不同施肥处理农田土壤中噬菌体与细菌携带抗生素抗性基因的比较

doi:10.13560/j.cnki.biotech.bull.1985.2021-1544

摘要/Abstract

摘要：

为了揭示有机肥和化肥长期施用对农田土壤噬菌体携带的抗生素抗性基因（antibiotic resistance genes,ARGs）多样性和丰度的影响,并与土壤细菌携带的ARGs进行对比,本文将土壤抗生素抗性分为细菌和噬菌体两个部分,利用微滴数字PCR（droplet digital PCR,ddPCR）技术定量分析了土壤噬菌体和细菌DNA中25种ARGs亚型和I类整合子（intl1）的丰度。结果表明,土壤噬菌体中ARGs的检出率和总丰度以及intl1丰度均低于土壤细菌,其中噬菌体中检测到20种ARGs亚型,在不施肥、单施化肥和单施有机肥土壤的噬菌体中,目标ARGs的检出率分别为68%、72%和76%。土壤噬菌体中ARGs的总丰度在有机肥施用土壤中显著高于不施肥和化肥施用土壤（P<0.05）,其中多耐药类、大环内酯-林肯酰胺-链阳性菌素B（MLSB）类和β-内酰胺类抗性基因丰度占显著优势。除了β-内酰胺类抗性基因blaTEM,噬菌体中其他ARGs亚型的丰度均显著低于细菌（P<0.05）。噬菌体与细菌携带ARGs在不同施肥处理中均存在显著正相关（P<0.05）。冗余分析结果显示,施肥可能通过改变土壤pH、重金属和营养因子水平来影响细菌和噬菌体中ARGs的赋存特征。本研究结果表明,噬菌体是除细菌之外的农田土壤另一个重要ARGs储存库,施用有机肥能同时显著提高土壤细菌和噬菌体中ARGs的多样性和丰度。

关键词: 抗生素抗性基因, 噬菌体, 农田土壤, 微滴数字PCR, 冗余分析

Abstract:

This study is aimed to elucidate the effect of the long-term application of organic and chemical fertilizers on the diversity and abundance of antibiotic resistance genes（ARGs）carried by bacteriophages,and to compare them with ARGs carried by bacteria in farmland soil. Soil antibiotic resistance was divided into bacterial and bacteriophage fractions,and droplet digital PCR（ddPCR）was employed to analyze the abundance of 25 ARG subtypes and class I integrase gene（intl1）in DNA of soil bacteria and bacteriophages. The total abundance and detection rate of ARGs and intl1 abundance carried by bacteriophages were evidently lower than those in soil bacterial fraction. Twenty ARG subtypes were detected in bacteriophage DNA. Detection rates of target ARGs in bacteriophages were 68%,72%,and 76% in unfertilized,chemically fertilized,and organically fertilized soils,respectively. The total abundance of ARGs in bacteriophages in the soil amended with organic fertilizer was the significantly higher than that in the soil amended with chemical fertilizer and the unfertilized soil（P<0.05）,and the gene abundances of multi-drug,macrolide-lincosamide-streptogramin group B（MLSB）,and β-lactam resistance dominated bacteriophages. Apart from β-lactam resistance gene blaTEM_,the abundance of other ARG subtypes in bacteriophages was significantly lower than that in soil bacteria（P<0.05）. The significantly positive correlations of ARG abundance were observed between soil bacteria and bacteriophages under different fertilization treatments（P<0.05）. The results of redundancy analysis showed that fertilization may affect the occurrence characteristics of ARGs in bacteria and bacteriophages by changing soil pH,heavy metal and nutrient factor levels. The findings of this study indicate that bacteriophages are another important reservoir of ARGs in farmland soil besides bacteria. The application of organic fertilizer simultaneously increased the diversity and abundance of ARGs carried by soil bacteria and bacteriophages.

Key words: antibiotic resistance genes, bacteriophages, farmland soil, droplet digital PCR, redundancy analysis

胡雪莹, 张越, 郭雅杰, 仇天雷, 高敏, 孙兴滨, 王旭明. 不同施肥处理农田土壤中噬菌体与细菌携带抗生素抗性基因的比较[J]. 生物技术通报, 2022, 38(9): 116-126.

HU Xue-ying, ZHANG Yue, GUO Ya-jie, QIU Tian-lei, GAO Min, SUN Xing-bin, WANG Xu-ming. Comparison in Antibiotic Resistance Genes Carried by Bacteriophages and Bacteria in Farmland Soil Amended with Different Fertilizers[J]. Biotechnology Bulletin, 2022, 38(9): 116-126.

图/表 7

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处理Treatment	施肥量Fertilizer amount/（kg· m^-2）
不施肥（CK） Without fertilizer	0
单施化肥（IF） Inorganic fertilizer amendment	Urea 0.04、（NH₄）₂HPO₄ 0.05和K₂SO₄ 0.03
单施有机肥（OF） Organic fertilizer amendment	Compost from chicken manure 2.38

处理Treatment	施肥量Fertilizer amount/（kg· m^-2）
不施肥（CK） Without fertilizer	0
单施化肥（IF） Inorganic fertilizer amendment	Urea 0.04、（NH₄）₂HPO₄ 0.05和K₂SO₄ 0.03
单施有机肥（OF） Organic fertilizer amendment	Compost from chicken manure 2.38

抗生素抗性基因亚型 ARG subtypes	细菌Bacteria			噬菌体acteriophages
抗生素抗性基因亚型 ARG subtypes	CK	IF	OF	CK	IF	OF
strA	+	+	+	-	+	+
strB	+	+	+	+	+	+
aadA-01	+	+	+	+	+	+
blaOXA-20	-	-	-	-	-	-
blaTEM	+	+	+	+	+	+
blaCTX-M	-	+	+	-	-	+
ermA	+	+	+	-	-	-
ermB	+	+	+	-	-	-
mphA-01	+	+	+	+	+	+
oleC	+	+	+	+	+	+
sul1	+	+	+	-	-	-
sul2	+	+	+	+	+	+
tetA	+	+	+	+	+	+
tetW	+	+	+	+	+	+
tetM	+	+	+	+	+	+
tetX	+	+	+	+	+	+
acrA-05	+	+	+	+	+	+
emrD	+	+	+	+	+	+
mepA	+	+	+	+	+	+
mexF	+	+	+	+	+	+
qnrA	-	-	-	-	-	-
qnrS	+	+	+	-	+	-
vanHB	+	+	+	+	+	+
vanA	+	+	+	+	+	+
mcr-1	+	+	+	+	-	+
检出率Detection rate/%	88	92	92	68	72	76
intl1	+	+	+	+	+	+

抗生素抗性基因亚型 ARG subtypes	细菌Bacteria			噬菌体acteriophages
抗生素抗性基因亚型 ARG subtypes	CK	IF	OF	CK	IF	OF
strA	+	+	+	-	+	+
strB	+	+	+	+	+	+
aadA-01	+	+	+	+	+	+
blaOXA-20	-	-	-	-	-	-
blaTEM	+	+	+	+	+	+
blaCTX-M	-	+	+	-	-	+
ermA	+	+	+	-	-	-
ermB	+	+	+	-	-	-
mphA-01	+	+	+	+	+	+
oleC	+	+	+	+	+	+
sul1	+	+	+	-	-	-
sul2	+	+	+	+	+	+
tetA	+	+	+	+	+	+
tetW	+	+	+	+	+	+
tetM	+	+	+	+	+	+
tetX	+	+	+	+	+	+
acrA-05	+	+	+	+	+	+
emrD	+	+	+	+	+	+
mepA	+	+	+	+	+	+
mexF	+	+	+	+	+	+
qnrA	-	-	-	-	-	-
qnrS	+	+	+	-	+	-
vanHB	+	+	+	+	+	+
vanA	+	+	+	+	+	+
mcr-1	+	+	+	+	-	+
检出率Detection rate/%	88	92	92	68	72	76
intl1	+	+	+	+	+	+