A novel plasmid-mediated extended-spectrum beta-lactamase not derived from TEM- or SHV-type enzymes.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
R Labia
Publications and source records attributed to R Labia.
Characterization of chromosomally encoded penicillinases in clinical isolates of Klebsiella pneumoniae.
Four Klebsiella pneumoniae strains, isolated in two geographically distant French hospitals, were found to produce constitutive beta-lactamases with an unusual isoelectric point for this species (8.1). The four enzymes were chromosomally encoded and related to the Ambler's class A plasmid-mediated SHV-type enzymes. The four enzymes were predominantly penicillinases, with good activity against amino- and ureidopenicillins. They poorly hydrolysed cephalothin, cephaloridine and cefoperazone and did not hydrolyse third-generation cephalosporins and aztreonam. The four enzymes were susceptible to the inhibitory effect of clavulanic acid. Finally, the strains were all found to produce an acetyl-esterase. The acetyl-esterases catalysized hydrolysis of the acetoxy group of cephalothin and cefotaxime although this did not affect their antibacterial activities. These esterases were not susceptible to the inhibitory effect of clavulanic acid and EDTA.
Chromosomes, Bacterial↗
Clinical isolates of Escherichia coli producing TRI beta-lactamases: novel TEM-enzymes conferring resistance to beta-lactamase inhibitors.
Two different strains of Escherichia coli exhibiting unusual patterns of resistance to beta-lactam antibiotics were isolated from patients at Cochin Hospital. Both isolates showed a low level of resistance to amoxycillin, ticarcillin and ureidopenicillins but were susceptible to cephalosporins, aztreonam and imipenem; beta-lactamase inhibitors potentiated the activities of the beta-lactams to only a limited extent. All resistance characteristics of the strains were transferable by conjugation to E. coli K12. Resistance was shown to be due to beta-lactamases of pI 5.20 and relative molecular masses of 24,000. The hydrolytic and inhibition profiles of these enzymes were similar to each other but differed from those of broad-spectrum beta-lactamases (TEM-1). The rates of hydrolysis (Vmax) of amoxycillin (c. 200%) were higher than that for TEM-1 (84%). Ticarcillin, ureidopenicillins and cephaloridine were hydrolyzed slowly. However, as for TEM-1, no hydrolysis was observed with cefoxitin, third generation cephalosporins, aztreonam and imipenem. The high Km values demonstrated the poor affinity of these enzymes for their substrates. Unlike TEM-1, they were poorly inhibited by beta-lactamase inhibitors. These two enzymes differed from each other as follows: (i) the concentrations of clavulanic acid required for 50% beta-lactamase inhibition were 31 mumol/L for one enzyme (E-SAL) and 9.4 mumol/L for the other (E-GUER); (ii) p-chloromercuribenzoate was a more active inhibitor of E-SAL then E-GUER. The titration curve method and DNA-DNA hybridization studies demonstrated that both enzymes were structurally related to TEM-1. The novel plasmid-encoded enzymes produced by the two isolates of E. coli appeared to be almost identical and to be derived from TEM-enzymes. On the basis of their presumed phylogeny and their biological properties, we propose that these beta-lactamases be given the generic name TRI (TEM Resistant to beta-lactamase Inhibitors).
Anti-Bacterial Agents↗
Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 beta-lactamase genes.
CAZ-2, CAZ-6, and CAZ-7 are plasmid-mediated beta-lactamases that are markedly active against ceftazidime. The corresponding structural genes were amplified by the polymerase chain reaction. Nucleotide sequences were determined by direct sequencing of the amplified products. Analysis of the nucleotide and the deduced amino acid sequences showed that CAZ-2, CAZ-6, and CAZ-7 are derived from TEM-2 by three, four, and two amino acid substitutions, respectively. All these substitutions are located at positions 102, 162, 235, 236, and 237 (Sutcliffe numbering), which are known to extend the substrate range of beta-lactamases. These substitutions are Lys-102, Ser-162, and Ser-236 in CAZ-2; Lys-102, Ser-162, Thr-235, and Lys-237 in CAZ-6; and Lys-102 and His-162 in CAZ-7. These results indicate that the nucleotide sequence of CAZ-2 is identical to that of TEM-8. The nucleotide sequence of CAZ-7 possesses the two mutations described in TEM-16 by the oligotyping method. In contrast, the combination of mutations encountered in CAZ-6 has not yet been described, and this enzyme was designated TEM-24.
