耐大环内酯类药物鸡毒支原体的诱导和耐药基因的突变分析
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摘要
在含有递增浓度红霉素或替米考星的液体培养基中连续传代培养鸡毒支原体S6和BG44T药物敏感菌株，使其逐渐产生耐药性，然后筛选出高度耐药菌株。红霉素对筛选出的S6耐红霉素株和BG447耐红霉素株的MIC≥436.7 μg／ml，替米考星对筛选出的S6耐替米考星和BG44T耐替米考星的MIC≥400μg／ml。耐红霉素的S6和BG44T菌株同时对替米考星、泰乐菌素、林可霉素有交叉耐药性。耐替米考星的S6和BG44T菌株对红霉素、泰乐菌素、林可霉素有交叉耐药性。
23SrRNAV区域基因突变与细菌产生大环内酯类耐药性有关。为了检测鸡毒支原体的23SrRNAV区域基因是否发生耐药突变，首先把鸡毒支原体上处于两个不同位置的23SrRNAV区域基因(论文中分别将这两处基因称为23rrn1和23rrn2)区分开来。对鸡毒支原体酶切产物进行琼脂糖凝胶电泳，将凝胶上的DNA转移到尼龙膜上，进行杂交，确定了含有23SrRNAV区域基因的限制片段的位置。将这些限制片段分离出来，进行PCR扩增和克隆。结果表明S6耐红霉素菌株的23rrn1在2058位发生了耐药相关突变，rrn2未发生耐药相关突变。S6耐替米考星株的23rrn1未发生耐药相关突变，23rrn2在2058、2504处发生了耐药相关突变。BG44T耐红霉素的23rrn1发生了2458位耐药相关突变，23rrn2发生了2057、2059位耐药相关突变。BG44T耐替米考星株的23rrn1未发生耐药相关突变，23rrn2发生了2504、2058位耐药相关突变。
Erythromycin-resistant mutants and tilmicosin-resistant mutants were obtained in vitro from mycoplasma gallisepticum(MG)S6 and BG44T by increasing concemtrations of erythromycin and tilmicosin.The MICs of erythromycin for erythromycin-resistant S6 and BG44T are more than 436.7 u g/ ml. The MICs of tilmicosin for tilmicosin-resistant S6 and BG44T are more than 400 u g/ ml. Erythromycin-resistant S6 and BG44T showed cross-resistance to tilmicosin,tylosin and lincomycin. Tilmicosin-resistant S6 and BG44T showed cross-resistance to erythromycin,tylosin and lincomycin.
Resistance by ribosomal mutations in domain V of 23S rRNA has been described.To determine the role of 23S rRNA in macrolide resistance in MG,we firstly differentiated the 23 S rRNA gene in the two ribosomal operons in MG(These two genes were named by 23rrnl and 23rrn2 respectively in mis article).MG DNA was digested by a restrictive endonuclease.electrophoresed in agarose gel, and transfered the DNA from the agarose gel to nylon membrane.To ascertain the positions of the two 23S rRNA genes,Southern hybridization was performed. 23S rRNA genes from the agarose gel were isolated, amplified, and cloned of 23S rRNA V genes were performed from isolated 23S rRNA genes.Cloned these genes.Within the 23rrnl, erythromycin-resistant S6 contained the 2058 mutation that was associated with resistance. For erythromycin-resistant S6, 23rrn2 was unchanged.And for tilmicosin-resistant S6, 23rrnl was unchanged and 23rrn2 harbored 2058,2504 mutation that was associated with resistance. Erythromycin-resistant BG44T occurred 2458 resistant point mutation in 23rrnl that was associated with resistance and 2057 , 2058 resistant point mutation that was associated with resistance in 23rrn2.Tilmicosin-resistant BG44T did not show resistant point mutation in 23rrnl and 2504,2058 point mutation that was associated with resistance in 23rrn2.
23SrRNAV区域基因突变与细菌产生大环内酯类耐药性有关。为了检测鸡毒支原体的23SrRNAV区域基因是否发生耐药突变，首先把鸡毒支原体上处于两个不同位置的23SrRNAV区域基因(论文中分别将这两处基因称为23rrn1和23rrn2)区分开来。对鸡毒支原体酶切产物进行琼脂糖凝胶电泳，将凝胶上的DNA转移到尼龙膜上，进行杂交，确定了含有23SrRNAV区域基因的限制片段的位置。将这些限制片段分离出来，进行PCR扩增和克隆。结果表明S6耐红霉素菌株的23rrn1在2058位发生了耐药相关突变，rrn2未发生耐药相关突变。S6耐替米考星株的23rrn1未发生耐药相关突变，23rrn2在2058、2504处发生了耐药相关突变。BG44T耐红霉素的23rrn1发生了2458位耐药相关突变，23rrn2发生了2057、2059位耐药相关突变。BG44T耐替米考星株的23rrn1未发生耐药相关突变，23rrn2发生了2504、2058位耐药相关突变。
Erythromycin-resistant mutants and tilmicosin-resistant mutants were obtained in vitro from mycoplasma gallisepticum(MG)S6 and BG44T by increasing concemtrations of erythromycin and tilmicosin.The MICs of erythromycin for erythromycin-resistant S6 and BG44T are more than 436.7 u g/ ml. The MICs of tilmicosin for tilmicosin-resistant S6 and BG44T are more than 400 u g/ ml. Erythromycin-resistant S6 and BG44T showed cross-resistance to tilmicosin,tylosin and lincomycin. Tilmicosin-resistant S6 and BG44T showed cross-resistance to erythromycin,tylosin and lincomycin.
Resistance by ribosomal mutations in domain V of 23S rRNA has been described.To determine the role of 23S rRNA in macrolide resistance in MG,we firstly differentiated the 23 S rRNA gene in the two ribosomal operons in MG(These two genes were named by 23rrnl and 23rrn2 respectively in mis article).MG DNA was digested by a restrictive endonuclease.electrophoresed in agarose gel, and transfered the DNA from the agarose gel to nylon membrane.To ascertain the positions of the two 23S rRNA genes,Southern hybridization was performed. 23S rRNA genes from the agarose gel were isolated, amplified, and cloned of 23S rRNA V genes were performed from isolated 23S rRNA genes.Cloned these genes.Within the 23rrnl, erythromycin-resistant S6 contained the 2058 mutation that was associated with resistance. For erythromycin-resistant S6, 23rrn2 was unchanged.And for tilmicosin-resistant S6, 23rrnl was unchanged and 23rrn2 harbored 2058,2504 mutation that was associated with resistance. Erythromycin-resistant BG44T occurred 2458 resistant point mutation in 23rrnl that was associated with resistance and 2057 , 2058 resistant point mutation that was associated with resistance in 23rrn2.Tilmicosin-resistant BG44T did not show resistant point mutation in 23rrnl and 2504,2058 point mutation that was associated with resistance in 23rrn2.
引文
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