Design of genus-specific semi-nested primers for simple and accurate identification of Enterobacter strains

Authors: JORDAN, SARA - Zurich University Of Applied Sciences; PLOTHER, JOEL - Zurich University Of Applied Sciences; DE MAAYER, PIETER - University Of Witwatersrand; Broders, Kirk; KVITKO, BRIAN - University Of Georgia; COUTINHO, TERESA - University Of Pretoria; SMITS, THEO - Zurich University Of Applied Sciences

Submitted to: BMC Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/27/2025
Publication Date: 7/28/2025
Citation: Jordan, S., Plother, J.F., de Maayer, P., Broders, K., Kvitko, B.H., Coutinho, T.A., Smits, T.H.M. 2025. Design of genus-specific semi-nested primers for simple and accurate identification of Enterobacter strains. BMC Microbiology. https://doi.org/10.1186/s12866-025-04175-1.
DOI: https://doi.org/10.1186/s12866-025-04175-1

Interpretive Summary: Bacteria in the genus Enterobacter are important clinical pathogens known to infect immuno-compromised patients and have developed anti-biotic resistance. This group of bacteria are also commonly found in many indoor and outdoor environments. There have been several new species added to this group as well as species previously thought to be Enterobacter, that turned out to belong to different groups of bacteria. The clinical importance, common occurrence, and potential for mis-identification has created the need for an accurate, low-cost identification tool to detect Enterobacter species. An ARS researcher in collaboration with researchers at Zurich University designed and validated a low-cost method for accurate identification of Enterobacter strains. The method was able to detect all strains belonging to Enterobacter, but did not recognize closely related species of bacteria. This tool was also used to assess the accuracy of strains in the ARS Culture Collection previously deposited at Enterobacter and found that 42 of the 120 strains were not Enterobacter. These strains were later identified by DNA sequencing, and several were found to be closely related bacterial species. This will greatly improve the accuracy of the ARS Culture Collection. These findings and the identification tool developed will be of value to clinicians, microbiologist working in quality control, and managers of culture collections needing a quick and inexpensive tool to identify Enterobacter strains.

Technical Abstract: 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. 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. 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.