Base Sequence↗
[Identification of a new penicillinase in a Klebsiella pneumoniae strain of which a mutant also produces an esterase hydrolyzing cephalothin and cefotaxime].
Klebsiella pneumoniae strain L 164 produces a penicillinase whose isoelectric point is 8.1, an unusual figure for this bacterial species. This strain exhibits resistance to conventional penicillins and a synergistic effect is seen with clavulanic acid. In contrast, susceptibility to cephalosporins is marked, as shown by the low minimum inhibitory concentrations (MICs). This phenotype is characteristic of strains with no acquired resistance. A first mutant with MICs for cephalothin and cefotaxime 8-fold to 16-fold those of the initial strain was obtained spontaneously. This mutant's MICs for the other beta-lactams were not substantially changed. In addition to the same penicillinase as the one produced by the parent strain, this mutant produced an acetyl-esterase capable of hydrolyzing the cephalosporins with an acetoxyl side-chain, i.e., cefalothin and cefotaxime, to deacetylated derivates which retain substantial antibacterial activity. Another mutant selected on an amoxicillin gradient produced ten times more penicillinase than the parent strain but no esterase. This second mutant exhibited very high MICs for penicillins and first and second generation cephalosporins. The MIC for cefotaxime was comparable to that seen with the esterase-producing mutant. Among the antimicrobials tested, only third generation cephalosporins and cefoxitin showed adequate activity.
Acetylesterase↗
Replacement of lysine 234 affects transition state stabilization in the active site of beta-lactamase TEM1.
Lysine 234 is a residue highly conserved in all beta-lactamases, except in the carbenicillin-hydrolyzing enzymes, in which it is replaced by an arginine. Informational suppression has been used to create amino acid substitutions at this position in the broad spectrum Escherichia coli beta-lactamase TEM-1, in order to elucidate the role of this residue which lies on the wall at the closed end of the active site cavity. The mutants K234R and K234T were constructed and their kinetic constants measured. Replacement of lysine 234 by arginine yields an enzyme with similar activity toward cephalosporins and most penicillins, except toward the carboxypenicillins for which the presence of the guanidine group enhances the transition state binding. The removal of the basic group in the mutant K234T yields a protein variant which retains a low activity toward penicillins, but losts drastically its ability to hydrolyze cephalosporins. Moreover, these two mutations largely decreased the affinity of the enzyme for penicillins (10-fold for K234R and 50-fold for K234T). This can be correlated with the disruption of the predicted electrostatic binding between the C3 carboxylic group of penicillins and the amine function of the lysine. Therefore, lysine 234 in the E. coli beta-lactamase TEM-1 is involved both in the initial recognition of the substrate and in transition state stabilization.
Amino Acid Sequence↗
Cefotaxime-hydrolysing activity of the beta-lactamase of Klebsiella oxytoca D488 could be related to a threonine residue at position 140.
The chromosomally encoded beta-lactamase of Klebsiella oxytoca D483 strain, active against all third-generation cephalosporins but ceftazidime, was purified to homogeneity. The pure protein was digested by trypsin, Staphylococcus aureus V8 protease or proteinase Asp-N. Amino acid sequences of the HPLC-separated proteolytic peptides were determined by manual Edman degradation. Overlapping fragments gave the alignment of the 263 residues of the beta-lactamase which presented 90% homology with the beta-lactamase of the K. oxytoca E23004 strain and about 40% homology with the other enzymes of the structural class A. The cefotaximase activity might result from interaction of a threonine residue at position 140 (position 165 in the numbering of Ambler) with the oxyimino group of the antibiotic.
