强力霉素抗性基因目标供体菌、受体菌和接合子的筛选及鉴定

Screening and identification of target donors, recipients and conjugates of Doxycycline resistance gene

目的为进一步研究环境中病原微生物目标供体菌强力霉素抗性基因(Dox)水平传播机制奠定基础。方法以强力霉素抗性基因为筛选指标,通过药敏试验从7个菌株中筛选出遗传型分别为Dox^RX^S的候选供体菌和Dox^SX^R的候选受体菌(X代表不是强力霉素的抗生素),将供、受体菌共培养后筛选出遗传型为Dox^RX^R的接合子。分别测定X对目标供体菌、Dox对目标受体菌的最小抑菌浓度。对目标供、受体菌从形态学、生理生化、分子生物学和BIOLOG方面进行鉴定。结果研究的7个病原菌均为多重耐药,最多能耐受15种抗生素,对链霉素、萘啶酮酸和磺胺二甲嘧啶的耐药率最高(100%),对庆大霉素、诺氟沙星和环丙沙星的耐药率最低(0%)。目标供体菌、受体菌和接合子Con-Ⅱ基因型分别为Dox^RKan^S、Dox^SKan^R和Dox^RKan^R。Kan对目标供体菌的最小抑菌浓度为0.310 0μg/mL,Dox对目标受体菌的最小抑菌浓度为0.048 8μg/mL。目标供体菌(Dox^RKan^S)鉴定为Escherichia coli O157:H7(E.coli O157:H7),目标受体菌TR-M30-1(Dox^SKan^R)鉴定为产酸克雷伯菌。结论目标供体菌为E.coli O157:H7(Dox^RKan^S),目标受体菌为TR-M30-1(Dox^SKan^R),接合子为Con-Ⅱ(Dox^RKan^R)。

Objective To lay a foundation for further study on the horizontal transmission mechanism of Doxycycline resistance gene(Dox)of target donor strains in the environment.Methods Candidate-donors(Dox^RX^S)and candidate-recipients(Dox^SX^R)were selected(X represents any of the 18 antibiotics tested except for Dox)through the drug susceptibility test on 7 bacterial pathogens,in which the Dox was used as the criterion of selection.After the conjugation of donor and recipient bacteria,the zygotes(Dox^RX^R)were isolated.The minimum inhibitory concentration of two antibiotics to the target donor and recipient bacteria was detected.The target donor and recipient bacteria were identified using morphological,physiological,biochemical,molecular methods and BIOLOG method.Results All of the seven pathogens could tolerate multiple antibiotics(up to 15 antibiotics).The drug resistance rates to Streptomycin(aminoglycosides),Nalidixic acid(quinolones)and Sulfamethazine(sulfonamides)were 100%,but those to Gentamicin(aminoglycosides),Norfloxacin(quinolones)and Ciprofloxacin(quinolones)were 0%.The phenotypes of target donor,target recipient and zygote(named as Con-II)were Dox^RKan^S,Dox^SKan^R and Dox^RKan^R respectively.The minimum inhibitory concentration of Kan to target donor was 0.3100μg/mL and that of Dox to target recipient was 0.0488μg/mL.The target donor with phenotype of Dox^RKan^S was identified as Escherichia coli O157:H7.The target recipient TR-M30-1(Dox^SKan^R)was identified as Klebsiella acidophilus.Conclusion The target donor strain,target recipient strain and zygote are E.coli O157:H7(Dox^RKan^S),TR-M30-1(Dox^SKan^R)and Con-II(Dox^RKan^R)respectively.