JAC Advance Access originally published online on August 27, 2008
Journal of Antimicrobial Chemotherapy 2008 62(5):991-997; doi:10.1093/jac/dkn339
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Original research |
Natural D240G Toho-1 mutant conferring resistance to ceftazidime: biochemical characterization of CTX-M-43
1 Department of Biomedical Sciences and Technologies, University of L'Aquila, L'Aquila, Italy 2 Department of Chemistry, Chemical Engineering and Materials, University of L'Aquila, L'Aquila, Italy
* Corresponding author. Tel: +39-0862-433489; Fax: +39-0862-433433; E-mail: perilli{at}univaq.it
Received 1 February 2008; returned 29 April 2008; revised 3 July 2008; accepted 29 July 2008
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Objectives: The aim of this article is biochemical and kinetic characterization of CTX-M-43, a natural Asp-240
Gly mutant of CTX-M-44 (ex Toho-1), from a clinical isolate of Acinetobacter baumannii isolated in a Bolivian hospital. Methods: Steady-state kinetic parameters (Km and kcat) were determined for a large pattern of substrates. Analysis of inactivators and transient inactivators was performed to determine the efficiency of acylation (k+2/K) and the deacylation constant (k+3). Molecular modelling of Michaelis complex of ceftazidime, cefotaxime and ceftibuten, obtained from molecular mechanics calculations, was carried out.
Results: CTX-M-43 showed a general increase in affinity towards all cephalosporins tested, with respect to CTX-M-44. Carbapenems acted as inactivators with a good acylation efficiency for meropenem and ertapenem and significant deacylation constant for imipenem. MICs of imipenem obtained at a higher bacterial inoculum of recombinant Escherichia coli were increased.
Conclusions: Kinetic data and molecular modelling of Michaelis complex confirmed that Asp-240Gly allows a better accommodation of the bulky C7β aminothiazol-oxyimino substituent, resulting in a general increase in the enzyme affinity towards oxyimino cephalosporins. The ascertained potentialities of CTX-M-type enzymes, supported by the kinetic data and the behaviour of the recombinant E. coli at different bacterial inocula towards carbapenems, make a possible evolution of those enzymes towards a carbapenemase activity plausible.
Keywords: Acinetobacter baumannii , antibiotic resistance , CTX-M-type enzymes , class A
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Nowadays, it is well established that the production of acquired β-lactamases is the predominant cause of resistance to oxyimino-cephalosporins in Gram-negative bacteria. Most of them, belonging to class A extended-spectrum β-lactamases (ESBLs), were derived from TEM- or SHV-type β-lactamases through several mutations, ascribed mainly to a selective pressure induced by extensive use, and sometimes abuse and misuse of β-lactam antibiotics.1–4 Among class A β-lactamases, CTX-M ESBLs have an efficient hydrolytic activity towards oxyimino cephalosporins, but exhibit lower activity towards penicillins than non-ESBL enzymes. With this respect, they are characterized by a much greater hydrolytic activity against cefotaxime than ceftazidime and by a different profile to inhibitors.5,6 Also among CTX-M enzymes, the spectrum of activity is unusually heterogeneous. These enzymes have unique amino acid sequences, with only
