Convergence of Carbapenem and Colistin Resistance in Intestinal Escherichia coli ST167/ST2659 Driven by IncX3 bla_NDM-5 and IncI2-Associated Mcr-1.1 Mobility

Introduction

Carbapenem-resistant Enterobacterales have become a major global threat, with New Delhi metallo-beta-lactamases (NDM) representing one of the most clinically challenging carbapenemase families due to limited therapeutic options.^1–3 Among NDM variants, bla_NDM-5 is widely disseminated and has been repeatedly linked to epidemic plasmid backbones—particularly IncX3,⁴ a compact and highly transmissible plasmid type frequently reported as a major vehicle for interspecies spread of bla_NDM genes.⁵ The success of IncX3-mediated dissemination raises particular concern when bla_NDM-5 converges with other last-line resistance mechanisms within the same bacterial host or within linked mobile genetic elements.⁶

In parallel, the discovery of the plasmid-mediated colistin resistance gene mcr-1 marked a watershed moment by enabling horizontal transfer of polymyxin resistance, effectively threatening one of the final therapeutic lines against carbapenem-resistant infections.⁷ Since then, numerous mcr-1 variants have been detected across human, animal, food, and environmental settings, with mcr-1.1 being among the most prevalent alleles.^5,6,8 The mcr-1 gene is commonly found on several plasmid families, including IncI2 and IncX4, which facilitate dissemination within Enterobacterales.⁹

A growing body of evidence suggests that the intestinal tract is a critical ecological niche where high-density bacterial communities, antibiotic exposures, and mobile genetic elements interact to promote horizontal gene transfer.¹⁰ Colonization by multidrug-resistant organisms not only precedes infection in vulnerable hosts but also enables silent amplification and onward transmission.¹¹ From an antimicrobial stewardship and infection control perspective, colonizing reservoirs deserve particular attention when they carry co-transferable plasmids encoding resistance to both carbapenems and colistin, because co-selection under β-lactam pressure may stabilize colistin-resistance plasmids (and vice versa), increasing the probability of emergence of extensively drug-resistant or even pan-resistant strains.^12,13

Despite increasing reports of bla_NDM and mcr-1 co-carriage, the true risk is often underestimated when studies stop at gene detection without resolving plasmid architectures, localization, and functional transferability.^14,15 Whether such plasmids are simultaneously transferable, are questions that directly shape transmission risk and control strategies.

Here, we investigated two fecal colonizing E. coli isolates carrying bla_NDM-5 and mcr-1.1 recovered during routine intestinal colonization screening of hospitalized patients. To our knowledge, plasmid-level characterization of coexisting IncX3-borne bla_NDM-5 and IncI2-associated mcr-1.1 in gut-colonizing E. coli, together with functional validation of transferability, remains rarely described. Using combined short-read and long-read sequencing to achieve plasmid-level resolution, together with S1-PFGE/Southern blot localization and conjugation assays, we demonstrate a high-risk plasmidome in an ST167 isolate featuring redundant IncX3 bla_NDM-5 plasmids and dual transferable mcr-1.1 IncI2 plasmid, collectively forming a fully conjugative last-line resistance package in the gut reservoir. Such plasmid-resolved colonization data may help identify high-risk transmissible resistance platforms relevant to targeted surveillance and infection-control strategies.

Materials and Methods

Bacterial Isolates and Identification

Two non-duplicate E. coli isolates (L1771 and L2386) were recovered from fecal samples collected during routine intestinal colonization screening of hospitalized patients, a practice implemented to identify silent reservoirs of multidrug-resistant organisms, at the First Affiliated Hospital of Zhejiang University School of Medicine in 2025. Species identification was performed using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and confirmed by whole-genome sequencing (WGS)-based taxonomic assignment.

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing was performed to determine the resistance profile of the isolates. Minimum inhibitory concentrations (MICs) of amikacin, aztreonam, cefoxitin, cefotaxime, ceftazidime, ciprofloxacin, ertapenem, imipenem, meropenem, tetracycline, tobramycin, and trimethoprim-sulfamethoxazole were determined by broth microdilution. MICs of colistin and tigecycline were interpreted according to EUCAST clinical breakpoints, whereas the remaining agents were interpreted according to current CLSI criteria. Escherichia coli ATCC 25922 was used as the quality-control strain.

