Introduction

The coliforms Escherichia coli (EC), Escherichia fergusonii (EF), and Citrobacter freundii (CF) are gram-negative facultative anaerobes belonging to the Enterobacteriaceae family. Although these three species are common gut commensals in humans and animals, they also function as opportunistic pathogens, capable of causing a wide range of diseases under certain conditions [1-3]. Escherichia spp. are motile with flagella and some are pathogenic, including EC, EF, Escherichia hermannii, and Escherichia albertii [4]. EC is well-known for its role in a variety of infections, including those affecting the intestines, urinary tract, and lungs, and is a leading cause of sepsis [1]. EF is an emerging pathogen that has garnered attention owing to its increasing incidence in clinical settings, particularly under conditions such as wound infections and hemolytic uremic syndrome. This increase in EF-related infections is compounded by growing concerns over resistance to antimicrobial treatments [5,6].

In contrast, Citrobacter spp., particularly CF, are recognized as major foodborne pathogens. They are known to cause severe infections such as meningitis, brain abscesses, and central nervous system infections. CF stands out as the predominant species within the Citrobacter genus in clinical environments, where it poses a substantial threat, especially in nosocomial (hospital-acquired) infections [3,7,8].

Accurate bacterial identification is crucial to avoid unnecessary treatment and drug misuse by clinicians and patients, thereby ensuring efficient illness treatment. For researchers working with bacteria, the initial identification of isolates is a critical step toward achieving their goals. 16S rRNA sequencing is commonly used for the genetic identification of bacteria. However, EC, EF, and CF share identical 16S rRNA genes, making them difficult to distinguish [9,10]. Our previous study also indicated difficulties in differentiating EC and EF based solely on 16S rRNA sequencing and/or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [11].

MALDI-TOF MS is a powerful technique widely employed for identification and differentiation of bacterial species in hospitals. In this method, microbial proteins (typically ribosomal proteins) are ionized, and their mass-to-charge ratios are analyzed, generating unique spectral fingerprints for rapid and accurate identification [12]. However, there are limitations in identifying EC, EF, CF, Shigella spp., Salmonella spp., and Mycobacterium spp. [9,10]. Another major diagnostic machine in hospitals, VITEK 2 (bioMérieux, Marcy-l'Étoile, France), which identifies bacteria based on biochemical characteristics and antimicrobial susceptibility tests, struggles to differentiate these bacterial species due to variable biochemical characteristics, such as in EC (sorbitol and lactose fermentation) and EC O157:H7 (sorbitol non-fermentation and lactose fermentation), potentially leading to false-positive results [9,11].

In our previous study, we demonstrated that sequencing the adenylate kinase (adk) gene, which is commonly targeted in multilocus sequence typing (MLST) of Escherichia species, allows for accurate differentiation between EC and EF [11]. Building on these findings, the current study aimed to address the challenge of distinguishing EC, EF, and CF by exploring genetic variations within the adk gene. Drawing from the insights of our earlier work, which highlighted the effectiveness of adk sequencing in discriminating between EC and EF, we extended our investigation to include CF. Moreover, we developed a new polymerase chain reaction (PCR) method combined with restriction digestion of the adk gene, enabling straightforward differentiation of these three species through agarose gel electrophoresis of the resultant DNA fragments. This innovative approach was designed to enhance the efficiency and simplicity of the genetic identification and confirmation of EC, EF, and CF in hospital and research laboratory settings.

Methods

Ethics statement: This study was approved by the Institutional Review Board (IRB) of Kyungpook National University Hospital (IRB No: KNUH 2021-03-011-002).

1. Adenylate kinase gene sequence analysis

Archived adk gene sequences from EC, EF, and CF were retrieved from the National Library of Medicine (https://www.ncbi.nlm.nih.gov/guide/sequence-analysis/). The sequences were aligned and restriction enzyme sites were mapped to differentiate EC, EF, and CF (Fig. 1).

2. Bacterial strains and isolates

EF KCTC 22525^T was purchased from the Korean Collection for Type Cultures (Daejeon, Korea). EC ATCC 25922 was purchased from the American Type Culture Collection (Manassas, VA, USA). We used 83 bacterial isolates from our previous study [11], along with one additional EC strain, for a total of 84 isolates (79 EC and five CF) obtained from the fecal samples of patients with inflammatory bowel disease or ischemic colitis. The isolation, identification, and characterization of these clinical isolates were described in our previous study [11]. The adk gene sequence of the additional strain used in this study is provided in the Supplementary Material.

