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) ␤-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 ␤-lactam MICs (e.g., cefepime MIC, 16 and <0.5 g/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 g/ml), but the cefepime MICs were higher for CMY-33 than CMY-2 (8 versus 0.25 g/ml, respectively). The k cat /K m or inhibitor complex inactivation (k inact )/K i app (M ؊1 s ؊1 ) indicated that CMY-33 possesses an extended-spectrum ␤-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 (ϳ4-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. E nterobacteriaceae can manifest resistance to third-generation cephalosporins as a result of the production of extended-spectrum ␤-lactamases (ESBLs), chromosomal AmpC (cAmpCs), or plasmid-mediated AmpCs (pAmpCs) (1, 2). In general, ESBLs are inhibited by the commercially available ␤-lactamase inhibitors but hydrolyze well the fourth-generation cephalosporin cefepime (FEP). On the other hand, AmpCs are not inhibited by inhibitors and do not hydrolyze FEP (1, 3-5). Therefore, FEP is suggested for the treatment of infections caused by AmpC producers (6-8).In the past, AmpC variants with enhanced hydrolytic efficiency against FEP were sporadically reported in Enterobacter spp. (8-11), Serratia marcescens (12), and Escherichia coli (13-17). These chromosomal extended-spectrum AmpC ␤-lactamases (cESACs) possess specific amino acid insertions, deletions, duplications, or substitutions in the H-10 helix (also named the R2-loop) that allow better accommodation and hydrolysis of FEP in the serine active site (1,4,11,14). More ...

Increased Hydrolysis of Oximino-β-Lactams by CMY-107, a Tyr199Cys Mutant Form of CMY-2 Produced by Escherichia coli

“…This, in turn, could mask the effects of ESAC production. Nevertheless, the occurrence of CMY-107, as well as the CMY-94 (Leu293Ser), CMY-95 (Leu293Ser/Val211Ala), and CMY-33 (Leu293-Ala294 deletion) ESACs, mutant forms of CMY-2 that emerged during ceftazidime, aztreonam, and cefepime chemotherapy (8,10), suggests that prolonged treatment of patients colonized with CMY-2-expressing members of the family Enterobacteriaceae with oximino-␤-lactams may lead to the selection of ES mutants. Spread of the respective genes is also evident, as the Tyr199Cys mutant form of CMY-2 was first identified in a surveillance study in France during 2004 to 2008 (26).…”

Conjugative plasmidic AmpC detected in Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae human clinical isolates from Portugal

“…Coudron, Rodríguez-Martínez et al, and Pires et al [5,6,8] describe that the AmpC enzyme is inhibited by boronic acid and cloxacillin. It was tested in our isolates, and boronic acid demonstrated to be a suitable inhibitor of resistance.…”

“…The cloxacillin inhibition method was performed using Muller-Hinton agar (MHA) added to cloxacillin (200 μg/mL Sigma-Aldrich) to disk diffusion method, as described previously. It was positive when isolates demonstrated susceptibility to the antimicrobials to which were resistant in the method without cloxacillin [6,8].…”

“…These enzymes confer resistance to cephamycin as cefoxitin to the oxyimino-cephalosporins (ceftazidime and cefotaxime, for instance), and these are not inhibited by clavulanate, sulbactam, and tazobactam. Boronic acid and cloxacillin are considered AmpC inhibitors used in phenotypical tests to confirm the enzyme presence [5][6][7][8][9].…”

Comparative Genomics of Escherichia coli Sequence Type 219 Clones From the Same Patient: Evolution of the IncI1 blaCMY-Carrying Plasmid in Vivo

“…The accumulation of antibiotic resistance, loss of nonessential genes, metabolic alterations, and virulence factor attenuation all occur when pathogenic organisms adapt to the host (Viberg et al, 2017 ). Antibiotic exposures also facilitate the genomic evolution of antibiotic resistance-associated genes (Pires et al, 2015 ; Lin et al, 2016 ). Here, we report a clinical case that illustrates the dynamic nature of an IncI1 plasmid within the host, as bla CMY−111 was changed to bla CMY−4 in E. coli .…”