Hall Leslie, Jude Kurt P, Clark Shirley L, Wengenack Nancy L
Department of Laboratory Medicine and Pathology, Mayo Clinic.
J Vis Exp. 2011 Jun 24(52):3094. doi: 10.3791/3094.
The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioMérieux, Hazelwood, MO). The agar proportion method, while considered the "gold" standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is ≥1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.
快速检测抗菌药物耐药性对于控制耐药结核分枝杆菌（Mtb）数量的增加至关重要。传统上，Mtb的药敏试验（AST）是通过比例琼脂法或在BACTEC（Becton Dickinson，Sparks，MD）、VersaTREK（TREK Diagnostics，Cleveland，OH）或BacT/ALERT（bioMérieux，Hazelwood，MO）等仪器上进行的宏肉汤试验来进行的。琼脂比例法虽然被认为是AST的“金”标准，但劳动强度大，需要通过在含药琼脂上进行菌落计数并与不含药琼脂进行比较来计算耐药性。如果与不含药培养基相比，含药培养基上的生长率≥1%，则该菌株被认为对该药物耐药。宏肉汤法需要仪器以及一线药物（异烟肼、乙胺丁醇、利福平、吡嗪酰胺）的测试断点（“临界”）药物浓度。这里描述的方法以96孔微量滴定板形式（[MYCOTB（TREK Diagnostics）]）在市场上有售，其中包含用于治疗结核病的12种抗菌药物的浓度递增组合，包括一线药物（异烟肼、利福平、乙胺丁醇）和二线药物（阿米卡星、环丝氨酸、乙硫异烟胺、卡那霉素、莫西沙星、氧氟沙星、对氨基水杨酸、利福布汀、链霉素）。由于吡嗪酰胺需要酸性测试条件，因此一线药物吡嗪酰胺不包含在微量滴定板中。与传统琼脂比例法（21天）相比，微量滴定系统的优点包括设置简便且周转时间更快（14天）。此外，可以使用肉汤或固体培养基制备的接种物来设置平板。由于微量滴定板形式是新的，并且由于Mtb在实验室中带来独特的安全挑战，本方案将描述如何安全地设置、孵育和读取微量滴定板。
