Jordan Sara, Pothier Joël F, de Maayer Pieter, Broders Kirk, Kvitko Brian H, Coutinho Teresa A, Smits Theo H M
Environmental Genomics and Systems Biology Research Group, Institute for Environment and Natural Resources, Zürich University for Applied Sciences (ZHAW), Wädenswil, Switzerland.
School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2000, South Africa.
BMC Microbiol. 2025 Jul 28;25(1):456. doi: 10.1186/s12866-025-04175-1.
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.
肠杆菌属属于肠杆菌科，在临床和环境方面都具有重要意义。该属经历了频繁的分类学变化，即使在属水平上鉴定分类单元也具有挑战性。本研究旨在设计肠杆菌属特异性引物，可用于简单的PCR鉴定大量假定的肠杆菌分离株。
基于最初的89个基因组，采用比较基因组学方法来鉴定在肠杆菌基因组上普遍存在但在肠杆菌科其他成员基因组中不存在的基因。在4276个肠杆菌RefSeq基因组中进一步证实了这些基因的存在。虽然未鉴定出严格的属特异性基因，但hpaB基因在肠杆菌属之外显示出有限的分布。针对hpaB及其侧翼基因hpaC（hpaBC）设计了半巢式引物，并在单管PCR反应中对123株菌株进行了评估。所有呈阳性反应的分类单元都属于肠杆菌属。对于肠杆菌菌株，PCR产生了110bp和370bp的两个扩增子，而仅显示110bp扩增子的菌株被分类为肺炎勒克菌。对美国农业部农业研究局培养物保藏中心（NRRL）保藏的120株登记为肠杆菌属的菌株进行的盲测显示，三分之一的菌株属分配错误。将hpaBC片段序列的基因树与常用于单基因条形码或多位点序列分析（MLSA）的标记基因进行比较，进一步证明了其在初步物种鉴定中的潜力。
巢式PCR检测法是一种快速且经济高效的肠杆菌属物种初步鉴定方法。由于引物设计基于大规模基因组比较，包括目前未描述的物种分支，即使该属内发生分类学变化，它仍然有效。
