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

Study Background and Research Question

Carbapenem-resistant Enterobacter cloacae (CREC) is emerging as a formidable threat within the broader context of multidrug-resistant Gram-negative bacteria. With rising prevalence in clinical settings, CREC poses significant challenges due to limited therapeutic options and the potential for widespread resistance gene dissemination. The COVID-19 pandemic has further complicated antimicrobial stewardship, leading to increased antibiotic consumption and altered transmission risks. Despite the global attention on carbapenem-resistant Enterobacteriaceae (CRE), detailed molecular epidemiology and transmission mechanisms of carbapenemase-encoding genes (CEGs) in CREC, particularly in high-density healthcare environments, remain insufficiently characterized. The present study addresses this gap by investigating the distribution, localization, and transfer dynamics of CEGs in CREC isolates from eight Guangdong teaching hospitals between December 2022 and June 2024 according to the reference study.

Key Innovation from the Reference Study

The central innovation of this study lies in its comprehensive mapping of CEG prevalence, gene localization (chromosomal vs. plasmid), and transmission efficiency in a real-world, pandemic-impacted hospital network. Notably, the research goes beyond routine surveillance by integrating plasmid conjugation assays, molecular genotyping, and epidemiological analysis to reveal both the molecular and clinical determinants of resistance gene spread. By focusing on blaNDM-1, blaIMP, and blaKPC-2 genes and their genetic vehicles, the study elucidates the mechanisms underlying CREC multidrug resistance and the high frequency of successful horizontal gene transfer.

Methods and Experimental Design Insights

The investigators collected 54 non-duplicate CREC isolates from eight tertiary teaching hospitals over an 18-month period. Key methodological steps included:

• Plasmid Elimination and Detection: Variable temperature Sodium Dodecyl Sulfate (SDS) plasmid elimination was combined with PCR to detect the presence and location of CEGs.
• Antibiotic Susceptibility Testing: Broth microdilution was employed to assess resistance to a panel of antibiotics, discriminating CEG-positive from CEG-negative phenotypes.
• Conjugation and Mobility Analysis: Plasmid conjugation experiments quantified the horizontal transfer efficiency of resistance genes. Success rates for blaNDM-1, blaIMP, and blaKPC-2 were individually determined.
• Genotyping: Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) and cluster analysis using NTSYS software identified genotypic diversity and potential clonal expansion.
• Epidemiological Correlation: Patient demographics, clinical department, and specimen type were correlated with CEG detection rates.

Protocol Parameters

• Plasmid elimination: Variable temperature SDS; optimize conditions based on isolate thermal tolerance.
• PCR detection: Use gene-specific primers for blaNDM-1, blaIMP, blaKPC-2; confirm with sequencing when ambiguous.
• Conjugation frequency assessment: Standardize donor:recipient ratio (1:1) and select appropriate markers for transconjugant verification.
• Antibiotic susceptibility testing: Broth microdilution; include carbapenems, cephalosporins, fluoroquinolones, and aminoglycosides for comprehensive resistance profiling.
• Genotyping: ERIC-PCR with at least three technical replicates to ensure cluster reliability.

Core Findings and Why They Matter

The study uncovered a high prevalence (85.19%) of CEGs among CREC isolates, with the blaNDM-1 gene being predominant. Specifically, 33.33% of isolates harbored blaNDM-1 on both chromosomes and plasmids, while 46.30% carried it exclusively on plasmids. The presence of blaIMP and blaKPC-2 was less frequent, often localized to plasmids. The resistance rate to multiple antibiotics—including imipenem, cefepime, gentamicin, ceftazidime/avibactam, ciprofloxacin, and levofloxacin—was significantly higher in CEG-positive strains, underscoring the clinical impact of these genes.

Importantly, conjugation experiments demonstrated a strikingly high transfer success rate (95.65%) for CEGs, particularly for blaNDM-1 (95.45%) and blaIMP (100%), highlighting the robust potential for horizontal spread. Six mobile genetic element types were identified, with ISEcp1 being the most common (87.04%). Genotyping revealed significant diversity, with 17 distinct genotypes and two dominant types (E and G) spread across different hospitals and departments. Epidemiological analysis showed higher CEG detection in male and elderly patients, with respiratory medicine and sputum samples being the most common clinical contexts. These findings collectively point to both the genetic versatility and epidemiological adaptability of CREC in hospital settings as detailed in the study.

Comparison with Existing Internal Articles

Several internal resources provide detailed guidance on antibiotic resistance research, particularly in the context of Gram-negative bacteria and multidrug resistance:

• Aztreonam: Applied Workflows for Gram-Negative Resistance Assays offers practical insights for modeling resistance phenotypes and troubleshooting in Enterobacteriaceae studies, including the role of monocyclic β-lactam antibiotics such as Aztreonam. This complements the reference study by providing laboratory workflow strategies that can be adapted for CREC resistance modeling.
• The article Aztreonam in Multidrug Resistance Research: New Mechanistic Horizons explores the use of Aztreonam in dissecting transmission dynamics and pharmacological impacts, directly aligning with the reference study’s focus on horizontal gene transfer and resistance mechanisms.
• For those investigating metabolic and off-target effects, Aztreonam: Mechanisms, Metabolic Impact, and Advanced Research Uses expands on Aztreonam’s influence on hepatic cytochrome P450 enzymes and bone marrow progenitor cells, aspects that are crucial in the broader pharmacological evaluation of new antibiotic regimens.

Collectively, these articles bridge laboratory technique optimization with the translational insights provided by the Guangdong CREC study, supporting a more integrated approach to resistance research.

Limitations and Transferability

While the study provides robust molecular and epidemiological data, certain limitations must be acknowledged:

• Geographic Focus: The findings are specific to Guangdong Province and may not directly extrapolate to other regions with differing antibiotic stewardship practices.
• Sample Size and Temporal Window: Although 54 isolates across eight hospitals provide valuable diversity, the study’s temporal coverage (2022–2024) captures a unique pandemic-influenced period that may not represent baseline transmission dynamics.
• Gene Coverage: The primary focus on blaNDM-1, blaIMP, and blaKPC-2 leaves open the possibility that other resistance determinants could play regionally significant roles.
• Clinical Correlation: While patient data were analyzed, direct clinical outcomes (e.g., treatment efficacy, morbidity) were not systematically addressed, limiting translational conclusions.

Nonetheless, the workflow and analytical framework are transferable to other Gram-negative resistance studies, and the methodological rigor supports adaptation to broader hospital surveillance programs.

Research Support Resources

For researchers aiming to investigate antibiotic activity against Gram-negative aerobic bacteria or model the inhibition of bacterial cell wall synthesis, the use of high-purity standards and reference compounds is essential. Aztreonam (SKU A5931), a first-in-class synthetic monocyclic β-lactam antibiotic, is well-suited for such studies. Its established activity profile against Gram-negative bacteria and demonstrated effects on both bone marrow progenitor cell inhibition and hepatic cytochrome P450 enzymes (product information) make it a valuable tool for replicating and extending resistance assays, pharmacological profiling, or mechanistic investigations akin to those described in the Guangdong CREC study. Aztreonam is supplied by APExBIO for research use only and should be handled according to stability and solubility guidelines for optimal performance in laboratory workflows.