Characterizing Carbapenemase Gene Transmission in CREC: A Guangdong Multi-Hospital Study (2022–2024)

Study Background and Research Question

Carbapenem-resistant Enterobacteriaceae (CRE) continue to pose a mounting global health concern, particularly as therapeutic options dwindle. Enterobacter cloacae complex, a common nosocomial pathogen, holds the third-highest detection rate among CRE in China, trailing only Klebsiella pneumoniae and Escherichia coli. The COVID-19 pandemic introduced multifaceted challenges—amplified antibiotic use, disruption of healthcare services, and increased complexity of infections—that further catalyzed the emergence and spread of multidrug-resistant bacteria. However, detailed molecular investigations into carbapenemase-encoding genes (CEGs), their chromosomal or plasmid localization, and real-world transmission dynamics in CREC during this period have been limited.

The reference study (Chen et al., 2025) set out to characterize the molecular epidemiology and transmission mechanisms of CEGs in CREC strains collected from eight teaching hospitals in Guangdong province, China, between December 2022 and June 2024. The research aimed to address the following key questions: What is the prevalence of key carbapenemase genes, particularly blaNDM-1, blaIMP, and blaKPC-2? How are these genes distributed across chromosomal and plasmid DNA? What are the patterns of transmission, resistance, and clinical epidemiology associated with these CEGs?

Key Innovation from the Reference Study

The study’s central innovation lies in its integrated molecular and epidemiological approach, combining plasmid elimination, PCR genotyping, mobile genetic element analysis, and conjugation experiments across a large, geographically diverse clinical sample. With 54 non-redundant CREC isolates sourced from multiple departments and hospitals, the research offers an unusually granular view of CEG prevalence, mobility, and clinical correlation during a period of heightened antimicrobial use. Notably, it reveals the predominance of plasmid-borne blaNDM-1 genes and quantifies their extensive horizontal transferability within clinical settings. This work advances the understanding of CREC’s capacity for rapid multidrug resistance dissemination, particularly in high-risk populations and healthcare environments affected by pandemic conditions.

Methods and Experimental Design Insights

To achieve a robust molecular characterization, the study utilized a multi-pronged experimental design:

• Sample Collection: 54 CREC isolates were obtained from eight tertiary teaching hospitals between December 2022 and June 2024, representing various departments and patient demographics.
• Plasmid Elimination and PCR: The variable temperature sodium dodecyl sulfate (SDS) method facilitated selective plasmid elimination, followed by targeted PCR amplification to detect blaNDM-1, blaIMP, and blaKPC-2 genes in both chromosomal and plasmid fractions.
• Mobile Genetic Element Identification: Six distinct mobile genetic elements (MGEs) were screened, with ISEcp1 emerging as the most prevalent, indicating pronounced gene mobility.
• Conjugation Experiments: Plasmid conjugation assays directly tested the transferability of carbapenemase genes, with subsequent PCR confirmation in recipient strains.
• Antibiotic Susceptibility Testing: Broth microdilution was employed to assess resistance profiles to a panel of antibiotics, correlating CEG presence with multidrug resistance phenotypes.
• Genotyping and Epidemiological Analysis: ERIC-PCR and NTSYS software were used to cluster genotypes and analyze epidemiological variables (age, gender, department, specimen type).

This comprehensive workflow enabled not only genetic detection but also the functional assessment of gene transfer and phenotypic resistance, providing a multidimensional perspective on CEGs in CREC.

Core Findings and Why They Matter

The study’s findings have significant implications for understanding and combating antimicrobial resistance in clinical settings:

