NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Key Laboratory of Respiratory Disease Pathogenomics, Chinese Academy of Medical Sciences, Beijing, China.
Microbiol Spectr. 2022 Aug 31;10(4):e0101422. doi: 10.1128/spectrum.01014-22. Epub 2022 Jul 26.
With looming resistance to fluoroquinolones in Mycoplasma genitalium, public health control strategies require effective antimicrobial resistance (AMR) diagnostic methods for clinical and phenotypic AMR surveillance. We developed a novel AMR detection method, MG-AsyHRM, based on the combination of asymmetric high-resolution melting (HRM) technology and unlabeled probes, which simultaneously performs M. genitalium identification and genotypes eight mutations in the gene that are responsible for most cases of fluoroquinolone resistance. These enhancements expand the traditional HRM from the conventional detection of single-position mutations to a method capable of detecting short fragments with closely located AMR positions with a high diversity of mutations. Based on the results of clinical sample testing, this method produces an accordance of 98.7% with the Sanger sequencing method. Furthermore, the specificity for detecting S83I, S83N, S83R, and D87Y variants, the most frequently detected mutations in fluoroquinolone resistance, was 100%. This method maintained a stable and accurate performance for genomic copies at rates of ≥20 copies per reaction, demonstrating high sensitivity. Additionally, no specific cross-reactions were observed when testing eight common sexually transmitted infection (STI)-related agents. Notably, this work highlights the significant potential of our method in the field of AMR testing, with the results suggesting that our method can be applied in a range of scenarios and to additional pathogens. In summary, our method enables high throughput, provides excellent specificity and sensitivity, and is cost-effective, suggesting that this method can be used to rapidly monitor the molecular AMR status and complement current AMR surveillance. Mycoplasma genitalium was recently added to the antimicrobial-resistant (AMR) threats "watch list" of the U.S. Centers for Disease Control and Prevention because this pathogen has become extremely difficult to treat as a result of increased resistance. M. genitalium is also difficult to culture, and therefore, molecule detection is the only method available for AMR testing. In this work, we developed a novel AMR detection method, MG-AsyHRM, based on the combination of asymmetrical HRM technology and unlabeled probes, and it simultaneously performs M. genitalium identification and genotypes eight mutations in the gene that are responsible for most cases of fluoroquinolone resistance. The MG-AsyHRM method is a high-throughput, low-cost, simple, and culture-free procedure that can enhance public health and management of M. genitalium infections and AMR control, providing a strong complement to phenotypic AMR surveillance to address the spread of fluoroquinolone resistance.
随着生殖道支原体对氟喹诺酮类药物的耐药性日益严重，公共卫生控制策略需要有效的抗菌药物耐药性（AMR）诊断方法来进行临床和表型 AMR 监测。我们开发了一种新的 AMR 检测方法 MG-AsyHRM，它基于不对称高分辨率熔解（HRM）技术和未标记探针的结合，可同时进行生殖道支原体鉴定和负责大多数氟喹诺酮类药物耐药的 基因中的 8 个突变的基因分型。这些增强功能将传统 HRM 从常规的单一位点突变检测扩展到能够检测具有高度突变多样性的紧密相邻 AMR 位置的短片段的方法。基于临床样本测试结果，该方法与 Sanger 测序方法的符合率为 98.7%。此外，检测最常见的氟喹诺酮类耐药突变 S83I、S83N、S83R 和 D87Y 的特异性为 100%。该方法在基因组拷贝率为 20 个拷贝/反应以上时保持稳定和准确的性能，表现出高灵敏度。此外，在检测八种常见性传播感染（STI）相关病原体时，未观察到特定的交叉反应。值得注意的是，这项工作突出了我们的方法在 AMR 检测领域的巨大潜力，结果表明，我们的方法可以应用于多种场景和其他病原体。总之，我们的方法实现了高通量，提供了出色的特异性和灵敏度，并且具有成本效益，表明该方法可用于快速监测分子 AMR 状态，并补充当前的 AMR 监测。生殖道支原体最近被美国疾病控制与预防中心（CDC）的抗菌药物耐药性（AMR）威胁“观察名单”所收录，因为这种病原体由于耐药性的增加而变得极难治疗。此外，生殖道支原体也难以培养，因此，分子检测是 AMR 检测的唯一方法。在这项工作中，我们开发了一种新的 AMR 检测方法 MG-AsyHRM，它基于不对称 HRM 技术和未标记探针的结合，同时进行生殖道支原体鉴定和负责大多数氟喹诺酮类药物耐药的 基因中的 8 个突变的基因分型。MG-AsyHRM 方法是一种高通量、低成本、简单且无需培养的程序，可以增强公共卫生和生殖道支原体感染的管理以及 AMR 控制，为解决氟喹诺酮类耐药性的传播提供了对表型 AMR 监测的有力补充。
