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

BMC Microbiology volume 25, Article number: 456 (2025) Cite this article

Abstract

Background

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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.

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Background

The genus Enterobacter encompasses Gram-negative, rod-shaped, facultatively anaerobic bacteria that belong to the family Enterobacteriaceae [1]. Members of this genus are ubiquitous in nature and are frequently isolated from various sources including soil, water, sewage, plants, and from the intestines of animals and humans. In the latter hosts, it is known as an opportunistic, yet important, pathogen [1]. As such Enterobacter spp. are included in the “ESKAPE” group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species), which were described as the most important antibiotic resistant bacterial pathogens causing nosocomial infections [2,3,4].

The genus Enterobacter was first proposed by E. Hormaeche and P. R. Edwards in 1960 based on biochemical characteristics [5]. With the advent of molecular tools, the taxonomy of this genus has been subjected to frequent changes. During the last decades several species were removed from this genus and assigned to other genera or were described as heterotypic synonyms [4,5,6,7]. One multi-locus sequence analysis (MLSA) study reclassified twelve species to the genera Lelliottia, Pluralibacter, Kosakonia and Cronobacter [8]. Similarly, new Enterobacter species continue to be described [9,10,11,12]. According to the list of prokaryotic names with standing in nomenclature (LPSN), the genus currently consists of 27 validly described species, 19 of which have been published in the last decade [13, 14]. Additionally, E. adelaidei was recently described as novel species but has not yet been validated by the International Journal of Systematic and Evolutionary Microbiology (IJSEM) [15]. The closest neighbors of the genus Enterobacter are Leclercia and Lelliottia, which together form the “Enterobacter clade” [16]. Recently, two new genera, Huaxiibacter and Silvania, were described as belonging to this clade [17, 18].

The frequent taxonomic changes within and around this genus and the close relationship of novel genera makes it challenging to reliably identify Enterobacter strains. Sequencing of the 16S rRNA gene is known to not be of limited use as sequences of different genera can not be separated sufficiently [19], while the databases have many misidentified organisms included, partly also due to taxonomic revisions [20]. The common use of biochemical panels in diagnostics, like BD Phoenix™ or VITEK-2® (bio-Merieux) are also of limited used as it can lead to false Enterobacter genus assignments [21, 22].

As an alternative to sequencing approaches, matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) is a powerful tool for identifying bacterial species, which is particularly used in routine diagnostics [23]. However, this method also has its limitations and can lead to misidentifications, especially when using pattern-matching approaches with manufacturer-provided databases, as these are usually overrepresented by clinical isolates and do not cover all species or even genera. For example, the current version of the SARAMIS® database only covers seven Enterobacter species and does not include the closely related genera Huaxiibacter and Silvania. Biomarker-based databases based on molecular masses of ribosomal proteins calculated from genome sequences have a high potential for identification [24, 25]. The most optimal approach currently is the use of whole-genome sequencing (WGS). Both approaches, MALDI-TOF MS and WGS require expensive devices and extended know-how and are therefore not an option for many labs,

As such, this study aimed to develop a simple PCR assay for the detection and assignment of isolates that are members of the genus Enterobacter. While specific PCR primers are available for some Enterobacter species [26, 27], none exist that target the entire genus, especially not after the recent large taxonomic changes. By using comprehensive comparative genomics approaches for target gene selection, we have developed a robust and cost-effective tool for Enterobacter identification.

