Vertical distribution of antibiotic resistance genes and bacterial communities in soil of livestock manure stacking site
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摘要: 本文通过对养殖场猪粪和鸡粪堆放地0~100 cm土壤样品的采集和分析, 研究了长期堆放畜禽粪便对土壤中抗生素抗性基因(简称“抗性基因”)和细菌群落结构垂直分布的影响。定量PCR结果表明, 与对照土壤相比, 猪粪和鸡粪堆放增加了0~100 cm土壤中四环素类抗性基因(tetC、tetG、tetL、tetW)、磺胺类抗性基因(sulI、sulII)以及整合酶基因(intI1)的检出率和检出丰度, 说明粪肥堆放造成堆放地土壤中抗性基因污染。聚类分析结果表明, 抗性基因和intI1基因的丰度随土壤深度呈递减趋势, 且主要集中在0~30 cm土层, 表明堆放地土壤中抗性基因存在向下层土壤迁移的风险。相关性分析表明, intI1基因分别与抗性基因呈显著正相关, 说明intI1基因可能在抗性基因传播中起着重要作用。同时高通量测序结果表明, 与对照土壤相比, 猪粪和鸡粪堆放显著降低了0~10 cm和10~30 cm土层细菌群落结构的多样性, 0~30 cm土层中, 猪粪和鸡粪堆放地土壤中细菌群落结构与对照土壤的差异要高于深层土壤。此外, 方差分解分析结果表明, 土壤化学性质和细菌群落结构均影响了土壤中抗性基因的垂直分布, 且细菌群落结构的变化是其主要的影响因素。本研究可为控制畜禽粪便堆放地土壤中抗性基因污染提供科学依据。Abstract: Livestock manure has been regarded as an important reservoir of antibiotics and antibiotic resistance genes (ARGs). However, most livestock manure is stacked directly in the farm, which causes a potential threat to the surrounding soil and groundwater safety. In order to study the effect of long-term livestock manure stacking on the vertical distribution of ARGs and bacterial communities, 0−100 cm soil samples were collected from pig and chicken manure stacking sites, respectively. Real-time quantitative PCR results showed that pig and chicken manure stacking increased the detection ratio and abundance of tetracycline resistance genes (tetC, tetG, tetL, tetW) and sulfonamide resistance genes (sulI, sulII), and an integrase gene (intI1) in soil samples. This demonstrated that livestock manure stacking could lead to the contamination of ARGs in the surrounding soil. According to the cluster analysis, the abundance of ARGs and intI1 gene were decreased with increasing soil depth and mainly concentrated in the 0−30 cm soil layer, which posed a risk of migration of ARGs into the deep soil. In addition, intI1 gene had a significant and positive correlation with ARGs abundance, indicating that intI1 gene may play an important role in disseminating ARGs. Furthermore, the high-throughput sequencing results showed that both pig and chicken manure stacking significantly reduced and changed the diversity of bacterial communities in the 0−10 cm and 10−30 cm soil layers, compared with the control soil. The difference of bacterial community structure between livestock manure stacking site soil and control soil was higher in 0−30 cm than in deep soil. What’s more, both soil chemical properties and bacterial community affected the vertical distribution of ARGs in soil, with the shift of bacterial community structure representing the major driver shaping the ARGs distribution based on variation partitioning analysis. Taken together, our results provide insight into the control of ARGs pollution in livestock manure stacking stie soil around the farms.
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图 1 不同处理不同深度土壤tetW、tetC、tetG、tetL、sulI、sulII和intI1基因的相对丰度(基因拷贝数/16S rRNA基因拷贝数)
每一行代表一个处理, 每一列代表一个基因。基因相对丰度的数据经过lg转换。CK: 对照; PM: 猪粪堆放地; CM: 鸡粪堆放地。A row is one treatment, and a column is one target gene. The data is a lg scale of gene relative abundance. CK: control; PM: pig manure stacking site; CM: chicken manure stacking site.
