title: In Vivo Evolution of CMY-2 to CMY-33 β-Lactamase in Escherichia coli Sequence Type 131: Characterization of an Acquired Extended-Spectrum AmpC Conferring Resistance to Cefepime.
abstract: Cefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance have been reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identical Escherichia coli isolates obtained from a patient. The CMY-2-producing E. coli isolate (CMY-2-Ec) was isolated from a wound. Thirty days later, one CMY-33-producing E. coli isolate (CMY-33-Ec) was detected in a bronchoalveolar lavage fluid sample. Two weeks before the isolation of CMY-33-Ec, the patient received cefepime. CMY-33-Ec and CMY-2-Ec were identical by repetitive extragenic palindromic-PCR (rep-PCR), being of hyperepidemic sequence type 131 (ST131) but showing different β-lactam MICs (e.g., cefepime MIC, 16 and ≤ 0.5 μg/ml for CMY-33-Ec and CMY-2-Ec, respectively). Identical CMY-2-Ec isolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed in E. coli strain DH10B, both CMYs conferred resistance to ceftazidime (≥ 256 μg/ml), but the cefepime MICs were higher for CMY-33 than CMY-2 (8 versus 0.25 μg/ml, respectively). The kcat/Km or inhibitor complex inactivation (kinact)/Ki app (μM(-1) s(-1)) indicated that CMY-33 possesses an extended-spectrum β-lactamase (ESBL)-like spectrum compared to that of CMY-2 (e.g., cefoxitin, 0.2 versus 0.4; ceftazidime, 0.2 versus not measurable; cefepime, 0.2 versus not measurable; and tazobactam, 0.0018 versus 0.0009, respectively). Using molecular modeling, we show that a widened active site (∼ 4-Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the first in vivo demonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum, resembling an ESBL. The prevalence of CMY-2-Ec isolates is rapidly increasing worldwide; therefore, awareness that cefepime treatment may select for resistant isolates is critical.
authors.affiliation: Institute for Infectious Diseases, University of Bern, Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
authors.forename: João
authors.initials: J
authors.lastname: Pires
authors.affiliation: Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Taracila
authors.affiliation: Research Service, Louis Stokes VA Medical Center, Cleveland, Ohio, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Bethel
authors.affiliation: Division of Infectious Diseases, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Doi
authors.affiliation: Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
authors.forename: Sara
authors.initials: S
authors.lastname: Kasraian
authors.affiliation: Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
authors.forename: Regula
authors.initials: R
authors.lastname: Tinguely
authors.affiliation: Department of Infectious Diseases, Bern University Hospital and University of Bern, Bern, Switzerland.
authors.forename: Parham
authors.initials: P
authors.lastname: Sendi
authors.affiliation: Research Service, Louis Stokes VA Medical Center, Cleveland, Ohio, USA Division of Infectious Diseases and HIV Medicine, Department of Medicine, University Hospitals Case Medical Center, Cleveland, Ohio, USA Infectious Diseases Section, Medical Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio, USA Department of Pharmacology, Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA bonomo@va.gov aendimiani@gmail.com.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
authors.affiliation: Institute for Infectious Diseases, University of Bern, Bern, Switzerland bonomo@va.gov aendimiani@gmail.com.
authors.forename: Andrea
authors.identifier: http://orcid.org/0000-0003-3186-5421
authors.initials: A
authors.lastname: Endimiani
mesh_terms: D000368:Aged; D000900:Anti-Bacterial Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015780:Carbapenems / Q000494:pharmacology / Q000627:therapeutic use; D000077723:Cefepime; D002443:Ceftriaxone / Q000494:pharmacology / Q000627:therapeutic use; D002511:Cephalosporins / Q000494:pharmacology* / Q000627:therapeutic use; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000188:drug therapy / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests; D062105:Molecular Docking Simulation; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D015780:Carbapenems; D002511:Cephalosporins; D029968:Escherichia coli Proteins; D002443:Ceftriaxone; D000077723:Cefepime; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; C000605810:beta-lactamase CMY-2, E coli; C000610591:beta-lactamase CMY-33, E coli