**Background:** Enterobacter hormaechei, a member of the Enterobacter cloacae complex (ECC), is an emerging multidrug-resistant pathogen associated with high mortality rates (up to 40%) in vulnerable populations. Despite its clinical importance, the molecular mechanisms of E. hormaechei pathogenesis and immune evasion are poorly understood. Serum complement-mediated killing is a key host defense against Gram-negative bacteria, and many pathogens produce capsular polysaccharide (CPS) to evade complement. This study investigated whether and how Enterobacter resists serum-mediated killing.
**Methods:** The authors screened 96 clinical Enterobacter isolates for growth in human serum. One highly serum-sensitive ST78 isolate, NR3055, was selected for further study. Serum-resistant (SR) clones of NR3055 were selected in vitro by plating on agar with human serum. Comparative genomics (Illumina and Oxford Nanopore sequencing) identified a premature stop codon in the wzy gene of the CPS biosynthesis locus in NR3055. The wild-type wzy gene was complemented into NR3055 (NR3055::wzy). Strains were characterized by transmission electron microscopy (TEM), serum killing assays, whole human blood killing assays, C3b deposition by flow cytometry, neutrophil opsonophagocytosis assays, and a murine peritonitis model. Recurrent infection and passive immunization experiments were performed to assess anti-CPS immune protection.
**Key Results:** Among 96 clinical isolates, most were highly serum-resistant, but four were highly serum-sensitive. NR3055 bacterial burden was reduced by five logs within 2 h in 50% human serum. Eight in vitro-selected SR clones were highly resistant to serum and whole human blood killing. TEM revealed a CPS-like structure surrounding NR3055 SR2 cells that was mostly absent on NR3055 cells; the capsule-width to cell-diameter ratio was approximately five times larger on SR2. Comparative genomics identified a single nucleotide variant in all SR clones: a repair of a premature stop codon in wzy (c.629C>A; p.Ser210*). Among 979 publicly available E. hormaechei assemblies, 151 had capsule type Enterobacter-NL68, mostly in ST78 (94 assemblies); putative loss-of-function mutations in cps loci were rarely detected. Complementation of wzy (NR3055::wzy) restored serum resistance, with uninhibited growth in 5% fresh human serum, while NR3055 was killed by 5 orders of magnitude within 2 h. NR3055 cells showed 97% sedimentation in liquid culture vs. 8.6% for SR2 and 5% for NR3055::wzy. In a murine peritonitis model, 10^8 CFU of NR3055 was avirulent, while NR3055 SR2 and NR3055::wzy were highly virulent (P < 0.001). At 2 and 16 h postinfection, NR3055::wzy-infected mice had significantly higher bacterial burdens in blood, peritoneal lavage, spleen, kidneys, liver, lungs, and heart compared to NR3055-infected mice (P ≤ 0.001 to P ≤ 0.0001). C3b deposition was significantly increased on NR3055 compared to SR2 and NR3055::wzy (P ≤ 0.0001). NR3055 SR2 evaded neutrophil uptake, while NR3055 showed opsonization-dependent uptake that increased over time. In C3-deficient mice, 93% succumbed to NR3055 infection within 50 h, compared to only 10% of wild-type mice (P ≤ 0.0001). In recurrent infection models, 90% of mice preinfected with unencapsulated NR3055 succumbed to lethal challenge with NR3055::wzy, while almost all mice preinfected with encapsulated NR3055::wzy were protected (P < 0.001). Passive immunization with serum from NR3055::wzy-exposed mice conferred protection against lethal challenge compared to naive serum (P ≤ 0.1).
**Clinical Implications:** This study establishes CPS as a critical virulence factor for E. hormaechei, demonstrating that Wzy-dependent CPS production enables evasion of complement-mediated killing and is required for virulence and dissemination in vivo. The finding that recurrent infection with CPS-producing E. hormaechei induces protective immunity, and that this protection is transferable via serum, suggests that CPS could be a viable target for vaccine development against these high-priority ESKAPE pathogens. Given the increasing antimicrobial resistance in Enterobacter spp., these findings provide crucial information for designing new therapeutic and prophylactic interventions.