低浓度恩诺沙星对离体恒化器模型中人体肠道菌群的影响

Effects of Enrofloxacin on Human Intestinal Mocrofloa in Chemostat Model

【目的】应用离体恒化器模型评价低浓度恩诺沙星对人体肠道菌群的影响,为制定微生物学日允许摄入量(ADI)提供依据。【方法】四套接种人体肠道菌群的离体恒化器模型中连续添加0、0.2、2及20mg·L-1恩诺沙星8d,采用细菌培养计数和PCR-DGGE方法研究细菌数量、耐药性、菌群结构和定植抗力的变化。【结果】2及20mg·L-1恩诺沙星可抑制肠道总厌氧菌、总需氧菌和兼性厌氧菌、大肠杆菌、肠球菌的生长(P<0.05或P<0.01);0.2、2及20mg·L-1恩诺沙星均使总需氧和兼性厌氧菌、大肠杆菌耐药百分率增加(P<0.05或P<0.01),停药7d后,总需氧和兼性厌氧菌、大肠杆菌耐药百分率呈下降趋势,但与对照组相比仍处于较高水平(P<0.01)。20mg·L-1恩诺沙星对肠道菌群结构的影响较大,使PCR-DGGE图谱发生明显变化,条带数、菌群多态性和相似性参数降低(P<0.05或P<0.01);2和20mg·L-1恩诺沙星可使肠道菌群对外源沙门氏菌的定植抗力明显下降。【结论】0.2mg·L-1恩诺沙星(相当于3.67μg·kg-1体重)可对离体恒化器模型中的人体肠道菌群耐药性产生显著影响,说明中国现行规定的恩诺沙星和环丙沙星的ADI(6.2μg·kg-1体重)可能会对人体肠道菌群产生影响,主要为选择出耐药需氧和兼性厌氧菌。

【Objective】 The effects of low concentrations of enrofloxacin on human intestinal microflora were evaluated by using chemostat models of the human large bowel,which would be helpful for establishing the microbiological ADI of enrofloxacin.【Method】 Four steady state microbial communities were established in chemostats using inocula from human feces.Three chemostats were exposed to enrofloxacin（0.2,2,and 20 mg·L^-1,respectively） for 8 days;one served as a no-drug control.Changes of bacterial populations,the relative levels of resistant aerobic and facultative anaerobes to ciprofloxacin,colonization resistance against exogenous Salmonella,and the microflora structure after drug exposure were assayed by plate counting method and PCR-DGGE.【Result】 Results showed that 2 mg·L^-1 and 20 mg·L^-1 enrofloxacin significantly reduced flora populations of total aerobic and facultative anaerobes,Escherichia.coli,Enterococcus,and the total anaerobic bacteria（P〈0.05 or P〈0.01）.The percentages of resistant aerobic and facultative anaerobes and E.coli were increased significantly after treatment with 0.2,2 and 20 mg·L^-1 enrofloxacin（P〈0.05 or P〈0.01）.Seven days after treatment,the percentages of resistant aerobic and facultative anaerobes,and E.coli were decreased,but still higher than the control group（P〈0.01）.The number,Shannon＇s diversity index and Sorenson＇s similarity index of PCR-DGGE lanes decreased significantly after treatment with 20 mg·L^-1 enrofloxacin（P〈0.05 or P〈0.01）.The colonization barrier against Salmonella was disrupted by 2 mg·L^-1 and 20 mg·L^-1 enrofloxacin.【Conclusion】 0.2 mg·L^-1 enrofloxacin（equal to 3.67 μg·kg^-1 b.w.） can increase the percentage of resistant aerobic flora to ciproflxacin.Thus the ADI（6.2 μg·kg^-1 b.w.） of enrofloxacin and ciprofloxacin established by Ministry of Agriculture may affect human intestinal microflora by selecting resistant aerobic flora.