinical significance and research value. However, traditional methodologies for ARGs identification and profiling largely depend on the molecular characterization of cultivable bacteria, hardly providing a comprehensive landscape of the resistome in a given ecological niche. To cope with the increasing necessity to uncover the ARGs in uncultivable bacteria, the functional metagenomics emerged as a promising toolkit that combines the phenotypic profiling with next high-throughput sequencing, it does not depend on the culture of specific bacteria with target genes, and thus it is deemed as a powerful alternative to reveal all novel functional genes. In the present review, we systematically summarized the latest advances in the field of functional metagenomics and dated their recent applications in unveiling the unknown ARGs from environments. This review offers a solid theoretical foundation for in-depth understanding the resistant mechanism of antibiotics.

Key words: functional metagenomics, environmental microbiome, novel resistance genes, antibiotic

鲁兆祥, 王夕冉, 连新磊, 廖晓萍, 刘雅红, 孙坚. 基于功能宏基因组学挖掘抗生素耐药基因研究进展[J]. 生物技术通报, 2022, 38(9): 17-27.

LU Zhao-xiang, WANG Xi-ran, LIAN Xin-lei, LIAO Xiao-ping, LIU Ya-hong, SUN Jian. Advances in the Discovery of Novel Antibiotic-resistant Genes Based on Functional Metagenomics[J]. Biotechnology Bulletin, 2022, 38(9): 17-27.

图/表 3

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抗生素类别 Class of antibiotic	药物 Drug	耐药率 Resistance rate/%	参考文献 Reference
β-内酰胺类 β-lactam antibiotics	头孢曲松 Ceftriaxone	81	[10]
	头孢他啶 Ceftazidime	87.5
	美罗培南 Meropenem	99.7
	氨苄西林 Ampicillin	97.5	[11]
	头孢哌酮-舒巴坦Cefperazone-sulbactam	98.9
	哌拉西林-他唑巴坦Piperacillin-tazobactam	98.9
	头孢吡肟 Cefepime	100	[21]
四环素类 Tetracyclines	四环素 Tetracycline	85.0	[16]
	替加环素 Tigecycline	98.9	[11]
大环内酯类 Macrolides	红霉素 Erythrocin	74.1	[22]
	螺旋霉素I Spiramycin I	48.1
	阿奇霉素 Azithromycin	74.1
	克拉霉素 Clarithromycin	74.1
林可酰胺类Lincoamide antibiotics	林可霉素 Lincomycin	77.8
酮内酯类 Ketolide antibiotics	泰立霉素 Telithromycin	77.8
氨基糖苷类 Aminoglycosides	阿米卡星 Amikacin	100	[21]
	庆大霉素 Gentamicin	31.4	[11]
多磷类 Polyphosphorous	磷霉素 Fosfomycin	66.7
多黏菌素类 Polymyxins	黏菌素 Colistin	73.3
磺胺类药物 Sulfonamides	复方磺胺甲恶唑 Sulfadimidine	53.3
喹诺酮类 Quinolones	环丙沙星 Ciprofloxacin	93.3
	左氧氟沙星 Levofloxacin	100	[21]

抗生素类别 Class of antibiotic	药物 Drug	耐药率 Resistance rate/%	参考文献 Reference
β-内酰胺类 β-lactam antibiotics	头孢曲松 Ceftriaxone	81	[10]
	头孢他啶 Ceftazidime	87.5
	美罗培南 Meropenem	99.7
	氨苄西林 Ampicillin	97.5	[11]
	头孢哌酮-舒巴坦Cefperazone-sulbactam	98.9
	哌拉西林-他唑巴坦Piperacillin-tazobactam	98.9
	头孢吡肟 Cefepime	100	[21]
四环素类 Tetracyclines	四环素 Tetracycline	85.0	[16]
	替加环素 Tigecycline	98.9	[11]
大环内酯类 Macrolides	红霉素 Erythrocin	74.1	[22]
	螺旋霉素I Spiramycin I	48.1
	阿奇霉素 Azithromycin	74.1
	克拉霉素 Clarithromycin	74.1
林可酰胺类Lincoamide antibiotics	林可霉素 Lincomycin	77.8
酮内酯类 Ketolide antibiotics	泰立霉素 Telithromycin	77.8
氨基糖苷类 Aminoglycosides	阿米卡星 Amikacin	100	[21]
	庆大霉素 Gentamicin	31.4	[11]
多磷类 Polyphosphorous	磷霉素 Fosfomycin	66.7
多黏菌素类 Polymyxins	黏菌素 Colistin	73.3
磺胺类药物 Sulfonamides	复方磺胺甲恶唑 Sulfadimidine	53.3
喹诺酮类 Quinolones	环丙沙星 Ciprofloxacin	93.3
	左氧氟沙星 Levofloxacin	100	[21]

