Department of Phthisiology and Pulmonology, Samara State Medical University, Samara, Russia.
Professional Center for Education and Research in Genetic and Laboratory Technologies, Samara State Medical University, Samara, Russia.
Int J Mycobacteriol. 2024 Jul 1;13(3):252-257. doi: 10.4103/ijmy.ijmy_135_24. Epub 2024 Sep 14.
Mycobacterium abscessus complex (MABSc) causes chronic infection in patients with concomitant structural changes in the respiratory tract, which is especially important for patients with cystic fibrosis. To isolate an MABSc culture from clinical material, a variety of nutrient media are used. For species determination of microorganisms isolated on these media, additional identification methods are used, for example, polymerase chain reaction, sequencing, or mass spectrometry. The latter method is relatively easy to implement but requires improvement, due to the identification inaccuracy of nontuberculosis mycobacterias in general. Consequently, a set of nutrient media may be important for subsequent identification by mass spectrometry.
The study was conducted on 64 strains of MABSc representatives: 56 strains were obtained from patients with cystic fibrosis and 8 strains from patients with pulmonary pathology unrelated to cystic fibrosis. The obtained MABSc strains were transplanted to the universal chromogenic medium and the selective medium for the Burkholderia cepacia complex (BCC) isolation. Species identification was carried out by mass spectrometry based on matrix-activated laser time-of-flight desorption/ionization (MALDI-ToF MS). Microbial identification is based on a comparison of the obtained mass spectra with reference spectra from the database. Microorganisms were identified based on the coincidence degree (Score value). Sample preparation for microbial identification by mass spectrometry was carried out by an extended direct application method. Fragments of the rpoB and hsp65 genes with lengths of 752 bp and 441 bp, respectively, were used as molecular markers for subspecific identification of MABSc strains.
A comparison of the peaks obtained after mass spectrometry of MABSc strains isolated on the studied nutrient media showed significant differences between these indicators selective medium for the BCC isolation with the supplement of iron polymaltose hydroxide (III) and universal chromogenic medium (P < 0.001) and selective medium for the BCC isolation with universal chromogenic medium (P < 0.001). Twenty-five strains of MABSc representatives were sequenced: results of subspecies determination in strains isolated on the universal chromogenic medium coincided with the results sequencing in 13 (86.6%) strains out of 15.
MALDI-ToF mass spectrometry allows microbial identification in a short time and with minimal cost, but it does not yet allow the proper identification of the subspecies of certain microbial groups, such as MABSc. Cultivation methods need optimization and new approaches to the extraction process of the bacterial protein fraction.
脓肿分枝杆菌复合体（MABSc）可导致伴有呼吸道结构改变的患者发生慢性感染，这对囊性纤维化患者尤为重要。为了从临床标本中分离 MABSc 培养物，使用了多种营养培养基。对于在这些培养基上分离的微生物的种属鉴定，使用了额外的鉴定方法，例如聚合酶链反应、测序或质谱。后一种方法相对容易实施，但由于一般来说非结核分枝杆菌的鉴定不准确，因此需要改进。因此，对于随后的质谱鉴定，一组营养培养基可能很重要。
本研究对 64 株 MABSc 代表菌株进行了研究：56 株来自囊性纤维化患者，8 株来自与囊性纤维化无关的肺部病理患者。将获得的 MABSc 菌株移植到通用显色培养基和用于分离伯克霍尔德菌属复合群（BCC）的选择性培养基上。通过基质辅助激光解吸/电离时间飞行（MALDI-ToF MS）的质谱进行物种鉴定。微生物鉴定基于与数据库中的参考光谱进行比较。根据吻合度（Score 值）对微生物进行鉴定。微生物的质谱鉴定样品制备采用扩展直接应用方法进行。使用长度分别为 752bp 和 441bp 的 rpoB 和 hsp65 基因片段作为 MABSc 菌株亚种鉴定的分子标记。
对研究营养培养基上分离的 MABSc 菌株进行质谱分析后获得的峰的比较表明，这些指标之间存在显著差异，选择性培养基补充铁多聚糖氢氧化铁（III）与通用显色培养基（P < 0.001）和选择性培养基与通用显色培养基（P < 0.001）。对 25 株 MABSc 代表菌株进行测序：在通用显色培养基上分离的菌株的亚种鉴定结果与 15 株中的 13 株（86.6%）的测序结果一致。
MALDI-ToF 质谱允许在短时间内以最小的成本进行微生物鉴定，但仍不能正确鉴定某些微生物群体的亚种，如 MABSc。培养方法需要优化，并且需要新的细菌蛋白部分提取方法。
