猪阴沟肠杆菌16SrRNA甲基化酶耐药基因分析

 【目的】分析猪阴沟肠杆菌GZL41B中4种质粒介导的16SrRNA甲基化酶耐药基因及其水平传播方式;【方法】采用PCR及序列分析方法检测鉴定猪阴沟肠杆菌中16SrRNA甲基化酶耐药基因。以大肠杆菌E.coli Rif+488(耐利福平)为受体菌进行接合试验研究16SrRNA甲基化酶耐药基因的水平传播方式。用微量稀释法测定原菌株及其接合子对18种抗菌药物的敏感性。用PCR及序列测定法分析比较来自同一猪腹泻直肠拭子阴沟肠杆菌GZL41B和大肠杆菌GZL41H的侧翼序列并对172株动物源大肠杆菌中16SrRNA甲基化酶耐药基因流行情况进行调查。【结果】从阴沟杆菌中成功扩增出目的片段,该片段经限制性内切酶HindⅢ酶切后,出现与预期的531 bp和194 bp相符的两段片段,序列测定结果证实该基因为rmtB,GenBank登录号为EF017943。以E.coli Rif+488(耐利福平)为受体菌,成功地获得了接合子,该接合子经鉴定为大肠杆菌。原菌株和接合子对卡那霉素、阿米卡星、妥布霉素、西梭米星、萘替米星、庆大霉素等4,6-二脱氧链霉胺类高度耐药,其MIC均≥256 μg?mL-1。大肠杆菌GZL41H中,rmtB的上游为Tn3转座子的部分序列,下游为肠炎沙门氏菌基因岛SGI1上融合酶编码基因groEL/intI1的部分序列orf1,而来源相同的阴沟肠杆菌GZL41B未扩增到与大肠杆菌GZL41H相同的侧翼序列。172株动物源大肠杆菌中,25株菌(15%)检出rmtB,1株菌(0.6%)检出armA;【结论】16SrRNA甲基化酶耐药基因rmtB首次出现在猪阴沟肠杆菌中,该基因介导猪阴沟肠杆菌对4,6-二脱氧链霉胺类氨基糖苷抗生素的高度耐药,并能通过接合方式在不同种属细菌间进行传播。rmtB在阴沟肠杆菌和大肠杆菌中传播的遗传背景存在差异。动物源大肠杆菌中rmtB广泛流行。

169rRNA Methylase Resistant Gene in Enterobacter cloacae from Pig Farms

【Objective】 To analyze the plasmid-mediated 16SrRNA methylase genes and their horizontal transferable mode of Enterobacter cloacae GZL42B which was isolated from pig farm. 【Method】 Polymerase chain reaction (PCR) and sequencing were applied to detect and identify the 16SrRNA methylase resistant gene. Conjugation experiments were used to study the horizontal transferable mode of 16SrRNA methylase in E. cloacae by using the E.coli Rif+488(rifampin-resistant) as the recipient. MICs were evaluated by using the micro-dilution method. Comparison of the neighboring sequences of rmtB between E. cloacae GZL42B and Escherichia coli GZL42B which isolated from the same rectal swab of one pig and the prevalence of 16SrRNA methylases genes in 172 E. coli were investigated by PCR and sequencing. 【Result】 The fragment of rmtB was amplified succeessfully. The restriction fragments were corresponded with the expectant, and the rmtB was confirmed by using sequencing. The accession number of the rmtB sequence submitted to GenBank was EF017943. Transconjugant was obtained successfully by using the E. coli Rif+488 as the recipient. The MICs of the parental E. cloacae GZL42B and its transconjugant to kanamycin, amikacin, tobramycin, sisomicin, netilmicin and gentamycin were ≥256 μg?mL-1. The right-hand end of transposon Tn3 and the orf1 which was part of groEL/intI1 on genomic island SG1 of Salmonella enterica serovar Typhimurium were located on the upstream and downstream of rmtB in Escherichia coli GZL42H, respectively. But they were not amplified in E. cloacae GZL42B. Tweenty-five and one of all 172 isolates (15% and 0.6%) were rmtB and armA positive, respectively. 【Conclusion】 It was the first report that the 16SrRNA methylase rmtB emergenced in E. cloacae from pig farm. High resistance to 4,6- deoxystreptamine was mediated by rmtB and can be transferred to E. coli Rif+488. The rmtB can be transferred between different species of bacteria. The genetic basis for dissemination of rmtB was different between E. coli GZL42H and E. cloacae GZL42B. And the rmtB was highly prevalent in animal E. coli.