40% identity with other class A β-lactamases. In the last years, the rate of dissemination of CTX-M-type enzymes has increased significantly in clinical isolates of Gram-negative bacteria.5 To date, the CTX-M family comprises 69 enzymes spread worldwide and divided into six clusters based on their amino acid sequence similarities.5,6 Less than 90% of the identity is observed between members belonging to distinct clusters. In 1995, Ishii et al.7 reported the identification of a new non-TEM and non-SHV cephalosporinase designed as Toho-1, recently renamed CTX-M-44, belonging to the CTX-M-2 cluster. With respect to other CTX-M members, Toho-1 shows a lower hydrolytic activity against penicillins and a higher activity against cefotaxime.8
The crystal structure of Toho-18,9 showed an overall fold similar to non-ESBLs. However, an increase in flexibility of the β-strand B3 and 
loop and the residues Asn-104, Phe-167, Ser-237, Asp-240 and Arg-276 are involved in the oxyimino-cephalosporin hydrolysing activity.8–11 Asp-240Gly mutants of CTX-M, CTX-M-15,12 CTX-M-16,13 CTX-M-2714 and CTX-M-3215 recently described, exhibit an increased enzymatic activity against ceftazidime. The appearance of Asp-240Gly mutants in CTX-M-type enzymes with improved catalytic efficiency against ceftazidime suggests that those enzymes are evolving as a result of selective pressure caused by the extensive use of ceftazidime in clinical therapy. The peculiarity of the residues involved in this evolution suggests the potential of those enzymes under an evolutionary standpoint.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Bacterial strains
blaCTX-M-43 was identified in a clinical isolate of Acinetobacter baumannii (SJ-0008), as reported previously.16 The organism was isolated from the sputum of a 42-year-old patient from the intensive care unit of the Hospital Municipal Universitario San Juan de Dios in Santa Cruz de la Sierra, Bolivia. Molecular analysis of the A. baumannii isolate showed the simultaneous presence of blaCTX-M and blaTEM-1 genes.16
Amplification of the entire blaCTX-M gene was performed using the following specific primers for blaCTX-M-2 genes: CTX-M/B_for 5'-ATGATGACTCAGAGCATTCGCCGCT-3' and CTX-M/B_rev 5'-TCAGAAACCGTGGGTTACGATTTTCG -3'.
The DNA sequence was determined by direct sequencing of the products from two independent PCRs, using ABI Prism 310 (Applied Biosystems, Monza, Italy). The deduced amino acid sequence showed the presence of an Asp-240Gly amino acid substitution with respect to CTX-M-44 (ex Toho-1) (GenBank accession number D37830
[GenBank]
). In accordance with Bush and Jacoby nomenclature (www.lahey.org), the new enzyme was named CTX-M-43 (GenBank accession number DQ102702
[GenBank]
).
Primers for PCR cloning into the pBC-SK(+) vector were designed inserting upstream a XhoI restriction site before the initial methionine and downstream a BamHI site after the stop codon of blaCTX-M-43. Cloning into the pET-26(b) vector has been performed inserting upstream a NdeI restriction site before the initial methionine and a BamHI site after the stop codon of blaCTX-M-43. The primers used for cloning were as follows: CTX-M-43_XhoI_for 5'-GGGGCTCGAGATGATGACTCAGAGCATTCGC-3', CTX-M-43_NdeI_for 5'-GGGGGGCATATGATGACTCAGAGCATTCGC-3' and CTX-M-43_BamHI_rev 5'-GGGGGGGGATCCTCAGAAACCGTGGGTTAC-3'.
Recombinant plasmids named pBC-SK(+)/CTX-M-43 and pET-26(b)(+)/CTX-M-43 were electroporated with the MicroPulser electroporator system (Bio-Rad Laboratories, UK) into Escherichia coli HB101 and E. coli BL21 (DE3), respectively. Colonies were selected on LB agar plates supplemented with 30 mg/L chloramphenicol and 32 mg/L cefotaxime for E. coli HB101 and 50 mg/L kanamycin, 32 mg/L cefotaxime and IPTG (isopropyl β-D-1-thiogalactopyranoside) 0.4 mM for E. coli BL21 (DE3).
