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Characterization of a new plasmid-mediated extended-spectrum beta-lactamase from Serratia marcescens
C Kunugita1, F Higashitani, A Hyodo
1Anticancer and Antimicrobial Research Lab, Taiho Pharmaceutical Co, Ltd, Tokushima, Japan.
Abstract:
A new extended spectrum beta-lactamase was detected in Serratia marcescens 42039 that was isolated from urine of patients with complicated urinary tract infection in Japan. This stain produced three different beta-lactamase types (TEM-1, a cephalosporinase, and a new beta-lactamase: CKH-1). The TEM-1 and CKH-1 encoding genes were conjugated from S. marcescens 42039 to Escherichia coli K-12 at frequencies of 10(-5) to 10(-6). The MICs of beta-lactams against the transconjugant were: ampicillin > 1600, piperacillin 800, cephalothin 1600, ceftazidime 6.25, cefotaxime 100, and ceftriaxone 200 micrograms/ml. The CKH-1 enzyme was purified to more than 90% by ion-exchange chromatography. The molecular weight of purified CKH-1 was 30 K dalton and the isoelectric point was 8.2. Relative Vmax/Km values (cephaloridine = 100) of penicillin G, cephalothin, and oxyiminocephalosporins such as cefuroxime, ceftriaxone, and cefotaxime, were 256, 226, 116, 87, and 49, respectively. The I50 values of tazobactam, BRL-42715, and clavulanic acid against CKH-1 enzyme were 0.0011, 0.0002, and 0.097 microM respectively. The enzymatic activity of CKH-1 was not inhibited by EDTA and anti-TEM-1 serum. These findings indicate that CKH-1 is a member of the groups of class A beta-lactamases. This is the first report of a plasmid-mediated oxyiminocephalosporin hydrolyzing broad-spectrum beta-lactamase from clinical isolates of S. marcescens.
Insights
A novel extended-spectrum beta-lactamase, CKH-1, was identified in Serratia marcescens from complicated urinary tract infections. This enzyme confers resistance to various beta-lactams, indicating a new threat in antimicrobial resistance.
Area of Science:
- Microbiology
- Biochemistry
Background:
- Complicated urinary tract infections (cUTIs) pose a significant therapeutic challenge.
- The emergence of antimicrobial resistance, particularly beta-lactamase production in Gram-negative bacteria, is a growing global health concern.
- Serratia marcescens is an opportunistic pathogen frequently associated with healthcare-associated infections.
Purpose of the Study:
- To characterize a newly identified extended-spectrum beta-lactamase (ESBL) produced by a clinical isolate of Serratia marcescens.
- To investigate the enzymatic properties and resistance profile conferred by this novel beta-lactamase.
- To determine the genetic basis and transferability of the beta-lactamase genes.
Main Methods:
- Isolation and identification of Serratia marcescens from patient urine samples.
- Detection and characterization of beta-lactamase activity using biochemical assays.
- Gene conjugation experiments to transfer beta-lactamase encoding genes to Escherichia coli.
- Purification and kinetic analysis of the novel beta-lactamase (CKH-1).
- Determination of minimum inhibitory concentrations (MICs) for various beta-lactam antibiotics.
Main Results:
- A clinical isolate of Serratia marcescens (strain 42039) produced three beta-lactamases: TEM-1, a cephalosporinase, and a novel ESBL, CKH-1.
- Genes encoding TEM-1 and CKH-1 were successfully transferred to Escherichia coli K-12 via conjugation.
- The transconjugant strains exhibited high-level resistance to ampicillin, piperacillin, and cephalothin, with reduced susceptibility to ceftazidime, cefotaxime, and ceftriaxone.
- Purified CKH-1 enzyme (30 kDa, pI 8.2) demonstrated broad-spectrum activity, hydrolyzing cephalothin and oxyiminocephalosporins.
- CKH-1 showed potent inhibition by tazobactam and BRL-42715, classifying it as a Class A beta-lactamase.
Conclusions:
- CKH-1 represents the first reported plasmid-mediated oxyiminocephalosporin-hydrolyzing ESBL from clinical Serratia marcescens isolates.
- The discovery of CKH-1 highlights the evolving landscape of beta-lactamase resistance in S. marcescens.
- This finding underscores the need for continuous surveillance and development of strategies to combat emerging antimicrobial resistance.