• High Prevalence and Plasmid Localization: 85.19% (46/54) of isolates harbored CEGs. Notably, 33.33% carried the blaNDM-1 gene on both plasmids and chromosomes, while 46.30% had it exclusively on plasmids. Only a small fraction carried blaIMP or both blaNDM-1 and blaKPC-2 on plasmids (Chen et al., 2025).
• Efficient Horizontal Transfer: Conjugation experiments demonstrated that 95.65% of CEG-positive isolates could successfully transfer their resistance genes, with the blaNDM-1 and blaIMP genes showing nearly universal transfer success. This underscores the alarming potential for rapid dissemination of carbapenem resistance via plasmids.
• Mobile Genetic Elements Drive Spread: Six MGEs were identified, with ISEcp1 present in 87.04% of strains. Co-occurrence of multiple MGEs in a single strain—up to four types in 40.74% of cases—suggests a robust genetic machinery for gene mobilization and recombination.
• Multidrug Resistance Phenotype: CEG-positive isolates demonstrated significantly higher resistance rates to imipenem, cefepime, gentamicin, ceftazidime/avibactam, ciprofloxacin, and levofloxacin compared to CEG-negative isolates (P < 0.05). This supports the strong association between CEG carriage and broad-spectrum multidrug resistance.
• Genotypic and Epidemiological Diversity: ERIC-PCR grouped isolates into 17 genotypes, with types E and G being most prevalent and widely distributed across institutions. Higher CEG detection was observed in male and elderly patients, with respiratory medicine and sputum samples representing epidemiological hotspots.

Collectively, these results highlight the dual threat of high CEG prevalence and efficient plasmid-mediated transfer, reinforcing the need for vigilant surveillance and tailored antimicrobial strategies.

Comparison with Existing Internal Articles

The present findings align with prior internal analyses, such as the review "Transmission Dynamics of Carbapenemase Genes in CREC, 2022–2024", which also observed a predominance of plasmid-borne blaNDM-1 and emphasized the role of horizontal gene transfer in resistance spread. Similarly, another internal resource underscores how mobile genetic elements and diverse genotypes contribute to the rapid evolution of multidrug-resistant CREC in hospital environments. Together, these sources reinforce the conclusion that the interplay between plasmid genetics, mobile elements, and clinical epidemiology is central to the ongoing challenge of carbapenemase dissemination.

Moreover, internal workflow articles such as "Tigecycline: Glycylcycline Antibiotic for MDR Bacteria Research" discuss practical laboratory strategies for tackling multidrug resistance, especially in the context of infection models involving carbapenemase-producing Enterobacteriaceae. These resources provide scenario-driven best practices for experimental design and selection of effective antimicrobial agents, complementing the molecular epidemiological insights from the reference study.

Limitations and Transferability

While the study offers robust molecular and epidemiological data, several limitations merit consideration. Firstly, the sample size, while geographically diverse, is limited to eight teaching hospitals in Guangdong, potentially restricting generalizability to other regions or rural settings. The focus on pandemic-era isolates may also reflect unique selective pressures not present in non-pandemic conditions. Additionally, the study does not include functional infection models or clinical outcome data, which would further clarify the translational impact of these resistance mechanisms. Nevertheless, the transferability of findings to antimicrobial stewardship and infection control policies remains high, especially for institutions facing similar resistance trends.

Protocol Parameters

• Plasmid elimination (SDS method): Employ variable temperature SDS treatment to selectively eliminate plasmids before PCR genotyping of resistance genes.
• PCR detection: Use targeted primers for blaNDM-1, blaIMP, and blaKPC-2 on both chromosomal and plasmid DNA for comprehensive screening.
• Antibiotic susceptibility testing: Apply broth microdilution for quantifying resistance to carbapenems, cephalosporins, aminoglycosides, and fluoroquinolones.
• Conjugation assays: Perform plasmid transfer experiments using standard recipient strains, followed by PCR confirmation in transconjugants.
• Genotyping: Cluster isolates using ERIC-PCR and NTSYS for epidemiological tracking of dominant genotypes.

Research Support Resources

For researchers modeling multidrug-resistant Gram-negative infections or screening novel antimicrobial agents, robust tools are essential. Tigecycline (SKU A5226), the first clinically available glycylcycline antibiotic, has demonstrated potent activity against carbapenem-resistant Enterobacteriaceae, including both vancomycin-susceptible and resistant strains, methicillin-resistant Staphylococcus aureus (MRSA), and glycopeptide-intermediate S. aureus (GISA) infection models. Its broad-spectrum, bacteriostatic mechanism—via inhibition of the 30S ribosomal subunit—makes it a valuable resource for experimental workflows investigating resistance mechanisms described in the reference study. For further workflow and assay design guidance, consult APExBIO’s technical dossier or related scenario-driven internal articles.