The following antibiotics were used. The wavelengths (in nanometres) for each β-lactam antibiotic and its molar extinction coefficients (
) used to analyse the kinetic properties of the enzyme were as follows: benzylpenicillin (235 = –775 M–1/cm), moxalactam (270 = –6000 M–1/cm), cefazolin (260 = –7400 M–1/cm), cefalothin (273 = –6300 M–1/cm) and cefaloridine (260 = –10 000 M–1/cm) were purchased from Sigma Chemical Co. (St Louis, MO, USA); nitrocefin (482 = 15 000 M–1/cm) was from Oxoid (Basingstoke, UK); imipenem (300 = –9000 M–1/cm) and ertapenem (298 = –7500 M–1/cm) were from Merck Sharpe & Dohme Ltd (Pomezia, Italy); meropenem (297 = –6500 M–1/cm) was from AstraZeneca (Milan, Italy); piperacillin (235 = –822 M–1/cm) and tazobactam (233 = 3600 M–1/cm) were from Wyeth-Lederle (Catania, Italy); cefotaxime (260 = –7500 M–1/cm) was from Sanofi-Aventis (Milan, Italy); ceftazidime (260 = –9000 M–1/cm), clavulanate (265 = 2000 M–1/cm) and ampicillin (235 = –900 M–1/cm) were from GlaxoSmithKline (Verona, Italy); aztreonam (318 = –650 M–1/cm) and cefepime (260 = –10 000 M–1/cm) were from Bristol-Myers Squibb (Rome, Italy); ceftibuten (250 = –3700 M–1/cm) was from Schering-Plough (Milan, Italy); sulbactam (235 = 1780 M–1/cm) was from Pfizer (Rome, Italy) and carumonam (310 = –800 M–1/cm) was from Hoffman-La Roche (Basel, Switzerland).
The determination of MICs for E. coli HB101 pBC-SK(+)/CTX-M-43 and E. coli HB101 was performed by the conventional macrodilution broth procedure using a bacterial inoculum of 5 x 105 cfu/mL, as recommended by the CLSI.17 To evaluate the effect of inoculum on MICs, an in vitro susceptibility test was performed using a bacterial inoculum of 5 x 108 cfu/mL.
Production and purification of CTX-M-43 β-lactamase
An overnight culture of E. coli BL21 pET-26(b)(+)/CTX-M-43 grown in Luria–Bertani broth was diluted 10-fold with 6 L of the same medium containing kanamycin (50 mg/L). The crude extract was prepared as described previously.18 The first step of purification was performed in a Sepharose S fast-flow column, as described previously.19 Fractions containing β-lactamase activity were pooled, concentrated 20-fold using an Amicon concentrator (YM 10 membrane; Millipore, Bedford, MA, USA) and loaded onto a Superose 12 column (GE Healthcare, Milan, Italy) pre-equilibrated with 50 mM Tris–HCl buffer pH 7.0. Elution was performed with the same buffer at a flow rate of 1.0 mL/min. The total protein concentration was determined by the method of Bradford,20 with bovine serum albumin (BSA) as the standard. The β-lactamase activity was determined in 1 mL of 50 mM sodium phosphate buffer pH 7.0, at 30°C observing the hydrolysis rate of cefotaxime (100 µM). One unit of β-lactamase activity was defined as the amount of the enzyme that hydrolyses 1 mmol of the substrate/min.
Molecular weight and isoelectric point determination
The molecular weight was determined by SDS–PAGE,21 using a Mini-protean II electrophoresis system (Bio-Rad Laboratories, Richmond, CA, USA). Proteins were detected by staining the gel with Coomassie Brilliant Blue.20 Isoelectric focusing was carried out with a Multiphor II electrophoresis system (Pharmacia Biotech, Uppsala, Sweden) and a gel plate containing 5% Ampholine (pH range 3.5–9.5). Isoelectric point was determined by focusing 20 µg of purified enzyme. β-Lactamase activity was detected by zymogram technique using 250 µM nitrocefin.
Determination of kinetic parameters
Kinetic parameters of CTX-M-43 were determined by monitoring the variation in the absorbance of β-lactam antibiotics in 50 mM phosphate buffer (pH 7.0). All measurements were made with a microcomputer-linked Lambda 2 spectrophotometer (Applied Biosystems). The reactions were performed at 30°C. BSA at 20 mg/L was added to the dilute solution of β-lactamase to prevent denaturation. For the compounds behaving as substrates, the steady-state kinetic parameters, Km and kcat, were determined by Hanes–Woolf linearization or by non-linear least-square fit of the Michaelis–Menten equation.22–24 The reaction is described by the model:
|
|
For compounds rather stable to the action of the β-lactamase, the kcat values were determined from the initial rates calculated at saturating substrate concentrations, and the Km values were determined from experiments involving competition between the poor substrate and nitrocefin.
