Agbavor Bernadette, Arthur Rejoice Agyeiwaa, Agbanyo Abigail, Ahiatrogah Dzifa Kofi, Akenten Charity Wiafe, Adu-Asiamah Cynthia, Abass Kabiru Mohammed, Ofori Elizabeth, Amofa George, Boampong Kwadwo, Thye Thorsten, Dekker Denise, Addo Mathew Glover, Wansbrough-Jones Mark, Bull Tim John, Amoako Yaw Ampem, Phillips Richard Odame
Department of Theoretical and Applied Biology, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
Kumasi Centre for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
BMC Microbiol. 2025 Mar 5;25(1):118. doi: 10.1186/s12866-025-03835-6.
The isolation and culture of Mycobacterium ulcerans (Mu) as a primary diagnostic modality for Buruli ulcer (BU) disease are limiting due to their low sensitivity and slow-growing nature. M. ulcerans cultures can also be overgrown with other bacteria and fungi. Culture, however, remains an important tool for the study of persisting viable M. ulcerans, drug susceptibility tests, and other molecular assays to improve management of the disease. The challenge of contamination with other fast-growing bacteria necessitates decontamination of clinical samples prior to culturing, but current methods may be too harsh, resulting in low yields of M. ulcerans. We aimed to evaluate a Tika-Kic decontamination process for M. ulcerans that uses supplements to stimulate M. ulcerans growth to improve recovery.
Swab and Fine Needle Aspirate (FNA) samples were collected from 21 individuals with confirmed BU at baseline (week 0) and weeks 2 and 4 after initiating antibiotic treatment. Samples were decontaminated with Tika-Kic decontamination medium and the modified Petroff (NaOH) methods then inoculated each into Mycobacterium Growth Indicator Tube (MGIT) or Löwenstein Jensen (LJ) medium. Time to growth detection and confirmation by qPCR as well as the proportion of positive cultures for all three methods and the proportion of positive cultures for all three time points were documented. Common contaminating bacteria were also isolated and identified.
The proportion of M. ulcerans positive cultures obtained was higher for Tika-MGIT samples [14/43 (32%)] compared to Petroff-MGIT samples [10/43 (23%)] and Petroff-LJ samples [8/43 (19%)]. Baseline samples had a higher isolate proportion [17 (53%)] compared to samples collected after treatment initiation [9 (28%) for week 2 and 6 (19%) for week 4]. Contaminating bacteria isolated include Burkholderia cepacia, Pseudomonas aeruginosa, Pasteurella pneumotropica, Proteus mirabilis, Morganella morganii, Staphylococcus aureus and Enterococcus.
Our study shows an advantage for culturing Mycobacterium ulcerans from clinical samples using the Tika-Kic decontamination and growth medium. Further research is needed to refine sample processing to improve M. ulcerans recovery.
溃疡分枝杆菌（Mu）的分离培养作为布氏杆菌溃疡（BU）病的主要诊断方式，因其敏感性低和生长缓慢的特性而受到限制。溃疡分枝杆菌培养物也可能被其他细菌和真菌过度生长。然而，培养仍然是研究持续存活的溃疡分枝杆菌、药物敏感性试验以及其他改善疾病管理的分子检测的重要工具。由于存在被其他快速生长细菌污染的挑战，在培养前需要对临床样本进行净化处理，但目前的方法可能过于严苛，导致溃疡分枝杆菌产量较低。我们旨在评估一种用于溃疡分枝杆菌的Tika-Kic净化处理方法，该方法使用补充剂来刺激溃疡分枝杆菌生长以提高回收率。
从21名确诊为BU的个体在基线（第0周）以及开始抗生素治疗后的第2周和第4周采集拭子和细针穿刺抽吸（FNA）样本。样本用Tika-Kic净化培养基和改良的彼得罗夫（氢氧化钠）方法进行净化处理，然后分别接种到分枝杆菌生长指示管（MGIT）或洛温斯坦-詹森（LJ）培养基中。记录通过qPCR检测和确认生长的时间以及所有三种方法的阳性培养比例和所有三个时间点的阳性培养比例。还分离并鉴定了常见的污染细菌。
与彼得罗夫-MGIT样本[10/43（23%）]和彼得罗夫-LJ样本[8/43（19%）]相比，Tika-MGIT样本获得的溃疡分枝杆菌阳性培养比例更高[14/43（32%）]。与治疗开始后采集的样本相比，基线样本的分离比例更高[第2周为9（28%），第4周为6（19%）]，而基线样本的分离比例为17（53%）。分离出的污染细菌包括洋葱伯克霍尔德菌、铜绿假单胞菌、嗜肺巴斯德杆菌、奇异变形杆菌、摩根摩根菌、金黄色葡萄球菌和肠球菌。
我们的研究表明，使用Tika-Kic净化和生长培养基从临床样本中培养溃疡分枝杆菌具有优势。需要进一步研究优化样本处理以提高溃疡分枝杆菌的回收率。
