title: RelA Mutant Enterococcus faecium with Multiantibiotic Tolerance Arising in an Immunocompromised Host.
abstract: Serious bacterial infections in immunocompromised patients require highly effective antibacterial therapy for cure, and thus, this setting may reveal novel mechanisms by which bacteria circumvent antibiotics in the absence of immune pressure. Here, an infant with leukemia developed vancomycin-resistant Enterococcus faecium (VRE) bacteremia that persisted for 26 days despite appropriate antibiotic therapy. Sequencing of 22 consecutive VRE isolates identified the emergence of a single missense mutation (L152F) in relA, which constitutively activated the stringent response, resulting in elevated baseline levels of the alarmone guanosine tetraphosphate (ppGpp). Although the mutant remained susceptible to both linezolid and daptomycin in clinical MIC testing and during planktonic growth, it demonstrated tolerance to high doses of both antibiotics when growing in a biofilm. This biofilm-specific gain in resistance was reflected in the broad shift in transcript levels caused by the mutation. Only an experimental biofilm-targeting ClpP-activating antibiotic was able to kill the mutant strain in an established biofilm. The relA mutation was associated with a fitness trade-off, forming smaller and less-well-populated biofilms on biological surfaces. We conclude that clinically relevant relA mutations can emerge during prolonged VRE infection, causing baseline activation of the stringent response, subsequent antibiotic tolerance, and delayed eradication in an immunocompromised state.
  
  
  
  The increasing prevalence of antibiotic-resistant bacterial pathogens is a major challenge currently facing the medical community. Such pathogens are of particular importance in immunocompromised patients as these individuals may favor emergence of novel resistance determinants due to lack of innate immune defenses and intensive antibiotic exposure. During the course of chemotherapy, a patient developed prolonged bacteremia with vancomycin-resistant Enterococcus faecium that failed to clear despite multiple front-line antibiotics. The consecutive bloodstream isolates were sequenced, and a single missense mutation identified in the relA gene, the mediator of the stringent response. Strains harboring the mutation had elevated baseline levels of the alarmone and displayed heightened resistance to the bactericidal activity of multiple antibiotics, particularly in a biofilm. Using a new class of compounds that modulate ClpP activity, the biofilms were successfully eradicated. These data represent the first clinical emergence of mutations in the stringent response in vancomycin-resistant entereococci.
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Hospital, Memphis, Tennessee, USA.
authors.forename: Erin S
authors.initials: ES
authors.lastname: Honsa
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
authors.forename: Vaughn S
authors.identifier: 0000-0001-7726-0765
authors.initials: VS
authors.lastname: Cooper
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Hospital, Memphis, Tennessee, USA.
authors.forename: Mohammed N
authors.initials: MN
authors.lastname: Mhaissen
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Hospital, Memphis, Tennessee, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Frank
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Hospital, Memphis, Tennessee, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Shaker
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Hospital, Memphis, Tennessee, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Iverson
authors.affiliation: Department of Oncology, St. Jude Children's Hospital, Memphis, Tennessee, USA.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Rubnitz
authors.affiliation: Department of Pathology, St. Jude Children's Hospital, Memphis, Tennessee, USA.
authors.forename: Randall T
authors.initials: RT
authors.lastname: Hayden
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Hospital, Memphis, Tennessee, USA.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Lee
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Hospital, Memphis, Tennessee, USA.
authors.forename: Charles O
authors.initials: CO
authors.lastname: Rock
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Hospital, Memphis, Tennessee, USA.
authors.forename: Elaine I
authors.initials: EI
authors.lastname: Tuomanen
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Hospital, Memphis, Tennessee, USA Joshua.wolf@stjude.org jason.rosch@stjude.org.|Department of Pediatrics, University of Tennessee Health Science Center, Memphis, Tennessee, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Wolf
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Hospital, Memphis, Tennessee, USA Joshua.wolf@stjude.org jason.rosch@stjude.org.
authors.forename: Jason W
authors.initials: JW
authors.lastname: Rosch
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D016470:Bacteremia / Q000382:microbiology; D018441:Biofilms / Q000187:drug effects / Q000254:growth & development; D004361:Drug Tolerance; D016984:Enterococcus faecium / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D005260:Female; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology*; D006159:Guanosine Tetraphosphate / Q000378:metabolism; D006801:Humans; D016867:Immunocompromised Host; D007223:Infant; D008025:Ligases / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D050505:Mutant Proteins / Q000235:genetics*; D020125:Mutation, Missense; D017422:Sequence Analysis, DNA; D065507:Vancomycin-Resistant Enterococci / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000900:Anti-Bacterial Agents; D050505:Mutant Proteins; D006159:Guanosine Tetraphosphate; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases