Sabat Artur J, Gard Lilli, Fliss Monika A, Akkerboom Viktoria, Benus Robin F J, Lokate Mariette, Voss Andreas, Bathoorn Erik
Department of Medical Microbiology and Infection Prevention, University Medical Center Groningen, University of Groningen, Hanzeplein 1, Groningen, 9700 RB, The Netherlands.
Department of Medical Microbiology, Certe, Groningen, The Netherlands.
Antimicrob Resist Infect Control. 2025 Mar 24;14(1):23. doi: 10.1186/s13756-025-01538-1.
Vancomycin-resistant Enterococcus faecium (VREfm) poses a significant concern in healthcare settings, particularly during outbreaks. Traditional antibiotic susceptibility testing may fail to detect occult vancomycin resistance, and long culture times delay diagnosis. While whole genome sequencing (WGS) is the most accurate method for tracing infectious disease transmissions, its response times are not rapid enough to optimally support controlling of ongoing outbreaks. To address this limitation, we investigated the genomic diversity among outbreak isolates and developed outbreak-specific PCR tests for rapid VREfm carrier screening using strain-specific biomarkers identified through comparative genomics.
Total DNA from VREfm isolates was sequenced using Oxford Nanopore and Illumina platforms. Multi locus sequence types (MLST-ST) and core genome sequence type clusters (cgMLST-CT) were determined with Ridom SeqSphere + software. Comparative analysis of whole genomes was conducted using Lasergene software (DNASTAR).
A large VREfm outbreak involving 111 patients caused by E. faecium ST117-CT469 was identified in the Northern Netherlands, spanning from August 2021 to September 2024. A subset of 55 E. faecium ST117-CT469 isolates were evaluated by WGS and outbreak specific PCRs. Antibiotic susceptibility testing revealed occult vancomycin resistance in the outbreak strain. Comparative genomics identified unique markers specific to E. faecium ST117-CT469. Two PCR assays were developed for rapid outbreak detection: a traditional PCR assay distinguishing outbreak from non-outbreak strains based on amplicon size and a TaqMan real-time PCR assay. Both assays demonstrated 100% reproducibility and specificity. The TaqMan assay was able to detect as little as 5 fg of bacterial DNA in the presence of human DNA, equivalent to approximately one bacterial genomic copy. Sequence analysis of WGS data for all 55 outbreak isolates showed perfect nucleotide sequence conservation in the regions where the primers and probe hybridized. Sequence comparison against NCBI GenBank entries confirmed the perfect specificity of both PCR assays for detecting the ST117-CT469 outbreak strain.
These PCR tests maintain the accuracy and discriminatory power of WGS for identifying the ST117-CT469 outbreak strain but are more cost-effective, faster, and easier to use compared to WGS. They enhance VREfm outbreak management by providing an efficient method for rapid screening. Application of strain-specific PCR based on WGS data is currently the most effective screening method during large, ongoing outbreaks.
耐万古霉素屎肠球菌（VREfm）在医疗机构中是一个重大问题，尤其是在疫情爆发期间。传统的抗生素敏感性测试可能无法检测到隐匿的万古霉素耐药性，且培养时间长会延迟诊断。虽然全基因组测序（WGS）是追踪传染病传播的最准确方法，但其响应时间不够快，无法最佳地支持控制正在发生的疫情。为解决这一局限性，我们调查了疫情分离株之间的基因组多样性，并利用通过比较基因组学鉴定出的菌株特异性生物标志物，开发了用于快速筛查VREfm携带者的疫情特异性PCR检测方法。
使用牛津纳米孔和Illumina平台对VREfm分离株的总DNA进行测序。使用Ridom SeqSphere +软件确定多位点序列类型（MLST-ST）和核心基因组序列类型簇（cgMLST-CT）。使用Lasergene软件（DNASTAR）对全基因组进行比较分析。
在荷兰北部发现了一起由屎肠球菌ST117-CT469引起的涉及111名患者的大规模VREfm疫情，时间跨度为2021年8月至2024年9月。通过WGS和疫情特异性PCR对55株屎肠球菌ST117-CT469分离株进行了评估。抗生素敏感性测试显示疫情菌株存在隐匿的万古霉素耐药性。比较基因组学鉴定出了屎肠球菌ST117-CT469特有的独特标志物。开发了两种用于快速疫情检测的PCR检测方法：一种传统PCR检测方法，根据扩增子大小区分疫情菌株和非疫情菌株；一种TaqMan实时PCR检测方法。两种检测方法均显示出100%的重现性和特异性。TaqMan检测方法能够在存在人类DNA的情况下检测低至5 fg的细菌DNA，相当于约一个细菌基因组拷贝。对所有55株疫情分离株的WGS数据进行序列分析，结果显示引物和探针杂交区域的核苷酸序列完全保守。与NCBI GenBank条目进行序列比较，证实了两种PCR检测方法对检测ST117-CT469疫情菌株具有完美的特异性。
这些PCR检测方法在鉴定ST117-CT469疫情菌株方面保持了WGS的准确性和鉴别能力，但与WGS相比，成本效益更高、速度更快且更易于使用。它们通过提供一种高效的快速筛查方法，加强了VREfm疫情管理。基于WGS数据的菌株特异性PCR应用目前是大规模持续疫情期间最有效的筛查方法。
