多重耐药质粒RP4在膜生物反应器中的水平转移

HORIZONTAL TRANSFER OF ANTIBIOTIC-MULTIRESISTANT PLASMID RP4 IN MEMBRANE BIO-REACTOR

目的研究多重耐药质粒RP4在膜生物反应器(MBR)中水平转移情况,进而预测MBR处理含有耐药基因污水的生态风险。方法向正常运行的MBR中接种E.coli K12(RP4)Rif,利用含有不同抗生素的营养琼脂培养基选择性平板计数,得出可培养接合子数量和接合转移率;并利用实时定量PCR方法检测污泥中耐药质粒RP4;同时监测MBR对NH4+-N、CODCr的去除效率和污泥浓度(MLSS),以评价MBR运行效能。结果接种供体菌后,可培养接合子数量、转移频率由0逐渐升高,接种供体菌后第9 d,接合子数量增加到3.38×104cfu/ml、接合转移率达1.70×10-3/cell;随后,可培养接合子数量、接合转移率都呈现波动下降(于第23天分别下降至2.55×102cfu/ml、3.20×10-5/cell);与实时定量PCR检测结果基本一致。供体菌的引入对MBR去除NH4+-N、CODCr的效率都有一定影响,即先降低后逐渐升高,污泥浓度由2200 mg/L逐渐降低至约1100 mg/L。结论实验过程中,RP4质粒在反应器中发生了较高频率的接合转移,并对反应器NH4+-N、CODCr的去除效率都产生影响。

Objective To investigate the horizontal transfer of multiresistant plasmid in Membrane Bio-reactor （MBR） in order to predict the ecology hazards of MBR dealing with sewage containing antibiotic resistance genes, and to provide the scientific basis for controlling their proliferation. Methods The donor strain, Escherichia coli K12（RP4） Rif, was added into MBR whose performance was stable. In order to obtain the amount of cultivable transconjugants and transfer frequency of plasmid RP4, the samples were enumerated using nutrient agar containing different kinds of antibiotics. The quantities of plasmid RP4 which was carried by activated sludge bacteria was detected by real time-PCR. And to eval- uate the operation performance of MBR, the content of mixed liquid suspended solid （MLSS）, the removal efficiency of NH4 ＋ -N, and chemical oxygen demand by chromium method（CODcr） was detected as well. Results The number of cultivable transconjugants and transfer frequency from zero increased gradually when donor strain was added into the MBR. The amount of cultivable transconjugants increased to 3.38 ×10^4 cfu/ml, and transfer frequency to 1.7 × 10^-3/cell on the 9th day after inoculating donor strain, and declined with fluctuation later on （decreased to 2.55 × 10^2 cfu/ml, 3.20 ×10^-5/cell respectively on the 23rd day）. These results were consistent with the results of real time-PCR detection. After inoculating donor strain, the NH4 ^＋-N and CODcr removal efficiency decreased at first and then rose, and MLSS descended from 2200 mg/L to about 1100 mg/L progressively. Conclusions A higher frequency of horizontal conjugation mediated by antibiotic-multiresistant plasmid RP4 in the MBR was found, and the NH4^＋ -N and CODcr removal efficiency was decreased at first and then recovered.