Carbapenemase Gene Transmission in Enterobacter cloacae

Carbapenem-resistant Enterobacter cloacae (CREC) is an important antimicrobial resistance research target because resistance can arise from both mobile carbapenemase genes and expansion of successful bacterial lineages. The 2025 study by Chen, Yan, Liu, and colleagues examined these processes in isolates collected from eight teaching hospitals in Guangdong Province, China, during 2022–2024. Its central contribution is not simply documenting carbapenemase prevalence; it connects gene location, transferability, mobile genetic elements, antimicrobial phenotype, and strain relatedness in one regional dataset.

Study Background and Research Question

Carbapenem-resistant Enterobacterales are associated with limited treatment options and substantial infection-control challenges. Within China, CREC is reported as a major CRE species, and carbapenemase-encoding genes (CEGs) are frequently central to the resistance phenotype. The study was motivated by concerns that pandemic-era antibiotic exposure, disrupted healthcare services, and complex patient infections could intensify the emergence and spread of resistant organisms.

The investigators asked several related questions: How frequently do CREC isolates carry CEGs? Are the genes located on plasmids, chromosomes, or both? Can the resistance determinants move into a recipient strain under laboratory conditions? Which mobile genetic elements surround them, and do isolates from different hospitals show evidence of shared clonal backgrounds? These questions distinguish gene-level transmission from vertical propagation of a bacterial lineage.

Importantly, the work is a multicenter clinical-isolate investigation rather than a bacterial infection model. It therefore addresses the epidemiology and molecular biology of resistant organisms in healthcare-associated samples, not treatment efficacy or disease progression in an animal or cellular host.

Key Innovation from the Reference Study

The study’s strongest innovation is its integrated transmission framework. A positive PCR result alone cannot determine whether a resistance gene is chromosomal, carried on a transferable plasmid, or moving through a clonal population. Chen et al. combined variable-temperature SDS plasmid elimination with PCR to infer gene localization, broth microdilution to link genotype with phenotype, conjugation assays to test transfer, mobile-element profiling to examine genetic context, and ERIC-PCR with NTSYS analysis to estimate strain relatedness. The complete approach is described in the reference study.

This design is particularly useful for antimicrobial resistance research because it separates three levels of dissemination. Horizontal transfer is supported when a resistance determinant moves into a recipient during conjugation. Vertical dissemination is suggested when genetically related isolates share resistance features. A third level is genomic context: insertion sequences and other mobile elements can facilitate movement between plasmids and chromosomes or stabilize resistance regions within bacterial populations.

The study also avoids treating all carbapenemase genes as epidemiologically equivalent. It distinguishes blaNDM-1, blaIMP, and blaKPC-2, then evaluates their transfer behavior separately. That gene-specific analysis is more informative than reporting a single aggregate CEG prevalence.

Methods and Experimental Design Insights

Fifty-four CREC isolates were obtained from eight teaching hospitals in Guangdong between December 2022 and June 2024. The investigators first screened for CEGs by PCR and used variable-temperature SDS treatment to help determine whether detected genes were plasmid-associated or chromosomal. Because plasmid elimination is an indirect localization method, the value of the approach comes from interpreting it alongside PCR results and conjugation experiments rather than in isolation.

Antimicrobial susceptibility was measured by broth microdilution. The panel included imipenem, cefepime, gentamicin, ceftazidime/avibactam, ciprofloxacin, and levofloxacin. These measurements provided a phenotypic comparison between CEG-positive and CEG-negative CREC groups, while avoiding the assumption that a carbapenemase genotype predicts susceptibility to every antimicrobial class.

Plasmid conjugation experiments were then used to test whether CEGs could transfer to a recipient strain. PCR analysis of transconjugants verified the acquired genes. The researchers additionally characterized six mobile genetic elements, including ISEcp1, and used ERIC-PCR fingerprinting with NTSYS software to classify the 54 isolates into genotypes. Together, these methods support a layered interpretation: resistance gene presence, physical location, experimental mobility, genetic context, and population structure.

Protocol Parameters

• Sampling frame: Analyze CREC isolates collected from multiple teaching hospitals over a defined surveillance interval; the reference study used 54 isolates from eight hospitals collected between December 2022 and June 2024.
• Gene detection: Combine PCR screening with a plasmid-elimination workflow when estimating whether CEGs are plasmid-borne or chromosomal, as implemented in the published protocol framework.
• Phenotypic comparison: Use broth microdilution to compare antimicrobial susceptibility by CEG status rather than relying on genotype alone.
• Transfer testing: Confirm putative plasmid mobility through conjugation followed by PCR verification of recipient or transconjugant isolates.
• Strain relatedness: Use ERIC-PCR and computational clustering as a screening-level approach for relatedness, while treating the resulting genotypes as distinct from whole-genome phylogeny.
• Replication planning: Predefine controls for plasmid loss, donor growth, recipient selection, and PCR interpretation. These are workflow recommendations for reproducibility, not additional parameters reported by the reference study.

