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A novel gene linked to Imipenem resistance in E. coli isolate lacking known Imipenem-resistance genes

Abstract

Imipenem-resistant Escherichia coli strains represent a growing public health concern, posing a threat due to their resistance to last-resort antibiotics. Here, we present the discovery of the Imipenem-Linked Resistance Gene VIN (ILR-VIN) within E. coli isolates from Vietnam, revealing its absence in non-resistant E. coli and local bacterial species. ILR-VIN constitutes a previously unrecognized genetic element potentially linked to Imipenem resistance, with notable prevalence in Vietnamese E. coli strains.We conducted an in-depth examination of the genetic basis of Carbapenem resistance in E. coli strains causing urinary tract infections. In a set of 47 UTI strains, we identified five displaying Imipenem resistance, with four of them carrying known resistance genes. Interestingly, ECV219, despite exhibiting Imipenem resistance, lacked known resistance genes, suggesting an unreported resistance mechanism. Comparative genetic analysis revealed distinct genes in ECV219, indicating a novel Imipenem resistance gene. To assess its function, we conducted transformation experiments in E. coli Rosetta™(DE3)pLysS and performed bioinformatics analyses using BLASTp, InterProScan, and Pfam to characterize the gene’s structure and potential functions.Our study identifies ILR-VIN as a novel gene linked to Imipenem resistance in E. coli isolate lacking known Imipenem-resistance genes. Experimental evidence confirmed that ILR-VIN expression enhances bacterial survival under Imipenem stress, providing direct evidence of its role in resistance. This discovery highlights the importance of ongoing research into antibiotic resistance genes to develop effective treatment strategies against antibiotic-resistant bacterial infections.

Introduction

In recent years, the global rise of antibiotic resistance among bacterial pathogens has evolved into a formidable public health challenge1. A key component of this crisis is the increasing prevalence of Carbapenem-resistant bacteria, which pose a significant threat due to their resistance to last-resort antibiotics2. Among these, Imipenem-resistant Escherichia coli strains have garnered considerable attention for their clinical impact and potential to disseminate resistance genes3.

Carbapenems, a class of beta-lactam antibiotics, have long been a mainstay in treating severe bacterial infections. However, the emergence of Carbapenem resistance in E. coli has raised serious concerns, as this pathogen is one of the leading causative agents of urinary tract infections (UTIs) and other healthcare-associated infections4. The ability of Carbapenem-resistant E. coli to compromise the efficacy of antibiotic treatments necessitates a deeper understanding of the genetic mechanisms behind this resistance.

This study delves into the genetic determinants of Carbapenem resistance in E. coli strains isolated from patients with UTIs in Vietnam. With a focus on Imipenem resistance, we aim to identify and characterize resistance genes and explore potential novel mechanisms contributing to this resistance. Investigating the genetic underpinnings of Carbapenem resistance is crucial, not only for understanding the mechanisms at play but also for guiding effective treatment strategies5.

A notable observation within our dataset is the presence of a previously unidentified resistance gene, the Imipenem-Linked Resistance Gene detected in Vietnam (ILR-VIN), which has implications for Carbapenem resistance in E. coli. We present compelling evidence for the association of ILR-VIN with resistance to Carbapenem antibiotics, particularly Imipenem. This discovery underscores the need for continuous surveillance and molecular characterization of antibiotic resistance genes to adapt to the ever-evolving landscape of resistance.

In this paper, we discuss our comprehensive approach, beginning with the identification of Carbapenem-resistant E. coli strains in UTI cases and an analysis of their resistance profiles. We introduce the ILR-VIN gene, its prevalence in local strains, and its potential role in resistance. Comparative genetic analyses explore unique features of strains carrying this gene and unveil the potential existence of an unidentified resistance mechanism. The study culminates in a correlation analysis between ILR-VIN and Carbapenem resistance, highlighting its pivotal role in this context.

By shedding light on the genetic underpinnings of Carbapenem resistance in E. coli, our research aims to contribute to the development of targeted interventions and responsible antibiotic use. Furthermore, this study emphasizes the need for continued genomics-driven surveillance in clinical settings to address the evolving landscape of antibiotic resistance, a critical step in mitigating the global challenge of antibiotic-resistant bacterial infections.

