Novel Chimeric β-Lactamase CTX-M-64, a Hybrid of CTX-M-15-Like and CTX-M-14 β-Lactamases, Found in a Shigella sonnei Strain Resistant to Various Oxyimino-Cephalosporins, Including Ceftazidime

Abstract

The plasmid-mediated novel ␤-lactamase CTX-M-64 was first identified in Shigella sonnei strain UIH-1, which exhibited resistance to cefotaxime (MIC, 1,024 g/ml) and ceftazidime (MIC, 32 g/ml). The amino acid sequence of CTX-M-64 showed a chimeric structure of a CTX-M-15-like ␤-lactamase (N-and C-terminal moieties) and a CTX-M-14-like ␤-lactamase (central portion, amino acids 63 to 226), suggesting that it originated by homologous recombination between the corresponding genes. The introduction of a recombinant plasmid carrying bla CTX-M-64 conferred resistance to cefotaxime in Escherichia coli, and the activities of cefotaxime and ceftazidime were restored in the presence of clavulanic acid. Of note, CTX-M-64 production could also confer consistent resistance to ceftazidime, which differs from the majority of CTX-M-type enzymes, which poorly hydrolyze ceftazidime. These results were consistent with the kinetic parameters determined with the purified CTX- Shigellosis remains a public health concern throughout the world and has become an actual threat, particularly in developing countries, where 99% of the estimated 165 million annual episodes occur. Children under 5 years of age have been involved in more than half of the episodes and deaths (14). Shigellosis is more severe in malnourished children and elderly and immunocompromised people. Antibiotic treatment shortens the duration of clinical symptoms and pathogen excretion, prevents disease transmission, and reduces the risk of potential complications (18,27,34). However, empirical therapy with first-line antimicrobial agents, including ampicillin, trimethoprim-sulfamethoxazole, chloramphenicol, nalidixic acid, co-trimoxazole, and tetracycline, has become less effective due to the high prevalence of multidrug-resistant (MDR) clinical isolates among Shigella species (9, 28, 29, 32). For these MDR isolates, the therapeutic options for oral administration are fluoroquinolones for adults and oxyimino-cephalosporins for children.Plasmid-encoded class A extended-spectrum ␤-lactamase (ESBL) production is still uncommon among Shigella species, despite the worldwide spread and prevalence of ESBL-producing clinical isolates belonging to the family Enterobacteriaceae. Four CTX-M-type ␤-lactamases, CTX-M-2, CTX-M-3, CTX-M-14, and CTX-M-15, and several TEM-derived ESBLs have been reported for Shigella sonnei (1,11,15,25). S. sonnei strain UIH-1, characterized in this study, produced a novel CTX-Mtype ␤-lactamase, a hybrid of the CTX-M-15-like ␤-lactamase, which is a new CTX-M-15 variant (GenBank accession no. DQ256091), and the CTX-M-14 ␤-lactamase; and this chimeric enzyme conferred resistance to ceftazidime as well as to cefotaxime and ceftriaxone. MATERIALS AND METHODSClinical isolate. S. sonnei UIH-1 was identified with the API 20E system (bioMérieux) in combination with tests for the utilization of citrate with Christensen's citrate medium (4), sodium acetate, and mucate and by PCR detection of the invE and ipaH genes with specific primer sets (Takara Bio, Shiga, Japan)...

Residues Distal to the Active Site Contribute to Enhanced Catalytic Activity of Variant and Hybrid β-Lactamases Derived from CTX-M-14 and CTX-M-15

“…Bovine serum albumin (BSA) (100 g/ml) was added to the diluted enzymes and the reaction mixture to prevent enzyme degradation. For ceftazidime, which was a poor substrate, the K i values of mCTX-Ms enzymes were determined by a competitive inhibition assay using 100 M nitrocefin as a reporter substrate (5,15). The effect of temperature on the enzymatic activity was studied by determination of the kinetic constants of CTX-M-15 and CTX-M-55 toward cefotaxime using the same assay buffer and condition as aforementioned except at temperatures of 25°C, 40°C, and 60°C.…”

“…This suggests that recombination may take place between bla CTX-M- 14 and bla CTX-M-55 , producing various hybrid enzymes. Interestingly, all three published hybrids, CTX-M-64, CTX-M-123, and CTX-M-137, confer relatively high ceftazidime MICs compared to the majority of other CTX-M-type ESBLs (3)(4)(5). Conversely, the hydrolytic activities of CTX-M-55, CTX-M-123, and CTX-M-132 enzymes have not been described.…”

“…More than 95% identity is often observed within the same cluster, whereas less than 90% identity is detectable between clusters (2). Several hybrids of the CTX-M-1 and -9 groups, namely, CTX-M-64, CTX-M-123, CTX-M-137, and CTX-M-132, have also been identified recently (3)(4)(5). Formation of these hybrid CTX-M enzymes was suggested to be the result of recombination between bla CTX-M- 15 and bla CTX-M-14 , the two most dominant variants detectable worldwide (1,6).…”

“…Conversely, the hydrolytic activities of CTX-M-55, CTX-M-123, and CTX-M-132 enzymes have not been described. Although the kinetic parameters of CTX-M-14, -15, and -64 have been reported, only a few substrates have been characterized (5,8,9). In addition, kinetic parameters of these enzymes for ␤-lactamase inhibitors and animal-use-only cephalosporins, namely, ceftiofur, are unknown.…”

Genomic Portrait of the Evolution and Epidemic Spread of a Recently Emerged Multidrug-Resistant Shigella flexneri Clone in China

“…Resistance genes commonly present in Shigella spp. and Enterobacteriaceae, including the SRL, Tn7, gyrA, dfrA5, bla CTX-M , bla TEM , bla SHV , bla CMY , qnr, qepA, and aac(6=)-Ib-cr, were selected as references (9,(28)(29)(30)(31)) (see Table S1 in the supplemental material). The antibiotic resistance genes were searched using BLASTn, with an E value of 1 ϫ 10 Ϫ15 as the cutoff value for a significant match, based on the data generated by whole-genome sequencing.…”