Core Findings and Why They Matter

CEGs were detected in 46 of 54 isolates, corresponding to 85.19% of the collection. The dominant determinant was blaNDM-1. Eighteen isolates carried blaNDM-1 on both chromosomes and plasmids, while 25 carried it exclusively on plasmids. Two isolates contained only plasmid-borne blaIMP, and one isolate carried both plasmid-associated blaNDM-1 and blaKPC-2. These distributions show that the same resistance determinant may occupy different genomic compartments, with important consequences for persistence and transfer potential.

The conjugation results provided the clearest evidence of horizontal dissemination. CEG transfer succeeded for 44 of 46 CEG-positive isolates, or 95.65%. The transfer success rate was 42 of 44 for blaNDM-1, 2 of 2 for blaIMP, and 0 of 1 for blaKPC-2. The unequal results caution against assuming that every carbapenemase gene has the same mobility, even when detected in a similar clinical background. These quantitative findings are reported in the reference paper.

CEG-positive isolates had significantly higher resistance rates than CEG-negative isolates for imipenem, cefepime, gentamicin, ceftazidime/avibactam, ciprofloxacin, and levofloxacin. The ciprofloxacin result is best interpreted as a marker of multidrug-resistant background rather than proof that a carbapenemase directly causes fluoroquinolone resistance. Fluoroquinolone susceptibility can reflect additional target, permeability, efflux, and regulatory mechanisms that were not resolved by this study.

Mobile-element analysis found six element types, with ISEcp1 identified most frequently in 47 of 54 isolates, or 87.04%. The most common pattern contained four mobile-element types simultaneously, observed in 22 isolates, or 40.74%. Such genetic complexity matters because insertion sequences can be associated with mobilization and rearrangement of resistance regions, although the presence of an element does not by itself prove that a specific transfer event occurred.

ERIC-PCR divided the collection into 17 genotypes. Types E and G were each represented by 11 isolates, or 20.37%, and appeared in different departments across five hospitals. Two type E isolates shared a Dice coefficient of 100%, indicating indistinguishable fingerprints under that assay. The combination of widespread transferability and repeated strain patterns suggests that CREC dissemination may involve both mobile plasmids and clonal expansion. Neither mechanism alone adequately explains the observed epidemiology.

The clinical distribution also provides surveillance context. The highest detection frequencies were observed among male patients, elderly individuals, respiratory medicine cases, and sputum specimens, reported as 35 of 54, 39 of 54, 11 of 54, and 18 of 54, respectively. These values describe the composition of the study collection; they should not be interpreted as population-level risk estimates without denominator data for all patients tested.

Comparison with Existing Internal Articles

The internal article Dynamics of Carbapenemase Genes in Enterobacter cloacae During COVID-19 provides a broad epidemiological interpretation of the same Guangdong-focused evidence. It is useful as a contextual companion because it emphasizes pandemic-era pressures and surveillance implications, whereas the reference study supplies the experimental details for localization, conjugation, mobile elements, and ERIC-PCR typing.

A second related resource, Carbapenemase Gene Transmission in CREC, foregrounds the relationship between blaNDM-1, plasmid-mediated horizontal transfer, and clonal dissemination. Its framing aligns closely with the reference study’s central message. Researchers should still use Chen et al. as the primary source for the reported sample counts, transfer rates, and molecular findings, while treating the internal articles as interpretive summaries rather than substitutes for the original report.

Limitations and Transferability

The study has several limitations that affect how broadly its findings can be generalized. First, the sample is limited to 54 isolates from eight teaching hospitals in one province. The high CEG prevalence and transfer success may therefore reflect local referral patterns, laboratory selection, or plasmid ecology rather than the situation across China or other healthcare systems.

Second, the design does not provide a matched pre-pandemic comparator. Although the introduction frames the work around COVID-19-related changes in antibiotic use and healthcare delivery, the data do not by themselves establish that the pandemic caused the observed transmission pattern. A longitudinal design with standardized sampling before, during, and after the pandemic would be better suited to that causal question.

Third, plasmid elimination, PCR, conjugation, and ERIC-PCR each have resolution limits. SDS treatment can produce incomplete plasmid loss or affect bacterial fitness; conjugation demonstrates transfer under experimental conditions but not necessarily in patients; and ERIC-PCR can identify related fingerprints without resolving the complete chromosome and plasmid architecture. Whole-genome sequencing, long-read plasmid assembly, and genomic epidemiology would strengthen conclusions about gene neighborhoods, plasmid backbones, and transmission chains.

Finally, the study reports susceptibility differences but does not establish the independent contribution of each resistance mechanism to ciprofloxacin or other drug phenotypes. Future work could pair resistance-gene localization with expression analysis, plasmid copy-number assessment, and carefully controlled functional testing. These extensions would improve mechanistic interpretation while preserving the study’s central insight that gene transfer and clonal spread must be evaluated together.

Research Support Resources

For laboratories extending susceptibility benchmarking or resistance-selection workflows, researchers can use Ciprofloxacin (SKU A8399), a fluoroquinolone antibiotic and bacterial DNA gyrase inhibitor that also targets topoisomerase IV. Its fluoroquinolone mechanism of action produces DNA replication inhibition, making it relevant to antibacterial mechanism studies and antimicrobial resistance research. The product information reports greater than 98% purity, a molecular weight of 331.34, limited solubility in water, ethanol, and DMSO, and storage at −20 °C; solvent selection and fresh solution preparation should therefore be planned before use.