[Nature of the inhibition of some cephalosporinases by carbenicillin]. / Nature de l'inhibition de céphalosporinases par la carbénicilline

This paper deals with the kinetic inhibition of six cephalosporinases (cephalosporin amido-beta-lactamhydrolase, EC 3.5.2.8) by carbenicillin. In previous cases, the inhibition has appeared usually to be competitive and slowly reversible. This makes it possible to measure the two terms of the ratio Ki=k5/k4;k4 and k5 being respectively the velocity constants of formation and destruction of the enzyme-inhibitor complex. A program was prepared which made it possible to obtain these constants from only one experiment. With ampicillin and cloxacillin, we verified that the reaction is faster, and that only Ki can be measured. These facts suggest that special precautions should be taken in order to obtain signigicant values for the constants governing inhibition.

[Kinetics of beta-lactamase inhibition by clavulanic acid]. / Cinetique de l'inhibition de beta-lactamases par l'acide clavulanique.

The mechanisms of action of 3 R-factors on beta-lactamases (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) (TEM-1 pI = 5.4, TEM-2 pI = 5.6 and Pitton's type 2 pI = 7.7) have been kinetically analyzed for clavulanic acid inactivation. Clavulanic acid appears as a competitive and irreversible inhibitor (Kcat inhibitor) reacting in two steps: a, formation of a reversible enzyme . inhibitor complex (characterized by a Ki); b, evolution of the reversible complex into a new derivative (covalent, stable and inactive) by monomolecular kinetics characterized by a k6 (or Kcat) related to half-life. The kinetic constants are: TEM-1: Ki = 0.8 micrometer, k6 = 0.027 s-1; TEM-2: Ki = 0.7 micrometer, k6 = 0.03 s-1; type 2: Ki = 0.6 micrometer, k6 = 0.046 s-1. These results justify the 'progressive irreversible' character of the inhibition generally described.

Inhibition kinetics of three R-factor-mediated beta-lactamases by a new beta-lactam sulfone (CP 45899).

A new beta-lactam sulfone, CP 45899, has been proved to be a time-dependent irreversible inhibitor of three R-factor-mediated beta-lactamases (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6): TEM-1 (pI = 5.4), TEM-2 (pI = 5.6) and Pitton's type 2 (pI = 7.7). This inhibition occurs in two principal steps: (1) formation of a reversible enzyme-inhibitor complex (characterized by a Ki); (2) evolution of this complex into one, or more, inactive protein(s) (kinact). With the three beta-lactamases CP 45899 shows, respectively, Ki of 0.9, 0.8 and 1.8 microM and kinact of 1.2 . 10(-3), 0.8 . 10(-3) and 1 . 10(-3) s-1; the turnover numbers are: 525, 2280 and 1220. These results are compared to those previously obtained with clavulanic acid.

Kinetic studies of a beta-lactamase by a computerized microacidimetric method.

On-line computerized treatment of enzyme kinetic data allows the precise measurement of Michaelis--Menten constants (Km and V) from a single progress curve. This method has been used to determine the kinetic constants of a beta-lactamase extracted from an Escherichia coli strain. In the profile of enzymatic activity there obtained, Km and V are a function of the pH. From these results some information is derived about the mechanism of the enzyme--substrate binding.

Chromosomally encoded cephalosporin-hydrolyzing beta-lactamase of Proteus vulgaris RO104 belongs to Ambler's class A.

Proteus vulgaris RO104 strain produces a chromosomally encoded beta-lactamase that confers resistance to various beta-lactam antibiotics including methoxyimino third-generation cephalosporins. The beta-lactamase hydrolyzes first- and second-generation cephalosporins efficiently and cefotaxime to a lesser extent. Catalytic activity is inhibited by low concentrations of clavulanic acid and sulbactam. By its broad-spectrum substrate profile, beta-lactamase of Proteus vulgaris RO104 belongs to the group 2e defined by Bush. The protein purified to homogeneity by a four-step procedure was characterized by a pI of 8.31 and a specific activity of 1200 U/mg. The beta-lactamase was digested by trypsin, endoproteinase Asp-N and chymotrypsin. Amino-acid sequence determinations of the resulting peptides allowed the alignment of the 271 amino-acid residues of the protein which did not contain any cysteine residue. From amino-acid sequence comparisons, Proteus vulgaris RO104 beta-lactamase was found to share about 68% identity with the chromosomally mediated beta-lactamases of Klebsiella oxytoca D488 and E23004. Therefore, the cephalosporin-hydrolyzing beta-lactamase of Proteus vulgaris RO104 belongs to Ambler's class A.

