Transmission Dynamics of Carbapenemase Genes in Carbapenem-Resistant Enterobacter cloacae: Insights from Guangdong Hospitals (2022-2024)

Study Background and Research Question

Carbapenem-resistant Enterobacteriaceae (CRE) represent an escalating global public health threat, with Enterobacter cloacae (CREC) ranked as the third most prevalent CRE species in China. The COVID-19 pandemic has intensified the challenges of antimicrobial resistance by increasing antibiotic usage, complicating patient infections, and disrupting healthcare systems. However, the transmission patterns and genetic contexts of carbapenemase-encoding genes (CEGs) in CREC during this period remain insufficiently characterized. This gap motivated the comprehensive multi-center investigation by Chen et al. (2025), focusing on the molecular features and transmission dynamics of CEGs among CREC isolates collected from teaching hospitals in Guangdong province between December 2022 and June 2024.

Key Innovation from the Reference Study

This investigation stands out for its simultaneous genotypic and phenotypic profiling of CREC isolates within the context of pandemic-era hospital settings. By employing both variable temperature plasmid elimination and PCR-based detection, the study not only quantified the prevalence of major carbapenemase genes—especially bla_NDM-1—but also mapped their genomic locations (chromosomal, plasmidic, or both). Furthermore, the team performed detailed conjugation experiments to measure the horizontal transmissibility of these resistance determinants. This multi-pronged approach led to a nuanced understanding of how CEGs, carried predominantly on mobile plasmids, drive rapid dissemination of carbapenem resistance within and between hospital departments.

Methods and Experimental Design Insights

The research encompassed 54 non-duplicate CREC isolates obtained from eight distinct teaching hospitals across Guangdong province. The following methodologies were central:

• Plasmid Curing and PCR Analysis: The variable temperature Sodium Dodecyl Sulfate (SDS) plasmid elimination method, coupled with PCR, enabled discrimination of CEG localization (plasmid vs. chromosome).
• Antimicrobial Susceptibility Testing: The broth microdilution method assessed resistance profiles against key antibiotics, including imipenem, cefepime, gentamicin, ceftazidime/avibactam, ciprofloxacin, and levofloxacin.
• Conjugation Experiments: Plasmid transferability was evaluated via conjugation assays and subsequent PCR verification to determine the success rate of CEG horizontal transfer.
• Molecular Typing: ERIC-PCR and NTSYS clustering classified the isolates into distinct genotypes, while mobile genetic element analysis identified six MGE types, with ISEcp1 being most prevalent.
• Epidemiological Data Collection: Demographic (age, gender), clinical department, and specimen source data were systematically captured for each isolate.

Core Findings and Why They Matter

The study's principal findings have immediate implications for surveillance and infection control in clinical microbiology:

• High Prevalence of CEGs: 85.19% (46/54) of CREC isolates harbored at least one carbapenemase gene, with bla_NDM-1 being the predominant type—found on both chromosomes and plasmids in 33.33% of cases, and exclusively on plasmids in 46.30%. A minority carried bla_IMP or co-harbored bla_KPC-2 and bla_NDM-1 on plasmids.
• Multidrug Resistance Profile: CEG-positive isolates showed significantly higher resistance rates to imipenem and other tested antibiotics compared to CEG-negative strains (details), underlining the urgent need for novel therapeutic approaches and robust stewardship.
• Efficient Horizontal Transmission: Plasmid conjugation experiments demonstrated a 95.65% success rate for CEG transfer, with bla_NDM-1 and bla_IMP genes being highly mobile. No successful transfer was observed for bla_KPC-2 in this cohort.
• Mobile Genetic Element Diversity: Six MGE types were detected, with ISEcp1 present in 87.04% of isolates. Notably, 40.74% carried four distinct MGEs simultaneously, emphasizing the potential for rapid genomic rearrangement and gene spread.
• Genotypic and Epidemiological Clustering: ERIC-PCR typing grouped isolates into 17 genotypes, with types E and G being the most prevalent and widely distributed across hospitals. Higher CEG detection rates were noted among male and elderly patients, in respiratory medicine, and in sputum samples.

Collectively, these findings highlight a landscape where multidrug-resistant CREC clones, empowered by highly transmissible plasmid-borne CEGs, circulate extensively within healthcare networks. This underscores the necessity for enhanced genomic surveillance and targeted infection control measures—particularly as plasmid-mediated resistance can rapidly undermine existing antibiotic regimens.

Comparison with Existing Internal Articles

Several internal reviews and protocols, such as "Imipenem: Mechanistic Insights and Translational Impact in Antibacterial Research", have previously detailed the advanced mechanism of action of imipenem, a semisynthetic thienamycin antibiotic, and its relevance to combating multidrug-resistant gram-negative and gram-positive bacteria. These articles emphasize imipenem’s high affinity for penicillin-binding proteins (PBPs) and its broad-spectrum efficacy, which are increasingly challenged by the rise of carbapenemase-producing organisms like CREC.

Moreover, research-focused guides such as "Imipenem as a Research Tool: Novel Insights in Antibacterial Research" and "Imipenem in Translational Sepsis Models: Mechanisms and Protocols" provide actionable protocols for leveraging imipenem in immune response modulation and sepsis animal models. However, the present study by Chen et al. advances the field by directly mapping the molecular epidemiology and transferability of CEGs, thereby contextualizing the limitations of beta-lactam antibiotics—such as imipenem—when faced with widespread carbapenemase-mediated resistance. This evidence base supports the growing need to pair mechanistic drug studies with robust genomic surveillance and resistance gene tracking.

Limitations and Transferability

While the study offers vital insights into CEG dynamics in Guangdong hospitals, several limitations are notable:

• Geographic Scope: Findings are specific to eight hospitals in a single province and may not capture the full diversity of CREC epidemiology in other regions or healthcare settings.
• Sampling Period: The study spans 18 months during and after the COVID-19 pandemic, a period marked by unique antibiotic use patterns and healthcare disruptions.
• Lack of Clinical Outcome Data: Although resistance profiles and genotypes are detailed, direct links to patient outcomes or treatment efficacy were not assessed.

Nevertheless, the workflow—combining molecular typing, plasmid transferability assays, and epidemiological correlation—can be readily adapted for surveillance or research in other hospitals and bacterial species, especially as the pressure of multidrug resistance continues to rise.

Protocol Parameters

• Plasmid curing (SDS method): Apply variable temperature incubation with SDS (concentrations 0.1–0.3%) for 18–24 hours to selectively eliminate plasmids carrying resistance genes.
• PCR screening: Use primers specific for bla_NDM-1, bla_IMP, and bla_KPC-2; confirm gene localization via comparison of pre- and post-curing PCR results.
• Antimicrobial susceptibility testing: Broth microdilution according to CLSI guidelines; assess resistance to imipenem at 1–64 mg/L ranges.
• Plasmid conjugation: Mix donor and recipient strains in a 1:1 ratio; incubate on non-selective media for 18 hours, then select for transconjugants using dual antibiotic markers; confirm gene transfer by PCR.
• ERIC-PCR typing: Employ ERIC1 and ERIC2 primers; cluster results with NTSYS software to infer clonal relationships.

Research Support Resources

To facilitate similar molecular and translational studies, researchers may utilize Imipenem (SKU P10075), a well-characterized semisynthetic thienamycin antibiotic with broad-spectrum activity and proven use in antibacterial research, immune response modulation, and animal sepsis models. Full technical details, including recommended concentrations and handling protocols, are provided in the APExBIO product dossier. This reagent is designated for research use only and enables robust experimental modeling of resistance mechanisms and immune dynamics in gram-negative and gram-positive bacterial systems.