Accurate antibiotic susceptibility testing is essential for successful tuberculosis treatment. Recent studies have highlighted the limitations of MIC-based phenotypic susceptibility methods in detecting other aspects of antibiotic susceptibilities in bacteria. Duration and peak of antibiotic exposure, at or above the MIC required for killing the bacterial population, has emerged as another important factor for determining antibiotic susceptibility. This is broadly defined as antibiotic tolerance. Antibiotic tolerance can further facilitate the emergence of antibiotic resistance. Currently, there are limited methods to quantify antibiotic tolerance among clinical M. tuberculosis isolates. In this study, we develop a most-probable-number (MPN)-based minimum duration of killing (MDK) assay to quantify the spectrum of M. tuberculosis rifampicin susceptibility within subpopulations based on the duration of rifampicin exposure required for killing the bacterial population. MDK₉₀–₉₉ and MDK_99.99 were defined as the minimum duration of antibiotic exposure at or above the MIC required for killing 90 to 99% and 99.99% of the initial (pretreatment) bacterial population, respectively. Results from the rifampicin MDK assay applied to 28 laboratory and clinical M. tuberculosis isolates showed that there is variation in rifampicin susceptibility among isolates. The rifampicin MDK_99/99.99 time for isolates varied from less than 2 to 10 days. MDK was correlated with larger subpopulations of M. tuberculosis from clinical isolates that were rifampicin tolerant. Our study demonstrates the utility of MDK assays to measure the variation in antibiotic tolerance among clinical M. tuberculosis isolates and further expands clinically important aspects of antibiotic susceptibility testing.

中文翻译：

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用于确定临床结核分枝杆菌分离株利福平敏感性谱的基于最可能数的最短杀灭试验持续时间

准确的抗生素敏感性测试对于成功治疗结核病至关重要。最近的研究强调了基于 MIC 的表型敏感性方法在检测细菌中抗生素敏感性的其他方面的局限性。抗生素暴露的持续时间和峰值，等于或高于杀死细菌种群所需的 MIC，已成为确定抗生素敏感性的另一个重要因素。这被广泛定义为抗生素耐受性。抗生素耐受性可以进一步促进抗生素耐药性的出现。目前，量化临床结核分枝杆菌抗生素耐受性的方法有限隔离。在这项研究中，我们开发了一种基于最大可能数 (MPN) 的最小杀伤持续时间 (MDK) 测定法，以根据杀死细菌种群所需的利福平暴露持续时间来量化亚群中结核分枝杆菌利福平敏感性的谱. MDK ₉₀ – ₉₉和 MDK _99.99被定义为分别杀死 90% 至 99% 和 99.99% 的初始（预处理）细菌群所需的 MIC 或高于 MIC 的抗生素暴露最短持续时间。对 28 株实验室和临床结核分枝杆菌分离株进行的利福平 MDK 检测结果表明，分离株之间的利福平敏感性存在差异。利福平MDK _{99/ 99.99}分离株的时间从不到 2 天到 10 天不等。MDK 与来自对利福平耐受的临床分离株的更大的结核分枝杆菌亚群相关。我们的研究证明了 MDK 测定在测量临床结核分枝杆菌分离株之间抗生素耐受性变化方面的实用性，并进一步扩展了抗生素敏感性测试的临床重要方面。