Results and discussion

Comparative genomic analyses

To investigate the genus border of Enterobacter, a core gene phylogeny of 89 Enterobacteriaceae was generated (Fig. 1) using the pipeline UBCG2 (Up-to-date Bacterial Core Genes 2; [28]). The tree topology showed a clear separation of the “Enterobacter clade”, encompassing the genera Enterobacter, Huaxiibacter, Leclercia, Lelliottia, Silvania and “Pseudenterobacter”, from other taxa in the Enterobacteriaceae. The protein sequences of all 89 Enterobactericeae taxa were compared using OrthoFinder [29]. The pan-genome encompassed a total of 12,244 orthogroups, with 732 of these being core to all 89 taxa, while 2,112 proteins were core to all taxa in the “Enterobacter” clade. The core proteome of the 29 taxa in the genus Enterobacter (Fig. 1) comprised 2,368 proteins. None of these core proteins were, however, exclusive to Enterobacter spp., with between 13 and 645 orthologues in the other 60 taxa. As such, the 35 orthogroups with orthologues in < 25 of the non-Enterobacter taxa were analyzed further. Priority was then given to those proteins that were absent from the genera closest to Enterobacter in the core gene tree. No proteins were identified that were absent in all other members of the “Enterobacter clade”, however, one protein, HpaB (a 4-hydroxyphenylacetate 3-monooxygenase oxidase component) was absent from the genomes of the closely related Huaxiibacter chinensis 155,047T, Lelliottia amnigena LMG 2784T, Lelliottia nimipressuralis CCUG 25,894T and Silvania hatchlandensis H19S6T (Fig. 1). As such, sequences of hpaB, along with its neighboring genes hpaA and hpaC were selected for primer design.

Fig. 1
figure 1

Core genome tree based on 81 bacterial core genes in the type strains of 29 Enterobacter species/subspecies (blue) and 60 other Enterobacteriaceae used for the comparative genomics. Filled squares show (1) Presence of the target gene hpaB, (2) Expected small PCR amplicon 1 in members of the genus, (3) Expected long PCR amplicon 2 in members of the genus

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To confirm the presence of hpaABC on the genomes of a broader set of Enterobacter strains, the publicly available genomes of all Enterobacter strains (4,276 RefSeq assemblies) were downloaded from the NCBI database and subjected to local BLASTN analysis with the hpaABC sequence of E. cloacae ATCC 13,047T (CP001918.1 region 743,259 to 746,860) as query sequence. The region was absent from the genomes of only two Enterobacter strains, namely, Enterobacter roggenkampii FDAARGOS_523 (GCF_003812145.1) and Enterobacter cloacae Tembi-46 (GCA_024583295.1). As only 0.047% of the genomes are lacking the gene, it is assumed that it is part of the core genome of the genus and the lack thereof is due to genome incompleteness.

The BLASTN search with the hpaABC sequence of E. cloacae ATCC 13,047T against all non-Enterobacter genomes yielded in total 8,246 genomes, in which this fragment was present (Supplementary Table S1). This yielded 34 hits within the genus Leclercia, but none within the genera Huaxiibacter, Silvania, and Lelliottia. Other genera having the hpaABC region included other members of the Enterobacteriaceae, like Klebsiella, Escherichia or Salmonella.

Primer design and in silico testing

By removing redundant genomes from the set of 4,276 Enterobacter genomes, a subset of 1069 Enterobacter genomes was obtained, from which the hpaABC region was used for primer design. As no primer pair could be identified that excluded all other genera, a second forward primer was designed that should exclude Leclercia pneumoniae. The regions of the final primers hpa_287F (5’-GGCGCGCCATGATCTCY-3’), hpa_521F (5’-GCTGCRYTATTTCAGCAGCT-3’) and hpa_625R (5’-ATGGTCGACCGCTGYATGTC-3’) showed a high conservation among the Enterobacter genomes (Supplementary Figure S1), only having a limited degeneracy. These primer sequences covering the hpaB and hpaC genes were further evaluated in silico by fastPCR [30] analysis. To avoid that the primers would be able to detect fragments within other closely related organisms, the primer sequences were individually tested against all 8,346 non-Enterobacter (Supplementary TableS1). The reverse primer hpa_625R is relatively non-specific, with fastPCR matches to all 1,069 Enterobacter genome sequences and to 4,326 of the 8,346 (51.8%) non-Enterobacter sequences. By contrast, the forward primer hpa_521F had matches in 1,067 (99.8%) of the Enterobacter genomes as well as four matches (< 0.1%) in the non-Enterobacter genomes, the latter of which all belonged to the species L. pneumoniae. The second forward primer hpa_521F had matches in 1,041 (97.4%) Enterobacter strains and 16 (0.2%) non-Enterobacter taxa, with all of the latter designated as Kluyvera or Klebsiella species. The genomes designated as Klebsiella spp. demonstrated an inconclusive taxonomy check status in the NCBI quality analysis and likely also belong to the genus Kluyvera. Overall, it was predicted that only Enterobacter strains would yield two amplicons of 110 bp and at 370 bp sizes in a single PCR reaction (Fig. 2; Supplementary Figure S2). The smaller amplicon, hereafter designated as amplicon 1, is expected to occur for L. pneumoniae, while the larger amplicon, hereafter amplicon 2, would occur for some Kluyvera strains.

