title: OXA-372, a novel carbapenem-hydrolysing class D β-lactamase from a Citrobacter freundii isolated from a hospital wastewater plant.
abstract: OBJECTIVE
  The objective of this study was to characterize a novel class D carbapenemase, named OXA-372, identified in a carbapenem-resistant Citrobacter freundii strain (Cfr-FI-07) isolated from a hospital wastewater plant in central Italy.
  
  
  METHODS
  Cfr-FI-07 was isolated using a selective chromogenic medium for carbapenem-resistant Enterobacteriaceae. Carbapenemase production was confirmed by spectrophotometric assay. WGS was carried out using an Illumina MiSeq platform. The functional profile of OXA-372 was investigated by expression of the cloned gene in Escherichia coli and by analysis of kinetic parameters of the purified enzyme.
  
  
  RESULTS
  C. freundii Cfr-FI-07 produced carbapenemase activity, but tested negative for common carbapenemase genes. WGS confirmed the absence of known carbapenemase genes and revealed the presence of a novel class D β-lactamase (DBL) determinant, named blaOXA-372, encoding a protein distantly related to other DBLs. In E. coli, production of OXA-372 conferred resistance to penicillins, including temocillin, and reduced susceptibility to carbapenems, while susceptibility to expanded-spectrum cephalosporins was virtually unaffected. This substrate specificity was confirmed by kinetic characterization of the purified enzyme, which exhibited high catalytic efficiencies for carbapenems (kcat/KM values ≥ 0.22 s(-1) · μM(-1)). The blaOXA-372 gene was associated with a genetic platform of original structure consisting of a Tn402/Tn5053 hybrid transposon derivative, named Tn6255, inserted into a TnPa38-like transposon, named Tn6256, located on an IncA/C-IncN plasmid of approximately 140 kb.
  
  
  CONCLUSIONS
  OXA-372 is a novel class D carbapenemase, belonging to a new lineage of DBLs, encoded by a gene associated with mobile elements. Functional properties revealed similarities, but also some differences, compared with other class D carbapenemases.
authors.affiliation: Department of Medical Biotechnology, University of Siena, Siena, Italy Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Antonelli
authors.affiliation: Department of Medical Biotechnology, University of Siena, Siena, Italy.
authors.forename: Marco Maria
authors.initials: MM
authors.lastname: D'Andrea
authors.affiliation: Clinical Microbiology and Virology Unit, Florence Careggi University Hospital, Florence, Italy.
authors.forename: Guendalina
authors.initials: G
authors.lastname: Vaggelli
authors.affiliation: Department of Medical Biotechnology, University of Siena, Siena, Italy.
authors.forename: Jean-Denis
authors.initials: JD
authors.lastname: Docquier
authors.affiliation: Department of Medical Biotechnology, University of Siena, Siena, Italy Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy Clinical Microbiology and Virology Unit, Florence Careggi University Hospital, Florence, Italy gianmaria.rossolini@unifi.it.
authors.forename: Gian Maria
authors.initials: GM
authors.lastname: Rossolini
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D015780:Carbapenems / Q000494:pharmacology; D016971:Citrobacter freundii / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D062065:Wastewater / Q000382:microbiology*; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D015780:Carbapenems; D062065:Wastewater; D001618:beta-Lactamases; C063912:carbapenemase