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

João Pires, Magdalena Taracila, Christopher R. Bethel, Yohei Doi, Sara Kasraian, Regula Tinguely, Parham Sendi, Robert A. Bonomo, Andrea Endimiani

Research output: Contribution to journalArticle

6 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)7483-7488
Number of pages6
JournalAntimicrobial agents and chemotherapy
Volume59
Issue number12
DOIs
Publication statusPublished - 01-12-2015

Fingerprint

Escherichia coli
Ceftazidime
Plasmids
cefepime
Cefoxitin
Lactams
Bronchoalveolar Lavage Fluid
Cephalosporins
Gram-Negative Bacteria
Catalytic Domain
Hydrolysis
Polymerase Chain Reaction
Wounds and Injuries
Therapeutics
Infection

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

Cite this

Pires, João ; Taracila, Magdalena ; Bethel, Christopher R. ; Doi, Yohei ; Kasraian, Sara ; Tinguely, Regula ; Sendi, Parham ; Bonomo, Robert A. ; Endimiani, Andrea. / 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. In: Antimicrobial agents and chemotherapy. 2015 ; Vol. 59, No. 12. pp. 7483-7488.
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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-{\AA} 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.",
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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. / Pires, João; Taracila, Magdalena; Bethel, Christopher R.; Doi, Yohei; Kasraian, Sara; Tinguely, Regula; Sendi, Parham; Bonomo, Robert A.; Endimiani, Andrea.

In: Antimicrobial agents and chemotherapy, Vol. 59, No. 12, 01.12.2015, p. 7483-7488.

Research output: Contribution to journalArticle

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T2 - Characterization of an acquired extended-spectrum AmpC conferring resistance to cefepime

AU - Pires, João

AU - Taracila, Magdalena

AU - Bethel, Christopher R.

AU - Doi, Yohei

AU - Kasraian, Sara

AU - Tinguely, Regula

AU - Sendi, Parham

AU - Bonomo, Robert A.

AU - Endimiani, Andrea

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N2 - 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.

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