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

Racha Beyrouthy; Marion Barets; Elodie Marion; Cédric Dananché; Olivier Dauwalder; Frédéric Robin; Lauraine Gauthier; Agnès Jousset; Laurent Dortet, François Guérin; Thomas Bénet; Pierre Cassier; Philippe Vanhems; Richard Bonnet


Emerging Infectious Diseases. 2018;24(8):1505-1515. 

In This Article

Materials and Methods

Clinical and Epidemiologic Survey

The patients were admitted to the Edouard Herriot Hospital in Lyon, France, where a prospective surveillance of CPEs has been implemented since 2012. Case-patients were defined as persons hospitalized after CPE was diagnosed in >1 clinical sample during January 12, 2014–December 31, 2015 (Technical Appendix Table 1). We investigated contact patients and performed an environmental study (Technical Appendix).

Bacteria Isolation and Phenotypic Characterization

We isolated CPE on a chromogenic medium, chromID CARBA (bioMérieux, Marcy l'Etoile, France). We preincubated environmental CPE specimens for 24 h at 36°C in Trypticase soy broth (TSB; bioMérieux) supplemented with 0.5 mg/L ertapenem. The clinical (n = 7) and environmental (n = 2) isolates were identified by mass spectrometry (VitekMS; bioMérieux). We used the API 50CH system (bioMérieux) for biochemical testing and assessed antimicrobial susceptibility according to the EUCAST guidelines ( We detected carbapenemase by using the RAPIDEC CARBA-NP test (bioMérieux).[16] As previously described, we determined the sequence of blaVIM-4 by using the Sanger method.[17] We performed conjugation experiments at 25°C as previously described[18] and plasmid size determination by pulsed-field gel electrophoresis.[19] Biofilm formation and cell adhesion assays are described in the Technical Appendix.

WGS and Genome Assembly

We determined the whole-genome sequences of strains C45 and C309 by using a hybrid de novo assembly of 2 × 150-bp paired-end reads generated by using sequencing technology by Illumina (San Diego, CA, USA) and long reads generated by using Pacific Biosciences technology (Menlo Park, CA, USA). We determined WGS of the other strains by using a de novo assembly of 2 × 150-bp paired-end reads. We performed the assemblies by using SPAdes,[20] mapped the reads by using the Burrows-Wheeler aligner (BWA),[21] and polished the assembly by using Pilon.[22] The raw data were deposited in EMBL as project PRJEB22398 and the assemblies as LT991954–60. We report further analysis processes in the online Technical Appendix.