In compounds behaving as transient inactivators, a rather stable acyl-enzyme (ES*) was found to accumulate. The slow breakdown of ES*, due to a low value of k+3, regenerated the free enzyme. The values of the first-order rate constant Ki, characterizing the rate of ES* accumulation, were derived from the hydrolysis curves of nitrocefin as a reporter substrate, at 482 nm.25,26 Alternatively, the k+3 value was obtained by the direct hydrolysis of the poor substrate with large enzyme concentration. The hydrolysis rate was measured at the steady state. In that case, the kcat value was equal to k+3.25,26
In compounds behaving as inactivators, the k+3 value was obtained by monitoring the reactivation of the enzyme in the presence of the reporter substrate. The enzyme was first completed inactivated and then a small proportion of the reaction mixture was diluted to reduce the inactivator concentration to a negligible value.22,26 The values of k+2 and K were calculated by non-linear regression using the software LabPlot version 1.5.1. All measurements were made at least three times for each substrate, and reproducible results were obtained.
In order to provide qualitative insights into the structural features of the Michaelis complex of some of the investigated antibiotics, Molecular Mechanic calculations using GROMACS27 package with GROMOS9628 force fields were carried out. For this purpose, the crystal structure of the acyl-enzyme CTX-M-44 Glu-166Ala mutant (PDB code, 1IYO),9 in complex with cefotaxime, was used as a template. The aspartate residue at position 240 was mutated into a glycine residue using the MOLDEN program. A first potential energy minimization was carried out using the steepest descent algorithm. Then, a short (200 ps) molecular dynamics simulation was performed at 300 K in order to carry out a local search of the most stable structure. As this analysis is only mechanical, the most stable structure was defined as the one showing the lowest potential energy. The same procedure was repeated for the Michaelis complex with ceftazidime and ceftibuten. The results outlined in this article refer to the most stable structures.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
As shown in Table 1, the A. baumannii clinical isolate showed resistance to almost all β-lactam antibiotics tested. However, it showed susceptibility to imipenem and meropenem and the MICs of amoxicillin and cefotaxime decreased in the presence of 4 mg/L clavulanic acid. Tazobactam at 4 mg/L was able to restore susceptibility to piperacillin.
|
CTX-M-43 was purified from E. coli BL21 pET-26(b)(+)/CTX-M-43 by two chromatographic steps, which yielded a protein pure at 95% as evaluated by the SDS–PAGE analysis. The apparent molecular weight estimated for the CTX-M-43 enzyme was
28 kDa, and the isoelectric point was determined at 7.8. The steady-state kinetic parameters kcat and Km, determined for a set of substrates (Table 2), indicate that CTX-M-43 β-lactamase hydrolyses all cephalosporins tested, including ceftazidime. Cefazolin is the best substrate for CTX-M-43 among first-generation cephalosporins, with a catalytic efficiency of 4.2 µM–1/s. The kcat value for ceftazidime was not determined in our experimental condition, because of high Km value (1600 µM), even if a slight hydrolysis was detected. Ceftibuten, a non-oxyimino third-generation cephalosporin, and cefepime, a fourth-generation oxyimino cephalosporin, behaved as good substrates; for ceftibuten, it was possible to determine the kinetic parameters kcat and Km. Interestingly, monobactams such as aztreonam and carumonam behaved as good substrates, although aztreonam showed a better catalytic efficiency with respect to carumonam. With regard to moxalactam, an oxacephem, the Km value indicates a good affinity of the enzyme for this compound; however, the low catalytic efficiency observed is mainly due to the low kcat value, 6.0 x 10–3/s. Among all carbapenems tested (Table 3), imipenem behaved as a transient inactivator, leading to the accumulation of a rather stable acyl-enzyme complex, with a very low deacylation constant (2.1 x 10–4/s) and a low acylation efficiency (84 M–1/s). Meropenem and ertapenem were inactivators of CTX-M-43 with good acylation efficiencies (k+2/K), but with negligible deacylation constants (k+3). In this respect, ertapenem showed the best acylation efficiency (3.0 x 104 s–1/M). Classical inhibitors of class A enzymes (Table 3), sulbactam, tazobactam and clavulanate, acted as competitive inhibitors with Ki values of 0.6, 0.5 and 37 µM, respectively. No direct hydrolysis of the inhibitors was detected in our experimental conditions.