Amino Acid Sequence↗
The Michaelis-Menten equation: computing substrate concentration as a function of time without restrictions on the initial conditions.
We describe a novel algorithm for enzyme kinetics following the Michaelis-Menten equation, with the particular aim of computing the substrate concentration as a function of time without restrictions on the initial conditions. This algorithm, named 'tangent exponential' was demonstrated to converge for all initial conditions when the initial substrate concentration is positive. When the data are close to the solution, a quadratic convergence was demonstrated.
Algorithms↗
Chromosomally-mediated beta-lactamases produced by Levinea amalonatica with activity against methoxyimino-cephalosporins.
Levinea amalonatica strain A2370 was isolated from the blood culture of a patient hospitalized in Charles Nicolle hospital (Tunis) during July 1986 and had decreased susceptibility to cefotaxime. Isoelectric focusing of crude extracts from this strain demonstrated three bands of beta-lactamase activity which focused at pH 5.4, 5.5 and 6.05. The three bands were separated and analysed for their kinetic properties. One enzyme (pI 5.4) had the properties of a TEM-1 beta-lactamase and did not confer resistance to third-generation cephalosporins. The two other bands, named B1 and B2, had pIs of 6.05 and 5.5 respectively, hydrolysed cefotaxime and similar cephalosporins, and were produced constitutively. After prolonged storage (six months at -20 degrees C) of the highly purified B1 enzyme, a mixture of B1/B2, with the B2 band predominating, was obtained. This observation suggested that the B2 enzyme was derived from B1. These enzymes appeared to differ from the MJ-2 beta-lactamase described previously in L. amalonatica.
Anti-Bacterial Agents↗
Site-directed mutagenesis on TEM-1 beta-lactamase: role of Glu166 in catalysis and substrate binding.
Class A beta-lactamases are the major cause of bacterial resistance to beta-lactam antibiotics. In these active-site serine hydrolases, glutamic acid 166 has been hypothesized to act as a general acid-base catalyst. Replacing this residue by tyrosine in TEM-1 beta-lactamase yields an enzyme the activity of which is substantially lowered and strongly dependent on pH, thus confirming the alleged role of Glu166 in catalysis. This substitution also resulted in a spectacular change in substrate profile, the mutant enzyme being more active on cephalosporins than on penicillins. In fact, the E166Y enzyme behaves much like a class C enzyme, with high affinity and low hydrolytic activity towards second and third generation cephalosporins. Glu166 therefore seems to play a major part in defining the substrate profile of class A beta-lactamases.
Binding Sites↗
Identification of CTX-2, a novel cefotaximase from a Salmonella mbandaka isolate.
A strain of Salmonella mbandaka isolated from the feces of an Algerian infant showed a reduced susceptibility to cefotaxime (MIC, 8 mg/liter). This strain produced two transferable beta-lactamases of pIs 5.3 and 5.6. The novel beta-lactamase with a pI of 5.3 inhibited by clavulanic acid showed cefotaxime hydrolysis and was therefore designated CTX-2.
Anti-Bacterial Agents↗
Translocation of antibiotic resistance determinants including an extended-spectrum beta-lactamase between conjugative plasmids of Klebsiella pneumoniae and Escherichia coli.
