Molecular epidemiology of carbapenem-resistant Escherichia coli in a tertiary hospital located in the Dabie Mountains region, China

Introduction

Carbapenem-resistant Enterobacteriaceae (CRE), particularly carbapenem-resistant Klebsiella pneumoniae and Carbapenem-resistant Escherichia coli (CREc), pose a critical threat due to limited therapeutic options and high mortality rates (Kedišaletše et al., 2023; Macareño-Castro et al., 2022; Price et al., 2022). In China, CREc has rapidly expanded geographically. National-scale surveillance by the China Antimicrobial Surveillance Network (CHINET) indicates that Escherichia coli (E. coli) accounts for 18.9% of Enterobacterales isolates, with 1.8–2.0% of these strains being CREc (Hu et al., 2020). To investigate regional trends within this national framework, we analyzed data from the Anhui Bacterial Drug Resistance Surveillance Big Data Platform (Anhui Province Health Commission, 2023). This platform aggregates anonymized data from participating hospitals throughout Anhui Province. Our analysis, based on data retrieved in May 2023, revealed a particularly alarming trend in Lu’an City—a mountainous region of Anhui Province—where CREc rates underwent a 5.75-fold increase, rising from 0.4% in 2016 to 2.7% in 2020. Carbapenems remain the first-line therapeutic option for all E. coli isolates, including multidrug-resistant (MDR) strains (Demirci, Ünlü & Tosun, 2019). However, the emergence of CREc frequently leads to treatment failure, primarily mediated by carbapenemase production, with the gene encoding New Delhi Metallo-β-lactamase (bla_NDM) being the most frequently reported carbapenemase gene (Han et al., 2020; Li et al., 2021, 2024a). This resistance severely limits treatment options, necessitating the use of alternative antibiotics, including tigecycline, colistin, and fosfomycin. Among these, fosfomycin demonstrates particular promise due to its unique mechanism of action (irreversible inhibition of cell wall synthesis via MurA blockade) and retained activity against certain MDR pathogens. However, the clinical utility of fosfomycin is increasingly challenged by emerging resistance mechanisms. The predominant mechanism of fosfomycin resistance is the acquisition of fos genes, with fosA3 being the most globally widespread (Zhang et al., 2025).

Traditionally, antimicrobial resistance and virulence in E. coli were thought to be mutually exclusive. However, recent studies report a rising prevalence of hypervirulent MDR strains, including CREc, posing a critical public health challenge (Ba et al., 2024; Karbalaei et al., 2025; Loras et al., 2021; Medugu et al., 2025). Of particular concern is the emergence of virulence determinants-including adhesins (e.g., fimH), siderophores (e.g., iutA) and hemolysins (e.g., hlyA), in MDR E. coli populations. Co-expression of these virulence factors (VFs) with resistance genes enhances bacterial colonization, immune evasion, and tissue damage, resulting in worse clinical outcomes (Karbalaei et al., 2025; Shahin et al., 2019; Yousefi et al., 2023).

With the dissemination and accumulation of bla_NDM-carrying CREc isolates in China, comprehensive long-term molecular epidemiological surveillance and researches have been conducted in economically developed cities or developed economies (Fu et al., 2023; Ko et al., 2023). However, data from the least developed regions and rural areas are still lacking, particularly regarding integrated analyses of VFs, sequence types (STs) in these settings. To address this gap, we set out to analyze the susceptibility of antimicrobial agents, distribution of antibiotic resistance genes, plasmid replicons, VFs, serotypes, and STs in Dabie Mountains region, China. Portions of this text were previously published as part of a preprint (Wang et al., 2023, Research Square, DOI: 10.21203/rs.3.rs-3910839/v1).

Materials and Methods

Results

Genetic characteristics and relatedness of 33 CREc

MLST analysis of seven housekeeping genes (adk-fumC-gyrB-icd-mdh-purA-recA) classified the 33 CREc isolates into 24 STs (Table S1). ST410 (n = 4) was the most prevalent, followed by ST156 (n = 3), ST692 (n = 3), ST167 (n = 2), and ST2 (n = 2) (Fig. 1). The remaining 19 isolates belonged to 19 independent STs.

Among the 33 CREc isolates, 30 (90.9%) harbored bla_NDM variants, including bla_NDM-5 (n = 24), bla_NDM-13 (n = 4), bla_NDM-1 (n = 1), and bla_NDM-6 (n = 1). Notably, bla_NDM-6 was found in ST361, while bla_NDM-13 was found in ST744, ST1196, ST2064, and ST2179. The remaining ST206 isolate (ECO28), did not carry any known carbapenemase genes (Fig. 1). Additionally, two isolates of ST662 and ST131 carried the gene encoding Klebsiella pneumoniae Carbapenemase (bla_KPC-2).

