Factors that contribute to the success of primary isolation of Mycoplasma genitalium from clinical samples.

IF 2
Jose L Huaman, Catriona S Bradshaw, Teck-Phui Chua, Erica L Plummer, Jennifer A Danielewski, Lenka A Vodstrcil, Jorgen S Jensen, Suzanne M Garland, Natasha Wild, Gerald L Murray
{"title":"Factors that contribute to the success of primary isolation of <i>Mycoplasma genitalium</i> from clinical samples.","authors":"Jose L Huaman, Catriona S Bradshaw, Teck-Phui Chua, Erica L Plummer, Jennifer A Danielewski, Lenka A Vodstrcil, Jorgen S Jensen, Suzanne M Garland, Natasha Wild, Gerald L Murray","doi":"10.1099/jmm.0.002040","DOIUrl":null,"url":null,"abstract":"<p><p><b>Introduction.</b> <i>Mycoplasma genitalium</i>, a small and slow-growing bacterium, has gained notoriety due to rapidly increasing rates of antibiotic resistance.<b>Gap Statement.</b> <i>M. genitalium</i> is difficult to culture, limiting efforts to understand its biology and the mechanisms of antimicrobial resistance.<b>Aim.</b> To understand factors that influence success in primary isolation of <i>M. genitalium</i> from clinical samples.<b>Methodology.</b> Neat urine or swabs (high vaginal and anal, in universal transport medium) were collected from patients with confirmed or suspected <i>M. genitalium</i> 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<sub>2</sub> for 8 weeks and observed daily, with qPCR used to monitor growth.<b>Results.</b> In total, 127 TMA-positive samples were subjected to qPCR, and <i>M. genitalium</i> 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 <i>M. genitalium</i> 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).<b>Conclusion.</b> The initial bacterial load emerged as a critical determinant of isolation success. This emphasizes the importance of optimizing sample collection and <i>M. genitalium</i> isolation procedures.</p>","PeriodicalId":94093,"journal":{"name":"Journal of medical microbiology","volume":"74 7","pages":""},"PeriodicalIF":2.0000,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12231096/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of medical microbiology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1099/jmm.0.002040","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0

Abstract

Introduction. Mycoplasma genitalium, a small and slow-growing bacterium, has gained notoriety due to rapidly increasing rates of antibiotic resistance.Gap Statement. M. genitalium is difficult to culture, limiting efforts to understand its biology and the mechanisms of antimicrobial resistance.Aim. To understand factors that influence success in primary isolation of M. genitalium from clinical samples.Methodology. Neat urine or swabs (high vaginal and anal, in universal transport medium) were collected from patients with confirmed or suspected M. genitalium 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% CO2 for 8 weeks and observed daily, with qPCR used to monitor growth.Results. In total, 127 TMA-positive samples were subjected to qPCR, and M. genitalium 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 M. genitalium 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).Conclusion. The initial bacterial load emerged as a critical determinant of isolation success. This emphasizes the importance of optimizing sample collection and M. genitalium isolation procedures.

从临床样品中成功分离生殖支原体的因素。
介绍。生殖支原体是一种生长缓慢的小细菌,由于对抗生素的耐药性迅速增加而臭名昭著。差距的声明。生殖支原体难以培养,限制了对其生物学和抗微生物药物耐药性机制的了解。了解影响从临床样本中成功分离生殖支原体的因素。从在墨尔本性健康中心就诊的确诊或疑似生殖器支原体感染的患者身上收集干净的尿液或拭子(阴道和肛门,在通用运输介质中)。在实验室分析之前,将标本保存在-80°C。通过转录介导扩增(TMA)试验进行初步诊断,随后通过定量PCR (qPCR)检测阳性的样品被洗涤两次,并用选择性抗生素混合物(环己亚胺和Thayer-Martin Medium I)接种到Vero细胞单层中。37°C, 5% CO2,培养8周,每天观察,qPCR监测生长情况。共对127份tma阳性样本进行qPCR检测,53.5%(68/127)的样本中检测到生殖支原体基因组DNA (gDNA)。与Vero细胞共培养后,从26.5%(18/68)的gdna阳性样本中获得分离物。不同类型标本的分离率不同,阴道高拭子标本中有12.5%(3/24)检出生长,尿液标本中有37.5%(15/40)检出生长。肛门拭子未检出分离株。成功培养的比例受样品中初始生殖支原体负荷的影响,这转化为Vero细胞单层的接种量大小。低接种量(10,000 geq)分离不成功。初始细菌负荷成为分离成功的关键决定因素。这强调了优化样本收集和生殖支原体分离程序的重要性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
自引率
0.00%
发文量
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信