3. DNA extraction

All strains were cultured overnight at 37°C on brain heart infusion agar (BD Biosciences, Franklin Lakes, NJ, USA) before DNA extraction. Then, two or three colonies of each bacterial strain were placed in sterile 1.5-mL Eppendorf tubes containing 100 µL RNase-free H₂O (WELGENE Inc., Gyeongsan, Korea), boiled for 10 minutes in a water bath, cooled on ice, and centrifuged at 10,000×g for 2 minutes before storage at −20°C. Aliquots of 1.5 µL of template DNA were used for PCR amplification.

4. Polymerase chain reaction of the adenylate kinase gene

PCR amplification of adk was performed as previously described [11] using the oligonucleotide primers adkF (5′-ATT CTG CTT GGC GCT CCG GG-3′) and adkR (5′-CCG TCA ACT TTC GCG TAT TT-3′) (Bioneer Corp., Daejeon, Korea). Briefly, the primer set was used to amplify adk from all 86 isolates, including EF KCTC 22525^T and EC ATCC 25922. PCR amplification was performed in a final reaction volume of 50 μL containing 0.25-μL TaKaRa Taq (5 U/μL), 5-μL 10× PCR buffer (Mg²⁺), 4-μL deoxyribonucleotide triphosphates (2.5 mM each), 0.2 μM of each primer, and 1.5-μL DNA template under the following cycling conditions: 95°C for 2 minutes; 30 cycles of 1 minute at 95°C, 1 minute at 54°C, and 2 minutes at 72°C; followed by 5 minutes at 72°C. Amplification of the 583-base pair (bp) products was confirmed using 1% agarose gel electrophoresis. The PCR products were purified using the Solg Gel & PCR Purification Kit (SolGent Co. Ltd., Daejeon, Korea) according to the manufacturer’s instructions. Sequencing was performed by SolGent Co., Ltd. using a 3770XL DNA Analyzer and a BigDye Terminator Cycle Sequencing Kit v.3.1 (Applied Biosystems, Carlsbad, CA, USA).

5. Digestion of adenylate kinase polymerase chain reaction amplicons with restriction endonucleases

Three restriction endonucleases, BtgI, BtsIMutI, and AgeI (New England Biolabs, Ipswich, MA, USA), were used to digest the adk PCR amplicons from all 86 strains. The digestion reactions were meticulously prepared on ice in 1.5-mL Eppendorf tubes. The final reaction volume was 50 µL, which contained 10-µL PCR products, 34-µL RNase-free H₂O, 5-µL 10× rCutSmart Buffer (New England Biolabs), and 1 µL of restriction enzyme.

Reaction mixtures containing BtgI and AgeI, both Time-Saver-qualified restriction enzymes, were incubated at 37°C for 15 minutes. In contrast, reaction mixtures containing BtsIMutI were incubated at 55°C for 1 hour and promptly cooled on ice. Subsequently, the digestion products were analyzed using 2.05% agarose gel electrophoresis and visualized using SYBR Gold Nucleic Acid Gel Stain (Invitrogen, Waltham, MA, USA).

Results

1. Differential restriction sites in adenylate kinase gene sequences of Escherichia coli, Escherichia fergusonii, and Citrobacter freundii

Analysis of adk gene sequences revealed differences in nucleotide (nt) 93 and 96 among the three species. Those of EC, EF, and CF were T/C, G/T, and C/T, respectively, creating BtsIMutI (5ʹ-CAGTGNN-3ʹ), BtgI (5ʹ-CCRYGG-3ʹ), and AgeI (5ʹ-ACCGGT-3ʹ) target sites, respectively (Fig. 1).

The size of the amplicons generated by PCR with the adkF and adkR primers was 584 bp using template DNA from all three species. The adk amplicon of EC contained unique target sites for BtsIMutI (nt 80) and AgeI (nt 436), that of EF contained unique target sites for BtsIMutI (nt 80) and AgeI (nt 436) and two BtsIMutI sites (nt 167 and 187), and that of CF contained a unique AgeI site (nt 82) but no BtsIMutI or BtgI target sites. Theoretically, BtgI, BtsIMutI, and AgeI digestion of the EC adk amplicon produces one (584 bp), two (504 and 80 bp), and two (436 and 148 bp) DNA bands, respectively. Likewise, BtgI, BtsIMutI, and AgeI digestion of the EF adk amplicon produces two (504 and 80 bp), three (397, 167, and 20 bp), and two (436 and 148 bp) DNA bands, respectively. BtgI or BtsIMutI digestion of the CF adk amplicon generates one DNA band in both cases without DNA fragmentation, whereas AgeI digestion produces two DNA bands (502 and 82 bp) (Fig. 2).