Whole-genome sequencing of 33 isolates identified 198,764 SNPs for phylogenetic analysis. Twelve of the 33 isolates showed close genetic relatedness. The first isolate (ECO01), was primarily collected in 2018, followed by five isolates in 2019 and six in 2021 (Fig. 2).

The core genetic environment of carbapenemase gene bla_NDM and bla_KPC in the 33 CREc isolates

Among the 33 CREc isolates, 30 carried bla_NDM and two carried bla_KPC-2. A detailed comparison of their genetic environments was performed (Fig. 3). The core genetic environments of bla_NDM and bla_KPC-2 were derived from Tn125 and Tn6296, respectively. Among the 33 isolates, seven distinct truncated ΔTn125 derivatives (ΔTn125-1 to -7) were identified, containing bla_NDM-1/5/6/13 variants with the following distribution: (1) ΔTn125-1 (n = 13, ECO04/06/07/09/11/13/14/15/18/19/27/31/32), (2) ΔTn125-2 (n = 4, ECO10/12/21/30), (3) ΔTn125-3 (n = 8, ECO01/02/03/05/23/25/32/33), (4) ΔTn125-4 (n = 1, ECO20), (5) ΔTn125-5 (n = 3, ECO17/26/29), (6) ΔTn125-6 (n = 1, ECO16), and (7) ΔTn125-7 (n = 1, ECO08) (Fig. 3A); while one (ECO07) bla_KPC-2-carrying isolate belonged to ΔTn6296-1 and another (ECO24) to ΔTn6296-2 (Fig. 3B).

These seven variants of Tn125 (ΔTn125-1 to ΔTn125-7) exhibited truncation at three downstream sites: cutA (periplasmic divalent cation tolerance protein), trpF (N-5′-phosphoribosyl anthranilate isomerase), and groEL (60 kDa chaperonin 2) in ΔTn125-1 to -4, ΔTn125-5/6, and ΔTn125-7, respectively (Fig. 3A); in addition, except for ΔTn125-1, the rest six Tn125 variants also displayed the truncation or insertion of the tnpA (transposase) in ISAba125.

Two variants of Tn6296, which were identified in the bla_KPC-2-carrying isolates, exhibited truncations at the tnpA(transposase) and tnpR (resolvase) sites in the downstream region (Fig. 3B).

Discussion

Our investigation of CREc antimicrobial resistance patterns and virulence characteristics provides critical insights for infection control implementation. During the study period from 2018 to 2022, we observed a dynamic trend in CREc detection rates, increasing from 0.8% in 2018 to a peak of 3.8% in 2020, followed by a decline to 0.7% in 2022. Notably, the relative surge in CREc prevalence during 2020 may potentially correlate with concurrent infections during the COVID-19 pandemic (Chatterjee et al., 2023).

The CREc infections were predominantly observed in the surgery department (45.4%), particularly in urology. While previous studies have reported mortality rates as high as 30.0% in CREc infections (Kong et al., 2024), only one fatality (3.0%) occurred in our cohort, possibly reflecting the less severe disease presentation in our patients. Notably, 24.2% (8/33) of patients discontinued treatment, seven of whom were cancer patients over 50 years old, suggesting that underlying malignancies may contribute to treatment non-adherence.

bla_NDM-mediated carbapenem hydrolysis represents the predominant resistance mechanism among CREc isolates (Li et al., 2021). Molecular subtyping revealed bla_NDM-5 as the dominant variant (accounting for 80.0% of all bla_NDM carriers), consistent with China’s predominant bla_NDM epidemiology (Li et al., 2021). Secondary bla_NDM subtypes included: bla_NDM-13 (n = 4), bla_NDM-6 (n = 1), and bla_NDM-1 (n = 1). Notably, These variants (bla_NDM-13 and bla_NDM-6) have been infrequently reported in China (Lv et al., 2016; Wang et al., 2023; Zhang et al., 2023), highlighting the need for continuous surveillance to track their dissemination and assess their potential clinical impact. Although no carbapenemase genes were detected in ECO28, this strain showed high expression of acrA, acrB, and tolC, which may be associated with reduced susceptibility to multiple antibiotics, including β-lactams, due to the overexpression of this tripartite efflux pump system (Lian et al., 2024).

