Characterizing Carbapenemase Gene Spread in Enterobacter cloacae: Insights from Guangdong Hospitals (2022–2024)

Study Background and Research Question

The emergence of carbapenem-resistant Enterobacteriaceae (CRE) poses a severe global public health challenge, with Enterobacter cloacae (CREC) recognized as one of the most prevalent CRE species in China, following Klebsiella pneumoniae and Escherichia coli. The COVID-19 pandemic has further complicated antibiotic stewardship due to increased antimicrobial usage and healthcare disruptions, potentially accelerating the dissemination of multidrug-resistant bacteria. Despite the well-established clinical significance of CREC, high-resolution studies exploring the molecular epidemiology, transmission dynamics, and genetic contexts of carbapenemase-encoding genes (CEGs) in CREC—especially in the context of the pandemic—have been limited.

The reference study by Chen et al. (BMC Microbiology, 2025) addresses this knowledge gap by systematically investigating the prevalence, genetic diversity, and transmissibility of CEGs in CREC isolates obtained from eight teaching hospitals across Guangdong Province between December 2022 and June 2024.

Key Innovation from the Reference Study

The central innovation of this study lies in its integrative, multi-hospital sampling design during the COVID-19 pandemic, combined with comprehensive molecular and phenotypic characterization of CREC isolates. Notably, the study dissects CEG localization on chromosomes versus plasmids, quantitatively assesses their transfer efficiencies, and correlates these genetic features with clinical and epidemiological variables. This approach provides a granular view of the genetic vehicles promoting resistance and reveals the dominant transmission pathways underpinning the rapid spread of carbapenem resistance in clinical settings.

Methods and Experimental Design Insights

• Sampling and Isolate Collection: A total of 54 non-duplicate CREC isolates were collected from eight tertiary teaching hospitals in Guangdong over an 18-month period. Sampling spanned multiple departments and specimen types, enabling broad representation of clinical contexts.
• Genetic and Phenotypic Characterization: Sodium dodecyl sulfate (SDS) plasmid elimination, PCR amplification, and broth microdilution assays were employed to determine CEG presence, chromosomal/plasmid localization, and antimicrobial susceptibility profiles.
• Plasmid Conjugation and Transferability: Plasmid conjugation experiments, coupled with PCR validation, quantified the efficiency of horizontal gene transfer for major carbapenemase genes.
• Molecular Typing: Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) and NTSYS cluster analysis were used to assess genotypic diversity and infer potential epidemiological linkages.
• Mobile Genetic Element Analysis: Six classes of insertion sequences and transposons associated with CEGs were identified, with ISEcp1 as the most prevalent.

Protocol Parameters

• Sample collection window: December 2022 – June 2024; non-duplicate, clinical CREC isolates from multiple departments and specimen types.
• Plasmid elimination and detection: Variable temperature SDS method for plasmid curing, followed by PCR targeting blaNDM-1, blaIMP, and blaKPC-2.
• Antimicrobial susceptibility testing: Broth microdilution in alignment with CLSI/EUCAST standards for imipenem, cefepime, gentamicin, ceftazidime/avibactam, ciprofloxacin, and levofloxacin.
• Plasmid conjugation assay: Filter mating experiments, with confirmation of CEG transfer in recipient strains by PCR and phenotypic resistance assessment.
• Genotype clustering: ERIC-PCR fingerprinting with cluster analysis using NTSYS software to define epidemiological relationships.

Core Findings and Why They Matter

According to the reference study, CEGs were detected in 85.19% of CREC isolates, with the blaNDM-1 gene being the most widespread. Specifically, 33.33% of isolates harbored blaNDM-1 on both chromosomes and plasmids, while 46.30% carried it exclusively on plasmids. The blaIMP gene was detected on plasmids in 3.70% of cases, and 1.85% of isolates contained both blaNDM-1 and blaKPC-2 on plasmids. Notably, resistance rates to multiple antibiotics—including imipenem, cefepime, and fluoroquinolones—were significantly higher in CEG-positive strains (P<0.05), emphasizing the clinical challenge posed by these multidrug-resistant pathogens.

The study further demonstrated that horizontal gene transfer of CEGs via plasmid conjugation was highly efficient: 95.65% overall transfer rate, with 95.45% for blaNDM-1 and 100% for blaIMP. In contrast, blaKPC-2 transfer was not observed in this cohort. Six types of mobile genetic elements were identified, with ISEcp1 present in 87.04% of isolates, indicating its central role in facilitating gene mobility. Epidemiologically, CEGs were more frequently detected in elderly male patients, particularly in respiratory medicine departments and in sputum specimens, aligning with known risk factors for CREC acquisition and transmission.

Comparison with Existing Internal Articles

This reference study extends prior work on the epidemiology and molecular mechanisms of carbapenem resistance. For example, "Transmission Dynamics of Carbapenemase Genes in CREC: Insights from Guangdong" provides a complementary genomic overview, but the current study delivers finer-scale data on the chromosomal versus plasmid localization of resistance genes and their conjugative mobility during the pandemic. Additionally, the "Ertapenem (sodium salt): Broad-Spectrum Carbapenem Antibiotic" review discusses the use of ertapenem as a reference compound in resistance modeling, highlighting the clinical importance of monitoring carbapenemase activity and resistance gene spread, as modeled in the present study.

Further, the multi-hospital study on transmission dynamics during COVID-19 corroborates the observed high rates of horizontal gene transfer, supporting the urgent need for laboratory modeling of resistance mechanisms using robust reference antibiotics such as ertapenem sodium salt.

Limitations and Transferability

While the study offers valuable insights, certain limitations must be acknowledged. The sample size (n=54) and geographic focus (Guangdong Province) may limit the generalizability of the findings to broader clinical settings, especially outside pandemic or high-antibiotic-use contexts. The study's design does not address the impact of infection control interventions or temporal trends beyond the 18-month window. Additionally, while plasmid transfer was demonstrated in vitro, in vivo transfer dynamics and the clinical impact of specific mobile genetic elements require further investigation.

Nonetheless, the methodology—including molecular typing, plasmid transfer assays, and resistance phenotyping—provides a transferable framework for surveillance and laboratory modeling in other regions or with different Enterobacteriaceae species. The high prevalence and mobility of blaNDM-1 in particular highlight the ongoing risk of rapid dissemination of resistance traits in hospital environments.

Research Support Resources

Researchers aiming to replicate or extend these molecular resistance studies can benefit from using reference compounds that accurately model clinical resistance selection pressures. Ertapenem (sodium salt) (SKU C3451) from APExBIO is a well-characterized broad-spectrum carbapenem antibiotic, exhibiting potent activity against a wide range of Gram-positive and Gram-negative bacteria. Its established pharmacokinetics and defined mechanism—as a penicillin-binding protein inhibitor—make it suitable for antibiotic resistance research and for quantifying the efficacy of resistance gene transfer in laboratory settings. For detailed protocols, researchers should consult both the product information and recent methodological literature.