2. Adenylate kinase polymerase chain reaction-restriction fragment length polymorphism of Escherichia coli, Escherichia fergusonii, and Citrobacter freundii isolates

EC and EF reference strains (ATCC 25922 and KCTC 22525^T, respectively) were subjected to adk PCR/restriction digestion. Similar to previous theoretical results, intact adk amplicons and DNA band patterns of the amplicons following digestion by the three restriction enzymes were generated as expected (Fig. 3). A clinical isolate of CF (C1-Y-1) was subjected to adk PCR, and the amplicon was digested with the three restriction enzymes. The band patterns and DNA sizes were the same as those predicted theoretically (Fig. 3).

Eighty-four clinical bacterial isolates (79 EC and five CF) were subjected to adk PCR/restriction digestion. The adk amplicons from EC isolates were selectively digested by BtsIMutI, whereas those from CF isolates were specifically digested by AgeI. This differential enzymatic digestion enabled a clear differentiation between EC and CF isolates based on the presence of unique restriction sites. The results of the adk PCR/restriction digestion assay consistently displayed the anticipated band patterns, which aligned with the aforementioned reference strain results (Fig. 4, Supplementary Fig. 1).

Discussion

This study employed a molecular approach to differentiate between EC, EF, and CF isolates by analyzing unique restriction sites within the adk gene sequences. The nt at positions 93 and 96 varied among the three species, creating distinctive restriction sites for BtsIMutI, BtgI, and AgeI restriction enzymes (Figs. 1, 3).

Theoretical agarose gel images predicted the outcomes of BtsIMutI, BtgI, and AgeI digestion of adk PCR amplicons, revealing specific banding patterns for each species (Fig. 2). The empirical agarose gel electrophoresis results, following adk PCR/restriction digestion of the EC and EF reference strains, as well as the CF clinical isolates, aligned precisely with the predicted patterns (Figs. 3, 4). The consistency between the theoretical predictions and experimental outcomes establishes the reliability of this molecular strategy for species identification.

The application of this method to 84 clinical bacterial isolates, including EC and CF, confirmed its efficacy in discriminating between the two species based on their unique restriction sites (Fig. 4, Supplementary Fig. 1). The selective digestion of EC adk amplicons by BtsIMutI and CF adk amplicons by AgeI demonstrated the specificity of the assay for differentiating between closely related bacterial strains. The band patterns observed for the clinical isolates closely mirrored those observed for the reference strains, validating the utility of our adk PCR/restriction digestion assay for a broad range of isolates.

In clinical diagnosis, EF is primarily identified using three methods: (1) biochemical characterization using API 20NE (bioMérieux), as well as adonitol and sorbitol fermentation assays; (2) utilization of the VITEK 2 GN ID card identification system (bioMérieux); and (3) 16S rRNA gene sequencing. Notably, none of these methods can accurately discriminate between enterotoxigenic EC and EF. Although enterotoxigenic EC ferment adonitol, EC O157:H7 shares phenotypic traits with EF, particularly in sorbitol fermentation. Furthermore, EC and EF exhibit substantial DNA hybridization similarity (64%) [2,11,13,14]. To address these challenges, researchers have developed molecular diagnostic tools for identifying EF [9,14]. However, despite these advancements, the differentiation of EC, EF, and CF remains unclear. The need for more precise diagnostic methods underscores the complexity of distinguishing between closely related bacterial species.

However, this newly developed adk gene PCR-restriction fragment length polymorphism (PCR-RFLP) method accurately distinguished EC, EF, and CF by using PCR for only one gene, followed by restriction digestion of the PCR product. The results were visualized and confirmed instantly by analyzing the agarose gel images. For confirmation, this method does not require sequencing or phylogenetic analysis.

The molecular specificity of this method offers advantages over other methods, providing a rapid and accurate means of species identification and differentiation of EC, EF, and CF. Although this study has the limitation of using clinical isolates of EF, the distinct banding patterns generated by the restriction enzymes facilitated straightforward visual differentiation of EC, EF, and CF. We note that our PCR-RFLP approach examines only the adk gene locus; therefore, other variants from point mutations, horizontal gene transfer, or natural sequence variability within the adk gene could lead to incorrect species identification. In addition, some bacterial strains may have altered restriction sites, resulting in unexpected or ambiguous banding patterns. Future studies should include larger and more diverse isolates to validate and extend this approach. Moreover, the accuracy of the assay is highly dependent on the efficiency of restriction enzyme digestion, which can be influenced by factors such as enzyme activity, pH, temperature, and DNA quantity. These technical variables can lead to inconsistent results, particularly in laboratories with different levels of technical expertise or equipment.

