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.