Prediction of the Evolution of Ceftazidime Resistance in Extended-Spectrum β-Lactamase CTX-M-9

Abstract

A random mutagenesis technique was used to predict the evolutionary potential of ␤-lactamase CTX-M-9 toward the acquisition of improved catalytic activity against ceftazidime. Thirty CTX-M mutants were obtained during three rounds of mutagenesis. These mutants conferred 1-to 128-fold-higher MICs of ceftazidime than the parental enzyme CTX-M-9. The CTX-M mutants contained one to six amino acid substitutions. Mutants harbored the substitutions Asp240Gly and Pro167Ser, which were previously observed in clinical CTX-M enzymes. Additional substitutions, notably Arg164His, Asp179Gly, and Arg276Ser, were observed near the active site. The kinetic constants of the three most active mutants revealed two distinct ways of improving catalytic efficiency against ceftazidime. One enzyme had a 17-fold-higher k cat value than CTX-M-9 against ceftazidime. The other two had 75-to 300-fold-lower K m values than CTX-M-9 against ceftazidime. The current emergence of CTX-M ␤-lactamases with improved activity against ceftazidime may therefore be the beginning of an evolutionary process which might subsequently generate a great diversity of CTX-M-type ceftazidimases.The most prevalent mechanism of resistance against ␤-lactams in gram-negative bacilli is the production of ␤-lactamases belonging to structural class A (1). Class A ␤-lactamases are active-site serine enzymes which cleave the amide bond in the ␤-lactam ring via an acyl-enzyme intermediate.Oxyimino cephalosporins, such as ceftazidime or cefotaxime, are highly resistant to this hydrolysis by class A penicillinases such as TEM-1, TEM-2, and SHV-1. However, the extensive use of these ␤-lactams has resulted in the emergence of extended-spectrum ␤-lactamases (ESBLs). The first ESBLs were derived from the TEM-1/2 and SHV-1 ␤-lactamases by critical amino acid substitutions which confer hydrolytic activity against oxyimino cephalosporins. The major substitutions are located in two elements of the binding site: the ␤3 strand at position 238 and the omega loop at positions 164 and 179 (27).Non-TEM, non-SHV ESBLs designated CTX-M enzymes were identified in the early 1990s (2, 36). The frequency of CTX-M enzymes has increased sharply worldwide since 1995, and they now form a growing family that comprises more than 40 enzymes (3). Most CTX-M enzymes exhibit a much greater hydrolytic efficiency against cefotaxime than against ceftazidime, unlike TEM-and SHV-type ESBLs. However, seven CTX-M mutants harboring point mutations which improve enzymatic efficiency against ceftazidime have been reported recently, suggesting that CTX-Ms are altering their substrate specificity in response to continued antibiotic selective pressure. Five mutants harbor substitution Asp240Gly (4,5,16,29,31), and two mutants harbor substitutions Pro167Ser/Thr (33, 38). These substitutions have not been previously observed in natural TEM or SHV ESBLs, suggesting that CTX-M enzymes have a singular evolutionary potential. In this work, a random mutagenesis technique was applied to the CTX-M-9-encoding gene to predict wh...

High Prevalence and Significant Association of ESBL and QNR Genes in Pathogenic Klebsiella pneumoniae Isolates of Patients from Kolkata, India

“…Mutation at codon 167 resulted in Glycine ? Arginine substitution in the constituent omega loop (161 to 179 codon), situated at the floor of catalytic active site of CTX-M enzyme [26]. Both codons 242 and 259 encode amino acids constituent of a/b domain of CTX-M protein.…”

Exploring the Inhibition of CTX-M-9 by β-Lactamase Inhibitors and Carbapenems

“…coli BL21(DE3) cells (Novagen) containing the pET 9a (ϩ) expression vector were grown in superoptimal broth (SOB) containing 50 g/ml kanamycin (11,16). These cells were grown with agitation at 37°C until an optical density at 600 nm of 0.8 was attained.…”

“…The bla CTX-M-9 gene was cloned into the pET9a (ϩ) plasmid vector (Novagen) as described previously and was maintained in E. coli BL21(DE3) cells (11,16).…”

Convergent In Vivo and In Vitro Selection of Ceftazidime Resistance Mutations at Position 167 of CTX-M-3 β-Lactamase in Hypermutable Escherichia coli Strains

“…This mutation is found in the naturally occurring enzymes of the CTX-M-2 and CTX-M-9 clusters (CTX-M-35 and CTX-M-19) and, as shown by Welsh et al (30), can be readily selected with CAZ following the in vitro mutagenesis of CTX-M-2. It is interesting to note that our in vitro experiments as well as similar studies by other authors (10,19,24,30) yielded only the Pro3Ser mutation at position 167, whereas a variety of substitutions at this position have been found in the naturally occurring CTX-M ceftazidimases (e.g., Thr in CTX-M-23 and CTX-M-42, Ser in CTX-M-52, and Gln in CTX-M-54). Although the reason for this remains unclear, it may be at least partially explained by the fact that a nucleotide transition required for the Pro167Ser substitution is more likely to occur in almost all types of mutators, including the mutD5 strain used in our experiments and the mutS strains employed in the other studies (19,24).…”

“…Recently, however, several CTX-M variants have been reported to contain point mutations at amino acid position 167 or 240 (Ambler's numbering scheme [1]) in association with increased CAZ-hydrolyzing activity, which leads to a higher level of CAZ resistance (2,3,5,8,21,22,25,27,28). Substitutions of these amino acids in CTX-M enzymes have been shown in both clinical isolates (3,8,25,28) and mutants selected in vitro by CAZ exposure (10,22,24,30). In recent studies by Karisik et al (19) and Novais et al (24), it has been demonstrated that mutations at position 167 in CTX-M-3, which lead to increased CAZ resistance, are readily selected in vitro when mutator host strains are used.…”