Furthermore, the use of clinical isolates underscores the applicability of this method in real-world scenarios, emphasizing its potential as a valuable tool for clinical diagnostics and epidemiological studies. In addition, the PCR-RFLP approach was designed based on sequence variation in the adk gene, which is one of the seven loci used in MLST for Enterobacteriaceae. This approach enables differentiation between closely related members of the Enterobacteriaceae family, such as Shigella and Salmonella, as these members also harbor conserved adk sequences with species-specific polymorphisms [15].

In conclusion, our novel adk PCR/restriction digestion assay is robust and highly specific for effectively differentiating EC, EF, and CF isolates. The success of this technique in theoretical predictions and experimental applications, coupled with its adaptability to clinical isolates, makes it a promising molecular tool for precise differentiation of bacterial species. This innovative approach simplifies the discrimination of EC, EF, and CF, and has significant implications for the advancement of bacterial typing methods. Its contribution to the ongoing efforts to unravel microbial diversity and epidemiology further accentuates its potential as an asset for molecular diagnostics.

Supplementary materials

Article information

Conflicts of interest

No potential conflict of interest relevant to this article was reported.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education and the Korean Government Ministry of Science and ICT (MSIT), grant numbers NRF-2017R1D1A3-B06032486 and NRF-2022R1F1A1073686, respectively.

Author contributions

Conceptualization: YJC, BK, MSR, JHS, JK, SK; Data curation: RHD, SK; Formal analysis, Funding acquisition, Supervision: JK, SK; Investigation: YJC, BK, MSR, JHS; Methodology: RHD, YJC, BK, MSR, JHS, SK; Project administration, Validation: JK; Visualization: RHD, YJC, BK, MSR, JHS, SK; Writing-original draft: RHD; Writing-review & editing: YJC, BK, MSR, JHS, JK, SK.

Fig. 1.

Regions of the adk sequences of Escherichia coli, Escherichia fergusonii, and Citrobacter freundii; nucleotides 93 (red) and 96 (blue) differ among the three species, resulting in BtsIMutI, BtgI, and AgeI digestion sites, respectively. adk, adenylate kinase.

Fig. 2.

Theoretical agarose gel images of the results of digestion of adk PCR amplicons from Escherichia coli, Escherichia fergusonii, and Citrobacter freundii using BtsIMutI, BtgI, or AgeI restriction enzymes; schematic diagrams of adk PCR amplicons (584 bp each) of E. coli, E. fergusonii, and C. freundii are presented with BtsIMutI, BtgI, and AgeI target sites; lane 1, 1 kb plus DNA ladder; lane 2, undigested PCR amplicons; lane 3, BtgI digestion; lane 4, BtsIMutI digestion; lane 5, AgeI digestion. adk, adenylate kinase; PCR, polymerase chain reaction; bp, base pair.

Fig. 3.

Agarose gel electrophoresis results of adk PCR/restriction digestion using Escherichia coli and Escherichia fergusonii reference strains (ATCC 25922 and KCTC 22525^T, respectively) and a Citrobacter freundii clinical isolate (C1-Y-1); lane 1, 1 kb plus DNA ladder; lane 2, adk amplicon from E. fergusonii; lane 3, BtgI digestion of adk amplicon from E. fergusonii; lane 4, BtsIMutI digestion of adk amplicon from E. fergusonii; lane 5, AgeI digestion of adk amplicon from E. fergusonii; lane 6, blank; lane 7, adk amplicon from E. coli; lane 8, BtgI digestion of adk amplicon from E. coli; lane 9, BtsIMutI digestion of adk amplicon from E. coli; lane 10, AgeI digestion of adk amplicon from E. coli; lane 11, 1 kb plus DNA ladder; lane 12, adk amplicon from C. freundii; lane 13, BtgI digestion of adk amplicon from C. freundii; lane 14, BtsIMutI digestion of adk amplicon from C. freundii; lane 15, AgeI digestion of adk amplicon from C. freundii; adk, adenylate kinase; PCR, polymerase chain reaction; bp, base pair.

Fig. 4.

Agarose gel electrophoresis results of AgeI digestion of adk gene amplicons from five clinical isolates of Citrobacter freundii. Agarose gel electrophoresis results of uncut (lanes 2, 4, 6, 8, and 10) and AgeI-digested (lanes 3, 5, 7, 9, and 11) DNA fragments of adk gene amplicons from five clinical isolates of C. freundii (C1-E-1, C1-Y-1, EB-B-4, EB-C-3, and Y2-B-107). Lanes 1 and 12 are 1 kb DNA ladders. adk, adenylate kinase.

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References

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