Materials and methods

Study design and setting

The research, conducted from 2018 to 2020 at Nghe An’s Obstetrics and Pediatrics Hospital and the Department of Genomics, Institute of Biomedicine & Pharmacy, Vietnam Military Medical University, involved the genetic analysis of bacterial specimens. It strictly adhered to ethical standards, in accordance with the principles of the Helsinki Declaration. Notably, the study, using non-identifiable bacterial samples from the microbiology department, required no direct patient interaction or use of identifiable patient data. The Institutional Review Board (IRB) of Nghe An’s Obstetrics and Pediatrics Hospital confirmed that neither an ethics approval number nor informed consent was necessary for this study, as it complied with their guidelines for research involving secondary data obtained from routine medical practices.

Sample collection and isolation

The study focused on 47 E. coli strains isolated from pediatric patients aged 2 months to 15 years who were presented with fever and sought medical care at Nghe An’s Obstetrics and Pediatrics Hospital between 2018 and 2020. All patients were diagnosed with and treated for febrile urinary tract infection (UTI) during this specific period. Diagnostic criteria for febrile UTI followed the guidelines set forth by the National Institute for Health and Clinical Excellence (NICE)6.

Exclusion criteria were applied to patients with febrile UTI who had severe illnesses requiring intensive care, life-threatening conditions, concurrent medical conditions, outpatient or lower-level antibiotic treatment during the current illness, uncooperative families, or polymicrobial urine cultures.

The urine culture technique was employed to identify bacteria, including urine microscopy to detect leukocyturia as a sign of UTI. Bacterial isolation and culturing were conducted, and samples with ≥ 105 colony-forming units per milliliter (CFU/mL) were considered positive.

The VITEK®2 Compact system was used for bacterial identification, employing colorimetric methods for bacterial identification and extended-spectrum beta-lactamase (ESBL) detection. Antibiotic susceptibility testing was carried out to determine the minimum inhibitory concentration (MIC) for each antibiotic.

Bacterial strains displaying antibiotic resistance, particularly E. coli, were collected and preserved in BHI medium with 20% glycerol at -80 °C for further analysis in the Department of Genomics, Institute of Biomedicine & Pharmacy, Vietnam Military Medical University.

Next-generation sequencing

After bacterial isolation, identification, and antibiotic susceptibility testing, total DNA extraction from the isolated strains was carried out. The QIAamp DNA Mini Kit was used for DNA extraction, which involved multiple steps such as cell lysis, proteinase digestion, DNA binding to a column, washing, and elution. Extracted DNA was stored at -20 °C for later use. DNA concentration was quantified and normalized to prepare samples for genomic sequencing. The DNA was fragmented into segments of 300–500 nucleotides, and indexes were added to differentiate samples within a single sequencing run.

Illumina’s Next-Generation Sequencing technology was employed for whole-genome sequencing. The complete E. coli genome was sequenced using Illumina’s Miseq v2 Reagent Kit with 300 cycles. Paired-end sequencing was performed to obtain comprehensive genomic data.

Bioinformatic analysis

A comprehensive bioinformatics analysis was conducted using a pipeline that seamlessly integrates advanced and reliable tools for comprehensive bacterial genome analysis. This included FastQC for sequence quality assessment, Trimmomatic for sequence cleaning, Shovill for genome assembly, Prokka for gene annotation, and Abricate for profiling antibiotic resistance and virulence genes. Databases such as Card, Megares, Resfinder, Argannot, NCBI, and Plasmidfinder were used for this purpose. Additionally, PubMLST was employed for sequence type (ST) determination, and Kraken2 for species identification. After thorough analysis of individual samples within the dataset, the Roary tool was used to examine core and accessory genome components. Gene sequences within each cluster were translated into protein sequences, facilitating subsequent gene alignment for identifying protein variations and nucleotide mutations. Finally, Iqtree was used to construct phylogenetic trees for each gene cluster.

This comprehensive and high-throughput analysis pipeline was executed using Python 3.6 and adhered to standardized protocols available on GitHub (https://github.com/amromics/amrviz). This rigorous bioinformatics approach ensured meticulous examination of genomic data, providing valuable insights into the genetic makeup of E. coli strains, including their core and accessory genes, antibiotic resistance, and virulence gene profiles.

Nucleotide BLAST analysis: We performed nucleotide BLAST analysis using the bacteria filter on NCBI. This analysis allowed us to identify bacterial samples in the NCBI database with sequence regions resembling the sequence of interest.

BLASTp analysis: To investigate the uniqueness of the ILR-VIN gene, we conducted a BLASTp analysis to compare its protein sequence with established Carbapenem resistance genes. The specific genes used for comparison included blaKPC, blaNDM, blaVIM, and blaOXA. The protein sequence of ILR-VIN was aligned against these well-known Carbapenemase proteins to assess sequence similarity and potential functional relationships.