Detection of Resistance Genes and Plasmid Replicon Typing

Presence of bla_NDM-5 and mcr genes was initially screened by PCR using previously published primers^16,17 and confirmed by WGS, which identified the isolates as carryingbla_NDM-5 and mcr-1.1. Plasmid replicon types were inferred using PlasmidFinder and confirmed using complete plasmid assemblies.¹⁸ Additional resistance genes were identified using CARD (https://card.mcmaster.ca/) with default identity/coverage thresholds, followed by manual curation.¹⁹

Whole-Genome Sequencing, Hybrid Assembly, and Annotation

Genomic DNA was extracted using a commercial kit (Qiagen) following the manufacturer’s instructions. Short-read sequencing was performed on an Illumina platform (NovaSeq) with paired-end reads, and long-read sequencing was performed using Oxford Nanopore Technologies. Quality filtering and adapter trimming were conducted using standard pipelines (fastp for Illumina; Guppy for Nanopore).

Hybrid assemblies were generated using Unicycler.²⁰ Circular contigs were confirmed by read mapping and inspection of overlaps. Genome annotation was performed using Prokka.²¹ Multilocus sequence typing (MLST) was assigned using the Achtman scheme.

S1 Nuclease PFGE and Southern Blot Hybridization

Plasmid profiles were assessed by S1 nuclease PFGE. Briefly, bacterial cells were embedded in agarose plugs, lysed, treated with proteinase K, and digested with S1 nuclease to linearize plasmids. PFGE was performed using a contour-clamped homogeneous electric field (CHEF) system with conditions optimized for separation of plasmids in the 20–300 kb range. DNA was visualized by ethidium bromide staining.

For Southern blotting, DNA was transferred to nylon membranes. Gene-specific probes targeting bla_NDM-5 and mcr-1.1 were generated by PCR amplification using specific primers, followed by gel purification of the amplicons. The purified PCR products were then labeled with digoxigenin according to the manufacturer’s instructions. Membranes were hybridized with the labeled probes, followed by washing and chemiluminescent detection according to the standard laboratory protocol. Hybridization signals were detected using chemiluminescence according to the probe labeling system protocol. Plasmid sizes were estimated using appropriate DNA size markers.

Conjugation Assays

Conjugation experiments were performed using filter-mating methods with E. coli J53 as the recipient. Donor and recipient cells were mixed at an appropriate ratio (1:1 to 1:4), incubated on filters placed on non-selective agar at 37°C for 12–18 h, and then plated on selective media containing sodium azide plus either carbapenem (for selecting bla_NDM-5 plasmids) or colistin (for selecting mcr-1.1 plasmids). Putative transconjugants were confirmed by PCR verification of transferred plasmids. Transferability was assessed qualitatively based on recovery of transconjugants on selective media, PCR confirmation of the corresponding resistance genes/plasmids, and concordant antimicrobial susceptibility profiles. Because this study was designed as a descriptive plasmid-resolved analysis of two isolates, conjugation frequencies were not quantified.

Comparative Genomics and Plasmid Visualization

Complete plasmid sequences were compared against closely related plasmids in public databases using BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Plasmid maps and genetic environments surrounding bla_NDM-5 and mcr-1.1 were visualized using Easyfig.²² IS elements were annotated with ISfinder where necessary.²³

Results

Isolate Characteristics and Antimicrobial Susceptibility

Two fecal colonizing E. coli isolates carrying both bla_NDM-5 and mcr-1.1 were analyzed: L1771 (ST2659) and L2386 (ST167). Both isolates exhibited colistin resistance with MICs of 4 mg/L. Carbapenem MICs indicated high-level resistance: L1771 showed imipenem MIC 32 mg/L and meropenem MIC 32 mg/L, while L2386 showed imipenem MIC 16 mg/L and meropenem MIC 16 mg/L (Table 1). In addition to resistance to colistin and carbapenems, both isolates displayed multidrug-resistant phenotypes involving cephalosporins, ciprofloxacin, tetracycline, tobramycin, and trimethoprim-sulfamethoxazole, while tigecycline remained active against both isolates. These phenotypes were consistent with the presence of bla_NDM-5 and mcr-1.1, respectively.

Table 1 Antimicrobial Susceptibility Profiles of E. coli L1771, L2386, and Their Transconjugants, Supporting Transferability of the Corresponding Resistance Plasmids

Localization of Mcr-1.1 and bla_NDM-5 by S1-PFGE and Southern Blot

In L1771, bla_NDM-5 was located on an IncX3 plasmid, whereas mcr-1.1 was encoded on the chromosome. This unique genetic configuration in L1771 highlights a distinct mechanism for mcr-1.1 dissemination, contrasting with plasmid-borne mcr-1.1 in L2386. The co-existence of bla_NDM-5 and mcr-1.1 in the same isolate, with colistin MIC 4 mg/L, underscores the potential for horizontal gene transfer and co-selection under selective pressure. These results in agreement with the sequencing-based plasmid inventory (Figure 1).

