Design of genus-specific semi-nested primers for simple and accurate identification of Enterobacter strains
Abstract 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 prim...
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2025-07-01
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| Online Access: | https://doi.org/10.1186/s12866-025-04175-1 |
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| author | Sara Jordan Joël F. Pothier Pieter de Maayer Kirk Broders Brian H. Kvitko Teresa A. Coutinho Theo H. M. Smits |
| author_facet | Sara Jordan Joël F. Pothier Pieter de Maayer Kirk Broders Brian H. Kvitko Teresa A. Coutinho Theo H. M. Smits |
| author_sort | Sara Jordan |
| collection | DOAJ |
| description | Abstract 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. |
| format | Article |
| id | doaj-art-164d2b46ecf541ec82fa1a8f34a74402 |
| institution | DOAJ |
| issn | 1471-2180 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Microbiology |
| spelling | doaj-art-164d2b46ecf541ec82fa1a8f34a744022025-08-20T03:04:15ZengBMCBMC Microbiology1471-21802025-07-0125111110.1186/s12866-025-04175-1Design of genus-specific semi-nested primers for simple and accurate identification of Enterobacter strainsSara Jordan0Joël F. Pothier1Pieter de Maayer2Kirk Broders3Brian H. Kvitko4Teresa A. Coutinho5Theo H. M. Smits6Environmental Genomics and Systems Biology Research Group, Institute for Environment and Natural Resources, Zürich University for Applied Sciences (ZHAW)Environmental Genomics and Systems Biology Research Group, Institute for Environment and Natural Resources, Zürich University for Applied Sciences (ZHAW)School of Molecular and Cell Biology, University of the WitwatersrandAgricultural Research Service, Mycotoxin Prevention and Applied Microbiology Research Unit, USDA, National Center for Agricultural Utilization Research, N. UniversityDepartment of Plant Pathology, University of GeorgiaDepartment of Microbiology and Plant Pathology, Centre for Microbial Ecology and Genomics/Forestry and Agricultural Biotechnology Institute (FABI), University of PretoriaEnvironmental Genomics and Systems Biology Research Group, Institute for Environment and Natural Resources, Zürich University for Applied Sciences (ZHAW)Abstract 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.https://doi.org/10.1186/s12866-025-04175-1EnterobacteriaceaeLeclercia pneumoniaePCRDiagnostics |
| spellingShingle | Sara Jordan Joël F. Pothier Pieter de Maayer Kirk Broders Brian H. Kvitko Teresa A. Coutinho Theo H. M. Smits Design of genus-specific semi-nested primers for simple and accurate identification of Enterobacter strains BMC Microbiology Enterobacteriaceae Leclercia pneumoniae PCR Diagnostics |
| title | Design of genus-specific semi-nested primers for simple and accurate identification of Enterobacter strains |
| title_full | Design of genus-specific semi-nested primers for simple and accurate identification of Enterobacter strains |
| title_fullStr | Design of genus-specific semi-nested primers for simple and accurate identification of Enterobacter strains |
| title_full_unstemmed | Design of genus-specific semi-nested primers for simple and accurate identification of Enterobacter strains |
| title_short | Design of genus-specific semi-nested primers for simple and accurate identification of Enterobacter strains |
| title_sort | design of genus specific semi nested primers for simple and accurate identification of enterobacter strains |
| topic | Enterobacteriaceae Leclercia pneumoniae PCR Diagnostics |
| url | https://doi.org/10.1186/s12866-025-04175-1 |
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