Jordan, SaraPothier, Joel F.De Maayer, PieterBroders, KirkKvitko, Brian H.Coutinho, Teresa A.Smits, Theo H. .2025-08-292025-08-292025-07Jordan, S., Pothier, J.F., de Maayer, P. et al. Design of genus-specific semi-nested primers for simple and accurate identification of Enterobacter strains. BMC Microbiology 25, 456 (2025). https://doi.org/10.1186/s12866-025-04175-1.1471-2180 (online)10.1186/s12866-025-04175-http://hdl.handle.net/2263/104055DATA AVAILABILITY : Data generated or analyzed during this study are included in this published article and its supplementary information files. The MALDI-TOF MS data are provided in the Zenodo archive https://doi.org/10.5281/zenodo.13992849 and will be made publicly available on acceptance of the manuscript.BACKGROUND : The genus Enterobacter, in the family Enterobacteriaceae, is of both clinical and environmental importance. This genus has undergone frequent taxonomic changes, making it challenging to identify taxa even at genus level. This study aimed to design Enterobacter genus-specific primers that can be used for simple PCR identification of large sets of putative Enterobacter isolates. RESULTS : Comparative genomic approaches were employed to identify genes that were universally present on Enterobacter genomes but absent from the genomes of other members of the family Enterobacteriaceae, based on an initial set of 89 genomes. The presence of these genes was further confirmed in 4,276 Enterobacter RefSeq genomes. While no strictly genus-specific genes were identified, the hpaB gene demonstrated a restricted distribution outside of the genus Enterobacter. Semi-nested primers were designed for hpaB and its flanking gene hpaC (hpaBC) and evaluated on 123 strains in single-tube PCR reactions. All taxa showing positive reactions belonged to the genus Enterobacter. For Enterobacter strains the PCR yielded two amplicons at 110 bp and at 370 bp, while strains only displaying the 110 bp amplicon were classified as Leclercia pneumoniae. A blind-test on 120 strains accessioned as Enterobacter sp. from the USDA-ARS culture collection (NRRL), revealed that one third of the strains had an incorrect genus assignment. Comparison of gene trees of the hpaBC fragment sequences with marker genes frequently used for single-gene barcoding or multi-locus sequence analysis (MLSA) further demonstrated its potential for preliminary species identification. CONCLUSIONS : The nested PCR assay represents a rapid and cost-effective approach for preliminary identification of Enterobacter species. As the primer design was based on large-scale genomic comparison, including currently undescribed species clades, it will remain valid even after taxonomic changes within the genus.en© The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.EnterobacteriaceaeLeclercia pneumoniaePolymerase chain reaction (PCR)DiagnosticsArticle