Figure 1 S1-PFGE and Southern blot hybridization localize blaNDM-5 and mcr-1.1 on multiple plasmids. (A) S1 nuclease PFGE profiles showing linearized plasmids from L2386 and L1771. (B) Southern blot hybridization using mcr-1.1-specific probe. (C) Southern blot hybridization using blaNDM-5-specific probe. Signals were consistent with blaNDM-5 being located on IncX3 plasmids in both isolates, with mcr-1.1 located on an IncI2 plasmid in L2386 and on the chromosome in L1771. Plasmid sizes were estimated using a DNA size marker (M).

Plasmid-Resolved Resistome and High-Risk Plasmidome Architecture in L2386 (ST167)

Long-read sequencing and hybrid assembly achieved plasmid-level resolution. In L2386, bla_NDM-5 was located on IncX3 plasmid (~48 kb). In the same isolate, mcr-1.1 was carried on an IncI2 plasmid (~65 kb) (Table 2). The IncI2 mcr-1.1 plasmid additionally co-carried bla_CTX-M-199, further increasing the likelihood of co-selection (Figure 2). Collectively, L2386 harbored a two-plasmid last-line resistance package linking carbapenem and colistin resistance with ESBL determinants.

Table 2 Plasmid Inventory and Resistance Genes of E. coli L1771 (ST2659) and E. coli L2386 (ST167)

Figure 2 Comparative genetic environment of mcr-1.1 in L1771 and L2386. BLASTn-based comparison of the mcr-1.1-containing region on the IncI2 plasmid in L2386 with the chromosomal mcr-1.1-containing region in L1771. Conserved segments are highlighted to illustrate similarities and differences in the local genetic context of mcr-1.1 between the two isolates. The backbone conservation is highlighted, showing homology across the mcr-1.1 genetic environment in L2386 and L1771, suggesting widespread mobilization potential within this lineage.

bla_NDM-5 and ESBL Co-Occurrence in L1771 (ST2659)

In L1771, bla_NDM-5 co-occurred with bla_CTX-M-65 and bla_TEM-1B, indicating ESBL–carbapenemase convergence in an additional intestinal colonizing lineage distinct from ST167. The chromosome encoding of mcr-1.1 in L1771, together with colistin MIC 4 mg/L, suggests an analogous risk for intestinal dissemination even in different genetic backgrounds (Table 1).

Conjugation Demonstrates Full Mobility of the Two-Plasmid Resistance Package in L2386

Conjugation assays demonstrated that both plasmids in L2386 were transferable (bla_NDM-5 IncX3 plasmid and mcr-1.1 IncI2 plasmid) (Figures 2 and 3), establishing functional evidence that the entire last-line resistance package is conjugatively mobilizable (Table 1). Transconjugants selected under carbapenem or colistin pressure carried corresponding resistance determinants as confirmed by PCR. These results indicate substantial potential for horizontal transfer and co-selection in gut-associated microbial communities.

Figure 3 Comparative plasmid map of IncX3-borne blaNDM-5 plasmids in E. coli L1771 and L2386. The IncX3 plasmid in L2386 (~48 kb) and in L1771 (~46 kb) both carry blaNDM-5 and share a highly similar backbone, including associated insertion sequence elements (IS5) and adjacent resistance-related regions. The conserved structural context supports the mobilization potential of blaNDM-5 in both isolates.

Discussion

In this study, we report a detailed genomic and phenotypic characterization of two gut-colonizing E. coli isolates (L2386 and L1771) co-harboring bla_NDM-5 and mcr-1.1. While both isolates exhibited resistance to carbapenems and colistin, they exhibited significant differences in the genetic architecture of their resistance determinants. L2386 carried bla_NDM-5 on IncX3 plasmid and mcr-1.1 on IncI2 plasmid, demonstrating a high-risk multi-plasmid resistance package that is fully transferable through conjugation. In contrast, L1771 harbored bla_NDM-5 on an IncX3 plasmid while mcr-1.1 was chromosomally encoded, making it non-transferable by plasmids.

IncX3 plasmids are widely recognized as successful vectors for bla_NDM dissemination.^24,25 Figure 3 shows the circular maps of the bla_NDM-5-bearing IncX3 plasmids in L1771 and L2386. These plasmids are linked by shared backbone regions, supporting the high conversation of bla_NDM-5 in two isolates. The conjugation assays confirm the transferability of both the bla_NDM-5 and mcr-1.1 plasmids from L2386 to E. coli J53, indicating the mobilization potential of this multi-plasmid resistance package.

