Cross-Genera Amplification of prs/hlyA by Multiplex PCR Resulted in Misidentification of Enterococcus faecium as Listeria monocytogenes

Abstract

ImportanceMolecular confirmation of Listeria monocytogenes typically employs a multiplex PCR method that targets both genus-specific prs and species-specific hlyA genes. This study assessed the specificity of this assay within Nigeria, where local microbial diversity may influence performance outcomes. MethodsOut of eight phenotypically presumptive L. monocytogenes food isolates tested, six produced the expected prs and hlyA amplicons, with five classified as serogroup 1/2b. However, all six isolates tested negative for the crucial virulence regulator prfA, necessitating further investigation. ResultsDefinitive 16S rRNA gene sequencing revealed that only two of the six PCR-positive isolates were identified as L. monocytogenes, while the remaining four were identified as Enterococcus faecium. This results in a false-positive rate of 66.7% (4/6) for the assay in this particular context. Phylogenetic analysis corroborated the taxonomic distinction, exhibiting a robust clustering of the four E. faecium isolates with reference strains. In contrast, the two confirmed L. monocytogenes isolates formed a separate sub-clade, indicating regional divergence and further underscoring the assays inability to differentiate between L. monocytogenes and Enterococcus species. ConclusionThese findings highlight a significant lack of specificity, as the prs/hlyA primers exhibited cross-reactivity with non-target E. faecium. The anomalous negative result for prfA served as a critical diagnostic indicator. Consequently, the positive outcomes from this widely utilized confirmatory assay should be regarded as presumptive and necessitate additional verification.