Quantifying the Adaptive Potential of an Antibiotic Resistance Enzyme

For a quantitative understanding of the process of adaptation, we need to understand its “raw material,” that is, the frequency and fitness effects of beneficial mutations. At present, most empirical evidence suggests an exponential distribution of fitness effects of beneficial mutations, as predicted for Gumbel-domain distributions by extreme value theory. Here, we study the distribution of mutation effects on cefotaxime (Ctx) resistance and fitness of 48 unique beneficial mutations in the bacterial enzyme TEM-1 β-lactamase, which were obtained by screening the products of random mutagenesis for increased Ctx resistance. Our contributions are threefold. First, based on the frequency of unique mutations among more than 300 sequenced isolates and correcting for mutation bias, we conservatively estimate that the total number of first-step mutations that increase Ctx resistance in this enzyme is 87 [95% CI 75–189], or 3.4% of all 2,583 possible base-pair substitutions. Of the 48 mutations, 10 are synonymous and the majority of the 38 non-synonymous mutations occur in the pocket surrounding the catalytic site. Second, we estimate the effects of the mutations on Ctx resistance by determining survival at various Ctx concentrations, and we derive their fitness effects by modeling reproduction and survival as a branching process. Third, we find that the distribution of both measures follows a Fréchet-type distribution characterized by a broad tail of a few exceptionally fit mutants. Such distributions have fundamental evolutionary implications, including an increased predictability of evolution, and may provide a partial explanation for recent observations of striking parallel evolution of antibiotic resistance.

为定量解析适应性演化过程，我们需要明晰其“原始材料”——即有益突变的发生频率与适合度效应。当前多数实验证据表明，有益突变的适合度效应服从指数分布，这与极值理论针对冈贝尔域（Gumbel-domain）分布的预测相符。本研究聚焦细菌酶TEM-1 β-内酰胺酶中48种独特有益突变对头孢噻肟（cefotaxime, Ctx）抗性及适合度的影响分布，这些突变通过筛选随机诱变产物以获得更高Ctx抗性的方式获得。本研究的贡献可分为三点：其一，基于300余份测序分离株中独特突变的发生频率，并校正突变偏倚，我们保守估计该酶中可提升Ctx抗性的单步突变总数量为87个[95%置信区间：75–189]，占全部2583种可能碱基替换的3.4%。在48种突变中，10种为同义突变，38种非同义突变的多数分布于催化位点周围的结合口袋中。其二，我们通过测定不同Ctx浓度下的菌株存活率，量化突变对Ctx抗性的影响，并通过将繁殖与存活建模为分支过程，推导其适合度效应。其三，我们发现两类指标的分布均服从弗雷歇型（Fréchet-type）分布，其特征为存在少数极端适配突变构成的宽尾区域。这类分布具有重要的演化生物学意义，包括提升演化过程的可预测性，同时可为近期观测到的抗生素抗性显著平行演化现象提供部分解释。