title: A Silenced vanA Gene Cluster on a Transferable Plasmid Caused an Outbreak of Vancomycin-Variable Enterococci.
abstract: We report an outbreak of vancomycin-variable vanA(+) enterococci (VVE) able to escape phenotypic detection by current guidelines and demonstrate the molecular mechanisms for in vivo switching into vancomycin resistance and horizontal spread of the vanA cluster. Forty-eight vanA(+) Enterococcus faecium isolates and one Enterococcus faecalis isolate were analyzed for clonality with pulsed-field gel electrophoresis (PFGE), and their vanA gene cluster compositions were assessed by PCR and whole-genome sequencing of six isolates. The susceptible VVE strains were cultivated in brain heart infusion broth containing vancomycin at 8 μg/ml for in vitro development of resistant VVE. The transcription profiles of susceptible VVE and their resistant revertants were assessed using quantitative reverse transcription-PCR. Plasmid content was analyzed with S1 nuclease PFGE and hybridizations. Conjugative transfer of vanA was assessed by filter mating. The only genetic difference between the vanA clusters of susceptible and resistant VVE was an ISL3-family element upstream of vanHAX, which silenced vanHAX gene transcription in susceptible VVE. Furthermore, the VVE had an insertion of IS1542 between orf2 and vanR that attenuated the expression of vanHAX Growth of susceptible VVE occurred after 24 to 72 h of exposure to vancomycin due to excision of the ISL3-family element. The vanA gene cluster was located on a transferable broad-host-range plasmid also detected in outbreak isolates with different pulsotypes, including one E. faecalis isolate. Horizontally transferable silenced vanA able to escape detection and revert into resistance during vancomycin therapy represents a new challenge in the clinic. Genotypic testing of invasive vancomycin-susceptible enterococci by vanA-PCR is advised.
authors.affiliation: Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø-The Arctic University of Norway, Tromsø, Norway.
authors.forename: Audun
authors.initials: A
authors.lastname: Sivertsen
authors.affiliation: Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North-Norway, Tromsø, Norway.
authors.forename: Torunn
authors.initials: T
authors.lastname: Pedersen
authors.affiliation: Department of Medical Microbiology, St. Olavs University Hospital, Trondheim, Norway.
authors.forename: Kjersti Wik
authors.initials: KW
authors.lastname: Larssen
authors.affiliation: Department of Medical Microbiology, St. Olavs University Hospital, Trondheim, Norway Department of Laboratory Medicine, Children's and Women's Health, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Kåre
authors.initials: K
authors.lastname: Bergh
authors.affiliation: Unit for Infection Control, St. Olavs University Hospital, Trondheim, Norway.
authors.forename: Torunn Gresdal
authors.initials: TG
authors.lastname: Rønning
authors.affiliation: Unit for Infection Control, St. Olavs University Hospital, Trondheim, Norway Department of Laboratory Medicine, Children's and Women's Health, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Andreas
authors.initials: A
authors.lastname: Radtke
authors.affiliation: Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø-The Arctic University of Norway, Tromsø, Norway Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North-Norway, Tromsø, Norway kristin.hegstad@uit.no.
authors.forename: Kristin
authors.initials: K
authors.lastname: Hegstad
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D016983:Enterococcus / Q000187:drug effects / Q000235:genetics*; D005838:Genotype; D005810:Multigene Family / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014640:Vancomycin / Q000494:pharmacology*
chemical_list: D001426:Bacterial Proteins; D014640:Vancomycin