Erwinia amylovora can rapidly become highly resistant to applications of the antibiotic streptomycin through a single nucleotide mutation in the rpsL gene, which causes an amino acid substitution that prevents inhibitory binding of streptomycin while preserving the functionality of the ribosome. Several viable mutations are described in the literature, but screenings in the orchards consistently revealed that the one leading to the substitution of lysin by arginine at position 43 (K43R) is by far the most abundant. In this work, we tested the rate of emergence of the various spontaneous mutants, their tolerance to different antibiotic concentrations and their general fitness in competition experiments. Furthermore, we developed a molecular assay based on the SNaPshot™ Multiplex Kit from Thermo Fisher Scientific that allows swift determination of rpsL mutations using a single-base primer extension approach directly from colonies on plate. Or results indicate that the prevalence of variant K43R is largely due to fact that it retains the highest environmental fitness even in absence of antibiotic pressure. Sequence analysis of two resistant colonies that did not respond as expected in the SNaPshot™ assay revealed the existence of two yet undescribed rpsL mutations resulting in conditional-lethal streptomycin-dependent (SmD) phenotypes, which are unable to grow in absence of the antibiotic.

中文翻译：

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导致解淀粉欧文菌高链霉素耐药性的 rpsL 突变的表征和直接分子鉴别

通过 rpsL 基因中的单核苷酸突变，解淀粉欧文氏菌可以迅速对抗生素链霉素的应用产生高度抗性，这会导致氨基酸取代，防止链霉素的抑制性结合，同时保留核糖体的功能。文献中描述了几种可行的突变，但果园中的筛选一致表明，导致第 43 位精氨酸 (K43R) 处的溶素取代溶素的突变是迄今为止最丰富的。在这项工作中，我们测试了各种自发突变体的出现率、它们对不同抗生素浓度的耐受性以及它们在竞争实验中的一般适应性。此外，我们基于 Thermo Fisher Scientific 的 SNaPshot™ Multiplex Kit 开发了一种分子检测方法，可以使用单碱基引物延伸方法直接从板上菌落快速确定 rpsL 突变。或者结果表明，变异 K43R 的流行主要是因为它即使在没有抗生素压力的情况下也能保持最高的环境适应性。对 SNaPshot™ 检测中未按预期响应的两个抗性菌落的序列分析显示，存在两个尚未描述的 rpsL 突变，导致条件致死链霉素依赖 (SmD) 表型，在没有抗生素的情况下无法生长。或者结果表明，变异 K43R 的流行主要是因为它即使在没有抗生素压力的情况下也能保持最高的环境适应性。对 SNaPshot™ 检测中未如预期响应的两个抗性菌落的序列分析显示，存在两个尚未描述的 rpsL 突变，导致条件致死链霉素依赖 (SmD) 表型，在没有抗生素的情况下无法生长。或者结果表明，变异 K43R 的流行主要是因为它即使在没有抗生素压力的情况下也能保持最高的环境适应性。对 SNaPshot™ 检测中未如预期响应的两个抗性菌落的序列分析显示，存在两个尚未描述的 rpsL 突变，导致条件致死链霉素依赖 (SmD) 表型，在没有抗生素的情况下无法生长。