The key innovation in this study lies in the full plasmid resolution of L2386, revealing the IncX3 bla_NDM-5 plasmids alongside one IncI2 mcr-1.1 plasmids. The IncI2 plasmid carrying mcr-1.1 also co-encode bla_CTX-M-199, which contributes to the ESBL phenotype observed in L2386. Figure 3 highlights the complex genetic environment of bla_NDM-5 and underscores the resistance gene co-localization on IncX3 plasmids. The presence of these plasmids in L2386 is particularly concerning, as these two plasmids are fully conjugative, enabling the spread of both carbapenem and colistin resistance across different bacterial species.

In contrast, L1771 presents a distinct genetic configuration with mcr-1.1 encoded on the chromosome and bla_NDM-5 located on an IncX3 plasmid. This finding is significant because chromosomally encoded mcr-1.1 cannot transfer horizontally as easily as plasmid-borne mcr-1.1. The non-transferability of mcr-1.1 in L1771 is further confirmed by conjugation experiments, where only the bla_NDM-5-bearing plasmid was transferred to the recipient E. coli strain J53. This chromosomal location of mcr-1.1 in L1771 contrasts with L2386, where mcr-1.1 is plasmid-borne and readily transferred. Despite this difference in mobility, L1771 still represents a potential reservoir for mcr-1.1 through clonal spread or via chromosomal integration in other species, particularly under co-selection with bla_NDM-5 and other beta-lactam resistance genes.

The co-existence of carbapenem and colistin resistance genes on mobilizable plasmids is a critical finding of this study. L2386, with its IncX3-borne bla_NDM-5 and IncI2-borne mcr-1.1, provides a perfect model for studying co-selection under selective pressure, especially in gastrointestinal reservoirs where mobile genetic elements (MGEs) frequently mediate horizontal gene transfer. Our data confirms that both bla_NDM-5 and mcr-1.1 plasmids are transferable, making L2386 a potent source of multi-drug resistance for hospital-associated pathogens.

The intestinal tract is a high-density ecosystem where bacterial contact, selective pressure, and mobile elements interact^26,27. Our findings suggest that gut colonization can serve as a launchpad for dissemination of last-line resistance plasmids, with implications for screening strategies, infection control, and antimicrobial stewardship. From a clinical operational perspective, reliance on culture-based detection of colonization may underestimate the complexity and mobility of resistance if plasmid backbones and transferability are not assessed.

Our study also underscores the importance of plasmid-resolved surveillance for better tracking of carbapenem and colistin resistance in clinical settings. The IncX3 plasmids observed in L2386 and L1771 provide a detailed reference for understanding plasmid-mediated spread of bla_NDM-5 while the IncI2 plasmid highlights the diverse vehicles for mcr-1.1 dissemination. Given that mcr-1.1 is associated with IncI2, plasmid tracking in hospitalized patients and community-based surveillance programs should prioritize IncI2-borne mcr-1.1 plasmid for early detection and control measures.

While this study provides plasmid-resolved genomic insights into mcr-1.1 and bla_NDM-5 co-harbouring E. coli, it should be interpreted as a high-resolution descriptive analysis of two gut-colonizing isolates, designed to resolve plasmid architecture and transfer potential rather than estimate prevalence or broader epidemiologic burden. Further research could explore for the fitness cost of co-harbouring these plasmids in intact microbiota and the potential for horizontal gene transfer of bla_NDM-5 and mcr-1.1 in other species under antibiotic selective pressure. In addition, broader surveillance of plasmid-borne bla_NDM-5/mcr-1 combinations across geographic regions and clonal lineages would help define their epidemiological distribution and dissemination potential.

Conclusion

Our study demonstrates that gut-colonizing E. coli can harbor bla_NDM-5 and mcr-1.1 in distinct genomic configurations, including a transferable IncX3-borne bla_NDM-5 plasmid and a transferable IncI2-borne mcr-1.1 plasmid in L2386, as well as chromosomally encoded mcr-1.1 in L1771. The complete plasmid sequences provided in this genome note offer valuable insights for tracking last-line resistance and underscore the value of plasmid-resolved surveillance for assessing potential dissemination in clinical and colonization settings. Future work should determine how widely such configurations are distributed across bacterial lineages and settings, and clarify their transfer dynamics under different selective pressures.