Novel Enterobacter Lineage as Leading Cause of Nosocomial Outbreak Involving Carbapenemase-Producing Strains

We investigated unusual carbapenemase-producing Enterobacter cloacae complex isolates (n = 8) in the novel sequence type (ST) 873, which caused nosocomial infections in 2 hospitals in France. Whole-genome sequence typing showed the 1-year persistence of the epidemic strain, which harbored a blaVIM-4 ST1-IncHI2 plasmid, in 1 health institution and 2 closely related strains harboring blaCTX-M-15 in the other. These isolates formed a new subgroup in the E. hormaechei metacluster, according to their hsp60 sequences and phylogenomic analysis. The average nucleotide identities, specific biochemical properties, and pangenomic and functional investigations of isolates suggested isolates of a novel species that had acquired genes associated with adhesion and mobility. The emergence of this novel Enterobacter phylogenetic lineage within hospitals should be closely monitored because of its ability to persist and spread.


Pangenome Construction
To determine the pangenome of the hormaechei metacluster, 244 genomes were annotated by Prokka (14) and the corresponding dataset was analyzed with the Roary package (15). The homologous genes were functionally annotated with the Clusters of Orthologous Groups (COG) database (16) using EggNOG (17). Functional enrichment analysis was performed with the stats package (https://github.com/SurajGupta/rsource/tree/master/src/library/stats/R) in R to calculate odds ratios and Fisher exact tests with pvalues corrected for multiple testing by the Bonferroni adjustment. A p value less than 0.05 and an odds ratio higher than 1.5 were considered statistically significant.

List of ECC Genomes Included in this Study
The Genbank accession numbers (hsp60 cluster, phylogenomic group): AFHR01 (E,VII),

Biofilm Formation and Epithelial Cell Adhesion Assays
The initiation of biofilm formation was assayed by the ability of cells to adhere to the wells of 96-well microtiter dishes made of polyvinylchloride plastic as previously described (18).
Biofilm formation was detected after 3h of incubation at room temperature by determining the extent of crystal violet-stained cells attached to a surface at 595 nm. The commensal E. coli K-12 strain TG1 carrying a F-conjugative plasmid that promotes biofilm formation (19) was used as a positive control strain for biofilm formation.
HT-29 intestinal epithelial cells were purchased from ATCC and maintained in an atmosphere containing 5% CO2 at 37°C in Dulbecco's modified Eagle's medium. They were seeded at a density of 2×10 5

Clinical Data
The index case (patient 1, P1) was a 67-year-old woman hospitalized for a kidney transplant. Following surgery, a CPE designated C45 was isolated from a urine sample.
However, P1 did not develop an infection and was only colonized. Five months after she was discharged, a second CPE strain designated C46 was isolated from the urine of a 72-year-old man (patient 2, P2) admitted to the intensive care unit (ICU) for endarterectomy and tracheotomy. During his 106 days of hospitalization, the patient developed septic shock. Patient 3 (P3) was a 52-year-old woman admitted to the ICU for an undetermined recurrent septic shock following bowel resection and laparotomy. Two weeks after the isolation of strain C46 from P2, a CPE strain designated C47 was identified from the urine of P3. Patient 4 (P4) was a 69-yearold man hospitalized for a kidney transplant. Two weeks after the isolation of C47 from P3, a CPE strain designated C48 was isolated from the urine of P4. Patient 5 (P5) was an 87-year-old man admitted to the medical unit and then to the ICU for a consciousness disorder. A CPE strain designated C308 was isolated from a urine sample of P5 3 months after the isolation of strain C48. Two months after the isolation of C308, CPE strain C310 was isolated from a skin sample taken from an 84-year-old woman admitted to the medical unit for the treatment of a necrotic ulcer (patient 6, P6). Patient 7 (P7) was an 82-year-old man admitted to the surgical unit to undergo lithotrity. One month after the isolation of C310, CPE strain C309 was isolated from the operative peritoneal fluid of P7.