Additional bioinformatics analysis: To further elucidate the potential function of the ILR-VIN protein, we conducted comprehensive bioinformatics analyses using InterProScan and Pfam to identify conserved protein domains and families.

- InterProScan Analysis: InterProScan was used to scan the ILR-VIN protein sequence against a comprehensive database of protein families, domains, and functional sites. This analysis aimed to identify any conserved domains that could provide insights into the protein’s function.

- Pfam analysis: The ILR-VIN protein sequence was also analyzed using Pfam, a database of protein families represented by multiple sequence alignments and hidden Markov models (HMMs). This analysis sought to detect any significant matches to known protein families, which could suggest functional attributes of ILR-VIN.

Transformation experiments for ILR-VIN gene analysis

- Preparation of competent cells: (1) Strains and media: Rosetta™(DE3)pLysS Competent Cells were utilized for the transformation experiments. Rosetta™(DE3)pLysS is a highly versatile E. coli strain designed to improve the expression of heterologous genes. This property makes Rosetta™(DE3)pLysS an ideal choice for studying the ILR-VIN gene. The strain was cultured using Laura Bertani (LB) broth, comprising 10 g Bacto-tryptone, 5 g yeast extract and 10 g NaCl per liter of distilled water, adjusted to pH 7; (2) Competent cell preparation: Colonies of Rosetta™(DE3) Competent Cells were obtained by plating on LB agar. An overnight culture was prepared by inoculating 2 mL of LB medium with a single colony and shaking at 200–250 rpm at 37 °C. The overnight culture was then diluted 1:100 in fresh LB broth and incubated with shaking at 37 °C until reaching an OD600 of 0.4–0.6. The culture was chilled on ice for 10–15 min, followed by centrifugation at 4 °C and 4000 x g for 10 min to harvest the cells. The cell pellet was resuspended in cold 0.1 M CaCl2, incubated on ice for an hour, and centrifuged again. The final cell pellet was resuspended in cold 0.1 M CaCl2 with 60% glycerol and stored at -80 °C or used immediately for transformation.

- Plasmid transformation: (1) Transformation protocol: Plasmid pET28a(+) carrying the ILR-VIN gene sequence was mixed with competent cells and incubated on ice for 30 min. A heat shock at 42 °C for 45 s followed by immediate cooling on ice for 2 min was performed. The bacterial suspensions were then incubated with LB medium for 1 h at 37 °C with shaking. Aliquots of the culture were then plated on LB agar plates containing Kanamycin (50 µg/mL) to ensure the selection of transformed colonies. The plasmid pET28a(+) includes a bacterial resistance sequence against Kanamycin, aiding cell survival in this antibiotic environment; (2) Verification of transformation: Transformed E. coli formed colonies in the medium containing Kanamycin, confirming successful transformation. After overnight incubation, colonies formed on Kanamycin-containing LB medium were subcultured. Plasmid DNA was extracted from these colonies using a QIAamp DNA Mini Kit. The presence of the ILR-VIN gene was confirmed by DNA sequencing.

- Protein expression: For protein expression, overnight cultures of transformed Rosetta™(DE3)pLysS cells were diluted 1:100 into fresh LB medium containing Kanamycin (50 µg/mL) and incubated at 37 °C with shaking at 200 rpm until an optical density at 600 nm (OD600) of 0.6–0.8 was achieved. At this point, IPTG was added to a final concentration of 0.5 mM to induce ILR-VIN expression, and the cultures were subsequently incubated at 25 °C with shaking at 200 rpm for an additional 24 h. Control cultures without IPTG were grown under identical conditions to serve as uninduced controls.

- Cell lysis and protein preparation: After induction, cells were harvested by centrifugation at 4,000 × g for 10 min at 4 °C. The cell pellet was resuspended in lysis buffer (50mM Tris-HCl, pH 7.0) supplemented with protease inhibitors PMSF at a final concentration of 0.1mM. The suspension was then incubated in a water bath at 37 °C for 15 min. The lysate was clarified by centrifugation at 13,000 × g for 10 min at 4 °C.

- SDS-PAGE analysis

To assess the expression of ILR-VIN protein in IPTG-induced and uninduced cells, crude lysates were first analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis was performed at a constant voltage until the dye front reached the bottom of the gel. Protein samples were prepared in 5X sample buffer (25% (v/v) glycerol; 14.4mM β-mecraptoethanol; 2% (w/v) SDS; 0.1 (w/v) bromophenol blue; 60mM Tris - HCl pH 6.8) and denatured by heating at 95 °C for 15 min. Following electrophoresis, the gel was stained with Coomassie Brilliant Blue to visualize protein bands.

