不同构型人工湿地基质中土著菌的耐药性及整合子丰度调查

Investigation of Antibiotic Resistance of Indigenous Bacteria and Abundance of Class I Integron in Matrix of Constructed Wetlands of Different Configurations

采用Kirby-Bauer纸片琼脂扩散法分析了夏、冬季节9个不同构型人工湿地中的葡萄球菌(Staphylococcus)、假单胞菌(Pseudomonas)两种土著菌对7种常用抗生素的耐药性,并通过荧光定量PCR检测人工湿地基质中Ⅰ型整合子(int1)的丰度.结果表明,共分离出的522株葡萄球菌和543株假单胞菌,约84%的菌株对所测试的抗生素具有耐药性,多重耐药率达到68%以上,MRI指数平均为0.22,与某些环境中人源或动物源细菌耐药水平相当,表明人工湿地基质土著菌具有较高水平的耐药性;两种土著菌对氨苄西林和复方新诺明的耐药率较高,四环素、庆大霉素和环丙沙星的耐药率均极低(<3%),而对头孢他啶和氯霉素的耐药性呈现差别.湿地基质中int1基因的浓度为1.14×105~5.66×105copies·g-1,其相对丰度为0.54%~3.68%.季节和湿地工艺对细菌耐药和整合酶基因分布有较大影响.夏季2种细菌的抗生素耐药率、多重耐药指数和int1丰度均显著高于冬季;下行垂直流湿地中的抗生素耐药率、多重耐药指数(MRI)最高,而在水平潜流湿地中int1丰度最高.研究表明人工湿地基质的土著菌长期暴露在一定浓度抗生素和耐药肠道菌的生活污水环境下获得了耐药性,人工湿地中抗生素耐药菌和耐药基因的污染及环境风险不容忽视.

Environmental indigenous bacteria( Staphylococcus and Pseudomonas) were isolated from 9 different constructed wetlands( CWs) in summer and winter. The antibiotic resistance analysis of the isolated bacteria was conducted by Kirby-Bauer disc agar diffusion method. And the quantitative PCR assay was used to quantify the abundance of class I integron( int1) in the matrix of CWs.The results indicated that over 84% of isolates among the 522 Staphylococcus strains and 543 Pseudomonas strains had antibiotic resistance and above 68% of isolates had multi-antibiotic resistance,the average of MRI index was 0. 22. Antibiotic resistance of indigenous bacteria in CWs was at the same resistance level of human or animal bacteria in certain environment,indicating that indigenous bacteria from constructed wetlands had relatively high level of resistance. Staphylococcus and Pseudomonas strains had higher resistances to Ampicillin( AMP) and Sulfamethoxazole( SXT),and extremely low resistance rates of lower than 3% to Tetracycline( TE),Gentamicin( CN) and Ciprofloxacin( CIP). To Ceftazidime( CAZ) and Chloramphenicol( C),the two kinds of indigenous bacteria showed distinctly different resistances. Quantitative PCR revealed that the abundance of int1 in CWs was 1. 14 ×105-5. 66 × 105copies·g- 1,and its relative abundance was 0. 54%-3. 68%. Both of season and wetland type had important impact on antibiotic resistance and abundance of int1. The antibiotic resistance rate and the multiple resistance index( MRI) for the indigenous bacteria and the abundance of int1 in summer were higher than those in winter. Among three types of CWs,the antibiotic resistance rate and the MRI value were the highest in the downward vertical flow,while the int1 abundance was the highest in the sub-surface flow. The research indicated that indigenous bacteria acquired antibiotic resistance due to long term exposure to antibiotics of certain concentrations and antibiotic-resistant intestinal bacteria in sewage environment. The environmental risks of antibiotic resistant bacteria and resistant genes in CWs should not be ignored.