Forty-three clinical isolates of enterobacteria were selected for the production of the new plasmid-mediated expanded-spectrum beta-lactamase CTX-1. The geometric means of MICs were ranged as follows: ticarcillin, greater than 4096 mg/l; ticarcillin + clavulanic acid (2 mg/l), 64-87 mg/l; LY 163892, 8.0-69.1 mg/l; cefotaxime, 5.7-26.4 mg/l; temocillin, 8.0-21.8 mg/l; Ro 158074, 4.0-18.7 mg/l aztreonam, 1.0-14.4 mg/l and BMY 28142, 1.4-2.8 mg/l. Moxalactam, imipenem and CM 40876 were resistant to hydrolysis and MICs were lower than 2.0 mg/l. A high protective effect on cefotaxime (MIC less than or equal to 0.5 mg/l) was obtained by sulbactam (4 mg/l). Escherichia coli transconjugants from each species showed similar levels of MICs.

Anti-Bacterial Agents↗

PubMedDate not supplied

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.

Base Sequence↗

PubMedDate not supplied

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.

Blood Bactericidal Activity↗

PubMedDate not supplied

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.

Anti-Bacterial Agents↗

PubMedDate not supplied

[Distinction between the primary structures of TEM-1 and TEM-2 beta-lactamases].

TEM-1 and TEM-2 beta-lactamases were shown to contain a disulphide bond. When the amino acid compositions of the two proteins were determined, the TEM-2 enzyme exhibited one more lysine residue and one less glutamine (or glutamic acid) residue than the TEM-1 enzyme. This substitution was located at the 14th N-terminal residue, as shown by structural analysis of Staphylococcus aureus protease peptides separated by high performance liquid chromatography. From these results, the Ambler and Scott sequence can be attributed to TEM-2 and the Sutcliffe sequence to TEM-1.

Amino Acid Sequence↗

PubMedDate not supplied

Purification and biochemical properties of Pitton's type 2 beta-lactamase (SHV-1).

A five-step procedure has been developed for purifying Pitton's type 2 plasmid-mediated beta-lactamase (PIT-2, also called SHV-1) from cultures of a hyperproducing variant of an Escherichia coli K12 strain carrying the plasmid p453. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a single protein band with a molecular weight of about 27,500. The amino acid composition of the protein and the amino acid sequence of the NH2-terminal region have been determined. PIT-2 enzyme contains 272 amino acid residues with 2 cysteines. Studies of the S-carboxymethylated protein (previously reduced or unreduced) suggest that these two residues are presumably in the form of free sulphydryl groups in the native protein. Conversely, TEM-2 beta-lactamase contains 2 cysteine residues which are in the form of a disulphide bond. Comparison of PIT-2 with other beta-lactamases was made using the difference index (DI) of Metzger et al. The PIT-2 enzyme appeared more closely related to the TEM-type penicillinases (DI of 6.5).

Amino Acid Sequence↗

PubMedDate not supplied

Klebsiella pneumoniae and other Enterobacteriaceae producing novel plasmid-mediated beta-lactamases markedly active against third-generation cephalosporins: epidemiologic studies.

Analysis of the enzymes produced by clinical isolates of multiresistant Klebsiella pneumoniae from hospitals in France revealed two novel broad-spectrum beta-lactamases. The first, characterized by an isoelectric point of 6.3 and a high hydrolytic activity on cefotaxime, is designated CTX-1. This beta-lactamase was encoded by a 95-kilobase plasmid (incompatibility group 7M) and cotransferred with resistance to tetracyclines, sulfonamides, and aminoglycosides (AAC [6']-IV). From 1984 to June 1987, 490 CTX-1-producing strains of Enterobacteriaceae were isolated. The second plasmid-mediated beta-lactamase (CAZ-1) was isolated in 1987 from three K. pneumoniae strains more resistant to ceftazidime than to other third-generation cephalosporins. This broad-spectrum beta-lactamase differed from CTX-1 by its isoelectric point--close to 5.6--and its high hydrolytic activity on ceftazidime and was encoded by a 150-kilobase plasmid. It was demonstrated that these expanded-spectrum beta-lactamases are TEM derivatives.

Cephalosporins↗

PubMedDate not supplied

Interactions of new plasmid-mediated beta-lactamases with third-generation cephalosporins.

The kinetic constants of three recently identified plasmid-mediated beta-lactamases--SHV-2, CTX-1, and CAZ-1--markedly active against third-generation cephalosporins were analyzed in comparison with three better-characterized beta-lactamases--two plasmid-mediated enzymes, TEM-2 and PIT-2/SHV-1, and R-30, a beta-lactamase from Klebsiella oxytoca that has few similarities to the newer enzymes. All of these enzymes are synthesized constitutively, demonstrate efficient hydrolysis of penicillins, are highly susceptible to the action of clavulanic acid and sulbactam, and have no detectable activity against the cephamycins and imipenem. With the methoxyimino cephalosporins, including those of the third generation, the rates of hydrolysis observed for the SHV-2, CTX-1, and CAZ-1 enzymes are high and show no relation to those observed for the other presently known beta-lactamases. Structure-activity relations suggest that the oxime substituent of these cephalosporins is a major structural factor in the catalytic process observed with the three new beta-lactamases.

Cephalosporins↗

PubMedDate not supplied

Moxalactam: an oxa-beta-lactam antibiotic that inactivates beta-lactamases.