Fig. 2
figure 2

Agarose gel electrophoresis of PCR amplicons: (1) Enterobacter cloacae ATCC13047T, (2) Enterobacter cancerogenus LMG 2693T, (3) Enterobacter asburiae DSM17506T, (4) Enterobacter soli LF7T, (5) Enterobacter ludwigii ECWSU1, (6) Enterobacter kobei AZ3a, (7) Enterobacter mori 19UT013, (8) Enterobacter hormaechei subsp. steigerwaltii 20TX0131, (9) Enterobacter bugandensis 20TX0138, Enterobacter roggenkampii 20CA0095, 11. Enterobacter wuhouensis 20GA0343, 12. Leclercia pneumoniae LMG 5336, 13. Leclercia pneumoniae ATCC 27,994, 14. Leclercia pneumoniae ATCC 27,993, 15. Escherichia coli SM06, 16. Kluyvera sp. 9062RM, 17. Pseudocitrobacter sp. CIP 102,343, 18. Cronobacter sakazakii ATCC29544T, 19. Siccibacter turicensis LMG23730T, 20. Phytobacter diazotrophicus ATCC 27,981, 21. Buttiauxella sp. LMG 5339, 22. Pantoea agglomerans ATCC 27,155T, 23. Franconibacter pulveris LMG24057T, 24. negative control; M: 1 Kb Plus DNA Ladder (Invitrogen)

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Evaluation of the primer set against a test set of known species

The designed semi-nested PCR primer set comprising two forward and one reverse primer was tested on a set of 123 strains comprising 66 Enterobacter strains belonging to ten distinct species, as well as 57 non-Enterobacter taxa belonging to 20 distinct genera (Table 1). All strains were confirmed by MALDI-TOF MS in combination with identification based on the calculated molecular masses of ribosomal proteins by MabritecCentral for their identification. Average nucleotide identities (ANI) to the respective type strains was done for a selection of Enterobacter strains (indicated in Supplementary Table S2) to confirm the identification results.

Table 1 Overview of PCR results in the test set of known species (detailed list in Supplementary table S2). A + or – sign indicates that all tested stains were positive or negative, respectively. Variable results were not obtained
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Enterobacter strains positive in the PCR expectedly yielded two amplicons 1 and 2, while strains only displaying the amplicon 1 were classified as L. pneumoniae (Table 1; Fig. 2; Supplementary Figure S3). Strains showing no or only the amplicon 2 did not belong to the genus Enterobacter. No side products were observed in any of the reactions. Primers were also evaluated in a qPCR approach, which might be more convenient than gel visualization for the analysis of many strains. The melting curves for the multiplexed setting were not clearly interpretable due to the presence of non-specific amplicons (Supplementary Figure S4). When the primer sets (hpa_287F and hpa_625R; hpa_521F and hpa_625R) were evaluated separately, clear identification of the Enterobacter strains was possible, similar to the conventional PCR approach. If applied in a qPCR approach, it is recommended to screen first with the primer pair hpa_521F and hpa_625R, and in a second step to use hpa_287F and hpa_625R on all positive strains from the first screen.

