土霉素暴露对小麦根际抗生素抗性细菌及土壤酶活性的影响

Influence of oxytetracycline exposure on antibiotic resistant bacteria and enzyme activities in wheat rhizosphere soil

通过培养的方法研究了土霉素暴露和小麦根际抗性细菌的数量、种类、分布特征及土壤酶活性之间的剂量效应关系。结果表明,土霉素暴露下小麦根际单一抗生素抗性细菌数量和抗土霉素-链霉素双重抗性细菌数都明显增加,且与暴露剂量呈正效应关系;同时,土壤磷酸酶、脱氢酶活性下降,但与土霉素的剂量效应关系不明显。从土霉素暴露的土壤中分离到50株抗性细菌,经形态观察、RFLP分组和16S rDNA序列测定与分析,将它们聚集在Actinobacteria、Bacilli、Alphaproteobacteria、Gammaproteobacteria和Sphingobacteria类群。其中放线菌最多(15株),占抗性菌总数的30%;其次是Bacillus属细菌(9株)和Pseudomonas属细菌(8株),分别占18%和16%。同时,具有抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌在土霉素暴露的样品中均被分离到,分别占抗性菌株总数的16%、8%和4%。值得注意的是,随着土霉素暴露剂量的增加,小麦根际优势促生菌Bacillus属细菌的抗性检出率逐步降低;但具有抗生素抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌的检出率却明显增加,提示可能会进一步增大其机会致病性。

Oxytetracycline is a tetracycline that is widely used to treat animals during poultry farming in China. Approximately 25% to 90% of antibiotics administered to animals are subsequently excreted, after which they enter the soil as manure. Due to the persistence and accumulation of such antibiotics in soil, oxytetracycline pollution may lead to a series of changes in soil microbial concentrations, microbial species, microbial antibiotic resistance, microbial community structure, and soil enzyme activities. In this study, relationships between antibiotic exposure dosages and the concentrations, species and distribution characteristics of antibiotic resistant bacteria and the activities of soil enzymes in wheat rhizosphere soil were investigated. To accomplish this, isolation and liquid cultivation methods were employed in the presence of different exposure dosages of oxytetracycline. Exposure to various concentrations of oxytetracycline resulted in a significant increase in the concentrations of oxytetracycline resistant bacteria to one to three times that of control samples that were not exposed to antibiotics. Exposure also led to an increase in the concentration of oxytertracycline-streptomycine double resistant bacteria to one to two times that of control samples that were not exposed to antibiotics. Additionally, themajority of double resistant bacteria （60%-80%） were colored bacteria. As the microbial community structures changed in response to oxytetracycline exposure, the activities of some soil enzymes such as alkaline phosphatase, acidic phosphatase and dehydrogenase in the wheat rhizosphere decreased; however, there was no clear relationship between the change in enzymes and oxytetracycline dose. A total of 50 strains of dominant antibiotic resistant bacteria were isolated from wheat rhizosphere soil samples treated with different doses of oxytetracycline. These bacteria were assigned to five groups, Actinobacteria, Bacilli, Alphaproteobacteria, Gammaproteobacteria and Sphingobacteria, based on morphological observations, RFLP grouping and their full length 16S rRNA gene sequences. Among them, Actinobacteria （15 strains） was the most commonly observed genus, accounting for 30% of the total antibiotic resistant isolates. This was followed by Bacillus sp. （9 strains） and Pseudomonas sp. （8 strains）, which accounted for 18% and 16% of the total antibiotic resistant isolates, respectively. Additionally, several important human opportunistic pathogenic bacteria with antibiotic resistance were frequently isolated from antibiotic treated soil, including Pseudomonas sp., Sphingomonas sp. and Stenotrophomonas sp. , which accounted for 16%, 8% and 4% of the total resistant isolates, respectively. Moreover, the dominant bacteria with antibiotic resistance in the wheat rhizosphere were Bacillus sp.. These organisms were generally isolated from samples of soil that had been treated with low doses of oxytetracycline （0, 100 μg/L）, while very few were isolated from samples of soil that had been treated with 2000 μg/L. Taken together, these findings suggest that exposure to high concentrations of antibiotics would inhibit the wheat growth-promoting bacteria in the rhizosphere, thereby indirectly inhibiting the growth of wheat. Conversely, human opportunistic pathogenic bacteria with antibiotic resistance （Pseudomonas sp. , Sphingomonas sp. and Stenotrophomonas sp. ） were generally isolated from soil samples treated with high doses of oxytetracycline exposure （2000 μg./L）. These antibiotic resistant opportunistic pathogenic bacteria and their antibiotic resistant genes pose a risk because they have the potential to spread within plants and humans.