Close amino acid sequence relationship between the new plasmid-mediated extended-spectrum beta-lactamase MEN-1 and chromosomally encoded enzymes of Klebsiella oxytoca.

Isolated from an Escherichia coli strain MEN-1 is a plasmid-mediated beta-lactamase that confers resistance to methoxy imino third-generation cephalosporins. The protein purified to homogeneity was digested by trypsin, chymotrypsin and endoproteinase Asp-N. Amino acid sequence determinations of the resulting peptides gave rise to the alignment of the 263 residues of the beta-lactamase. From amino acid sequence comparison MEN-1 was found to share more than 72% identity with the chromosomally mediated beta-lactamases of Klebsiella oxytoca. Therefore, MEN-1 is the first transferable extended-spectrum beta-lactamase which is not directly derived from the widespread TEMs or SHV-1 penicillinases with which it presents less than 39% identity.

Characterization and amino acid sequence analysis of a new oxyimino cephalosporin-hydrolyzing class A beta-lactamase from Serratia fonticola CUV.

Serratia fonticola CUV produces two isoenzymes (forms I and II) with beta-lactamase activity which were purified by a five-step procedure. The isoenzymes had identical kinetic parameters and isoelectric point (pI = 8.12). They were characterized by a specific activity towards benzylpenicillin of 1650 U/mg. The beta-lactamase hydrolyzed benzylpenicillin, amoxycillin, ureidopenicillins, first- and second-generation cephalosporins. Carboxypenicillins and isoxazolylpenicillins were hydrolyzed to a lesser extent. Towards cefotaxime and ceftriaxone (third-generation cephalosporins), the S. fonticola enzyme exhibited catalytic efficiencies much higher than those of MEN-1 and extended-spectrum TEM derivative beta-lactamases. The beta-lactamase from S. fonticola was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam. The purified isoenzymes were digested by trypsin, endoproteinase Asp-N and chymotrypsin. Amino acid sequence determinations of the resulting peptides allowed the alignment of 267 amino acid residues (Swiss-Prot, accession number P 80545) for form I beta-lactamase. Form II is five residues shorter than form I at its N-terminus. From amino acid sequence comparisons, S. fonticola CUV beta-lactamase was found to share more than 69.3% identity with the chromosomally encoded beta-lactamases of Klebsiella oxytoca, Proteus vulgaris, Citrobacter diversus and the plasmid-mediated enzymes MEN-1 and Toho-1. Therefore, the oxyimino cephalosporin-hydrolyzing beta-lactamase of S. fonticola belongs to Ambler's class A. Contribution of the serine at ABL 237 in the broad-spectrum activity of these beta-lactamases is discussed.

Clinical inhibitor-resistant mutants of the beta-lactamase TEM-1 at amino-acid position 69. Kinetic analysis and molecular modelling.

The kinetic parameters of three IRT (Inhibitor-Resistant-TEM-derived-) beta-lactamases (IRT-5, IRT-6 and IRT-I69) were determined for substrates and the beta-lactamase inhibitors: clavulanic acid, sulbactam and tazobactam, and compared with those of TEM-1 beta-lactamase. The catalytic behaviour of the beta-lactamases towards substrates and inhibitors was correlated with the properties of the amino acid at position ABL69. The three IRT beta-lactamases contain at that position a residue Ile, Leu and Val, amino acids whose side-chain are branched. Molecular modelling shows that the methyl groups of Ile-69 (C gamma 2) and Val-69 (C gamma 1) produced steric constraints with the side chain of Asn-170 as well as the main chain nitrogen of Ser-70, a residue contributing to the oxyanion hole. We suggest that hydrophobicity could be the main factor responsible for the kinetic properties of Met69Leu (IRT-5), as no steric effects could be detected by molecular modelling. Hydrophobicity and steric constraints are combined in Met69Ile and Met69Val, IRT-I69 and IRT-6, respectively.

Syntheses, in vitro antibacterial and antifungal activities of a series of N-alkyl, 1,4-dithiines.

A series of dithiines were synthesized by cyclization of 4-(alkylamino)-4-oxobutanoic acids under the action of SOCl2. Their in vitro antibacterial and antifungal activities have been evaluated against reference strains and versus reference compounds. The so-called 'isoimides' 2a, 2b were totally inactive whereas some imides had low MICs for few bacteria and for few fungal microorganisms.

Single amino acid substitution between SHV-1 beta-lactamase and cefotaxime-hydrolyzing SHV-2 enzyme.