Confirmation of the amplicons and marker gene resolution

To confirm the specificity of the PCR product and evaluate its potential for species identification, amplicon 2 of the PCR reactions (hpa_287F and hpa_625R) was sequenced for eleven of the 123 strains (representing ten distinct Enterobacter sp. and Kluyvera sp. 9062RM which was positive for amplicon 2) (Supplementary Table S3). The only strain with unclear results was 20GA0343, which was identified as E. wuhouensis based on MALDI-TOF MS and ANI. The best BLASTN matches for this strain were E. huaxiensis 090008 and E. wuhouensis AV1 with a sequence coverage of 99% and 100%, and nucleotide identity of 95.20% and 93.43%, respectively. For the other strains, BLASTN against the Enterobacter genomes gave consistent best hits that were in accordance with the MALDI-TOF MS/MabritecCentral identification. The five strains with available genomes all displayed 100% nucleotide identity for the sequenced amplicons with the corresponding genome sequences.

To further evaluate the potential of the hpaBC gene fragment sequence for Enterobacter species identification, its performance as phylogenetic marker gene was compared with other commonly used marker genes (dnaJ, atpD, gyrB, infB, rpoB and hsp60). Single gene and MLSA trees were generated using the gene sequences derived from the 4,276 publicly available Enterobacter spp. genomes. For all single gene trees (including the hpaBC gene fragment), individual strains from a certain species clustered with those of other species, while some species were separated in multiple clades, including for the hpaBC gene fragment (Fig. 3; Supplementary Figure S5). While there are differences between the marker genes, none of them can clearly separate all species. The dnaJ gene, which has been reported to allow precise Enterobacter species assignments [31], also does not provide a good resolution for some of the species. A MLSA on the basis of the concatenated gyrB, rpoB, infB and atpD gene sequences showed a more accurate resolution, however, it also does not allow clear separation of all species (Fig. 3). For example, E. asburiae SD4L was initially identified as E. cloacae based on the individual gyrB, rpoB, infB and atpD genes, but it was reclassified to E. asburiae after the complete genome was sequenced [32, 33].

Fig. 3
figure 3

Multi-locus sequence analysis (A; 10,515 bp) and single gene trees of the hpaBC PCR amplicon 2 (B; 370 bp) from 4276 genomes representing all species of Enterobacter, including clades of currently undescribed species

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Despite the limited resolution, sequencing of single or multiple genes may still represent a good approach if more accurate methods are not accessible. Some relevant species, including E. cloacae, E. kobei and E. ludwigii, are clearly separated in all gene trees (Fig. 3, Supplementary Figure S5), including the phylogeny based on the hpaBC gene fragment. As such, the hpaBC fragment, along with other single marker genes, can be used for preliminary species identification, but the results should be treated with caution.

Strain identification and PCR validation

The developed PCR was tested on 120 strains (catalogued as “Enterobacter spp.”) obtained from the USDA-ARS culture collection (NRRL) (Supplementary Table S4). The assay was performed as a blind assay using only lab-internal strain numbers for all strains to avoid influence based on the previous identification.

All strains were first identified with MALDI-TOF MS in combination with the MabritecCentral database (Supplementary Figure S6). Of these, 107 could be assigned to the species level, while eleven strains were only assigned at genus level with MALDI-TOF MS. One strain (B-51110) gave low matches with Kosakonia and Phytobacter, while the other strain (B-455) gave no match to any known species. The latter strain may potentially be a Gram-positive bacterium, for which the direct smear method is not suitable for identification [34]. The identification was confirmed for 23 isolates by WGS data (Supplementary Table S4).

Forty-four of the strains maintained in the NRRL pre-date the establishment of the genus Enterobacter and were thus originally deposited as Aerobacter. Furthermore, 15 strains were catalogued as species that are no longer part of the genus, namely Enterobacter (Klebsiella) aerogenes, Enterobacter (Lelliottia) nimipressuralis, Enterobacter amnigenus (Lelliottia amnigena) and Enterobacter (Kosakonia) cowanii. It was assumed that these strains would give negative results for the Enterobacter PCR.

