Characterization and Transmission of Carbapenemase Genes in CREC During the COVID-19 Era

Study Background and Research Question

The global rise in carbapenem-resistant Enterobacteriaceae (CRE) presents a formidable challenge to public health and hospital infection control. Among CRE, Enterobacter cloacae has gained prominence in China due to its increasing detection rates and broad distribution across clinical departments. The COVID-19 pandemic has further complicated this landscape, with heightened antibiotic use and healthcare disruptions potentially accelerating the emergence and transmission of multidrug-resistant organisms. Despite its clinical urgency, detailed molecular epidemiology of carbapenemase-encoding genes (CEGs) in carbapenem-resistant E. cloacae (CREC) remains limited, especially in the context of pandemic-driven healthcare dynamics. The recent multicenter study by Chen et al. addresses these knowledge gaps by systematically analyzing the genetic and epidemiological signatures of CEGs in CREC isolates from eight teaching hospitals in Guangdong Province between December 2022 and June 2024.

Key Innovation from the Reference Study

This work delivers a high-resolution snapshot of CEG prevalence, chromosomal and plasmid localization, and transmission dynamics in CREC during a period of intensified antimicrobial pressure. Notably, the study leverages a combination of variable temperature SDS-based plasmid elimination, PCR, and broad genotyping approaches to dissect the interplay between mobile genetic elements and resistance gene dissemination. The integration of epidemiological data—such as patient demographics, clinical departments, and specimen types—enables nuanced insight into risk factors and transmission hotspots, advancing current understanding beyond previous single-center or cross-sectional surveys.

Methods and Experimental Design Insights

Fifty-four CREC isolates were collected from diverse clinical departments and patient populations. To determine the presence and location of carbapenemase-encoding genes, isolates underwent SDS-based plasmid curing followed by PCR amplification of key resistance genes: bla_NDM-1, bla_IMP, and bla_KPC-2. The study further applied broth microdilution to assess antimicrobial susceptibility profiles, enabling direct comparison of resistance phenotypes between CEG-positive and -negative groups. Conjugation experiments evaluated the horizontal transferability of plasmid-borne CEGs, while ERIC-PCR and cluster analysis (via NTSYS software) resolved the clonal relationships among isolates. Mobile genetic elements were mapped to elucidate their contribution to gene mobility and persistence.

Core Findings and Why They Matter

• Prevalence and Genetic Context: CEGs were detected in 85.19% (46/54) of CREC isolates. Among them, 33.33% carried bla_NDM-1 on both chromosomes and plasmids, while 46.30% carried it exclusively on plasmids. Only 3.70% harbored bla_IMP on plasmids, and a single isolate contained both bla_NDM-1 and bla_KPC-2 on plasmids (Chen et al.).
• Resistance Phenotype: CEG-positive CREC displayed significantly higher resistance rates to antibiotics such as imipenem, cefepime, gentamicin, ceftazidime/avibactam, ciprofloxacin, and levofloxacin compared to CEG-negative isolates (P<0.05).
• Transmission Potential: Plasmid conjugation experiments revealed a 95.65% (44/46) success rate for CEG transfer. Breakdown by gene showed transfer rates of 95.45% for bla_NDM-1 and 100% for bla_IMP, but none for bla_KPC-2.
• Mobile Genetic Elements: Six types of mobile elements were identified, with ISEcp1 being most prevalent (87.04%). Co-occurrence of four different mobile elements was observed in 40.74% of isolates, underscoring the complexity of resistance gene mobility.
• Genotypic Diversity and Epidemiology: Seventeen ERIC-PCR genotypes were resolved, with types E and G comprising 20.37% each, distributed across multiple hospitals and departments. Epidemiological analysis highlighted higher CEG detection rates among males (64.81%), elderly patients (72.22%), respiratory medicine cases (20.37%), and sputum samples (33.33%).

These findings collectively demonstrate that multidrug resistance in CREC is predominantly driven by plasmid-borne bla_NDM-1 and facilitated by mobile genetic elements, with robust potential for both horizontal (plasmid-mediated) and vertical (clonal expansion) transmission. The stratification by patient and specimen characteristics offers actionable intelligence for infection control and targeted surveillance.

Comparison with Existing Internal Articles

Recent internal literature underscores the relevance of plasmid selection assays and molecular tools in tracking and dissecting resistance gene dynamics. For example, "Chloramphenicol in Multidrug Resistance Research: Mechanisms & Protocols" discusses the application of chloramphenicol—a well-established bacterial protein synthesis inhibitor—for selecting and maintaining multidrug-resistant plasmids in laboratory workflows. This aligns with the reference study's emphasis on the centrality of plasmid-borne resistance genes in CREC. Similarly, "Chloramphenicol in Modern Plasmid Assays: Precision, Resistance, and Protocol Evolution" bridges the mechanistic basis of translation inhibition by 2,2-dichloro-N-[(1R,2R)-1,3-dihydroxy-1-(4-nitrophenyl)propan-2-yl]acetamide (chloramphenicol) with advanced protocols for monitoring resistance gene transfer and expression.

While these internal resources focus on optimizing workflows and troubleshooting in the laboratory, the referenced multicenter clinical study provides the crucial epidemiological context—linking molecular mechanisms to real-world trends in resistance gene spread and persistence. Together, they illustrate the continuum from bench to bedside in antimicrobial resistance research, with plasmid selection assays serving as both investigative and diagnostic tools for tracking multidrug resistance evolution.

Limitations and Transferability

The study by Chen et al. is strengthened by its multicenter design and comprehensive molecular characterization. However, several limitations should be acknowledged. First, the temporal window (2022–2024) captures pandemic-specific dynamics, which may not fully generalize to pre- or post-pandemic conditions. Second, the majority of isolates were from tertiary hospitals in Guangdong, potentially limiting broader geographic applicability. Finally, while horizontal gene transfer was convincingly demonstrated in vitro, in vivo dynamics may diverge due to host and environmental factors. Despite these caveats, the study’s workflow and findings are highly transferable to other hospital settings, particularly where plasmid-mediated resistance is suspected.

Protocol Parameters

• Plasmid selection assay: Chloramphenicol is commonly used at 25 μg/mL for stringent plasmids and 170 μg/mL for relaxed plasmids, as detailed in the product information.
• Dissolution: For optimal solubility, dissolve chloramphenicol in DMSO (≥16.16 mg/mL), water with gentle warming and ultrasonic treatment (≥16.25 mg/mL), or ethanol (≥33 mg/mL).
• Storage: Solutions should be stored at 4°C for short-term use; solid chloramphenicol is best kept at -20°C to maintain purity and activity.
• Stringency control in plasmid selection: Adjust antibiotic concentration based on plasmid copy number and selection pressure, as recommended in internal assay optimization articles.

Research Support Resources

For experimental validation of plasmid selection and resistance gene tracking, researchers can employ Chloramphenicol (SKU A2512), a high-purity bacterial protein synthesis inhibitor suitable for molecular biology research. Its robust inhibition of the bacterial 50S ribosomal subunit makes it a valuable tool for stringent plasmid selection and multidrug resistance studies. Full protocol parameters, solubility details, and storage recommendations are available from APExBIO's product documentation. This supports the laboratory workflows referenced in both the current study and associated internal literature.