- Ni-NTA affinity chromatography and SDS-PAGE analysis

The crude lysate from IPTG-induced cells was applied to a nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography column equilibrated with lysis buffer (50mM Tris-HCl, pH 7.0). The column was washed sequentially with wash buffers containing 60mM and 80mM Imidazole to remove nonspecific proteins. Finally, the ILR-VIN protein was eluted using an elution buffer containing 100mM Imidazole. The eluted protein fractions were then analyzed by SDS-PAGE under the same conditions as previously described.

Data analysis

The SPSS software was employed for data analysis. Various statistical tests, including Fisher’s exact test, were applied to assess relationships and differences between variables. Statistical significance was determined when the p-value (statistical value) was less than 0.05, indicating the presence of a statistically significant relationship or difference.

Result and discussion

Sequence analysis of Carbapenem-resistant genes

Our investigation into the Antibiotic Resistance (AR) profiles of the 47 Escherichia coli strains responsible for urinary tract infections (UTIs) in our study revealed a notable observation. Specifically, 5 out of the 47 samples demonstrated a distinct Carbapenem-Resistant (CR) phenotype, as denoted by their sample codes: ECV219, ECV305, ECV306, ECV307, and ECV312. These findings are consistent with previous studies2,3 highlighting the emergence of Carbapenem resistance.

To gain deeper insights into the genetic basis of these AR profiles, we employed advanced bioinformatics tools to scrutinize the sequences of AR genes within the strains. As a result, we successfully identified the presence of the blaNDM-5 gene in these strains, a known genetic marker associated with the CR phenotype7.

Intriguingly, our subsequent statistical analysis, employing Fisher’s exact test, uncovered a compelling pattern. The results of these analyses underscored a robust statistical association between the presence of the blaNDM-5 gene and the manifestation of the CR phenotype. Notably, Fisher’s exact test (p = 0.000196, < 0.01 significance level) supports this relationship.

However, within the subset of these five CR strains, a single sample, ECV219, presented an anomaly. Despite exhibiting Imipenem resistance, a clear hallmark of the CR phenotype, ECV219 did not carry the blaNDM-5 gene or any other genes known to be associated with Carbapenem resistance. This unexpected finding led us to propose a hypothesis. We postulate that ECV219 may conceal an Imipenem (Carbapenem) resistance gene previously undocumented, thus warranting further investigation to elucidate the genetic underpinnings of this unique antibiotic resistance profile.

Genetic disparities and potential Imipenem resistance gene

In an effort to delve deeper into the intricacies of ECV219’s unique antibiotic resistance profile, we conducted a comparative analysis with other E. coli strains from the E. coli collection within our study. Our objective was to scrutinize these strains for potential genetic disparities, particularly genes unique to ECV219, which might elucidate the origins of its distinctive resistance profile.

Remarkably, within this collection, ECV219 shared a common sequence type (ST43) with two other samples, namely ECV220 and ECV310. However, while these three samples exhibited congruent ST sequence types, ECV219 stood out by displaying Imipenem (Carbapenem) resistance, a characteristic not shared by the other two samples. Moreover, a genetic inspection of the AR genes in these strains unveiled another striking distinction: ECV219 differed both in its antibiotic resistance gene repertoire and the number of resistance genes present, setting it apart from its counterparts.

A more detailed comparative gene analysis among these three samples underscored the specific genetic markers unique to ECV219. Specifically, four distinct genes - aadA5, mph(A), sul1, and dfrA17 - were identified in ECV219 that were conspicuously absent in the other strains. It is essential to note that these genes were not associated with Carbapenem antibiotics; rather, they were found to be implicated in resistance against other classes of antibiotics. Most intriguingly, all four of these genes resided within the same sequencing contig, designated as contig I0.

Based on these findings, we hypothesize the existence of an unidentified gene linked to Imipenem resistance. This gene is posited to be located within contig I0, though further investigations are required to pinpoint its exact location and unravel the precise genetic mechanisms contributing to the unique antibiotic resistance profile exhibited by ECV219.

Characterization of a carbapenem resistance-associated gene region

We aimed to identify E. coli strains within our sample set carrying all four genes (aadA5, mph(A), sul1, dfrA17) located on the same sequencing contig while lacking resistance to Carbapenem antibiotics. Our analysis of AR gene sequences successfully pinpointed ten samples meeting these criteria. Their corresponding contig information (I1, I2, …, I10) is presented in Table 1. Based on these findings, we propose that the Imipenem resistance gene, previously undetected, is an exclusive sequence residing solely in contig I0, distinct from other contigs (I1 to I10).