Antibiotic susceptibility testing showed that CREc isolates were most susceptible to tigecycline, polymyxin, and amikacin, aligning with the CHINET data, suggesting these antibiotics as the most effective treatments for CREc infections in China (Hu et al., 2020). We found that bla_CTX-M-55 was the predominant bla_CTX-M, followed by bla_CTX-M-14 and bla_CTX-M-15, current epidemiological data indicated that bla_CTX-M-55, previously considered a rare variant, has now surpassed bla_CTX-M-15 and bla_CTX-M-14 in prevalence to become the predominant genotype among CTX-M-type ESBLs (Yu et al., 2024; Zhang et al., 2019, 2021). Interestingly, four CREc isolates carrying ESBL genes remained susceptible to aztreonam, likely due to these enzymes’ limited hydrolytic activity against aztreonam. Although the acquired quinolone resistance gene qnr was detected in only eight isolates, nearly all isolates (32/33) exhibited resistance to ciprofloxacin and levofloxacin, indicating that qnr genes were not the primary drivers of fluoroquinolone resistance (Strahilevitz et al., 2009). Instead, mutations in the parC gene, found in 30 of the 33 isolates, were strongly associated with high-level fluoroquinolone resistance, highlighting the critical role of type II topoisomerase mutations in mediating resistance (Boueroy et al., 2023; Garoff et al., 2018). Fosfomycin, a broad-spectrum antimicrobial agent, is indicated for the treatment of both uncomplicated and complicated urinary tract infections (Moreno-Mellado et al., 2025). Additionally, it demonstrates excellent activity against infections caused by MDR and carbapenem-resistant bacteria, particularly when used in combination therapies (Hughes et al., 2020; Sojo-Dorado et al., 2022). However, reports of fosfomycin resistance have been increasingly documented in recent years (Martínez et al., 2025; Ríos et al., 2022). In our study, 36.4% (12/33) of strains exhibited fosfomycin resistance, with 11 harboring the fosA3 gene and 1 carrying fosA4. The fosA3 gene can be located on diverse plasmid types or the bacterial chromosome, and may co-transfer with ESBL genes and carbapenemase genes via mobile genetic elements through horizontal gene transfer mechanisms (Bi et al., 2017; Zhang et al., 2025).

The 33 CREc isolates in this study belonged to 24 distinct ST types, demonstrating considerable genetic diversity. Notably, SNP analysis revealed 12 closely related isolates (SNP difference ≤ 10). Although these strains were isolated from different wards over a 3-year period (2018–2021), their genetic relatedness suggests possible: (1) persistent colonization by resistant clones in the hospital environment, (2) horizontal transfer of resistance genes via mobile genetic elements (e.g., plasmids), or (3) intermittent interpersonal transmission of dominant clones. However, the absence of environmental surveillance data and detailed patient movement records necessitates further validation of these transmission routes.

Previous global reports have identified ST167, ST410, ST617, and ST131 as common among CREc isolates (Li et al., 2024a; Ma et al., 2023). ST410 is of particular concern as it is the third most prevalent strain and has frequently been associated with bla_NDM in multiple regions (Bi et al., 2017; Ko et al., 2023; Zhang et al., 2017). In our study, among the 30 CREc strains producing bla_NDM, 21 different STs were identified, with ST410 being the most prevalent, followed by ST156 and ST692. Previous studies indicated that ST410 has evolved into strains with high resistance and virulence (Ba et al., 2024). For instance, ST410 strains carrying the fosA gene-by virtue of its location on a conjugative plasmid-concurrently exhibit carbapenem and fosfomycin resistance, posing a dual threat to empirical therapy and representing a critical One Health concern (Güneri et al., 2022; Peng et al., 2022). In our study, genomic analysis revealed that all four ST410 strains harbored a conserved repertoire of virulence determinants, including fimH (mediating host colonization through type 1 fimbriae), csgA (critical for curli-dependent biofilm formation), hlyE (encoding a pore-forming hemolysin), and hha (modulating invasion-related gene expression), suggesting their collective contribution to the pathogen’s adhesive, invasive, and persistence mechanisms (Jans et al., 2024; Karbalaei et al., 2025; Krall et al., 2021). Therefore, vigilant monitoring of clinical CREc transmission, along with other resistance genes, is essential.

This study showed that among the 33 E. coli isolates we analyzed, 71 unique VFs were identified, encoding siderophores (iutA, iucC, irp2, fyuA), adhesins (fimH, afa, pap, sfa), and toxins (cnf1, hly, sat). In this study, the overall frequency of virulence genes ranged from 3.0% for sat to 97.0% for fimH. The fimH-encoded type 1 fimbriae are associated with biofilm formation, while the fimH adhesin mediates bacterial proliferation and invasion-key VFs in uropathogenic E. coli (Mayer et al., 2017). irp2 and fyuA, VFs of the E. coli iron uptake system, demonstrated significant association with hypervirulence and were detected in 36.4% (12/33) of CREc isolates (Hashimoto et al., 2021; Li et al., 2024b).