|
|
A general reduction in Km values was observed for some cephalosporins, comparing the kinetic parameters of CTX-M-43 with those of CTX-M-44, as shown in Table 2.
This observation is in accordance with a general increase in affinity towards cephalosporins induced by the Asp-240Gly substitution.29 In CTX-M-43, a 4-fold decrease in Km and kcat for cefotaxime preserves the kcat/Km value from significant alterations. For ceftazidime, we report a 7-fold reduction in the Km value with respect to CTX-M-44. Unfortunately, under our experimental conditions, the high value of Km made the accurate determination of the kcat value difficult. Interestingly, a 10-fold increase in the catalytic efficiency is observed in CTX-M-43 towards the fourth-generation cephalosporin, cefepime.
In order to confirm the observations achieved from kinetic experiments, MICs were determined for all the substrates at two different concentrations of the bacterial inoculum, 5 x 105 and 5 x 108 cfu/mL. E. coli HB101 pBC-SK(+)/CTX-M-43 showed resistance to all cephalosporins tested. The MICs relative to monobactams seem to reflect the different activity of the enzyme towards those antibiotics. In fact, a lower catalytic efficiency against carumonam might have an influence on the MIC. For carbapenems, it was possible to observe that the increase in the bacterial inoculum had no effect on MICs of meropenem and ertapenem. These data seem to confirm that those compounds, acting as inactivators with a negligible k+3 value, cannot be hydrolysed from CTX-M-43. Interestingly, imipenem behaved in a different way. In fact, increasing the bacterial inoculum, we observed an increase in the MIC value, from 0.25 to 1 mg/L. These data seem to be in agreement with the kinetic analysis performed, in which imipenem acted as a transient inactivator with a significant k+3 value.
Structural information obtained from the crystal structure of Toho-1 acyl-intermediate in complex with cefotaxime showed that aspartate-240 residue might establish hydrogen bonds with the amino group of the aminothiazolic substituent of the acyl-cefotaxime intermediate.8,9,11,29 Thus, the aspartate-240 might be involved in the oxyimino-cephalosporinase activity by fixing cefotaxime in the binding site.
In order to investigate the effect of glycine residue at position 240, molecular modelling of the Michaelis complex of CTX-M-43 in combination with ceftazidime, cefotaxime and ceftibuten was performed by molecular mechanics calculations. The hypothesis that substitution of the charged and bulky aspartate with the neutral and small glycine residue might favour the accommodation of the C7β substituents of oxyimino cephalosporins in the catalytic site seems to be confirmed. Specifically, the molecular model of CTX-M-43 shows that glycine residue at position 240 allows a better accommodation of the bulky C7β side chains of ceftazidime, cefotaxime and ceftibuten by expansion of the binding site (Figure 1). The absence of the electrostatic interaction between aspartate-240 and the amino group of the aminothiazolic substituent and the expansion of the binding site result in the increased affinity of the enzyme versus all cephalosporins tested, as supported from kinetics data.
|
Substitution Asp-240
Gly has never been observed in naturally occurring TEM and SHV. However, in a recent work,18 we described the kinetic characterization of TEM-149, a naturally occurring Glu-240Val mutant. In this enzyme, the catalytic efficiency versus ceftazidime increases as a result of the substitution of a charged and bulky residue with a hydrophobic residue. Nowadays, it is quite clear that CTX-M-type enzymes represent a versatile and adaptable class of enzymes. The data collected in the present work from in vivo activity and kinetic analysis show how CTX-M-type enzymes appear to be evolving in response to the extensive use of cephalosporins in clinical therapy. At the same time, the detectable deacylation constant for imipenem, in addition to the high acylation efficiency for meropenem and ertapenem, and the increase in MICs in response to bacterial inoculum make plausible the potentiality, from an evolutionary standpoint, of those enzymes to converge in the direction of a possible carbapenemase activity.