*Table 1 E. coli strains carrying aadA5, mph(A), sul1, and dfrA17 genes simultaneously on the same contig.*

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To explore this hypothesis, we further conducted sequence comparisons between contig I0 and other contigs, specifically I1 through I10, utilizing the Bioedit tool. The results disclosed that contigs I1 (ECV202), I2 (ECV203), I3 (ECV204), I4 (ECV212), I6 (ECV221), I7 (ECV319), and I8 (ECV321) share common sequences. Contig I1 and I7 were identified to have identical sequences among themselves, while contigs I2, I3, I4, I6, and I8 also possess sequences identical among themselves. Notably, the sequence alignments between those contigs (I1, I2, I3, I4, I6, I7, I8) displayed minimal similarity to contig I0.

Contig I0 of ECV219, on the other hand, exhibited significant sequence similarity with the contigs I5 from ECV213, contig I9 from ECV322, and contig I10 from ECV325. However, the lengths of these sequences were comparatively shorter than that of I0 (Table 1). Additionally, the alignment results revealed that the sequences of contigs I5, I9, and I10 were entirely identical. Upon close examination of the sequence data, particularly focusing on the 3’ end of the contigs, we identified a distinctive region in contig I0 compared to the other three contigs. This sequence variation was pinpointed between nucleotide positions 10,079 and 10,772 (Fig. 1). These nucleotide positions are measured according to the reference sequence of contig I0 in ECV219, which serves as the standard for comparison.

Fig. 1

figure 1

Sequence alignment of the contig I0 with contigs I5, I9, and I10 The sequence of the contig I0 from ECV219 is compared with contigs I5, I9, and I10 from other strains. While contig I0 showed significant sequence similarity with the mentioned contigs, it exhibited a distinctive region at the 3’ end. This sequence variation, highlighted between nucleotide positions 10,079 and 10,772, differentiates Contig I0 from the other three contigs. The nucleotide positions are based on the reference sequence of Contig I0 in ECV219, which is used as the standard for comparison.

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The contrasting sequence data between ECV219 and the other strains provide significant evidence supporting the hypothesis that the unique genetic region in ECV219 related to Carbapenem (Imipenem) resistance is likely to be confined to the contig I0 and is absent from other contigs (I1 to I10).

Identification of the novel Imipenem-linked resistance gene (ILR-VIN)

The distinct sequence region within contig I0 of the ECV219 strain, designated as “I0_3end” and spanning 694 base pairs (Table S1, Supplementary), underwent nucleotide BLAST analysis using the Enterobacterales filter on NCBI. This analysis enabled us to compile bacterial samples with sequence regions akin to the I0_3end sequence.

The results unveiled an E. coli strain possessing a sequence region exhibiting a 100% match with the I0_3end sequence. This matching region spans from nucleotide positions 131,101 to 131,794 within this strain’s genome (ID: CP018990_1). Within this unique region, we identified two genes: (1) A gene named “Integron integrase” (protein ID: AQV71290.1), stretching from nucleotide position 130,355 to 131,368, with a length of 1,014 base pairs; and (2) another gene referred to as the “Hypothetical protein” (protein ID: AQV71328.1), completely contained within the blast-identified sequence region, extending from nucleotide position 131,313 to 131,711 and having a length of 399 base pairs. Consequently, we have designated the previously unidentified hypothetical protein, which is potentially associated with Carbapenem antibiotic resistance, with a particular focus on Imipenem, as the Imipenem-Linked Resistance or “ILR-VIN” gene.

Our bioinformatics searches identified an integron integrase gene within this unique region, indicating potential mobility. This finding, combined with the data presented in Sect. 3, shows that the ILR-VIN gene is co-located with other antibiotic resistance genes on a single contig. Specifically, ILR-VIN resides alongside four other resistance genes: aadA5, mph(A), sul1, and dfrA17. This co-location suggests that ILR-VIN is likely situated on a plasmid, highlighting the potential for horizontal gene transfer and contributing to the spread of antibiotic resistance. Although the bioinformatics data suggest mobilizability, experimental validation through trans-conjugation assays is warranted in future research to confirm these findings.

To further investigate the uniqueness of the ILR-VIN gene, we performed BLASTp analysis to compare its protein sequence with well-known Carbapenem resistance genes such as blaKPC, blaNDM, blaVIM, and blaOXA. The results revealed no significant sequence similarity, indicating that ILR-VIN represents a novel variant with a distinct mechanism of action. This finding suggests that ILR-VIN may confer Carbapenem resistance through a unique pathway, different from the established Carbapenemases.

