Identification of a novel cephalosporinase (DHA-3) in Klebsiella pneumoniae isolated in Taiwan.

A strain of Klebsiella pneumoniae resistant to cefoxitin and oxyimino-cephalosporins, but susceptible to cefepime, was isolated from an adult patient hospitalised in Taichung, Taiwan. Isoelectric focusing revealed three beta-lactamases with isoelectric points of 5.4, 8.2 and 7.9, respectively. Following PCR with plasmid DNA templates and gene sequencing, these enzymes were shown to correspond to TEM-1, SHV-5 and a novel DHA-1-like enzyme (designated DHA-3). The bla genes for TEM-1 and SHV-5 were transferable, but the bla(DHA-3) gene was non-self-transferable in conjugation experiments. All three bla genes were successfully introduced by electrotransformation into an Escherichia coli recipient (DH5alpha), resulting in a similar resistance profile to that observed in the original donor strain. Other K. pneumoniae strains producing DHA-1-like enzymes have been identified previously in Taiwan, and this report suggests that DHA-type beta-lactamases are continuing to emerge in this country.

[Purification of beta-lactamases by affinity chromatography]. / Purification des beta-lactamases par chromatographie d'affinité

Affinity columns able to purifie beta-lactamases have been prepared either by linking covalently reversible inhibitors or substrates to agarose beds. The enzyme is eluted with a gradient of sodium chloride or released with the substrate. This method is a pertinent one for the purification of these enzymes and for the study of bacteria harbouring more than one beta-lactamase.

[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.

Purification and properties of cephalosporinase from Pseudomonas aeruginosa.

Cephalosporin beat-lactamase (cephalosporinase, CSase) was purified from a strain of Pseudomonas aeruginosa resistant to beta-lactam antibiotics. The purified enzyme preparation gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was about 34,000. The specific activity was 49.7 mumoles/minute/mg of protein of the purified enzyme for the hydrolysis of cephaloridine. The optimal pH and optimal temperature were about 8.0 and 40 degrees C, respectively. Its isoelectric point was 8.7. The enzyme activity was inhibited by iodine, some divalent ions, and some semisynthetic beta-lactam antibiotics, including cephamycin derivatives such as moxalactam and YM09330. Mouse antiserum obtained against the purified enzyme showed no cross-reaction with other types of beta-lactamase in neutralization test.

Diastereomeric 7-ureidoacetyl cephalosporins. III. Contribution of D- and L-isomers to the growth inhibiting activities of 7alpha-H and 7alpha-OCH3 derivatives for gram-positive and gram-negative bacteria.

A series of 7beta-ureidoacetyl, 7alpha-H and 7alpha-OCH3 cephalosporin antibiotics have shown broad-spectrum antibacterial activity in vitro. In the 7alpha-H but not in the 7alpha-OCH3 series, contrary to experience in the antibiotic field, the L-isomers were substantially more active than the D-isomers both in vitro and in vivo particularly, but not exclusively, against Enterobacteriaceae that produce potent chromosomal cephalosporinases. Enhanced resistance to and inhibition of beta-lactamase (s) appeared to be responsible for this effect. Studies in vitro specifically with 7beta-thienylureidoacetyl derivatives showed that D-isomers interacted with L-isomers in the 7alpha-OCH3 series in a synergistic manner against "cephalosporinase-type" enzyme producers while isomers in the 7alpha-H series did not. Examples were presented in which this favorable event resulted in improved efficacy of the racemic mixture over the pure D- or L-isomer alone in appropriate experimental infections.

beta-Lactamase and beta-lactam antibiotics resistance in acinetobacter anitratum (syn: A. calcoaceticus).

The cephalosporin beta-lactamase (cephalosporinase) produced by Acinetobacter was studied. The enzyme was partially purified by means of column chromatography and its properties were investigated. The enzyme was induced by benzylpenicillin, 6-aminopenicillanic acid and cephaloridine. Its molecular weight is 30,000 its optimal temperature 40C, and its optimal pH 7.25 similar to 7.50. Substrate specificity studies using various cephalosporins and penicillins, showed that the enzyme functioned as a cephalosporinase rather than penicillinase.

Plasmid-encoded ACC-4, an extended-spectrum cephalosporinase variant from Escherichia coli.

ACC-4, an omega loop mutant (Val(211)-->Gly) of the Hafnia alvei-derived cephalosporinase ACC-1, was encoded by an Escherichia coli plasmid. The genetic environment of bla(ACC-4) shared similarities with plasmidic regions carrying bla(ACC-1). Kinetics of beta-lactam hydrolysis and levels of resistance to beta-lactams showed that ACC-4 was more effective than ACC-1 against expanded-spectrum cephalosporins.

Use of chromatofocusing for separation of beta-lactamases. IX. Analytical chromatofocusing for the separation of a chromosomal cephalosporinase from Proteus vulgaris 1028.

Simultaneous purification and isoelectric point (pI) determination was carried out at analytical scale of the chromosomal cephalosporinase from the Proteus vulgaris 1028 strain. Comparison of the enzyme to the purification results with m-aminophenylboronic acid-agarose affinity chromatography with sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that minute amounts of accompanying proteins having identical pI values but different molecular masses were found in the chromatofocused preparation. The molecular mass of the enzyme was 24,000 dalton. The pI was found to be 8.3.

Purification and some properties of a cephalosporinase from Proteus vulgaris.

The purified cephalosporinase from Proteus vulgaris hydrolyzed a variety of cephalosporins, including cefuroxime, at a high level; its activity was inhibited by clavulanic acid.

[Isolation and properties of beta-lactamase of the cephalosporinase type from cells of Enterobacter aerogenes 6803]. / Vydelenie i svoistva beta-laktamazy tsefalosporinaznogo tipa iz kletok Enterobacter aerogenes 6803.

beta-Lastamase with the molecular weight of 32500 was isolated from the cells of clinical strain 6803 of Enterobacter aerogenes and purified. By the substrate profile determined microiodometrically beta-lactamase was classified as belonging to the cephalosporinase type. The activity of the electrophoretically homogenous enzyme was equal to 430 microM a minute per mg protein with respect to benzylpenicillin. The Km for benzylpenicillin, dicloxacillin, cephaloridin and cephalothin was 6.5410(-5), 3 X 10(-4), 2.1 X 10(-5) and 5.7 X 10(-5) M, respectively. The isoelectric point of the enzyme equal to 5.45 was estimated with the method of preparative isoelectrofocusing. The presence of the serine residue or residues was shown with the use of selective reagents applied to the functionally important groups. With the method of circular dichroism the ratio of alpha- and beta-structures in the enzyme molecule was determined, the slow hydrolysis of cephazolin was demonstrated and the values of Km and Kcat for this process were estimated.