Using the PCR assay designed in this study, 78 of the 120 strains were identified as Enterobacter, while no visible amplicon was observed on the agarose gel for the remaining 42 strains. All positive reactions matched with the MALDI-TOF MS identification as belonging to the genus Enterobacter (Table 2). These findings were consistent with the expected performance of the designed primer set and suggested its potential utility for Enterobacter identification.

Table 2 Overview of PCR results in the validation set (detailed list in Supplementary Table S4). A + or– sign indicates that all tested stains were positive or negative, respectively. Variable results were not obtained
Full size table

Overall, 144 Enterobacter strains out of 243 strains were correctly identified with the developed PCR assay, from which 42 Enterobacter and 40 non-Enterobacter were confirmed on the basis of WGS data, giving a 100% accuracy (Supplementary Table S5). However, it has to be considered that the set of tested strains suffers from sampling bias as many strains are from the same isolation source and therefore some species are overrepresented. Further validation of the PCR is needed, especially for the Enterobacter species that were not tested in this study. However, it is still regarded to be trustful due to the large scale of genomic comparisons that were performed for the primer design. As currently undescribed species clades of Enterobacter were also included, it is plausible to remain valid even after future taxonomic changes within the genus. It tested negative for a broad range of non-Enterobacter strains, including former Enterobacter members such as Kosakonia, Leclercia, and Lelliottia, as well as the closely related ESKAPE pathogen K. pneumoniae and strains from other genera that possess the target genes including Escherichia and Citrobacter.

Conclusions

The novel semi-nested PCR approach provides a reliable, rapid, and cost-effective way to identify Enterobacter strains to genus level. Subsequent sequencing of the PCR product allows a preliminary species assignment, but it should be noted that neither the hpaBC fragment, nor the other frequently used single copy marker genes individually, provide a perfect resolution for all Enterobacter species. The validity of the Enterobacter-specific PCR assay was further confirmed by evaluation of a comprehensive collection of potential Enterobacter species, where two thirds of NRRL strains (n = 78 of 120) listed in the catalogue as Enterobacter were now confirmed as belonging to the genus. The PCR assay is therefore suitable for the identification of large sets of putative Enterobacter strains and would be especially useful for less equipped laboratories which do not have access to MALDI-TOF MS or genome sequencing platforms.

Methods

Bacterial strains

The bacterial strains used in this study are listed in Supplementary Tables S2 and S4. Most of the strains were received as Enterobacter from the NRRL culture collection (https://nrrl.ncaur.usda.gov/) and from the Stop-The-Rot project surveys. All strains were streaked on Luria-Bertani (LB) agar and grown for 24–48 h at 28 °C, which is suited for both environmental and clinical isolates [1]. Subsequently, they were stored in 25% (vol/vol) LB-glycerol stocks at −80 °C.

Strain identification with MALDI-TOF MS combined with MabritecCentral

All evaluated strains were grown for 24 h on LB plates, and a loopful of cell material from a single colony was smeared onto target slides (Fleximass-SR48) in quadruplicate. The spots were overlaid with 1 µl sinapinic acid (40 mg/mL) matrix solution diluted in 60% acetonitrile and 0.3% trifluoroacetic acid and air-dried for a few minutes at room temperature. On each target plate, the reference strain Escherichia coli DH5α was used for system calibration and quality control. The mass spectra were generated using an Axima Performance instrument (Shimadzu, Kyoto, Japan), with detection in the linear positive mode at a laser frequency of 50 Hz and within a mass range 4 to 30 kDa. Peaks were picked from raw spectra as previously described [35]. The generated ascii files were uploaded to MabritecCentral (https://mabriteccentral.com) for species identification.

