Development of a strain-specific PCR as a diagnostic tool for surveillance, detection, and monitoring of...

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.