SHV-2 beta-lactamase was purified from an overproducing variant of a clinical isolate of Escherichia coli resistant to cefotaxime. Pure protein was digested by trypsin and Lys-C endoproteinase. Proteolytic peptides, isolated by reverse-phase HPLC, were submitted to manual Edman degradation and aligned by homology with the sequence of SHV-1 beta-lactamase. A putative amino acid sequence was deduced. Structural comparison revealed that SHV-2 differed from SHV-1 by only one amino acid, Gly----Ser, at position 213 of the mature protein.

ATR-FTIR spectroscopic investigation of E. coli transconjugants beta-lactams-resistance phenotype.

Hyphenation of attenuated total reflection Fourier transform infrared spectroscopy and cluster analysis has been used to characterise a susceptible Escherichia coli K12 strain and the transconjugants TEM-1, TEM-2, TEM-3, SHV-2, SHV-3, SHV-4. A good discrimination of the susceptible strain from the transconjugants was obtained. Although a limited success was achieved in the differentiation of SHV and TEM phenotypes in general, results obtained with TEM-2 and SHV-3 were convincing. Spectral differences observed are ascribed to the global effects of the conjugation process, particularly their repercussions in the nucleic acids and carbohydrate absorbing regions, rather than to beta-lactamase point-mutations.

[Kinetics of a new cephalosporinase from Pseudomonas aeruginosa]. / Etude cinétique d'une nouvelle céphalosporinase de Pseudomonas aeruginosa

Pseudomonas aeruginosa strain RL 39 produces an inducible cephalosporinase possessing an isoelectric point of 8,66. The kinetic constants have been measured by computerized microacidimetry. The results allow to differenciate this enzyme from the one produced by Ps. aeruginosa GN 918 (Yaginuma et al. (1973) Jap. J. Microbiol., 17, 141-149) showing a similar isoelectric point.

[Inducible cephalosporinases from Proteus morganii]. / Céphalosporinases inductibles de Proteus morganii

It has been shown that two Proteus morganii strains produce an inducible cephalosporinase. No significative difference was shown between them: they present the same isoelectric poit: 8,3 and very similar kinetic constants. The parameter tau, proportional to the half life of antibiotic at low concentration, in presence of beta lactamase, shows that cefamandole is the most stable cephalosporin studied.

Probing the active site of beta-lactamase R-TEM1 by informational suppression.

Using a new extended set of 13 amber suppressors in E coli, systematic amino-acid replacements were performed at positions 104(E) and 238(G) of TEM-1 beta-lactamase from PUC19. The enzyme is tolerant to most substitutions tested at position 104. Missense revertants E104K, E104S or E104Y exhibited only minor changes in enzyme activity with respect to wild-type TEM-1. Several substitutions at position 238 resulted in a new cefotaxime hydrolysing capacity, but to an extent that did not confer cefotaxime resistance for the bacteria producing the mutated enzymes. Only when the mutations at codons 104 and 238 were combined on the same gene, did a true cefotaxime resistant phenotype appear, mimicking the situation encountered with 3rd generation cephalosporins resistant clinical isolates.

[Identification of the beta lactamase R-TEM of Pseudomonas aeruginosa]. / Identification de la beta lactamase R-TEM chez Pseudomonas aeruginosa

This paper is dealing with the enzymatic problem raised by two strains of Ps. aeruginosa resistant to classical beta lactam antibiotics including carbenicillin. These two strains hydrolyse all these antibiotics. In both cases, we have shown the simultaneous biosynthesis of two enzymes: an inducible and chromosome cephalosporinase frequently found in this germ, and a constitutive beta lactamase, with a penicillinase activity which has been identified with the extrachromosomic beta lactamase R-TEM. These two enzymes have been separated by affinity chromatography, characterized by their kinetic constants given by computerized microacidimetry, and their isoelectric points which are respectively 9.2 for the cephalosporinase and 5.40 for the penicillinase R-TEM. Isoelectric focussing also shows the separation of these two enzymes.

Affinity of cefmenoxime for beta-lactamases: an analysis.

The interactions of cefmenoxime with beta-lactamases in comparison with cefotaxime, moxalactam, cefoperazone, and ceftazidime have been determined. On-line computerized microacidimetry allowed determination of the affinity of these compounds with the enzymes, which was characterized by Km values. The beta-lactamases that were used were two cephalosporinases and one penicillinase. Within these data, the cephalosporins could be classified into three groups: (1) those with high affinity for the cephalosporinases and very poor affinity for the penicillinase (cefmenoxime, cefotaxime, and moxalactam); (2) those with moderate affinity for the cephalosporinases and very poor affinity for the penicillinase (ceftazidime); (3) those with poor affinity for all enzymes (cefoperazone). In the case of the penicillinase (TEM-1), only cefoperazone was subject to some hydrolysis.