The extended-spectrum beta-lactamase CAZ-7, derived from TEMs, was produced by two different strains of the family Enterobacteriaceae, Klebsiella pneumoniae and Escherichia coli, isolated from the same patient. Both isolates were resistant to amikacin. In addition, the K. pneumoniae strain was TEM-1 producing and resistant to gentamicin. An E. coli HB101 transconjugant obtained from K. pneumoniae, selected on ceftazidime, showed that CAZ-7 and amikacin resistance were encoded by an 85-kb Inc7 or M plasmid, while an E. coli HB101 transconjugant obtained from E. coli under the same conditions showed that CAZ-7 and amikacin resistance were encoded by a greater than 150-kb Inc6 or C plasmid. Two other E. coli HB101 transconjugants obtained from K. pneumoniae, selected on gentamicin or chloramphenicol, showed that TEM-1 and gentamicin resistance could be encoded either by a greater than 150-kb Inc6 or C plasmid or by an 85-kb Inc7 or M plasmid. It was hypothesized that the genes for beta-lactam and aminoglycoside resistances were located on translocatable sequences. EcoRI digestion and hybridizations obtained with blatem, aacA4, and IS15 probes demonstrated that the CAZ-7 gene, amikacin resistance gene, and IS15 element were clustered on an approximately 20-kb fragment common to 85- and greater than 150-kb plasmids. E. coli HB101 transconjugants from K. pneumoniae and E. coli isolates were used to obtain translocations of CAZ-7 and amikacin resistance and of TEM-1 and gentamicin resistance between the 85- and greater than 150-kb plasmids. This study shows a typical example of in vivo gene dissemination involving transposable elements which translocate multiresistance genes, including an extended-spectrum beta-lactamase.
Ampicillin Resistance↗
Competitive interactions of drugs with their targets: a computerized improvement of Dixon's algorithm.
Many drugs are competitive and reversible enzyme inhibitors. When the target enzyme kinetics follows the Michaelis-Menten equation, the enzyme affinity of the inhibitor is characterized by a single parameter: the Ki value. This parameter is usually determined via Dixon's procedure: (i). the rate of reaction (V) is measured in the presence of a few concentrations of the inhibitor (I); (ii.) 1/V versus I gives a straight line, which allows a graphic determination of the inhibitory constants, or better via a least-square fit the linear regression. The introduction of appropriate weighting factors in the linear regression may improve the accuracy of the Ki determinations.
Algorithms↗
[Comparative activities of 15 beta-lactam antibiotics against 590 strains of Klebsiella pneumoniae according to the production of beta-lactamase].
In October 1988, all non repetitive strains of K. pneumoniae isolated in 17 hospitals have been studied. Among these 590 strains: 451 (76%) only produce the specific beta-lactamase of the species SHV-1 (pI 7,7) or SHV-1 type (pI 7,1), while 74 (12.5%) produce a TEM-1 or TEM-2 type beta-lactamase, and 65 (11%) an extended broad spectrum beta-lactamase: 22 CTX-1, 5 SHV-2, 4 SHV-3, 26 SHV-4, 8 SHV-5. The minimum inhibitory concentrations of the following antibiotics were performed by a liquid micro dilution technic: amoxicillin (AMX), amoxicillin + clavulanic acid (CL), 5 mg/l, ticarcillin (TIC), piperacillin (PIP), cefazolin (CEZ), cefamandole (CFM), cefoperazone (CFP), cefotaxime (CTX), cefotaxime + clavulanic acid 5 mg/l, cefotaxime + sulbactam (SUL) 5 mg/l, cefpirome (CPI), ceftazidime (CAZ), azthreonam (AZT), latamoxef (MOX), cefoxitin (FOX), cefotetan (CTT), temocillin (TMO), imipenem (IMI). The "wild" strains with SHV-1 beta-lactamase are resistant to AMX and have a decreased susceptibility to TIC and PIP, but are susceptible to other antibiotics. The TEM producing strains are more resistant to PIP and TIC, have a decreased susceptibility to CEZ and CFM but are susceptible to other antibiotics. For the extended broad-spectrum beta-lactamase producing strains, the MIC of penicillin antibiotics (AMX, TIC, PIP) are very high and also the MIC of CEZ, CFM and CFP. The MIC of CTX are higher for CTX-1 or SHV-4 producing strains, than for SHV-2, SHV-3, or SHV-5 producing strains. The combination with CL is more efficacious than the one with SUL to reduce the MIC of CTX in susceptibility area.(ABSTRACT TRUNCATED AT 250 WORDS)
Amoxicillin↗
Molecular evolution of ubiquitous beta-lactamases towards extended-spectrum enzymes active against newer beta-lactam antibiotics.
