Huaman Jose L, Bradshaw Catriona S, Chua Teck-Phui, Plummer Erica L, Danielewski Jennifer A, Vodstrcil Lenka A, Jensen Jorgen S, Garland Suzanne M, Wild Natasha, Murray Gerald L
Department of Obstetrics, Gynaecology and Newborn Health, University of Melbourne, Parkville, Victoria, Australia.
Centre for Women's Infectious Diseases, The Royal Women's Hospital, Parkville, Victoria, Australia.
J Med Microbiol. 2025 Jul;74(7). doi: 10.1099/jmm.0.002040.
, a small and slow-growing bacterium, has gained notoriety due to rapidly increasing rates of antibiotic resistance. is difficult to culture, limiting efforts to understand its biology and the mechanisms of antimicrobial resistance. To understand factors that influence success in primary isolation of from clinical samples. Neat urine or swabs (high vaginal and anal, in universal transport medium) were collected from patients with confirmed or suspected infections attending the Melbourne Sexual Health Centre. The specimens were stored at -80 °C prior to laboratory analysis. Initial diagnosis was by transcription-mediated amplification (TMA) assay, and samples subsequently testing positive by quantitative PCR (qPCR) were washed twice and inoculated into Vero cell monolayers with a selective antibiotic mixture (cycloheximide and Thayer-Martin Medium I). Cultures were incubated at 37 °C with 5% CO for 8 weeks and observed daily, with qPCR used to monitor growth. In total, 127 TMA-positive samples were subjected to qPCR, and genomic DNA (gDNA) was detected in 53.5% (68/127) of these samples. An isolate was obtained from 26.5% (18/68) of the gDNA-positive samples following co-culture with Vero cells. The isolation rate varied between sample types, with growth detected in 12.5% (3/24) of the high vaginal swabs and 37.5% (15/40) of the urine samples. No isolates were obtained from anal swabs. The proportion with a successful culture was influenced by the initial load in the sample, which translated into the inoculum size for the Vero cell monolayer. Isolation was unsuccessful with low inoculum (<2,000 genome equivalents, geq), partially successful (13.3%) with a moderate inoculum (2,500-9,500 geq) and highly successful (100%) in samples with a high inoculum (>10,000 geq). The initial bacterial load emerged as a critical determinant of isolation success. This emphasizes the importance of optimizing sample collection and isolation procedures.
一种小型且生长缓慢的细菌，由于抗生素耐药率迅速上升而声名狼藉。它难以培养，这限制了人们对其生物学特性和抗菌耐药机制的了解。为了了解影响从临床样本中初次分离该细菌成功率的因素，从墨尔本性健康中心确诊或疑似感染该细菌的患者中收集了纯净尿液或拭子（高阴道拭子和肛门拭子，置于通用运输培养基中）。在进行实验室分析之前，标本保存在 -80°C。初始诊断通过转录介导扩增（TMA）检测，随后通过定量PCR（qPCR）检测呈阳性的样本被洗涤两次，并接种到含有选择性抗生素混合物（放线菌酮和塞耶 - 马丁培养基I）的Vero细胞单层中。培养物在37°C、5% CO₂条件下孵育8周，每天观察，并使用qPCR监测生长情况。总共127个TMA阳性样本进行了qPCR检测，其中53.5%（68/127）的样本检测到该细菌的基因组DNA（gDNA）。在与Vero细胞共培养后，从26.5%（18/68）的gDNA阳性样本中获得了分离株。分离率因样本类型而异，在高阴道拭子中12.5%（3/24）检测到生长，在尿液样本中37.5%（15/40）检测到生长。从肛门拭子中未获得分离株。成功培养的比例受样本中初始细菌载量的影响，这转化为Vero细胞单层的接种量。接种量低（<2000基因组当量，geq）时分离不成功，接种量中等（2500 - 9500 geq）时部分成功（13.3%），接种量高（>10000 geq）的样本中高度成功（100%）。初始细菌载量成为分离成功的关键决定因素。这强调了优化样本采集和细菌分离程序的重要性。