样品来源 Source	抗生素种类及其耐药基因ARGs and associated antibiotics						参考文献Reference
样品来源 Source	酰胺醇类 Phenicols	氨基糖苷类 Aminoglycosides	β-内酰胺类 β-lactam antibiotics	四环素类 Tetracyclines	多药耐药 Multidrug resistance	其他 Others	参考文献Reference
土壤 Soil	AAT、aadA、A3PH、aphD、ARPA、KNT、FAT、HARK、APT、ARPB、aac6、aadB、 GAT、GRP、aac、strA	CAT、catB2、catB3、ceoB、CFE、CFRP、cmlA、cmx、CRD、CT、floR	ACT-1、ampc、CARB、L1、mecA、OXA、PSE-1、Sed1、TEM-21、VIM-18	tet32、tetA、tetA（31）、tetA（33）、tetA（39）、tetA（41）、tetA（B）、tetA（D）、tetA（G）、tetA（M）、tetA（P）、tetB、tetG、tetH、etL、tetM、tetO、tetQ、tetS、tetT、tetV、tetW、tetX、tetX2、tetY	acrA、acrB、adeA、adeB、AmrB、bpeF、ermFS、ermG、ermGM、ermT、ermX、mdfA、mdtF、mdtH、mdtL、mdtO、mef、mexA、mexB、mexD、mexE、mexF、mexG、MLSRP、msrA、oprN、Rpos、sdeY	AcRPB、AFRPB、AEP、AcRPA、AFRPA、fosB、FRP、rosA	[56]
			1,2-GDO				[57]
	3-NAT、6-NAT					ARTs	[52]
	AAT		Class A、B、C、D、Metallo beta-lactamase				[58]
			Bifunctional beta-lactamase				[52]
	2-NAT、2-OXO-DH、3-NAT、6-NAT、AAdT、ATIII、EEL、FAA、GBD、HAD、ICMT、ML、PM、NSDH、TL、tRNAs			AL、STK、otrC、tet（34）、tet（37）、tetV、tetX	ABC、RND		[59]
水 Water	aacA4、aadA、aadB、ereA、strA、strB	ceoB、cmlA	blaA	tet33、tet34、tetC、tetL、tetM、tetX	acrB、mexB、mexF	smeE、ermF、macB、bacA、sul1、sul2	[60]
	strA、strB			tetL		qnrA、mfeE mphB	[61]
	K⁺ transport ptn、3-NAT、PDH、GNAT、AAdt、A3PH、6-NAT、GT、MetRS		TEM beta-lactamase			RecX、RA	[62]
污泥 Sludge	aa6iia、aac6ib、aadD、ant2ia、ant3ia、ant6ia、aph33ib、aph6id、ereA	catB3、ceoB、cml_e3、cmle_8	bl2D_pse3、bl2D_oxa5、bl2_veb、bl2D_oxa10	tet32、tet33、tetA、tetC、tetG、tetO、tetPa、tetPb、tetV、tetW、tetX	acrB、ermB、ermC、ermQ、ermT、mdtF、mexB、mexD、mexF、mexW、mexY	smeB、smeE、lnuA、macB、mefA、rosA、rosB、bacA、arnA、sul1、sul2	[63]
	aac、aadA、AGRPA、aph		Class A BL、NPS-1、 OXA-1、OXA-10、OXA-2、VEB-5	tet39、tetA、tetC、tetG、tetM、tetO、tetP、tetS、tetW、tetX			[64]
	aadA、aadB、aphA-2、strA、strB	catB、cmlA、floR	blaCFX-a、laOXA-10、 blaVEB-5、blaVEB-3	tet33、tet36、tet39、tet40、tetA（P）、tetB（P）、tetG、tetM、tetO、tetW	acrB、adeA、ermB、mdtF、mexB、mexF、mexW	smeB、smeE、macB、bacA、sul1、sul2	[53]
			blaDWA、blaDWA2、blaDWA3、blaDWA4、blaDWB1				[65]