Production of beta-lactamases, and of the plasmid-encoded TEM- and SHV-type enzymes in particular, is the most common mechanism of resistance against beta-lactam antibiotics in Gram-negative bacteria. The two ubiquitous types of enzyme have a large spectrum of activity and preferentially hydrolyse the penicillins as well as some first- and second-generation cephalosporins. Recently, point mutations in the corresponding genes have been observed, apparently selected for, in the clinical setting, by originally 'beta-lactamase-stable' third-generation cephalosporins or by monobactams, which fall into the substrate range of the mutant or 'extended-spectrum' beta-lactamases. The point mutations are clustered in three areas, each adjacent to one of the seven evolutionarily conserved boxes described by Joris et al. (1988). The substituted amino acids at positions 102 (adjacent to the alpha-3 helix), 162 (adjacent to the alpha-7 helix) and 235, 236 and 237 (on the beta-3 strand) are located in close proximity to the active-site cavity and are thought to open up novel enzyme-substrate interactions, involving, in particular, the oxyimino moieties of the newer beta-lactam compounds.
Amino Acid Sequence↗
Nucleotide sequence of SHV-2 beta-lactamase gene.
The nucleotide sequence of plasmid-mediated beta-lactamase SHV-2 from Salmonella typhimurium (SHV-2pHT1) was determined. The gene was very similar to chromosomally encoded beta-lactamase LEN-1 of Klebsiella pneumoniae. Compared with the sequence of the Escherichia coli SHV-2 enzyme (SHV-2E.coli) obtained by protein sequencing, the deduced amino acid sequence of SHV-2pHT1 differed by three amino acid substitutions.
Amino Acid Sequence↗
Overcoming enzymatic resistance in bacteria: impact on future therapy.
Resistance of bacteria to antibiotics often involves inactivating enzymes. One approach developed to overcome this mechanism of resistance consists of combining an efficient but possibly unstable antibiotic with a powerful inhibitor of the inactivating enzyme. Attempts have been made with different antibiotics but significant success has only been obtained with the beta-lactams, clavulanic acid and sulbactam being the only compounds currently being used clinically. Sulbactam, a time-dependent irreversible inhibitor of plasmid-mediated penicillinases and of chromosomally mediated penicillinases and cephalosporinases, potentiates the antibacterial activity of beta-lactams but only exhibits a moderate antibacterial activity, which is related to its affinity for the lethal targets of the beta-lactams--the penicillin-binding proteins. In bacterial strains that produce either low amounts of beta-lactamase, or none at all, a synergistic effect can be observed when sulbactam is associated with a beta-lactam antibiotic that has a complementary affinity for the target sites.
Aminoglycosides↗
Use of beta-lactamase-producing anaerobes to prevent ceftriaxone from degrading intestinal resistance to colonization.
Six adult volunteers were given 1 g/d of intravenous ceftriaxone for 5 d (consecutive). Ceftriaxone and beta-lactamase activities were assayed in fecal samples obtained before and during drug administration, and anaerobic bacteria, Enterobacteriaceae, and fungi were counted. In two volunteers, no fecal beta-lactamase activity was detected, but ceftriaxone was present during treatment at concentrations of 1.8-2.0 mg/g of feces. Concomitantly, fecal counts of anaerobes in these volunteers dropped from 10.5 to less than 8 log10 colony-forming units (cfu)/g of feces, and those of Candida species increased more than 100-fold. However, in the feces of the four other volunteers, beta-lactamase activity was high during ceftriaxone administration, but no ceftriaxone was detected. In these volunteers, ceftriaxone administration was not followed by any significant change in counts of anaerobes or Candida species. This appeared to be due to the intraintestinal hydrolysis of ceftriaxone by resident beta-lactamase-producing anaerobes. In gnotobiotic mice associated with a human fecal flora containing no beta-lactamase-producing anaerobes, it was possible to prevent the deleterious effects of ceftriaxone on intestinal microbial composition and on colonization resistance (against a strain of Candida albicans and one of ceftriaxone-resistant Enterobacter cloacae) by feeding the animals with an association of four beta-lactamase-producing anaerobic strains.
Adult↗