The Enterobacter cloacae complex (ECC) is a ubiquitous group of opportunistic pathogens that thrive in hospital environments, water and soil, and readily colonize the human gastrointestinal, respiratory and urinary tracts (Wendel et al., 2022). ECC comprises several clinically relevant species, most notably Enterobacter hormaechei, Enterobacter cloacae, Enterobacter asburiae and Enterobacter roggenkampii among others (Wu et al., 2025), and can cause a spectrum of healthcare-associated infections including bloodstream, urinary tract, wound, peritonitis and hospital-acquired pneumonia (Emeraud et al., 2024). Bloodstream infection (BSI) is the most severe manifestation, associated with high mortality, prolonged hospitalization and substantial healthcare costs (Gupta et al., 2024; Zhou et al., 2024).

The principal mechanisms of ECC resistance to β-lactam antibiotics are chromosomal AmpC enzyme derepression, and production of extended-spectrum β-lactamases (ESBLs) and carbapenemases (Annavajhala et al., 2019; Yang et al., 2022). These genes can be horizontally transferred via plasmids and integrons, accelerating the spread of resistant clones (Moraz et al., 2021; Fadare and Okoh, 2021). Consequently, the relentless rise of multidrug-resistant (MDR) ECC poses a severe global health threat. Carbapenem-resistant Enterobacterales (CRE) were classified among the pathogens of critical priority by the World Health Organization in 2024 (Antimicrobial Resistance Collaborators, 2022; Miller and Arias, 2024) Nation-wide surveillance underscores this trend: CHINET data show that the prevalence of carbapenem-resistant Enterobacter cloacae complex (CRECC) in China rose from 0.7% in 2007 to ~8% in 2024, making it the third most common nosocomial CRE after Klebsiella pneumoniae and Escherichia coli (Chen et al., 2021; Davin-Regli et al., 2019; China Antimicrobial Surveillance Network, 2024).

Plasmid-mediated New Delhi metallo-β-lactamase (NDM) production is now the dominant resistance mechanism (Bush and Bradford, 2019). NDM enzymes hydrolyze penicillins, cephalosporins and carbapenems, exhibit only weak activity against aztreonam, and remain unaffected by classical β-lactamase inhibitors such as avibactam but are inhibited by EDTA (Nagulapalli Venkata et al., 2021). Consequently, NDM-producing ECC isolates are frequently susceptible only to tigecycline and polymyxins, with occasional aztreonam sensitivity. Epidemiological studies show that healthcare-associated outbreaks of NDM-producing ECC are driven by a restricted set of MLST clones-most notably ST78, ST171 and ST182 (Gomez-Simmonds et al., 2018)-that acquire bla_NDM on IncX3, IncHI2 or IncFII plasmids (Mavroidi et al., 2023). Once formed, these high-risk ST–plasmid pairings spread rapidly: the ST171-IncX3 complex has driven repeated hospital outbreaks across India, Japan and the United States (Wang et al., 2018), while ST182, tightly associated with IncFII, triggered the largest European ECC epidemic on record in Greece (Gartzonika et al., 2023). Collectively, these findings underscore that the intersection of a susceptible clone with a promiscuous, NDM-carrying replicon acts as a molecular trans-regional escalation switch for ECC, providing clear targets for genomic surveillance.

In China, nationwide molecular epidemiological and virulence data on CRECC-BSI remain scarce. Using whole-genome sequencing and comprehensive in-silico virulence/plasmid profiling, this retrospective, single-center study (n = 13 isolates) systematically delineates the resistance, virulence and transmission patterns of CRECC bloodstream isolates from a tertiary-care hospital in Shandong Province, providing an evidence base for targeted therapy and infection-control strategies.

BSI is a life-threatening condition caused by the invasion of pathogenic microorganisms into the circulation and remains one of the most severe complications in clinical practice. Its high mortality, coupled with the global spread of multidrug-resistant organisms, poses formidable challenges to public health (Temkin et al., 2024). Recent diagnostic and therapeutic advances have lagged behind the rapid emergence of resistant pathogens, underscoring the need for improved prevention and management strategies. CRECC-BSI is closely linked to prior CRECC colonization or infection, extensive antibiotic exposure (especially carbapenems) and invasive procedures that disrupt physiological barriers (Lumbreras-Iglesias et al., 2023). In this retrospective cohort of 13 patients, 84.6% were male, 84.6% underwent invasive interventions, and 62.9% were admitted to the ICU; these figures that align with the Italian multicenter CR-GNB BSI report (Falcone et al., 2023). Pulmonary (38.5%) and urinary-tract (23.1%) sources predominated, indicating that airway- and catheter-related infections are the main portals of entry in our hospital. Notably, 61.5% of patients had received carbapenems for ≥48 h before blood cultures became positive, a selective pressure that favors resistant clones.

