有机肥施用土壤-蔬菜系统中潜在人畜病原菌及其耐药性分析
Analysis of Potential Human and Zoonotic Pathogens and Their Resistance to Antibiotics in Organic Fertilizer Applied Soil-Vegetable System
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摘要: 畜禽粪便类有机肥中含有大量的人畜病原菌和抗生素抗性基因(antibiotic resistance genes, ARGs)，施入农田后部分病原菌仍能继续存活并繁殖，且可能污染所种植的蔬菜，尤其是一些具有耐药性的人畜病原菌经食物链传递后将对人体健康造成巨大威胁。然而，当前大量的研究仅基于单纯的分离培养或高通量测序技术，对有机肥施用土壤-蔬菜系统中的人畜病原菌及其携带的毒力因子基因(virulence factor genes, VFGs)与ARGs认识有限。因此，本研究针对典型的长期施用有机肥的土壤-蔬菜系统，采集有机肥、施肥土及该土壤上种植的叶类蔬菜，如鸡毛菜、菜心、油麦菜和白菜等，结合传统分离培养及全基因组测序(whole genome sequencing, WGS)技术，利用生物信息学方法对分离菌株进行分类学及基因组注释，探究人畜病原菌的分布以及VFGs和ARGs的携带情况。结果表明在该施用有机肥的土壤-蔬菜系统中分离检测到42株人畜病原菌，其中37株属于肠杆菌科，5株属于假单孢菌科，主要的菌株包括大肠杆菌、克雷伯氏菌、假单胞菌和柠檬酸杆菌等，其中，大肠杆菌在土壤环境中显著富集，假单胞菌和柠檬酸杆菌主要分布在蔬菜根际及叶际。此外，全基因组测序提供了病原菌遗传信息，表明了人畜病原菌可能具有潜在的攻击性和代谢活性，携带有效应子、粘附性、生物膜、免疫调节性、侵袭性及毒素相关的VFGs。更重要的是，这些人畜病原菌均携带多种ARGs，这些ARGs编码了对多重耐药类、β-内酰胺类、氨基糖苷类、磷霉素类和多黏菌素类等抗生素的抗性。综上，本研究揭示了有机肥施用导致的土壤-蔬菜系统中人畜病原菌的分布情况，详细解析了病原菌携带的VFGs与ARGs类型，指明了食用叶类蔬菜存在的潜在健康风险，为加强畜禽粪便有机肥中重要人畜病原菌的风险防控提供了理论依据。Abstract: Livestock manure-based organic fertilizers are known to harbor a variety of zoonotic pathogens and antibiotic resistance genes (ARGs). When applied to agricultural fields, some of these pathogens can persist, multiply, and potentially contaminate the cultivated vegetables. Notably, the transmission of antibiotic-resistant zoonotic pathogens through the food chain poses a substantial threat to human health. However, the current research predominantly relying on isolation culture or high-throughput sequencing techniques, it provides limited insights into the zoonotic pathogens and the virulence factor genes (VFGs) and ARGs they carry within the soil-vegetable system. Therefore, this study focused on a typical long-term soil-vegetable system applied with organic fertilizer. Organic fertilizer, fertilized soil, and leafy vegetables such as Brassica rapa subsp. pekinensis, Brassica rapa var. parachinensis, Lactuca sativa var. longifoliaf, and Brassica oleracea var. capitata planted in this soil were collected. By combining traditional isolation and cultivation methods with advanced whole genome sequencing (WGS) technology, we classified and annotated the genomes of the isolated strains to explore the prevalence of zoonotic pathogens and the presence of VFGs and ARGs. The results showed that 42 strains of zoonotic pathogens were isolated and detected in the soil-vegetable system applied with organic fertilizer, including Escherichia coli, Klebsiella spp., Pseudomonas spp., and Citrobacter spp. Notably, E. coli was found to be significantly enriched in the soil environment, while Pseudomonas spp. and Citrobacter spp. were predominantly distributed in the rhizosphere and phyllosphere of the vegetables. Additionally, WGS offers comprehensive genetic data of an organism, including genes that suggest high pathogenicity and metabolic activity in zoonotic pathogens. These pathogens carry VFGs associated with effectors, adhesion, biofilm, immune modulation, invasion, and toxin production. More importantly, these zoonotic pathogens also harbor multiple ARGs encoding resistance to diverse antibiotic classes, including multi-drug resistant, β-lactams, aminoglycosides, fosfomycin, and polymyxins. In summary, this study has unveiled the distribution of zoonotic pathogens within the soil-vegetable system after the application of organic fertilizer. It presents a detailed analysis of the VFGs and ARGs carried by these pathogens. The research has also highlighted the potential health risks linked to the consumption of leafy vegetables, providing a theoretical foundation for enhancing risk management and control measures against significant zoonotic pathogens in organic fertilizers derived from livestock manure.
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