样品来源 Source	抗生素种类及其耐药基因ARGs and associated antibiotics						参考文献Reference
样品来源 Source	酰胺醇类 Phenicols	氨基糖苷类 Aminoglycosides	β-内酰胺类 β-lactam antibiotics	四环素类 Tetracyclines	多药耐药 Multidrug resistance	其他 Others	参考文献Reference
土壤 Soil	AAT、aadA、A3PH、aphD、ARPA、KNT、FAT、HARK、APT、ARPB、aac6、aadB、 GAT、GRP、aac、strA	CAT、catB2、catB3、ceoB、CFE、CFRP、cmlA、cmx、CRD、CT、floR	ACT-1、ampc、CARB、L1、mecA、OXA、PSE-1、Sed1、TEM-21、VIM-18	tet32、tetA、tetA（31）、tetA（33）、tetA（39）、tetA（41）、tetA（B）、tetA（D）、tetA（G）、tetA（M）、tetA（P）、tetB、tetG、tetH、etL、tetM、tetO、tetQ、tetS、tetT、tetV、tetW、tetX、tetX2、tetY	acrA、acrB、adeA、adeB、AmrB、bpeF、ermFS、ermG、ermGM、ermT、ermX、mdfA、mdtF、mdtH、mdtL、mdtO、mef、mexA、mexB、mexD、mexE、mexF、mexG、MLSRP、msrA、oprN、Rpos、sdeY	AcRPB、AFRPB、AEP、AcRPA、AFRPA、fosB、FRP、rosA	[56]
			1,2-GDO				[57]
	3-NAT、6-NAT					ARTs	[52]
	AAT		Class A、B、C、D、Metallo beta-lactamase				[58]
			Bifunctional beta-lactamase				[52]
	2-NAT、2-OXO-DH、3-NAT、6-NAT、AAdT、ATIII、EEL、FAA、GBD、HAD、ICMT、ML、PM、NSDH、TL、tRNAs			AL、STK、otrC、tet（34）、tet（37）、tetV、tetX	ABC、RND		[59]
水 Water	aacA4、aadA、aadB、ereA、strA、strB	ceoB、cmlA	blaA	tet33、tet34、tetC、tetL、tetM、tetX	acrB、mexB、mexF	smeE、ermF、macB、bacA、sul1、sul2	[60]
	strA、strB			tetL		qnrA、mfeE mphB	[61]
	K⁺ transport ptn、3-NAT、PDH、GNAT、AAdt、A3PH、6-NAT、GT、MetRS		TEM beta-lactamase			RecX、RA	[62]
污泥 Sludge	aa6iia、aac6ib、aadD、ant2ia、ant3ia、ant6ia、aph33ib、aph6id、ereA	catB3、ceoB、cml_e3、cmle_8	bl2D_pse3、bl2D_oxa5、bl2_veb、bl2D_oxa10	tet32、tet33、tetA、tetC、tetG、tetO、tetPa、tetPb、tetV、tetW、tetX	acrB、ermB、ermC、ermQ、ermT、mdtF、mexB、mexD、mexF、mexW、mexY	smeB、smeE、lnuA、macB、mefA、rosA、rosB、bacA、arnA、sul1、sul2	[63]
	aac、aadA、AGRPA、aph		Class A BL、NPS-1、 OXA-1、OXA-10、OXA-2、VEB-5	tet39、tetA、tetC、tetG、tetM、tetO、tetP、tetS、tetW、tetX			[64]
	aadA、aadB、aphA-2、strA、strB	catB、cmlA、floR	blaCFX-a、laOXA-10、 blaVEB-5、blaVEB-3	tet33、tet36、tet39、tet40、tetA（P）、tetB（P）、tetG、tetM、tetO、tetW	acrB、adeA、ermB、mdtF、mexB、mexF、mexW	smeB、smeE、macB、bacA、sul1、sul2	[53]
			blaDWA、blaDWA2、blaDWA3、blaDWA4、blaDWB1				[65]