To elucidate the potential function of the ILR-VIN protein, we conducted bioinformatics analyses using InterProScan and Pfam to identify conserved protein domains and families. The analyses did not reveal any significant matches, suggesting that ILR-VIN may represent a novel protein with a unique function. This lack of similarity to known protein domains underscores the need for experimental validation to determine the role of ILR-VIN in Carbapenem resistance. Further structural and functional characterization through techniques such as X-ray crystallography, NMR spectroscopy, and functional assays will be essential to uncover the mechanisms by which ILR-VIN confers resistance.

The absence of significant matches in InterProScan and Pfam analyses indicates that ILR-VIN is likely a novel protein. This finding highlights the importance of continuing experimental investigations to determine its function and mechanism of action. Understanding ILR-VIN’s role in Carbapenem resistance could provide new insights into combating antibiotic-resistant infections.

Moreover, BLASTp analysis comparing the ILR-VIN protein with well-known Carbapenem resistance proteins such as blaKPC, blaNDM, blaVIM, and blaOXA revealed no significant sequence similarity, suggesting that ILR-VIN may represent a novel variant within the Carbapenemase family. These findings highlight the importance of further experimental validation to uncover the mechanisms by which ILR-VIN confers resistance.

Correlation analysis between the ILR-VIN gene and carbapenem resistance

In our study, we conducted a comprehensive correlation analysis to unravel the intricate relationship between the newly identified ILR_VIN gene and Carbapenem resistance among E. coli strains. The results of this analysis provide compelling evidence of the gene’s pivotal role in mediating resistance to Carbapenem antibiotics, particularly Imipenem.

Table 2 offers an overview of our correlation analysis between the ILR_VIN gene and the Carbapenem resistance phenotype. Notably, all Carbapenem antibiotics included in the study exhibit a robust correlation with the ILR_VIN gene. This strong association underscores the potential link between the presence of the ILR_VIN gene and resistance to Carbapenem antibiotics, a critical finding in understanding the mechanisms of resistance. Particularly striking is the high correlation observed between the ILR_VIN gene and resistance to Imipenem, one of the most essential Carbapenem antibiotics in the clinical setting. The Fisher’s exact test, with a p-value of < 0.05, lends high statistical significance to this relationship, further emphasizing the gene’s pivotal role in conferring Imipenem resistance to E. coli. These results not only enhance our understanding of the clinical implications of the ILR_VIN gene but also shed light on its potential as a key determinant in Carbapenem resistance, especially concerning Imipenem, which is instrumental in the treatment of severe bacterial infections.

The striking correlation between the ILR_VIN gene and Carbapenem resistance not only fortifies our initial hypothesis but also accentuates the intricate involvement of this hypothetical protein in E. coli’s ability to counteract the effects of Carbapenem antibiotics, especially Imipenem. Comprehending the genetic basis of this resistance mechanism assumes paramount importance, as it opens avenues for the development of targeted treatment strategies and underscores the necessity for responsible antibiotic use in clinical settings8. Furthermore, this discovery catapults us into a realm of research exploring the emergence and dissemination of antibiotic resistance genes, which is of critical importance in combating the ever-growing global menace of antibiotic-resistant bacterial infections.

Table 2 Correlation analysis between the gene and carbapenem resistance phenotype.

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The correlation analysis in our study indicates that ILR-VIN is associated with resistance to multiple Carbapenems, including Imipenem and Meropenem. While our findings support the gene’s broader role in Carbapenem resistance, further experimental validation using controlled expression systems is essential to determine the specific resistance profile conferred by ILR-VIN. It is also notable that ECV219 exhibited resistance specifically to Imipenem while remaining susceptible to other Carbapenem antibiotics, suggesting a unique relationship between ILR-VIN and Imipenem resistance.

In our pursuit of further evidence, we extended our investigation to the NCBI database, leveraging the ILR_VIN gene sequence. Our search aimed to identify bacterial strains harboring this gene and explore the reported Carbapenem resistance phenotypes associated with these strains. The results, as detailed in Table 3, revealed that 26 bacterial strains exhibited a complete match with the ILR_VIN gene sequence (updated: 14/10/2023), suggestive of a previously undiscovered gene. Importantly, within the subset of strains with resistance information available (n = 7), all seven displayed resistance to Carbapenem antibiotics, further solidifying the connection between the ILR_VIN gene and Carbapenem resistance.

Table 3 Bacterial strains with the ILR_VIN gene and carbapenem resistance detected from the NCBI database (updated: 14/10/2023).

