Roger Labia

Extended-spectrum beta-lactamases.

The elaboration of r-lactamase-mediated resistance in bacteria has involved increased prevalence in certain organisms (notably Staphylococclus aiureius in the 1940s) and spread to new hosts (such as Haemophilus influienzae and Neisseria gonorrhoeae in the 1970s and Enterococcusfaecalis in the 1980s) but not fundamental changes in the substrate spectra of the enzymes. Hence, penicillins, cephalosporins, and other ,B-lactam antibiotics which were such poor substrates for hydrolysis as to be virtually resistant to usual amounts of ,B-lactamase could be developed. Some pathogens could develop resistance by mutations allowing so much enzyme to be made that even poor substrates were inactivated or bound, but such resistance was limited to organisms with inducible chromosomal 1-lactamase and was not transmissible (27). Consequently, it came as an unwelcome surprise when Klebsiella isolates with plasmid-mediated resistance to broad-spectrum cephalosporins were found in the Federal isolates (Table 1). The genesis of these plasmid-determined extended-spectrum 1-lactamases and the contribution that they have made to our understanding of 1-lactamase structure-activity relationships are the subjects of this review.

β-Lactamases of Kluyvera ascorbata, Probable Progenitors of Some Plasmid-Encoded CTX-M Types

ABSTRACT Kluyvera ascorbata produces a β-lactamase that results in an atypical susceptibility pattern, including low-level resistance to penicillins, cephalothin, and cefuroxime, but this resistance is reversed by clavulanate. Ten nucleotide sequences of the corresponding gene, blaKLUA, were obtained and were found to have minor variations (96 to 100%). Otherwise, blaKLUA was found to be similar (95 to 100%) to some plasmid-encoded CTX-M-type β-lactamases. Finally, mobilization of blaKLUA on a plasmid was found to be mediated probably by a genetic mobile element like ISEcp1.

Biochemical properties of a carbapenem-hydrolyzing beta-lactamase from Enterobacter cloacae and cloning of the gene into Escherichia coli.

A clinical isolate of Enterobacter cloacae, strain NOR-1, exhibited resistance to imipenem and remained susceptible to extended-spectrum cephalosporins. Clavulanic acid partially restored the susceptibility of the strain to imipenem. Two beta-lactamases with isoelectric points (pI) of 6.9 and > 9.2 were detected in strain E. cloacae NOR-1; the higher pI corresponded to AmpC cephalosporinase. Plasmid DNA was not detected in E. cloacae NOR-1 and imipenem resistance could not be transferred into Escherichia coli JM109. The carbapenem-hydrolyzing beta-lactamase gene was cloned into plasmid pACYC184. One recombinant plasmid, pPTN1, harbored a 5.3-kb Sau3A fragment from E. cloacae NOR-1 expressing the carbapenem-hydrolyzing beta-lactamase. This enzyme (pI 6.9) hydrolyzed ampicillin, cephalothin, and imipenem more rapidly than it did meropenem and aztreonam, but it hydrolyzed extended-spectrum cephalosporins only weakly and did not hydrolyze cefoxitin. Hydrolytic activity was partially inhibited by clavulanic acid, sulbactam, and tazobactam, was nonsusceptible to chelating agents such as EDTA and 1,10-o-phenanthroline, and was independent of the presence of ZnCl2. Its relative molecular mass was 30,000 Da. Induction experiments concluded that the carbapenem-hydrolyzing beta-lactamase biosynthesis was inducible by cefoxitin and imipenem. Subcloning experiments with HindIII partial digests of pPTN1 resulted in a recombinant plasmid, designated pPTN2, which contained a 1.3-kb insert from pPTN1 and which conferred resistance to beta-lactam antibiotics. Hybridization studies performed with a 1.2-kb HindIII fragment from pPtN2 failed to determine any homology with ampC of E. cloacae, with other known beta-lactamase genes commonly found in members of the family Enterobacteriaceae (bla(TEM-1)) and bla(SHV-3) derivatives), and with previously described carbapenemase genes such as those from Xanthomonas maltophilia, Bacillus cereus, Bacteroides fragilis (cfiA), and Aeromonas hydrophila (cphA). This work describing the biochemical properties of a novel chromosome-encoded beta-lactamase from E. cloacae indicates that this enzyme differs from all the previously described carbapenemases. This is the first reported cloning of a carbapenem-hydrolyzing gene from a member of the family Enterobacteriaceae. Images

Computerized microacidimetric determination of β lactamase Michaelis—Menten constants

In spite of their sensitivity the microiodometric [2] hydroxamate [3] and other methods of characterisation of P lactamases [4] do not allow precise measurement of Km, although Vmax is obtained most accurately. Automation effects some improvement in the precision but reports of Km values in the literature remain sparse [5] . In the 1950’s a pHmetric determination of penicillin was proposed [6] but, nevertheless because of its low accuracy and sensitivity it was not developed to any great extent [7-l I]. . In the study of the enzyme produced by a strain of Escherichia coli resistant to ampicillin, penicillin G and V, we were able to increase the sensitivity of the pHmetric technique up to lpg/ml of ampicillin,. pHstat neutralisation of the penicilloic acid liberated by hydrolysis of penicillin provided an accurate measure of the degree of reaction at any instant. Computer analysis of the curve obtained from a single titration gave values of Km and Vm,.

Synthesis and antimicrobial activities of N-substituted imides.

