title: Evaluation of CTX-M steady-state mRNA, mRNA half-life and protein production in various STs of Escherichia coli.
abstract: OBJECTIVE
  High levels of β-lactamase production can impact treatment with a β-lactam/β-lactamase inhibitor combination. Goals of this study were to: (i) compare the mRNA and protein levels of CTX-M-15- and CTX-M-14-producing Escherichia coli from 18 different STs and 10 different phylotypes; (ii) evaluate the mRNA half-lives and establish a role for chromosomal- and/or plasmid-encoded factors; and (iii) evaluate the zones of inhibition for piperacillin/tazobactam and ceftolozane/tazobactam.
  
  
  METHODS
  Disc diffusion was used to establish zone size. RNA analysis was accomplished using real-time RT-PCR and CTX-M protein levels were evaluated by immunoblotting. Clinical isolates, transformants and transconjugants were used to evaluate mRNA half-lives.
  
  
  RESULTS
  mRNA levels of CTX-M-15 were up to 165-fold higher compared with CTX-M-14. CTX-M-15 protein levels were 2-48-fold less than their respective transcript levels, while CTX-M-14 protein production was comparable to the observed transcript levels. Nineteen of 25 E. coli (76%) had extended CTX-M-15 mRNA half-lives of 5-15 min and 16 (100%) CTX-M-14 isolates had mRNA half-lives of <2-3 min. Transformants had mRNA half-lives of <2 min for both CTX-M-type transcripts, while transconjugant mRNA half-lives corresponded to the half-life of the donor. Ceftolozane/tazobactam zone sizes were ≥19 mm, while piperacillin/tazobactam zone sizes were ≥17 mm.
  
  
  CONCLUSIONS
  CTX-M-15 mRNA and protein production did not correlate. Neither E. coli ST nor phylotype influenced the variability observed for CTX-M-15 mRNA or protein produced. mRNA half-life is controlled by a plasmid-encoded factor and may influence mRNA transcript levels, but not protein levels.
authors.affiliation: Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA.
authors.forename: Chelsie N
authors.initials: CN
authors.lastname: Geyer
authors.affiliation: Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA.
authors.forename: Randal C
authors.initials: RC
authors.lastname: Fowler
authors.affiliation: Veterans Affairs Medical Center, Minneapolis, MN, USA University of Minnesota, Minneapolis, MN, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: Johnson
authors.affiliation: Veterans Affairs Medical Center, Minneapolis, MN, USA University of Minnesota, Minneapolis, MN, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Johnston
authors.affiliation: Center for Global Infectious Diseases Research, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Weissman
authors.affiliation: Public Health England (PHE), West Midlands Public Health Laboratory, Heart of England NHS Foundation Trust, Bordesley Green East, Birmingham B9 5SS, UK Institute of Microbiology and Infection, School of Biosciences, School of Immunity and Infection, University of Birmingham, Edgbaston Campus, Birmingham B15 2TT, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Hawkey
authors.affiliation: Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA ndhanson@creighton.edu.
authors.forename: Nancy D
authors.initials: ND
authors.lastname: Hanson
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002511:Cephalosporins / Q000494:pharmacology; D052978:Disk Diffusion Antimicrobial Tests; D004926:Escherichia coli / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D005838:Genotype; D006801:Humans; D015151:Immunoblotting; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010878:Piperacillin / Q000494:pharmacology; D000077725:Piperacillin, Tazobactam Drug Combination; D020871:RNA Stability; D012333:RNA, Messenger / Q000032:analysis*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000078142:Tazobactam; D001618:beta-Lactamases / Q000032:analysis* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D012333:RNA, Messenger; C000594038:ceftolozane, tazobactam drug combination; D000077725:Piperacillin, Tazobactam Drug Combination; D010397:Penicillanic Acid; C456080:beta-lactamase CTX-M-14; C473109:beta-lactamase CTX-M-15; D001618:beta-Lactamases; D000078142:Tazobactam; D010878:Piperacillin