Figure 1. Relative abundances (gene copy number/16S rRNA gene copy number) of tetW, tetC, tetG, tetL, sulI, sulII and intI1 genes in different soil depths of different treatments
图 3 不同处理不同土层细菌α多样性(A)和基于Bray-Curtis距离的主坐标分析(PCoA)的细菌群落结构组成(B)
CK: 对照; PM: 猪粪堆放地; CM: 鸡粪堆放地。CK: control; PM: pig manure stacking site; CM: chicken manure stacking site.
Figure 3. Soil bacterial α diversity (A) and principal coordinate analysis (PCoA) based on the Bray-Curtis distance (B) showing the profile of bacterial community structure in different soil layers of different treatments
图 4 细菌群落结构和土壤化学性质对抗性基因分布的方差分解分析(VPA) (A)以及intI1基因丰度、抗性基因丰度和土壤化学性质间的Pearson相关性分析(B)
右图中, SOM、TN和TC分别为有机质含量、总氮含量和总碳含量。圆圈大小代表相关系数大小。蓝色表示正相关, 红色表示负相关。星号代表存在显著相关性。*: P<0.05; **: P<0.01; ***: P<0.001。In the right figure, SOM, TN and TC are contents of organic matter, total nitrogen and total carbon. The circle size represents the correlation coefficient. Blue indicates a positive correlation, and red indicates a negative correlation. The asterisk represents a significant correlation. *: P<0.05; **: P<0.01; ***: P<0.001.
Figure 4. Effects of bacterial community and soil chemical properties on the variations of resistance genes (A), and Pearson’s correlation among the intI1 gene abundance, resistance gene abundances, and soil chemical properties (B)
表 1 不同畜禽粪便堆放地的土壤化学性质
Table 1. Soil chemical properties in different treatments
土层深度
Soil depth (cm)处理
TreatmentpH 有机质含量
Organic matter content (g∙kg−1)总碳含量
Total carbon content (g∙kg−1)总氮含量
Total nitrogen content (g∙kg−1)碳氮比
C/N0~10 CK 7.87±0.07b 17.81±1.89b 15.00±0.20b 1.07±0.06a 14.08±0.63b PM 8.76±0.28a 29.48±5.18a 20.27±2.83a 1.43±0.42a 14.53±2.02b CM 8.00±0.17b 13.72±2.36b 19.60±1.56a 1.53±0.45a 17.08±4.16a 10~30 CK 7.90±0.05b 13.89±1.03a 13.30±0.44b 0.87±0.06a 15.38±0.85b PM 8.54±0.32a 17.21±2.90a 15.80±1.33ab 0.97±0.21a 16.64±2.03b CM 8.40±0.13a 7.94±2.02b 18.62±3.97a 0.65±0.25a 30.45±7.71a 30~50 CK 7.90±0.10a 6.92±1.12c 9.73±0.85b 0.53±0.06b 18.28±0.49a PM 8.00±0.19a 9.71±1.49b 13.13±2.20a 0.67±0.12b 19.75±1.72a CM 7.93±0.10a 14.17±0.88a 12.30±0.87ab 1.23±0.06a 10.00±1.01b 50~70 CK 7.88±0.08a 8.46±0.44a 10.77±0.74a 0.53±0.06b 20.40±3.14a PM 7.92±0.04a 8.73±0.92a 13.13±2.21a 0.60±0.10ab 21.90±1.39a CM 7.73±0.04b 7.93±1.84a 5.23±0.12b 0.77±0.15a 7.03±1.58b 70~100 CK 7.95±0.10b 7.96±0.30a 16.37±0.96a 0.40±0.00b 40.92±2.40a PM 8.15±0.12a 8.27±0.81a 18.67±3.76a 0.47±0.12ab 36.29±0.06a CM 7.68±0.08c 6.42±0.30b 4.63±0.06b 0.60±0.17a 8.12±2.05b CK: 对照; PM: 猪粪堆放地; CM: 鸡粪堆放地。不同字母代表同一土层各处理间差异显著(P<0.05)。CK: control; PM: pig manure stacking site; CM: chicken manure stacking site. Different letters represent significant differences among treatments in the same soil layer (P<0.05). -
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