Although all known class A carbapenemases are characterized for the presence of a disulphide bridge between the cysteine residues at positions 69 and 238, in CTX-M-43, the position 238 is occupied by a glycine. In fact, it is known that high flexibility of the β-strand B3, which contains the KTG-conserved motif, is one of the features contributing to the cephalosporinase activity of CTX-M enzymes. Ibuka et al.30 showed that a disulphide bridge at this position decreases drastically the flexibility of the β-strand B3, significantly reducing the activity against third-generation cephalosporins, whereas the catalytic efficiency against penicillins and first-generation cephalosporins was not affected. However, the carbapenemase activity of certain class A enzymes is the consequence of several strategic residues in combination with the presence of a disulphide bridge.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
This work was supported by grants to G. A. from PRIN 2004 and to G. C. from MURST ex 60% (Ministero dell'Istruzione, dell'Università e della Ricerca).
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
None to declare.
|
Supplementary data |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
A colour version of Figure 1 is available at JAC Online (http://jac.oxfordjournals.org/).
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
1 Bradford PA. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev (2001) 14:933–51.
2 Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for β-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother (1995) 39:1211–33.[Web of Science][Medline]
3
Paterson DL, Bonomo RA. Extended-spectrum β-lactamases: a clinical update. Clin Microbiol Rev (2005) 18:657–86.
4 Stürenburg E, Mack D. Extended-spectrum β-lactamases: implications for the clinical microbiology laboratory, therapy and infection control. J Infect (2003) 47:273–95.[CrossRef][Web of Science][Medline]
5
Bonnet R. Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother (2004) 48:1–14.
6
Livermore DM, Canton R, Gniadkowski M, et al. CTX-M: changing the face of ESBLs in Europe. J Antimicrob Chemother (2007) 59:165–74.
7 Ishii Y, Ohno A, Taguchi H, et al. Cloning and sequence of the gene encoding a cefotaxime-hydrolyzing class A β-lactamase isolated from Escherichia coli. Antimicrob Agents Chemother (1995) 39:2269–75.[Abstract]
8 Ibuka AS, Ishii Y, Galleni M, et al. Crystal structure of extended-spectrum β-lactamase Toho-1: insights into the molecular mechanism for catalytic reaction and substrate specificity expansion. Biochemistry (2003) 42:10634–43.[CrossRef][Web of Science][Medline]
9
Shimamura T, Ibuka A, Fushinobu S, et al. Acyl-intermediate structures of the extended-spectrum class A β-lactamase, Toho-1, in complex with cefotaxime, cephalothin, and benzylpenicillin. J Biol Chem (2002) 277:46601–8.
10
Bae IK, Lee BH, Hwang HY, et al. A novel ceftazidime-hydrolysing extended-spectrum β-lactamase, CTX-M-54, with a single amino acid substitution at position 167 in the omega loop. J Antimicrob Chemother (2006) 58:315–9.
11 Chen Y, Delmas J, Sirot J, et al. Atomic resolution structures of CTX-M β-lactamases: extended spectrum activities from increased mobility and decreased stability. J Mol Biol (2005) 348:349–62.[CrossRef][Web of Science][Medline]
12
Poirel L, Gniadkowski M, Nordmann P, et al. Biochemical analysis of the ceftazidime-hydrolysing extended-spectrum β-lactamase CTX-M-15 and of its structurally related β-lactamase CTX-M-3. J Antimicrob Chemother (2002) 50:1031–4.