Target gene selection

Genome sequences were obtained from the National Center for Biotechnology Information (NCBI) database [36]. The type strain genomes of 29 species and subspecies of Enterobacter and a selection of 60 genomes of type strains covering all recognized genera of the family Enterobacteriaceae were used for initial comparative genomics. A tree based on 81 bacterial core genes conserved among all 89 genomes was created with UBCG2 v2.0 [28] and visualized with iTOL 6.9.1 [37]. Orthofinder v2.5.4 [29] was used with default parameters to identify homologous genes that are abundant in all Enterobacter strains but lacking in most other Enterobacteriaceae [29].

All 4,413 available RefSeq assemblies assigned as Enterobacter were downloaded from the NCBI database on 23 September 2022. The completeness of these genomes was evaluated with BUSCO v5.4.3 [38]. Genome assemblies displaying < 97% BUSCO completeness were excluded for the primer design. In order to confirm the taxonomic assignment of the remaining 4,294 Enterobacter strains, their genomes were compared against the type strain genome dataset using fastANI v1.3 [39]. Eighteen of these genomes were not Enterobacter according to the ANI comparisons and therefore excluded from the selection. BLASTN v2.10.0 was used to confirm whether the genomes of the 4,276 Enterobacter strains contained the target gene.

Primer design

To reduce redundancy, a subset of 1,069 assemblies of the remaining 4,276 Enterobacter spp. was created by selecting only one strain per species for each NCBI BioProject. The sequences of the target gene hpaB (with the neighboring genes hpaA and hpaC) of this subset were used for primer design. A multiple sequence alignment (MSA) of the target sequence was generated with MUSCLE v3.8.1551 [40] and Degeprime v1.1.0 [41] was used to detect potential primer sequences. All potential primers that had an exact match with > 99% of the Enterobacter sequences were further tested in silico with fastPCR v6.9 [30] (with 0 allowed mismatches at the 3′ end) on all Enterobacteriaceae type strain genomes that contain the target gene. As no perfect primer pair could be identified, a second forward primer was designed based on the MSA that should exclude Leclercia pneumoniae. The final primers were tested again in silico on all 8,346 non-Enterobacter sequences that were identified with BLASTN using the hpaBC region from E. cloacae ATCC 13,047T (CP001918.1 region 743,259 to 745,351) as query sequence against NCBI nucleotide collection (nr/nt) excluding Enterobacter (taxid 547) on 06 June 2024.

PCR test and validation

All evaluated strains were grown on plates as described above and a loopful of cell material was resuspended in ultrapure water and boiled for 10 min at 95°C. The boiled cells were diluted tenfold and directly used as DNA template for the PCR. The primers hpa_287F (5’-GGCGCGCCATGATCTCY-3’), hpa_521F (5’-GCTGCRYTATTTCAGCAGCT-3’) and hpa_625R (5’-ATGGTCGACCGCTGYATGTC-3’) were ordered from Microsynth AG (Balgach, Switzerland).

The PCR mix for one reaction contained 4 µl KAPA2G Robust 2× mix, 0.4 µl hpa_287F (10µM), 0.2 µl hpa_521F (10µM), 0.4 µl hpa_625R (10µM), 1 µl diluted boiled cells and 2 µl nuclease-free water in a total volume of 8 µl. PCR amplification was performed in a T100 thermal cycler (Bio-Rad, Hercules, USA) with reaction conditions including an initial denaturation step at 95 °C for 3 min, followed by 30 cycles off amplification (95 °C for 15 s, 62 °C for 15 s, and 72 °C for 15 s) and a final extension step at 72 °C for 1 min.

Quantitative PCR (qPCR) was performed in a Light Cycler®480 II (Roche, Basel, Switzerland). One reaction mix contained 5 µl LightCycler 480 SYBR Green I Master mix, 0.5 µl of each primer (10µM), 2 µl diluted boiled cells and 1.5–2 µl nuclease-free water in a total volume of 10 µl. Cycling conditions were as follows: 95 °C for 5 min followed by 40 cycles of 95 °C for 15 s, 62 °C for 15 s and 72 °C for 15 s. A melting curve was performed after the amplification (raising 1 °C per second, from 65 °C to 97 °C).