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The discovery of 26 strains within the NCBI database, all sharing an identical gene sequence with the ILR_VIN gene, and uniformly demonstrating resistance to Carbapenem antibiotics, accentuates the gene’s central role in mediating resistance within this critical class of antibiotics. These findings not only corroborate the evidence from our primary analysis but also emphasize the gene’s significance in conferring resistance, highlighting the need for sustained research into this gene and its clinical implications.

Evaluation of ILR-VIN expression and its role in Imipenem resistance

To investigate the role of the ILR-VIN gene in conferring resistance to Imipenem, transformation experiments were conducted using E. coli Rosetta™(DE3)pLysS cells. This strain is optimized for robust protein expression from plasmids with a T7 promoter, due to its incorporation of complementary tRNAs that prevent ribosome stalling and the pLysS component, which minimizes background expression by producing low levels of T7 lysozyme. These features are critical for the expression of sensitive and potentially toxic proteins like ILR-VIN, allowing normal cell growth before IPTG induction. The plasmid pET28a(+), carrying the ILR-VIN gene under the control of a T7 promoter, was introduced into competent Rosetta™(DE3)pLysS cells, and successful transformation was confirmed by culturing cells under Kanamycin selection.

To evaluate the functional role of ILR-VIN, two experimental conditions were established. In the first group, transformed cells were induced with IPTG to trigger ILR-VIN expression, representing the experimental condition. In the second group, transformed cells were cultured without IPTG, serving as the uninduced control group in which ILR-VIN was not expressed. By comparing these two groups, the contribution of ILR-VIN to Imipenem resistance could be directly assessed. Both groups were exposed to varying concentrations of Imipenem (0, 1, 4, and 16 µg/mL) in LB medium for 24 h at 37 °C, and bacterial survival was measured by counting colony-forming units (CFUs).

To confirm the expression of ILR-VIN in IPTG-induced cells, SDS-PAGE analysis was performed. A distinct protein band with an approximate molecular weight of 18 kDa was detected in lysates from IPTG-induced cells, corresponding to the expected size of ILR-VIN. In contrast, no such band was observed in uninduced control samples. Further verification was conducted using Ni-NTA affinity chromatography. As shown in Fig. 2, ILR-VIN was successfully purified from IPTG-induced samples, with a prominent band detected in elution fractions containing 100 mM Imidazole. Elution fractions obtained at lower Imidazole concentrations (60 and 80 mM) did not contain detectable amounts of ILR-VIN, indicating that the protein remained bound to the column under these conditions.

Fig. 2

figure 2

SDS-PAGE analysis of ILR-VIN expression and purification. Lane 1 and Lane 2 represent the crude lysates of IPTG-induced and uninduced E. coli Rosetta™(DE3)pLysS cells, respectively. A distinct protein band corresponding to the expected molecular weight of ILR-VIN (~ 18 kDa) is observed in the induced sample (Lane 1, red arrow) but absent in the uninduced control (Lane 2). Lanes 1.1 and 1.2 show elution fractions from the Ni-NTA affinity column using 60 mM and 80 mM Imidazole, where ILR-VIN remains bound to the column. Fractions 1.3 to 1.6 represent the elution with 100 mM Imidazole, successfully recovering ILR-VIN as indicated by the prominent band near 18 kDa.

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Control cells, which were not induced with IPTG, exhibited robust colony formation in the absence of Imipenem (0 µg/mL), indicating normal bacterial growth under non-stress conditions. However, no colony formation was observed at any Imipenem concentration (1, 4, and 16 µg/mL), demonstrating complete susceptibility to the antibiotic (Fig. 3B). This finding confirmed that without ILR-VIN expression, the transformed E. coli cells were unable to survive exposure to Imipenem.

Fig. 3

figure 3

Survival of E.coli Rosetta™(DE3)pLysS Cells transformed with pET28a(+)/ILR-VIN under Imipenem stress. Colony-forming units (CFUs) of IPTG-induced cells expressing ILR-VIN (A) and uninduced control cells (B) after 24 hours of exposure to Imipenem (0, 1, 4, and 16 µg/mL). IPTG-induced cells exhibited enhanced survival at all Imipenem concentrations, while uninduced cells showed no colonies under Imipenem stress, confirming the role of ILR-VIN in resistance.

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In contrast, cells induced with IPTG to express ILR-VIN exhibited significantly enhanced survival under Imipenem treatment (Fig. 3A). At 1 µg/mL Imipenem, IPTG-induced cells formed robust colonies, indicating that ILR-VIN expression conferred measurable resistance. As the Imipenem concentration increased to 4 µg/mL, colony numbers declined, but viable cells persisted. Even at the highest Imipenem concentration tested (16 µg/mL), a small but detectable number of colonies were observed. These findings demonstrate that ILR-VIN expression provided protection against Imipenem and contributed to the bacterial cells’ survival under Imipenem stress.