13
Bonnet R, Dutour C, Sampaio JLM, et al. Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp-240Gly. Antimicrob Agents Chemother (2001) 45:2269–75.
14
Bonnet R, Recule C, Baraduc R, et al. Effect of D240G substitution in a novel ESBL CTX-M-27. J Antimicrob Chemother (2003) 52:29–35.
15
Cartelle M, del Mar Tomas M, Molina F, et al. High-level resistance to ceftazidime conferred by a novel enzyme, CTX-M-32, derived from CTX-M-1 through a single Asp240-Gly substitution. Antimicrob Agents Chemother (2004) 48:2308–13.
16
Celenza G, Pellegrini C, Caccamo M, et al. Spread of blaCTX-M-type and blaPER-2 β-lactamase genes in clinical isolates from Bolivian hospitals. J Antimicrob Chemother (2006) 57:975–8.
17 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement M100-S15 (2005) Wayne, PA, USA: CLSI.
18
Perilli M, Celenza G, De Santis F, et al. E240V substitution increases catalytic efficiency toward ceftazidime in a new natural TEM-type extended-spectrum β-lactamase, TEM-149, from Enterobacter aerogenes and Serratia marcescens clinical isolates. Antimicrob Agents Chemother (2008) 52:915–9.
19 Perilli M, Ettorre D, Segatore B, et al. Overexpression system and biochemical profile of CTX-M-3 extended-spectrum β-lactamase expressed in Escherichia coli. FEMS Microbiol Lett (2004) 241:229–32.[CrossRef][Web of Science][Medline]
20 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem (1976) 72:248–54.[CrossRef][Web of Science][Medline]
21 Laemmli UK. Cleavage of structural proteins during the assembly of bacteriophage T4. Nature (1970) 227:680–5.[CrossRef][Web of Science][Medline]
22 Copeland RA. Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis (2000) 2nd edn. New York: John Wiley & Sons.
23 Cornish-Bowden A. Fundamentals of Enzyme Kinetics (1995) 1st edn. London: Portland Press Ltd.
24 Segel IH. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems (1975) 1st edn. New York: John Wiley & Sons.
25 De Meester F, Joris B, Reckinger G, et al. Automated analysis of enzyme inactivation phenomena. Application to β-lactamases and DD-peptidases. Biochem Pharmacol (1987) 36:2393–403.[CrossRef][Web of Science][Medline]
26
Galleni M, Franceschini N, Quinting B, et al. Use of the chromosomal class A β-lactamase of Mycobacterium fortuitum D316 to study potentially poor substrates and inhibitory β-lactam compounds. Antimicrob Agents Chemother (1994) 38:1608–14.
27 Van der Spoel D, van Drunen R, Berendsen HJC. GROMACS. (1994) Groningen: Department of Biophysical Chemistry, BIOSON Research Institute.
28 Van Gusteren WF, Billeter SR, Eising AA, et al. Biomolecular Simulation: The GROMOS96 Manual and User Guide (1996) Zurich: Hochschulverlag AG an der ETH.
29 Delmas J, Chen Y, Prati F, et al. Structure and dynamics of CTX-M enzymes reveal insights into substrate accommodation by extended-spectrum β-lactamases. J Mol Biol (2008) 375:192–201.[Web of Science][Medline]
30 Ibuka AS, Matsuzawa H, Sakai H. An engineered disulfide bond between residues 69 and 238 in extended-spectrum β-lactamase Toho-1 reduces its activity toward third-generation cephalosporins. Biochemistry (2004) 43:15737–45.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Doublet, S. A. Granier, F. Robin, R. Bonnet, L. Fabre, A. Brisabois, A. Cloeckaert, and F.-X. Weill Novel Plasmid-Encoded Ceftazidime-Hydrolyzing CTX-M-53 Extended-Spectrum {beta}-Lactamase from Salmonella enterica Serotypes Westhampton and Senftenberg Antimicrob. Agents Chemother., May 1, 2009; 53(5): 1944 - 1951. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||