Tn125 is hypothesized to have derived from the Acinetobacter chromosome due to the integration of the bla_NDM gene, which was captured by two copies of ISAba125 (Poirel et al., 2012), giving rise to this ISAba125-composite transposon with the core bla_NDM genetic environment (bla_NDM-1-ble_MBL-trpF-dsbD-cutA-groES-groEL). It was an ancestral transposon as the transporter of bla_NDM and is the only bla_NDM -containing transposon in Enterobacterales (Grenier et al., 2024). bla_NDM and its Tn125 components (exhibiting various truncated forms) could be found within MDR regions of relevant bacterial plasmids or chromosomes (Ma et al., 2024).

In this study, seven variants of Tn125 (ΔTn125-1 to ΔTn125-7) were identified, exhibiting truncations at three downstream sites: cutA, trpF, and groEL. Tn125 frequently exists in various truncated forms (Ma et al., 2024). These ΔTn125 variants are structurally similar to those identified in multiple regions of China (Hu et al., 2022; Huo et al., 2024), suggesting that Tn125 undergoes consistent structural modifications and may follow a shared evolutionary pathway across China.

In contrast, only two Tn6296 derivatives carrying bla_KPC-2 were identified in 33 CREc isolates, and their genomic organization were more conserve than the Tn125 derivatives. Tn6296, first identified in the plasmid from Klebsiella pneumoniae (Jiang et al., 2010), was generated from insertion of the local bla_KPC-2 gene platform into Tn1722, leading to the truncation of mcp. The structures of two Tn6296 derivatives identified in this study were more compact due to the loss of downstream genes of Tn6376. Although such insertions or deletions in the exogenous insertion region bla_KPC-2 were frequently observed in Tn6296, however the core structure surrounding bla_KPC-2 (ISKpn27-bla_KPC-2-ISKpn6) remained intact in Tn6296 variants, indicating ISKpn27-bla_KPC-2-ISKpn6 may be the key functional unit responsible for carbapenem resistance (Song et al., 2024).

Among the 30 bla_NDM-carrying isolates, four harbored the rare bla_NDM-13 variant: ECO17 (ST744), ECO20 (ST1196), ECO26 (ST2064), and ECO29 (ST2179). One additional isolate (ECO22, ST361) carried the bla_NDM-6 variant. Compared to bla_NDM-1, bla_NDM-13 exhibits two amino acid substitutions, D95N and M154L, which significantly enhance its hydrolytic activity against cefotaxime (Shrestha et al., 2015). Previous reports have identified multiple copies of bla_NDM-13 within a single plasmid or chromosome, often associated with mobile genetic elements like IS30 or ISAba125, which may be linked to the transmission or stability of bla_NDM-13 (Huang et al., 2022; Lv et al., 2016). In this study, some of the strains carried a truncated tnpA gene and lacked genes such as dsbD, cutA, groES, and groEL. However, other studies have reported that these genes were intact in the genomes of the bla_NDM-13-carrying strains (Kim et al., 2019; Shrestha et al., 2015). This suggests that genetic rearrangements or the presence of mobile genetic elements may contribute to the variability in the genetic context of bla_NDM-13. The A233V substitution in bla_NDM-6 enhances its enzymatic fitness in the periplasm under Zn(II) starvation conditions compared to bla_NDM-1 (Sychantha et al., 2021). The presence of the ISAba125 family transposon upstream of bla_NDM-6 is consistent with previous studies, which documented that the promoter of bla_NDM is partially located within the inverted repeat upstream of ISAba125 (Poirel et al., 2012). Additionally, the genetic environment of bla_NDM-6 includes truncated tnpA transposase, as well as ble_MBL, trpF, dsbD, and a truncated cutA. Although tnpA is truncated, some studies have shown that this does not affect the plasmid’s ability to transfer between bacteria, allowing the bla_NDM-6 gene to be horizontally transferred via plasmids (Bahramian et al., 2019; Parvez et al., 2019).

This study has several limitations that should be taken into account. Firstly, the use of long-read sequencing limited the thorough analysis of rare variants of the bla_NDM gene, especially bla_NDM-6 and bla_NDM-13, which were not included in this study. Future research should explore the environmental structure and plasmid replicons to further understand these variants. Additionally, case-control studies should be incorporated to assess the potential risk factors associated with CREc infections. It is important to note that this was a single-center study with a relatively small sample size. Therefore, prospective, multicenter, large-scale clinical trials are essential to validate and extend our findings.

Conclusions

This study provides the first dataset on molecular epidemiology and genetic characteristics of CREc isolates from a tertiary hospital in the Dabie Mountains region, China, addressing the previously unmet need for CREc profiling in the least developed regions. The presence of bla_NDM-5 is the primary cause of carbapenem resistance in CREc. Notably, most of these strains also carry resistance genes to other antibiotics (e.g., fosA3) as well as various VFs (e.g., fimH/hlyE/csgA). These CREc-carrying multidrug resistance and virulence genes still display the diversified phylogenetic history in this area. Therefore, ongoing surveillance needs to be performed to prevent the further clonal expression of these CREc.

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