Characterizing Carbapenemase Genes in Carbapenem-Resistant Enterobacter cloacae: Evidence from Eight Guangdong Hospitals

Study Background and Research Question

Carbapenem-resistant Enterobacteriaceae (CRE) pose a significant challenge to global public health due to their rapid spread and limited treatment options. Enterobacter cloacae, among the top three CRE species in China, has shown a worrying increase in multidrug resistance rates, especially during the COVID-19 pandemic, when altered antibiotic usage patterns and healthcare disruptions may have facilitated resistance dissemination. The central research question addressed by Chen et al. (2025) was how carbapenemase-encoding genes (CEGs) are distributed, transmitted, and maintained in carbapenem-resistant Enterobacter cloacae (CREC) strains isolated from eight teaching hospitals in Guangdong, China, between December 2022 and June 2024.

Key Innovation from the Reference Study

The primary innovation of the Chen et al. study lies in its comprehensive molecular dissection of CEG prevalence, localization, and transfer dynamics in CREC during a period of heightened antibiotic pressure. Unlike prior regional surveys, this work systematically distinguished the chromosomal and plasmid localization of carbapenemase genes—especially blaNDM-1—and quantified their horizontal transfer capabilities among clinical isolates. The study also integrated epidemiological profiling, identifying key patient and departmental risk factors for CEG-positive CREC occurrence.

Methods and Experimental Design Insights

The researchers collected 54 non-duplicate CREC isolates from eight tertiary hospitals. Plasmid elimination was achieved via the variable temperature Sodium Dodecyl Sulfate (SDS) method, enabling separation of chromosomal and plasmid-encoded resistance. PCR assays targeted blaNDM-1, blaIMP, and blaKPC-2 genes, and their chromosomal or plasmid presence was confirmed via plasmid curing and conjugation experiments. Plasmid transmissibility was further evaluated through conjugation assays, while mobile genetic elements were characterized to understand genetic contexts promoting gene mobility.

Antimicrobial susceptibility was determined by the broth microdilution method, comparing resistance profiles between CEG-positive and CEG-negative groups. ERIC-PCR genotyping, analyzed with NTSYS software, provided clonal relatedness data, allowing for the mapping of intra- and inter-hospital transmission dynamics.

Core Findings and Why They Matter

Chen et al. found that 85.19% of CREC isolates harbored carbapenemase-encoding genes, with blaNDM-1 detected most frequently. Notably, 33.33% of isolates carried blaNDM-1 both chromosomally and on plasmids, while 46.3% had it exclusively on plasmids. A smaller subset harbored blaIMP or both blaNDM-1 and blaKPC-2 genes. The high proportion of plasmid-borne CEGs underscores the critical role of plasmid transfer in disseminating carbapenem resistance, particularly in hospital environments subject to high antibiotic pressure.

Plasmid conjugation experiments revealed a striking 95.65% success rate for horizontal CEG transfer, with 95.45% of blaNDM-1 and all blaIMP genes successfully mobilized. This efficiency highlights the potent potential for rapid, cross-strain resistance dissemination. Mobile genetic element analysis identified six types, with ISEcp1 present in 87.04% of strains, suggesting a major role in facilitating genetic mobility.

Clonal typing sorted the 54 strains into 17 genotypes, with two (types E and G) dominating multiple hospital departments, indicating both local clonal spread and broader horizontal gene transfer. Epidemiologically, CEG-positive isolates were most frequently found in elderly male patients, in respiratory departments, and in sputum samples. Resistance rates to key drugs—imipenem, cefepime, gentamicin, ceftazidime/avibactam, ciprofloxacin, and levofloxacin—were all significantly higher in CEG-positive isolates, confirming the clinical impact of plasmid-mediated resistance.

Comparison with Existing Internal Articles

This study deepens the landscape outlined by earlier internal reviews such as "Carbapenemase Gene Dynamics in CREC" which previously summarized the broad epidemiology and highlighted the dominance of blaNDM-1 in Guangdong hospitals. Chen et al.'s work stands out by providing granular data on gene localization and conjugation efficiency, crucial for understanding the mechanisms of resistance spread. In parallel, internal resources on chloramphenicol's role in plasmid-driven antibiotic resistance research contextualize the use of antibiotics as both therapeutic agents and molecular tools, reinforcing the importance of precise selection markers in resistance gene studies. Together, these resources underscore the intersection of clinical microbiology and molecular methodology in dissecting multidrug resistance.

Limitations and Transferability

While comprehensive in its molecular approach, the study is limited by its regional scope and sample size, potentially missing less common CEG variants or resistance mechanisms. Surveillance was confined to teaching hospitals in Guangdong, which may not represent the full diversity of resistance dynamics in other healthcare settings or geographic regions. Moreover, the study focused on a defined set of CEGs; other emerging carbapenemases or resistance pathways may be underrepresented. Nevertheless, the detailed protocol for plasmid curing, conjugation, and resistance profiling provides a transferable framework for similar epidemiological and molecular studies elsewhere.

Protocol Parameters

• CREC isolation: Collect non-duplicate clinical isolates from diverse hospital departments for representative sampling.
• Plasmid curing (SDS method): Apply variable temperature treatment with Sodium Dodecyl Sulfate to distinguish chromosomal from plasmid-borne genes.
• PCR detection: Use gene-specific primers to identify blaNDM-1, blaIMP, and blaKPC-2, with confirmation of location post-curing.
• Conjugation assay: Employ broth or filter mating with suitable recipient strains; monitor transfer efficiency by selecting for resistance phenotypes.
• Antimicrobial susceptibility testing: Conduct broth microdilution for key antibiotics, interpreting results according to CLSI or EUCAST guidelines.
• Genotyping: Implement ERIC-PCR and NTSYS clustering for clonal analysis.

Research Support Resources

Researchers aiming to study plasmid-driven resistance mechanisms or perform plasmid selection assays can benefit from using Chloramphenicol (SKU A2512), a well-characterized bacterial protein synthesis inhibitor that acts via the 50S ribosomal subunit. In molecular biology workflows—such as those dissecting gene transfer and resistance marker selection—chloramphenicol provides a stringent and reliable selection agent for both stringent and relaxed plasmids, facilitating the isolation and tracking of resistance determinants. High-purity preparations (as offered by APExBIO) help ensure experimental reproducibility, especially in studies paralleling those of Chen et al., where plasmid selection and resistance gene mobility are central.