Prammananan T, Sander P, Springer B, Böttger E C
Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.
Antimicrob Agents Chemother. 1999 Mar;43(3):447-53. doi: 10.1128/AAC.43.3.447.
Clinical resistance to aminoglycosides in general is due to enzymatic drug modification. Mutational alterations of the small ribosomal subunit rRNA have recently been found to mediate acquired resistance in bacterial pathogens in vivo. In this study we investigated the effect of 16S rRNA heterozygosity (wild-type [wt] and mutant [mut] operons at position 1408 [1408wt/1408mut]) on aminoglycoside resistance. Using an integrative vector, we introduced a single copy of a mutated rRNA operon (1408 A-->G) into Mycobacterium smegmatis, which carries two chromosomal wild-type rRNA operons; the resultant transformants exhibited an aminoglycoside-sensitive phenotype. In contrast, introduction of the mutated rRNA operon into an M. smegmatis rrnB knockout strain carrying a single functional chromosomal wild-type rRNA operon resulted in aminoglycoside-resistant transformants. Subsequent analysis by DNA sequencing and RNase protection assays unexpectedly demonstrated a homozygous mutant genotype, rRNAmut/rRNAmut, in the resistant transformants. To investigate whether RecA-mediated gene conversion was responsible for the aminoglycoside-resistant phenotype in the rRNAwt/rRNAmut strains, recA mutant strains were generated by allelic exchange techniques. Transformation of the recA rrnB M. smegmatis mutant strains with an integrative vector expressing a mutated rRNA operon (Escherichia coli position 1408 A-->G) resulted in transformants with an aminoglycoside-sensitive phenotype. Subsequent analysis showed stable heterozygosity at 16S rRNA position 1408 with a single wild-type allele and a single resistant allele. These results demonstrate that rRNA-mediated mutational resistance to aminoglycosides is recessive.
一般来说，对氨基糖苷类药物的临床耐药性是由于酶对药物的修饰作用。最近发现，小核糖体亚基rRNA的突变改变可在体内介导细菌病原体获得性耐药。在本研究中，我们调查了16S rRNA杂合性（1408位的野生型[wt]和突变型[mut]操纵子[1408wt/1408mut]）对氨基糖苷类耐药性的影响。我们使用一个整合载体，将一个突变的rRNA操纵子（1408 A→G）的单拷贝导入携带两个染色体野生型rRNA操纵子的耻垢分枝杆菌中；所得转化子表现出对氨基糖苷类药物敏感的表型。相反，将突变的rRNA操纵子导入携带单个功能性染色体野生型rRNA操纵子的耻垢分枝杆菌rrnB基因敲除菌株中，产生了对氨基糖苷类药物耐药的转化子。随后通过DNA测序和RNase保护试验进行的分析意外地表明，耐药转化子中存在纯合突变基因型rRNAmut/rRNAmut。为了研究RecA介导的基因转换是否是rRNAwt/rRNAmut菌株中氨基糖苷类耐药表型的原因，我们通过等位基因交换技术构建了recA突变菌株。用表达突变rRNA操纵子（大肠杆菌1408位A→G）的整合载体转化recA耻垢分枝杆菌rrnB突变菌株，得到了对氨基糖苷类药物敏感表型的转化子。随后的分析表明，在16S rRNA的1408位存在稳定的杂合性，有一个野生型等位基因和一个耐药等位基因。这些结果表明，rRNA介导的对氨基糖苷类药物的突变耐药性是隐性的。