The stark differences in survival between the IPTG-induced and uninduced cells underscore the importance of ILR-VIN expression in mediating resistance to Imipenem. The absence of colony formation in uninduced cells under antibiotic treatment confirmed that IPTG-driven ILR-VIN expression was a critical determinant of resistance. These results strongly support the hypothesis that ILR-VIN plays a functional role in mediating bacterial resistance to Imipenem.

These findings offer compelling evidence for the involvement of ILR-VIN in Imipenem resistance, underscoring the importance of further studies to unravel its precise mechanism of action. Future research should investigate whether ILR-VIN interacts with other resistance pathways, influences biofilm formation, or modulates efflux pump activity. Elucidating these mechanisms will deepen our understanding of bacterial resistance and inform the development of novel therapeutic strategies.

Taken together, the results provide an integrated perspective on the critical role of the ILR-VIN gene in Carbapenem resistance. By leveraging both in-house experimental data and external insights, this study contributes to the broader understanding of genetic factors underlying antibiotic resistance and paves the way for innovative strategies to combat the global challenge of antibiotic-resistant infections.

Conclusion

Our study identifies a novel gene, named ILR-VIN, discovered in an Imipenem-resistant E. coli strain that lacks any previously known Imipenem-resistance genes. Transformation experiments using E. coli Rosetta™(DE3)pLysS demonstrated successful expression of ILR-VIN under IPTG induction, which conferred measurable resistance to Imipenem. IPTG-induced cells expressing ILR-VIN showed significantly enhanced survival across various Imipenem concentrations, including 16 µg/mL, compared to uninduced controls, which exhibited complete susceptibility. These results confirm that ILR-VIN plays a functional role in mediating bacterial resistance to Imipenem.

This discovery highlights the critical importance of understanding novel resistance mechanisms and underscores the necessity of responsible clinical antibiotic use. While our findings provide strong evidence linking ILR-VIN to Imipenem resistance, further research is required to elucidate its precise mechanism of action and potential interactions with other resistance pathways. Investigating the broader implications of ILR-VIN, including its impact on biofilm formation and efflux pump activity, will enhance our understanding of bacterial resistance and aid in the development of innovative therapeutic strategies to address the growing global challenge of antibiotic-resistant bacterial infections.

Data availability

The datasets generated and/or analyzed during the current study are available in the NCBI’s BioProject, accessible via the accession number PRJNA1040333 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1040333).

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Acknowledgements

The authors would like to express their sincere gratitude to Ngoc Anh Thi Nguyen, Loan Thi Tran, Quyen Van Pham, Huong Quynh Thi Pham, Quyen Thi Nguyen, Hang Thi Pham, and Trang Thuy Dao for their exceptional technical assistance and insightful discussions throughout the research. Their contributions significantly contributed to the successful culmination of this study. Their contributions were pivotal in the successful completion of this study. This research is supported by the Vingroup Innovation Foundation (VINIF) under project code VINIF.2019.DA11.

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Author notes

Trang Thu Hoang and Huyen Thanh Thi Le contributed equally to this work.

Authors and Affiliations

Department of Genomics, Institute of Biomedicine & Pharmacy, Military Medical University, Hanoi, Vietnam

Trang Thu Hoang, Ung Dinh Nguyen, Thi Minh Ngoc Dao & Tho Huu Ho

Vinh Medical University, Nghean, Vietnam

Huyen Thanh Thi Le

Pediatric Department, Haiphong University of Medicine and Pharmacy, Haiphong, Vietnam

Sang Ngoc Nguyen

Vietnam Military Medical University, Hanoi, Vietnam

Tuan Ngoc Tran

Department of Microbiology, Vietnam Military Medical University, Hanoi, Vietnam

Cuong Hung Nguyen & Tho Huu Ho

Military Hospital 4, Nghean, Vietnam

Thang Quang Truong

Center for Biomedical Informatics, Vingroup Big Data Institute, Hanoi, Vietnam

Nam S. Vo

Faculty of IT, National University of Civil Engineering, Hanoi, Vietnam

Duc Quang Le

Amromics JSC, Nghean, Vietnam

Son Hoang Nguyen & Minh Duc Cao

Institute of Biotechnology (IBT), Vietnam Academy of Science and Technology (VAST), 18, Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

Thi Huyen Bui, Thu An Nguyen, Thi Lan Anh Pham & Thi Bich Thao Le

Authors

Trang Thu Hoang

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