N-aryl 3-halogenated azetidin-2-ones and benzocarbacephems, inhibitors of beta-lactamases.

N-(3-Carboxy-6-methylphenyl)-3-fluoroazetidin-2-one and a series of related N-aryl-3-halo- and -3,3-dihaloazetidinones 3, in which the halo substituent is a fluorine or a bromine atom, were prepared by using the Wasserman procedure of cyclization of beta-bromopropionamides as a key step. Their affinities for the TEM-1 beta-lactamase were determined and compared with those of a series of tricyclic azetidinones, the benzocarbacephems 2, and known beta-lactamase inhibitors. The beta-lactams 2 and 3 behave as competitive inhibitors and not as substrates of the enzyme; neither halogen substitution (series 3) nor ring strain (series 2) induces enzymatic hydrolysis.

Classification of beta-lactamases from Branhamella catarrhalis in relation to penicillinases produced by other bacterial species.

Branhamella catarrhalis strains resistant to commonly used penicillins, and presently isolated, produce a beta-lactamase. Most of these enzymes are chromosomally mediated, but a plasmid-mediated beta-lactamase has been described (enzyme BRO-1). With reference to isoelectric points, 7 different enzymes have been identified: 6 chromosomally mediated and 1 plasmid-mediated. Nevertheless, they have many common properties, such as being biosynthesised constitutively but with a low level of production. They have a penicillinase-type profile, and are strongly inhibited by clavulanic acid.

Relationships between beta-lactamase production and beta-lactam susceptibility of environmental strains of Yersinia kristensenii.

Strains of Yersinia kristensenii display high susceptibility to carbenicillin (MIC90 less than 8 micrograms/ml) in comparison with the majority of environmental strains of Yersinia closely related to Y. enterocolitica which are resistant to this antibiotic (MIC90 greater than 256 micrograms/ml). beta-lactamases of 39 strains of Y. kristensenii isolated from foods were analysed by isoelectric focusing and gel electrophoresis of ultrasonically disrupted uninduced cultures. beta-lactamase patterns showed the presence of only one out of three classes of enzymes of pI 6.7, 7.6 and 8.2, respectively, by strain. One beta-lactamase showed electrophoretic mobility different (EM + 2.0 cm/h) from that of all the other enzymes (EM + 1.6 cm/h) belonging to the class of pI 7.6. Induction by cefoxitin revealed the existence of inducible beta-lactamases in two out of eight selected strains. The substrate profile of these enzymes, which are probably chromosomally mediated, showed a predominant cephalosporinase activity. None of the type A and B beta-lactamases described by Cornelis and Abraham in Y. enterocolitica were found in any of the strains examined. The lack of beta-lactamase A (a penicillinase) accounts for the carbenicillin susceptibility of Y. kristensenii strains.

Sulbactam: secondary mechanisms of action.

Light and scanning electron microscopy showed that 0.25, 0.5 and 1 times the minimum inhibitory concentrations (MICs) of sulbactam (SULB) caused filament formation in different species of Enterobacteriaceae, while 2 and 4 times the MICs caused spheroplast formation and subsequent lysis. By using a competitive assay with 125I-penicillin X, SULB showed a primary affinity for the PBP 1a and PBP3 of Escherichia coli, as well as for the PBP1a of Proteus mirabilis. The bactericidal interaction of human polymorphonuclear leukocytes (PMN) and SULB against E. coli K-1 resistant to the bactericidal activity of human serum was studied in vitro; however, SULB concentrations showed variations in the medium according to human kinetic data. Under these conditions, bacterial growth occurred in Hanks balanced salt solution containing SULB, PMN, or SULB-PMN in combination. In addition, bactericidal activity was observed in serum, with a killing rate of 90% for PMN or SULB, and 95% for SULB-PMN in combination. The postantibiotic enhancement of PMN bactericidal function was assessed against E. coli K1 pretreated with 0.5 the MIC of SULB (32 micrograms/ml) for 0.5 hr. The 90% bacterial killing rate of PMN occurred by 1.5 hr for pretreated bacteria and by 2.5 hr for untreated bacteria. Furthermore, the luminol-enhanced chemiluminescence (CL) assay using an E. coli stimulus showed that SULB does not modify PMN activity.