Precise species identification and whole-genome sequencing analysis of <i>Enterobacter cloacae</i> complex causing bloodstream infections in China

The <i>Enterobacter cloacae</i> complex (ECC) is one of the major causes of hospital-acquired infections. However, achieving accurate species identification and comprehensive resistance profiling remains difficult in clinical practice, with a limited understanding of species-specific resistance patterns. ECC isolates were collected from patients with bloodstream infections at Peking Union Medical College Hospital between 2015 and 2020. Whole-genome sequencing (WGS) was performed to identify species, analyze antimicrobial resistance genes, and explore genomic variation in serial isolates. Multi-locus sequence typing (MLST) profiles were extracted from the WGS data. Phylogenetic analysis was conducted based on <i>hsp60</i> sequences. Eleven <i>hsp60</i> clusters were identified, with cluster VIII being the most prevalent (28/108). Average nucleotide identity (ANI)-based species classification showed <i>Enterobacter hormaechei</i> (31.5%) and <i>Enterobacter xiangfangensis</i> (15.7%) were dominant species. Five clade-cluster pairs (B-VIII, A-VI, G-XI, D-III, R-IX) accounted for 74% of isolates. A total of 90 sequence types (STs) were detected, including 29 novel STs. Resistance gene analysis revealed a high prevalence of <i>bla_ACT</i>, with distinct distribution patterns observed among different species. Twenty isolates were carbapenem-resistant, with three carrying <i>bla_NDM-1/5. Enterobacter roggenkampii</i> was the most common species (5/20) among all carbapenem-resistant isolates, and 83.3% of the isolates showed resistance to both carbapenems and colistin. Comparative genomics of longitudinal isolates from individual patients revealed adaptive single-nucleotide polymorphisms (SNPs) in <i>pco</i> genes. This study provides a detailed genomic characterization of ECC isolates from bloodstream infections, highlighting species diversity, resistance gene distribution, and potential within-host evolution. These insights advocate genome-based surveillance in managing ECC infections and understanding resistance evolution in clinical contexts.IMPORTANCE<i>Enterobacter cloacae</i> complex (ECC) is a major cause of hospital-acquired bloodstream infections, yet species-level identification and resistance profiling remain challenging. As one of the largest whole-genome sequencing (WGS)-based studies of ECC bloodstream isolates in northern China to date, we performed whole-genome sequencing of 108 ECC isolates, revealing high genetic diversity and identifying 29 novel sequence types. We clarified the correspondence between species, clades, and clusters and highlighted <i>Enterobacter roggenkampii</i> as a potential high-risk species linked to carbapenem and colistin resistance. Our findings not only improve the understanding of ECC population structure and resistance evolution in China but also provide valuable genomic data for future epidemiological surveillance and species-level diagnostics.