In the field of our research programs concerning novel antimicrobial agents, a series of N-substituted imides was synthesized. These compounds were obtained by cyclization of amido-acids in acetic anhydride/sodium acetate or hexamethyldisilazane/zinc bromide for the hydroxy-aromatic derivatives. The hydroxy-alkyl maleimides were directly prepared by condensation of the corresponding amino-alcohol with maleic anhydride in boiling toluene. Most of N-substituted maleimides showed an interesting antimicrobial activity towards bacteria from the ATCC collection (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) but the MIC values for P. aeruginosa were always high (128 microg/ml). The imides with alkyl substituents showed higher activities than aromatic analogues with MIC values in the range of 8-32 microg/ml. Comparatively, succinimides were practically inactive.

Beta-Lactamases: Determination of Their Isoelectric Points

Important discrepancies in isoelectric points (pI) of β-lactamases were observed depending on the experimental procedure used for their determination: isoelectric focusing (IEF) in sucrose density gradients or analytical IEF in thin-layer polyacrylamide gels (PA). The variations, negligable in the case of TEM-like β-lactamases, appeared to be important in the case of cephalosporinases and are related to an artifact which appears in PA-IEF. This has been clearly shown with β-lactamase preparations from the following bacterial strains: Pseudomonas aeruginosa NCTC 8203 (pI = 8.7), P. aeruginosa NCTC 10701 (pI = 9.4), and Proteus morganii NCTC 235 (pI = 8.3). The data previously obtained by PA-IEF were much lower. Images

Molecular Characterization of Chromosomal Class C β-Lactamase and Its Regulatory Gene in Ochrobactrum anthropi

ABSTRACT Ochrobactrum anthropi, formerly known as CDC group Vd, is an oxidase-producing, gram-negative, obligately aerobic, non-lactose-fermenting bacillus of low virulence that occasionally causes human infections. It is highly resistant to all β-lactams except imipenem. A clinical isolate, SLO74, and six reference strains were tested. MICs of penicillins, aztreonam, and most cephalosporins tested, including cefotaxime and ceftazidime, were >128 μg/ml and of cefepime were 64 to >128 μg/ml. Clavulanic acid was ineffective and tazobactam had a weak effect in association with piperacillin. Two genes, ampR and ampC, were cloned by inserting restriction fragments of genomic DNA from the clinical strain O. anthropi SLO74 into pBK-CMV to give the recombinant plasmid pBK-OA1. The pattern of resistance to β-lactams of this clone was similar to that of the parental strain, except for its resistance to cefepime (MIC, 0.5 μg/ml). The deduced amino acid sequence of the AmpC β-lactamase (pI, 8.9) was only 41 to 52% identical to the sequence of other chromosomally encoded and plasmid-encoded class C β-lactamases. The kinetic properties of this β-lactamase were typical for this class of β-lactamases. Upstream from the ampC gene, the ampR gene encodes a protein with a sequence that is 46 to 62% identical to those of other AmpR proteins and with an amino-terminal DNA-binding domain typical of transcriptional activators of the Lys-R family. The deduced amino acid sequences of theampC genes of the six reference strains were 96 to 99% identical to the sequence of the clinical strain. The β-lactamase characterized from strain SLO74 was named OCH-1 (gene, blaOCH-I).

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

Abstract A new β-lactam sulfone, CP 45899, has been proved to be a time-dependent irreversible inhibitor of three R-factor-mediated β-lactamases (penicillin amido-β-lactamhydrolase, EC 3.5.2.6): TEM-1 ( p I = 5.4 ), TEM-2 ( p I= 5.6 ) and Pittons type 2 ( pI = 7.7 ). This inhibition occurs in two principal steps: (1) formation of a reversible enzyme-inhibitor complex (characterized by a K i ): (2) evolution of this complex into one, or more, inactive protein(s) ( k inact ). With the three β-lactamases (CP 45899 shows, respectively, K i of 0.9, 0.8 and 1.8 μM and k inact 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.

TEM-89 β-Lactamase Produced by a Proteus mirabilis Clinical Isolate: New Complex Mutant (CMT 3) with Mutations in both TEM-59 (IRT-17) and TEM-3

ABSTRACT TEM-89 (CMT-3) is the first complex mutant β-lactamase produced by a clinical strain of Proteus mirabilis (strain Pm 631). This new enzyme, which has a pI of 6.28, is derived from TEM-3 and has a single amino acid substitution also encountered in TEM-59 (inhibitor-resistant TEM β-lactamase IRT-17): Ser-130 to Gly. TEM-89 hydrolyzed penicillins to the same extent that TEM-3 did but lost almost all hydrolytic activity for cephalosporins and, like TEM-59, was highly resistant to inhibitors.

EVIDENCE OF IN VIVO TRANSFER OF A PLASMID ENCODING THE EXTENDED-SPECTRUM BETA-LACTAMASE TEM-24 AND OTHER RESISTANCE FACTORS AMONG DIFFERENT MEMBERS OF THE FAMILY ENTEROBACTERIACEAE

ABSTRACT The epidemiological study of several multidrug-resistantEnterobacteriaceae isolated from five patients demonstrated in vivo dissemination of a 100-kb plasmid encoding the extended-spectrum β-lactamase TEM-24 from a clonal strain ofEnterobacter aerogenes to different strains ofKlebsiella pneumoniae, Escherichia coli, Proteus vulgaris, Proteus mirabilis, and Serratia marcescens.