Sanger sequencing.

PCR products were cleaned up with the NucleoSpin gel and PCR clean-up kit (Macherey-Nagel, Düren, Germany) and sent to Microsynth AG for Sanger sequencing with both the forward primer hpa_287F and reverse primer hpa_625R. The obtained sequences were trimmed and curated using UGENE v50.1 [42]. The consensus sequences were compared against the NCBI nucleotide collection (nr/nt) using BLASTN.

Single gene trees and MLSA

The full sequences of the marker genes atpD, dnaJ, gyrB, infB, rpoB and hsp60 as well the sequence of the hpaBC long amplicon (370 bp) were extracted from the 4,276 Enterobacter spp. genomes using BLASTN with the corresponding sequences from E. cloacae ATCC 13,047T (NC_014121.1; locustags: ECL_RS00020, ECL_RS01185, ECL_RS22690 ECL_RS25685, ECL_RS03995, ECL_RS02580). Each of the genes was missing in 1–2 genomes and incomplete in 0–2 genomes and these were therefore excluded from the respective trees. Multiple sequence alignments of each gene fragment were generated with MUSCLE v3.8.1551 [40]. For MLSA, the alignments of atpD, gyrB, infB and rpoB were concatenated (totalizing 10,515 bp). All phylogenies were created with fasttree v2.1 [43] and visualized with iTOL 6.9.1 [37, 44].

Data 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.

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Acknowledgements

The authors thank the EGSB team for support and valuable discussions and the HPC team of the School for Life Sciences and Facility Management at ZHAW for their computing resources and support.

Funding

Financial support was provided by the Swiss National Science Foundation (SNSF) – South-African National Research Foundation (NRF) Lead Agency project “EBDOmics: Comparative genomics of Enterobacter spp. causing Bulb Decay of Onions” (Project nr. 310030L_204333). JFP and THMS further acknowledge the support of the Department of Life Sciences and Facility Management of the Zurich University of Applied Sciences (ZHAW) in Wädenswil (Switzerland). TC and BK acknowledge financial support of the ‘Stop the rot’ USDA NIFA SCRI Onion Bacterial Project (2019-51181-30013). This research was funded in part by the United States Department of Agriculture– Agricultural Research Service to KB.

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Authors and Affiliations

  1. Environmental Genomics and Systems Biology Research Group, Institute for Environment and Natural Resources, Zürich University for Applied Sciences (ZHAW), Wädenswil, Switzerland

    Sara Jordan, Joël F. Pothier & Theo H. M. Smits

  2. School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2000, South Africa

    Pieter de Maayer

  3. Agricultural Research Service, Mycotoxin Prevention and Applied Microbiology Research Unit, USDA, National Center for Agricultural Utilization Research, N. University, Peoria, IL, 1815, 61604, USA

    Kirk Broders

  4. Department of Plant Pathology, University of Georgia, 120 Carlton St, Athens, GA, 30602, USA

    Brian H. Kvitko

  5. Department of Microbiology and Plant Pathology, Centre for Microbial Ecology and Genomics/Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa

    Teresa A. Coutinho

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  1. Sara Jordan
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  2. Joël F. Pothier
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  3. Pieter de Maayer
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  6. Teresa A. Coutinho
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  7. Theo H. M. Smits
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Contributions

SJ, TAC and THMS designed the study. SJ and JP performed experiments and analyses. SJ and THMS wrote the manuscript text. SJ prepared figures and tables. All authors read and approved the final manuscript.

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Correspondence to Theo H. M. Smits.

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Jordan, 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 Microbiol 25, 456 (2025). https://doi.org/10.1186/s12866-025-04175-1

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BMC Microbiology

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