In our results, all 13 isolates carried blaNDM (NDM-1 69.2%, NDM-5 30.8%); their geographical distribution differs markedly from the KPC predominance in North America and the VIM/OXA-48 prevalence in Europe, but aligns with the ongoing NDM surge reported in southern, north-eastern and north-western China (Chen et al., 2021; Han et al., 2023; Cai et al., 2024; Shi et al., 2017; Tian et al., 2020), underscoring pronounced regional divergence of CRECC resistance. In addition to carbapenemases, each isolate carried an AmpC β-lactamase of distinct subtype (ACT-16; n = 7, ACT-14; n = 2, MIR-2; n = 2, ACT-15; n = 1, ACT-5; n = 1, ACT-7; n = 1), while 50% harbored ESBLs (CTX-M-15 30.8%, CTX-M-3 23.1%), consistent with a previous report by Cai et al (Cai et al., 2024). Detection of two mcr-9-positive isolates further restricts polymyxin options and complicates therapeutic choices.

MLST revealed that ST171 accounted for 46.2% of isolates; in-hospital deaths were associated with this lineage, consistent with US and Japanese reports that designate ST171 as a hyper-virulent, highly transmissible clone (Pereira et al., 2019; Harada et al., 2017; Ghazawi et al., 2024). Although three isolate pairs differed by ≤5 SNPs and may represent short-term transmission links (Snitkin et al., 2012), the majority showed >20 SNP differences, indicating absence of a single-strain nosocomial outbreak. ST93, frequently reported in China, was not detected (Zhao et al., 2020). NCBI data reveal that Chinese bloodstream CRECC isolates exhibit diverse, sporadic STs, mirroring the polyclonal pattern observed here (Gomez-Simmonds et al., 2016). Based on the results of the conjugation assays, five donor strains harboring bla_NDM successfully transferred their resistance plasmids to recipient E. coli J53, yielding transconjugants with ≥8-fold MIC increases for IPM and MEM compared with the recipient. Despite donors harboring multiple replicons, every transconjugant carried only a single plasmid replicon. IncFII for bla_NDM-1 (ST171) or IncX3 for bla_NDM-5 (ST2085). These results confirm the superior conjugative fitness of both replicons. They also align with global surveillance data identifying IncFII and IncX3 as the primary vehicles for bla_NDM-1 and bla_NDM-5 dissemination, respectively (Carattoli, 2013; Mouftah et al., 2019). These data provide direct experimental evidence that plasmid-mediated carbapenem resistance can be functionally transferred within our hospital environment.

Our results shown every isolate harbored the type VI secretion system (T6SS), a key virulence module that directly contributes to bacterial pathogenicity and host interaction, thereby posing a major clinical threat (Ghazawi et al., 2024; Theriault et al., 2021). Additionally, the adherence system was detected at varying levels; by promoting cell adhesion and interacting with the host immune response, it amplifies inflammatory reactions. The diverse virulence repertoire of CRECC strains thus complicates both the treatment and control of bloodstream infections (Azam et al., 2023). The 13 CRECC isolates harbored multiple plasmid replicon types, with IncHI2, IncHI2A and IncX3 being the most prevalent — mirroring reports from North America and Europe (Wu et al., 2025). IncX3 was detected most frequently, in line with a recent nationwide survey in China; its small size and high stability facilitate rapid inter-strain transfer of resistance genes (Wang et al., 2018). These findings underscore the need for early rectal screening of carbapenem-resistant organisms in high-risk patients, minimization of invasive procedures, prompt device replacement with regular microbiological surveillance, and enhancement of host immunity to curtail the spread of CRECC.

Our institutional CRECC-BSI isolates exhibited diverse sequence types, a complex plasmid repertoire and multiple virulence genes. ST171 was the dominant clone, NDM-1 the principal carbapenemase, and IncX3/IncHI2 plasmids the key vehicles for resistance-gene dissemination. Enhanced surveillance of this high-risk lineage and interventions against plasmid-mediated horizontal transfer are essential to curb further spread of CRECC in health-care settings.

This study has several limitations. First, its retrospective, single-center design may introduce selection bias and limit the generalizability of our findings. Second, the relatively small sample size may not fully represent the national molecular epidemiology of CRECC. Future work should enlarge the isolate collection and integrate multicenter prospective data to obtain a comprehensive picture of CRECC trends and resistance mechanisms.