Moxalactam, like cefoxitin, cefuroxime, and cefotaxime, shows little or no interaction with penicillinases. Resistance of moxalactam to cephalosporinases, however, appears to be considerably greater than that of these other recently discovered beta-lactam antibiotics. Moxalactam is a beta-lactamase inactivator and inactivates most of the cephalosporinases produced by gram-negative bacteria. The process of inactivation is specific and follows time-dependent or progressive kinetics, as is the case for a class of enzyme inhibitors called "suicide substrates." Moxalactam is the first beta-lactam antibiotic shown to possess this property of inactivation of beta-lactamases, a property that explains its excellent resistance to beta-lactamases.

Bacteria↗

PubMedDate not supplied

Sulbactam: biochemical factors involved in its synergy with ampicillin.

Sulbactam is a time-dependent irreversible inhibitor of various beta-lactamases by reversible formation of a Michaelis-type enzyme-inhibitor complex and progressive evolution of this complex into inactivated protein(s). This process is either irreversible (true inactivation) or quasi-irreversible (stable acyl-enzyme). In this way, sulbactam efficiently protects ampicillin from degradation by beta-lactamases. Sulbactam itself exhibits a moderate antibacterial activity that is related to an affinity for the penicillin-binding proteins of various bacterial strains, which is similar to the affinity of penicillins such as ampicillin. However, sulbactam binding differs according to the bacterial species involved. In strains producing either low levels of beta-lactamase or none at all, a synergistic effect, minor but not negligible, can be observed when sulbactam is associated with a beta-lactam antibiotic with a complementary affinity for the target sites.

Ampicillin↗

PubMedDate not supplied

In vitro bactericidal activity of sulbactam plus ampicillin against methicillin-resistant Staphylococcus aureus.

By a microtiter checkerboard method, the in vitro bactericidal activity of sulbactam/ampicillin was tested against three groups of Staphylococcus aureus strains: group PR (penicillin-resistant through the production of penicillinase; methicillin-susceptible both at 30 degrees C in medium containing 5% NaCl and at 35 degrees C in medium without NaCl; n = 10); group MR1 (methicillin-resistant under the former conditions but not under the latter; n = 15); and group MR2 (methicillin-resistant under both sets of conditions; n = 20). Sulbactam/ampicillin was synergistic against all strains (fractional bactericidal index, less than or equal to 0.10). Moreover, there was a linear relation in each group between the different concentrations of the two antibiotics required for bactericidal activity. Thus, an equation of bactericidal synergy could be formulated for the combination. The bactericidal activity of sulbactam/ampicillin was assessed against one strain of each group and against a reference strain (S. aureus ATCC 6538 P) in an in vitro model simulating the kinetics of both antibiotics in human blood after intravenous administration of 2 g of ampicillin and 1 g of sulbactam. The observed rate of killing was approximately 99% except with the MR2 strains, against which the combination was bacteriostatic.

Ampicillin↗

PubMedDate not supplied

Immunological comparison of constitutive beta-lactamases of gram-negative bacteria by neutralization in zymogram gels: properties of anti-TEM-1 and anti-TEM-2 sera.

The zymogram technique was applied to a beta-lactamase neutralization assay with anti-TEM-1 and anti-TEM-2 sera. Both were shown to contain neutralizing antibodies directed towards various beta-lactamases of Gram-negative bacteria. The quantitative neutralization allowed classification into five groups of the 28 beta-lactamases used as standards and 61 from clinical isolates. In the first were enzymes such as TEM-1 and TEM-2 including TLE-1, SHV-1, SHV-2, penicillinases of Klebsiella pneumoniae and CTX-1. Partial neutralization distinguished two groups containing the CARB group of enzymes, which are different from PSE-2 and PSE-3, and the MAL penicillinases of Levinea malonatica, which are different from L. amalonatica enzymes. Broad spectrum beta-lactamases of K. oxytoca constituted a unique group of partially neutralized enzymes. Among the beta-lactamases not neutralized by either serum were the plasmid-mediated OXA-enzymes, various species-specific beta-lactamases and cephalosporinases. The antigenic similarities of the enzymes appeared to correlate with the extent of similarities of their catalytic properties, namely those of penicillinases. Such comparisons between the beta-lactamase groups provide an indirect approach to the physiological and structural analysis of established and recently evolved beta-lactamases.

Epitopes↗

PubMedDate not supplied

[Activity of sulbactam and clavulanic acid, alone and combined, on Acinetobacter calcoaceticus].

Sulbactam and clavulanic acid, both beta-lactamase inhibitors, were studied alone or associated on 85 isolates of Acinetobacter calcoaceticus. When sulbactam was tested alone the minimal inhibitory concentration (MIC) was less than 2 micrograms/ml with the carbenicillin-sensitive strains of A. Calcoaceticus and between 8 to 16 micrograms/ml with the carbenicillin-resistant strains. In the presence of clavulanic acid (5 or 10 micrograms/ml) the MIC of sulbactam on all the carbenicillin-resistant strains decreased to less than 4 micrograms/ml.

Acinetobacter↗

PubMedDate not supplied

[Affinity of N-formimidoyl-thienamycin (MK-0787) for beta-lactamases, analysis by a statistical method: correspondence analysis].

N-formimidoyl-thienamycin (MK-0787) is not subject to beta-lactamase hydrolysis. Nevertheless, this compound does show some affinity for the TEM-type penicillinases, cephalosporinases and a few other beta-lactamases. Surprisingly no affinity was found for the penicillinases of the CARB-type. The results thus obtained for affinity constants Ki and Km were submitted to a statistical analysis treatment, i.e. correspondence analysis and hierarchic ascendent classification. This confirms the fact that MK-0787 presents an unusual behaviour towards beta-lactamases.

Cephalosporinase↗