Mutational Replacement of Leu-293 in the Class C Enterobacter cloacae P99 β-Lactamase Confers Increased MIC of Cefepime

A multiresistant Serratia marcescens strain, HD, isolated from a patient with a urinary tract infection, was resistant to amino-, carboxy-, and ureidopenicillins, ceftazidime, and cefepime and was susceptible to cefotaxime and ceftriaxone, according to the guidelines of the NCCLS. No synergy was found between expanded-spectrum cephalosporins and clavulanic acid, according to the double-disk synergy test. The bla AmpC gene of the strain was amplified by PCR and cloned into Escherichia coli DH10B, giving rise to high-level resistance to ceftazidime, cefepime, and cefpirome. Sequencing analysis revealed that the bla AmpC gene from S. marcescens HD had a 12-nucleotide deletion compared to the bla AmpC gene from reference strain S. marcescens S3, leading to a 4-amino-acid deletion located in the H-10 helix of the ␤-lactamase. Kinetic analysis showed that this enzyme significantly hydrolyzed ceftazidime, cefepime, and cefpirome. This work underlined that resistance to the latest expanded-spectrum cephalosporins may be mediated by structurally modified AmpC-type ␤-lactamases.Serratia marcescens is a saprophytic, water-borne gram-negative rod (7) that possesses an inducible, chromosomally encoded AmpC-type ␤-lactamase (11) and is often involved in nosocomial infections (5,8,16,19). Infections caused by cephalosporinase-producing Enterobacteriaceae may be difficult to treat due to their ability to confer resistance to a variety of ␤-lactams, including the expanded-spectrum cephalosporins cefotaxime and ceftazidime. This resistance may arise from at least three different mechanisms in S. marcescens: high-level production of the chromosomal AmpC-type cephalosporinase (9), acquisition of an Ambler class A extended-spectrum ␤-lactamase (21), and acquisition of metallo-␤-lactamases (21). Cefepime and cefpirome, which contain quaternary nitrogen atoms at three different positions, remain active against overexpressed AmpC-producing enterobacteria. This activity results from both the stability of these molecules to attack by cephalosporinase (2, 6) and their rapid penetration through the outer membrane (15, 18).We analyzed an S. marcescens clinical isolate harboring a chromosomally encoded ␤-lactamase with ␤-lactam hydrolysis extended to a cefepime that possessed a 4-amino-acid deletion compared to a wild-type cephalosporinase. MATERIALS AND METHODSBacterial strains and plasmids. Clinical isolate S. marcescens HD was identified by using the API 20E system (bioMérieux, Marcy l'Etoile, France). Escherichia coli DH10B was used for the experiments (3). S. marcescens strain S3 was previously characterized as a wild-type AmpC producer (gift from H. Nikaido) (17).Cloning of ␤-lactamase genes. Whole-cell DNAs of S. marcescens isolates HD and S3 were extracted as described previously (3). On the basis of the ampC gene sequence of S. marcescens S3 (17), primers PRESM1 (5Ј-ATACCCTGCAACC TAAGAGC-3Ј) and PRESM2 (5Ј-ATCGCTGGTAGGGGCGCCTC-3Ј) were designed to amplify a 1,195-bp fragment corresponding to ampC genes without their own promoter seq...

Section: Discussionmentioning

confidence: 99%

Resistance to Cefepime and Cefpirome Due to a 4-Amino-Acid Deletion in the Chromosome-Encoded AmpC β-Lactamase of a Serratia marcescens Clinical Isolate

Mammeri

Poirel

Bemer³

et al. 2004

“…Previous hypotheses were raised stating that disruptions in the H-10 helix of cAmpCs (involving positions 282 to 296) are responsible for the ESBL phenotype. In particular, increased resistance to FEP, CAZ, CTX, and ATM was constantly observed in all previous cESACs (4,11,14); however, this is the first time that this phenomenon has been documented for a pESAC.…”

Section: Resultsmentioning

confidence: 89%

“…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)(4)(5). Therefore, FEP is suggested for the treatment of infections caused by AmpC producers (6)(7)(8).…”

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confidence: 99%

“…(8)(9)(10)(11), Serratia marcescens (12), and Escherichia coli (13)(14)(15)(16)(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 recently, plasmid-mediated ESACs (pESACs) derived from the most frequently detected pAmpC (CMY-2) were also identified (1).…”

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confidence: 99%

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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

Taracila

Bethel

et al. 2015

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 ...

