| Manure is an important reservoir of antibiotic resistant bacteria(ARB). Following manure application as fertilizer, ARB can be transferred to agricultural soils resulting in the high prevalence of resistant bacteria in soil. However, it is unclear whether ARB in manure can be transfered along manure-soil-vegetable chain during manure fertilization. In this study, we aimed to investigate the influence of manure application on abundance and diversity of antibiotic resistant endophytic bacteria in root, steam and leaves of pakchoi and celery. The results are as following:(1) The investigation of antibiotic resistant endophytic bacteria(AREB) in field vegetables. We detected the abundance and distribution of AREB in pakchoi and celery from the manure-fertilized or manure-free fields around Xinxiang City, Henan Province. The results showed that the AREB with Amoxicillin, Cephalexin or Tetracycline-resistance were detected in each part of celery and pakchoi(except leaf) from both manure-fertilized and manure-free fields, and their abundance ranged from 1.33×10 ~ 3.44×104 cfu·g-1. Notably, the abundance of the AREB manure-fertilized vegetables was 1~2 orders of magnitude higher than manure-free vegetables. Furthermore, the higest proportion of Amoxicillin, Cephalexin or Tetracycline-resistant bacteria to total cultivable endophytic bacteria in pakchoi and celery were 2.89, 3.61,10.61 and 9.16, 19.13, 10.65 folds higher than that of the control group, respectively, suggesting that the abundance of AREB has an intimate relationship with the application of manure. We also found that the ratio of endophytic bacteria resistant to β-lactams is highest in all AREB and reached 78.82% and 65.08% in pakchoi and celery, respectively. 16 S rDNA sequencing results showed that all the multiple antibiotic resistant endophytic bacteria(MAREB) in field vegetables were clustered into Pseudomonas sp. and Stenotrophomonas sp. It is especially noteworthy that among these MAREB, Pseudomonas putida and Stenotrophomonas maltophilia isolated from pakchoi are the opportunistic human pathogenic bacteria.(2) Influence of manure application on abundance of ARB in pakchoi and soil. We performed pot experiment to investigate the effect of manure, commercial organic fertilizer and multiple antibiotic resistant bacteria on the variation in the abundance of ARB from soil and plants. The results indicated that the ARB in pakchoi and soil increased with the application of manure or commercial organic fertilizer. The highest proportion of Cephalexin, Tetracycline or Ciprofloxaxin resistant endophytic bacteria to total cultivable endophytic bacteria in manure-fertilized pakchoi and soil was 5.35, 3.93, 1.25 and 1.65, 4.2, 1.44 folds higher than the control group, respectively. The highest proportion of multiple antibiotic resistant bacteria(MARB) in manure-fertilized soil was 4.35 higher than that of the control group..The highest proportion of ARB in pakchoi and soil treated with commercial organic fertilizer were 3.89, 2.17, 1.85 and 1.1, 1.58, 1.6 folds higher than that of the control group, respectively. All these results suggested that both the commercial organic fertilizer and manure application can increase the abundance of ARB in vegetables and soil, but the effect of the latter is more dramatic.(3) Influence of manure application on community structure of MARB in pakchoi and soil from pot experiment. The results from 16 SrDNA sequencing and the phylogenetic analysis showed that manure application could change the community structure of MAREB in vegetables. All the MAREB in the pakchoi with manure free were clustered into Stenotrophomonas sp. But all the MAREB in the pakchoi with manure or commercial organic fertilizer treatment were grouped into Brachybacterium sp. More importantly, the Brachybacterium sp. were also detected in the manure and soil before planting, suggesting that manure or commercial organic fertilizer might stimulate the tranfer of Brachybacterium sp. from soil to vegetables. The abundance of MARB in soil after harvest was significantly enchanced along with manure application than that before planting. We also found that although Escherichia coli with mutiply antibiotic resistance were added into the soil, they can not be detected in soil after harvest. Notably, many new MARB were isolated from soil after harvest. All these reuslts suggested that manure application probably performs a persistent effect on the community structrue of ARB in the soil by promoting the horizontal transfer of antibiotic resistant genes in the soil and increase the diversity of MARB. |
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