Biochemical And Genetic Characteristicsof CTX-M Hybrids

Extended spectrum β-lactamases（ESBLs） create serious therapeutic problems. Three CTX-M enzymes（CTX-M-64, CTX-M-123, and CTX-M-132） that are hybrids of members of the CTX-M-1 and CTX-M-9 groups have been detected in animals in China. The hybrid enzymes appear to display enhanced catalytic activity against most expanded-spectrum cephalosporins as compared to CTX-M-15 and CTX-M-14.These hybrid bla_CTX-M genes have all been suggested to be the result of recombination between bla_CTX-M-15 and bla_CTX-M-14, the most dominant variants detected worldwide. The objective of this study was to analyze kinetic contents of both parental and hybrid CTX-M enzymes, including CTX-M-15,-132,-123,-64,-14 and-55, and catalysis mechanism according to structures and to try to define how these hybrid genes may have arisen so as to provide a foundation for the further study of CTX-M enzymes.bla_CTX-M（full length） andmbla_CTX-M（without signal peptide regions） genes including CTX-M-15,-132,-123,-64,-14 and-55 were amplified by PCR, and inserted into p ET-28 b to construct the expression plasmid of bla_CTX-M genes. The positive recombination plasmids p ET-CTX-Ms and p ET-m CTX-Ms were transformed into E.coli BL21（DE3）, and the strains were used to determine the MICs and express protein. These six CTX-Msshowed similar expression levels, and MICs of E. coli strains producing the full-length CTX-Ms were tested. Regarding the MICs of different β-lactams for E. coli producing different CTX-Ms, CTX-M-14 mediated the lowest MICs to most of the β-lactams tested except ampicillin when compared to other CTX-Ms. Among the other five CTX-Ms, CTX-M-64 exhibited the highest MICs for cefotaxime, whereas CTX-M-64, CTX-M-132, CTX-M-123 and CTX-M-55 conferred higher MICs of ceftazidime and cefotaxime than those mediated by CTX-M-15. All three commercially available serine β-lactamase inhibitors, clavulanic acid, tazobactam, sulbactam were very effective when used in a combinatorial manner with cefotaxime, with the activity of cefotaxime exhibiting the highest degree of reduction（up to 70,000 folds） compared to cefotaxime alone.SDS-PAGE results showed that these protein were about 28 k Da and the optimal conditions to expree protein were IPTG 1.0mmol/L, cultured at 35℃ for 5h.The six m CTX-M enzymes with 99% purity in this study were obtained by affinity chromatography and gel filtration chromatography. Enzyme kinetic characterization of these CTX-Ms were consistent with the MIC data. CTX-M-14 exhibited the lowest catalytic activity towards the β-lactams tested except cephalothin. Whereas CTX-M-64 exhibited the highest catalytic activity on nitrocefin, cefuroxime, ceftiofur, ceftriaxone and cefotaxime. CTX-M-15 displayed slightly lower catalytic activities to most of the β-lactams tested except ampicillin when compared to CTX-M-55 and the other three hybrids. The IC50 and Ki of clavulanic acid, tazobactam and sulbactam showed that all these three inhibitors displayed nanomolar level of inhibition of these CTX-Ms, with sulbactam exhibiting higher IC50 and Ki than clavulanic acid, which was in turn slightly higher than tazobactam.Structural analysis showed that CTX-M-55 differs from CTX-M-15 by one substitution, A77 V, distal to the active site of enzyme. However, CTX-M-55 displayed enhanced catalytic activity（kcat/Km） against expanded-spectrum cephalosporins（ESCs）. CTX-M-55 exhibits higher structure stability most likely by forming hydrophobic interactions between A77 V and various key residues in different helices, thereby stabilizing the core architecture of the helix cluster and indirectly contributes to a more stable active site conformation, which in turn shows higher catalysis efficiency and more tolerant to temperature change. Analyses of the hybrids and their parental prototypes showed that evolution from CTX-M-15 to CTX-M-132, CTX-M-123 and CTX-M-64, characterized by gradual enhancement of catalytic activity to ESCs, was attributed to introduction of different substitutions to amino acids distal to the active site of CTX-M-15. Similarly, the increased hydrolytic activities against cephalosporins and sensitivity to β-lactamase inhibitors, clavulanic acid and sulbactam, of CTX-M-64 were partly due to the amino acids that were different from CTX-M-14 and located at both the C- and N-termini of CTX-M-64. These data indicate that residues distal to the active site of CTX-Ms contributed to their enhanced catalytic activities to ESCs.PCRwasused to analyzebla_CTX-Mhybrid genes in Escherichia coli isolates collected from human and ainimals in 2010²013, and the positivestrainswere studiedby E. coli multilocus sequence typing. Conjugation/transformation were performed to obtain transconjugants or transformants carrying a single plasmid with hybrid genes and their parental genes, and the sizes and replicon typeof plasmids were estimated respectivelyby S1-PFGE and PCR-based replicon typing.The complete sequences of plasmids carrying bla_CTX-M-15（p HNY2-1）, bla_CTX-M-64（p HNAH46-1）, bla_CTX-M-123（p HNAH4-1）were obtained through high-throughput sequencing method. The results showed that Inc I2 plasmids p HNY2-1 carrying bla_CTX-M-15, p HN1122-1carrying bla_CTX-M-55,p HNAH46-1 carrying bla_CTX-M-64, and p HNLDH19 carrying bla_CTX-M-132 are almost indentical and had the same ISEcp1-bla_CTX-M-123-Inc A/Cinsertion, which suggested these hybrid genes may generate frombla_CTX-M-15.Inc I1（ST108） plasmids p HNAH4-1carrying bla_CTX-M-123 had transposition unit, suggesting ISEcp1-mediated transfer of bla_CTX-M-123-Inc A/C-Inc I2 to an Inc I1 plasmid.The potential p HNAH46-1-like and p HNAH4-1-likewere detected by PCR sequencing and restriction fragment length polymorphism（RFLP）.Five additional E. coli of different sequence types from different provinces, farms and/or animals had bla_CTX-M-64 on a p HNAH46-1-like Inc I2 plasmid and nine had bla_CTX-M-123 on a p HNAH4-1-like Inc I1（ST108） plasmid.Thus epidemic Inc I plasmids may be responsible for spread of bla_CTX-M-64 and bla_CTX-M-123 between different animals and different locations in China.