“…Mutations in the AmpC cephalosporinase were recently reported to be the source of cefepime resistance in Escherichia coli, Enterobacter cloacae, and E. aerogenes (2,6,14,15,22). To evaluate the relationship between AmpC and cefepime resistance, the four E. aerogenes strains were transformed with plasmid pNH5 bearing an ampD gene (2).…”

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confidence: 99%

Occurrence of Efflux Mechanism and Cephalosporinase Variant in a Population of Enterobacter aerogenes and Klebsiella pneumoniae Isolates Producing Extended-Spectrum β-Lactamases

Tran

Dupont

Lavigne

et al. 2009

We investigated the occurrence of multidrug resistance in 44 Enterobacter aerogenes and Klebsiella pneumoniae clinical isolates. Efflux was involved in resistance in E. aerogenes isolates more frequently than in K. pneumoniae isolates (100 versus 38% of isolates) and was associated with the expression of phenylalanine arginine ␤-naphthylamide-susceptible active efflux. AcrA-TolC overproduction in E. aerogenes isolates was noted. An analysis of four E. aerogenes isolates for which cefepime MICs were high revealed no modification in porin expression but a new specific mutation in the AmpC ␤-lactamase.Enterobacter aerogenes and Klebsiella pneumoniae, two of the most prevalent nosocomial enterobacterial species, can frequently express a multidrug resistance (MDR) phenotype by the acquisition or high-level production of ␤-lactamases in combination with the overproduction of efflux pumps, associated or not with porin alterations in the outer membrane (1,3,7,9,13). Such modifications of envelope permeability, including efflux and influx (porin), in 28 Enterobacter aerogenes and 16 Klebsiella pneumoniae clinical isolates from Nîmes University Hospital, previously studied for extended-spectrum ␤-lactamase (ESBL) profiling and clonal identification, were investigated (11). Moreover, we ascertained the mechanism involved in cefepime resistance in four E. aerogenes isolates showing high-level resistance (MIC ϭ 128 mg/liter).Characterization of the MDR phenotype was carried out by determining the MICs of various structurally unrelated antibiotics selected for their capacities to be expelled and/or to enter cells via porins (Table 1). We used the twofold broth dilution method with Mueller-Hinton broth according to the CLSI guidelines: the tests were carried out with or without the broad-spectrum efflux pump inhibitor (EPI) phenylalanine arginine ␤-naphthylamide (PA␤N). This diamine compound, used at a low concentration (26.3 mg/liter), can increase the activities of chloramphenicol, tetracycline, norfloxacin, and sparfloxacin against various enterobacterial isolates which overproduce efflux pumps such AcrAB-TolC (4, 9, 16). The test results confirmed the existence of a PA␤N-susceptible efflux mechanism in all E. aerogenes and many K. pneumoniae strains, with marked decreases in the MICs of fluoroquinolones and chloramphenicol in the presence of PA␤N (Table 1). Efflux pump activity was identified when the PA␤N addition induced a threefold decrease in the MIC of an antibiotic molecule (4, 9). For the two fluoroquinolones, the PA␤N addition had better efficiency in decreasing the MICs of sparfloxacin than those of norfloxacin. The observed difference in PA␤N efflux inhibition between the two fluoroquinolones may reflect the respective locations of ligands inside the AcrB cavity and the flux competition (20). Yu et al. demonstrated, during previous cocrystalization analyses, the presence of different affinity sites located inside the AcrB pump and suggested that the benefit generated by the conjoint use of an EPI and an antibioti...