Combinations of Clavulanic Acid and other β-lactam Antibiotics against Strains of Pseudomonas aeruginosa, Serratia marcescens and other Bacteria

Combinations of clavulanic acid, a new β-lactamase inhibitor, with five cephalosporins and one cephamycin were tested against cell-free β-lactamases obtained from Serratia marcescens, Pseudomonas aeruginosa and an Enterobacter strain, 265A. Cefotaxime was the most resistant antibiotic and cephalothin the most sensitive antibiotic to β-lactamases. Low concentrations of clavulanic acid gave some protection against the Serratia and Pseudomonas enzymes. The most active source of β-lactamase was the 265A strain, against which only cefotaxime was highly resistant. Clavulanic acid had only a slight inhibitory effect on this enzyme, which was confirmed by an agar method, and potentiated slightly the activity of cephalothin and cefoxitin against two β-lactamase producing strains of Staphylococcus aureus. Lysis by cephalothin of one strain of S. marcescens was potentiated in the presence of clavulanic acid.     

Comparison of the phenotypes of the lpxA and lpxD mutants of Escherichia coli

Abstract We compared the phenotype of two thermosensitive Escherichia coli mutants defective in lipid A biosynthesis i.e. SM101 ( lpxA ) and CDH23-213 ( lpxD ). More than 40% of the periplasmic 27-kDa marker enzyme β-lactamase was released from SM101 at 28°C. At this temperature, the mutant still grew with a generation time (67 min), not much longer than that of the parent control strain (57 min). CDH23-213 released β-lactamase only at higher temperatures. SM101 and CDH23-213 were both unable to grow in hypo-osmotic conditions. Derivatives of SM101 and CDH23-213 with mdoA ::Tn 10 had identical phenotypes (including thermosensitivity and defective outer membrane permeability barrier to hydrophobic probes) to those of SM101 and CDH23-213, indicating that the potential loss of membrane-derived oligosaccharides (MDO) did not explain these phenotypic properties. A method for the estimation of lipid A synthesis rate was developed.     

Co-existence of β-lactamase and penicillin acylase in bacteria; detection and quantitative determination of enzyme activities

Twenty-six bacteria were examined for the presence of penicillin acylase and β-lactamase. A copper reducing assay, which was sensitive in the analytical range 2–20 μg/ml, was used for determination of penicilloates and a fluorescamine assay was used to determine 6-aminopenicillanic acid concentrations when both substances were produced by the action of the enzymes on a single substrate. Seventeen bacteria contained β-lactamases, six contained penicillin acylases and four contained both enzymes. Two bacteria contained a Type 1 penicillin acylase and four bacteria contained a Type II enzyme. No ampicillin acylases were detected. All β-lactamases were constitutive enzymes in those organisms where both enzymes co-existed. Bacillus subtilis and B. cereus produced inducible and extracellular β-lactamases. Acinetobacter calcoaceticus ATCC 21288 produced a constitutive β-lactamase which was detected extracellularly.     

Isolation and partial purification of a carbapenem-hydrolysing metallo-β-lactamase from Pseudomonas cepacia

Abstract A metallo-β-lactamase has been isolated from a clinical strain of Pseudomonas cepecia and partially purified using Cibacron blue F3GA coupled agarose. The resulting preparation showed a single band of β-lactamase activity (p I 8.45) after analytical isoelectric focusing. The enzyme was particularly effective in the hydrolysis of imipenem. Meropenem, biapenem, cephaloridine, ceftazidine, benzylpenicillin, ampicillin and carbenicillin were also hydrolysed, although at a lower rate. An unusual inhibition profile was noted. Inhibition by the metal ion chelators ethylenediaminetetraacetic acid and o -phenanthroline was reversed by addition of zinc, indicating a metallo-enzyme, whilst > 90% inhibition was attainable with 0.1 mM concentrations of tazobactam and clavulanic acid. A study of 8 other clinical isolates showed the enzyme to be present and inducible by imipenen in each case. This enzyme was assigned PCM-I ( Pseudomonas cepacia metalloenzyme I).     

Secretion by Streptomyces lividans of a cloned Gram-negative beta-lactamase

Abstract The OXA-2 β-lactamase gene was first found on a conjugative plasmid R46 from a clinical isolate of Salmonella typhimurium . To transfer the gene to Streptomyces lividans a shuttle vector was created by fusing an Escherichia coli plasmid carrying the OXA-2 β-lactamase gene with the S. lividans vector pIJ61. The OXA-2 β-lactamase gene was expressed in S. lividans , although with a much reduced efficiency; virtually all of the β-lactamase activity was found in the culture supernatant. The identity of the enzyme was established by substrate specificity and isoelectric focusing.     

A β-lactamase produced by a thermophilic Bacillus

Abstract A β-lactamase was purified from a thermophilic Bacillus strain, that had been isolated from a traditional hot bath in the Meknes area (Morocco). The properties of the enzyme were very similar to those of the β-lactamase produced by Bacillus licheniformis 749C but it exhibited a somewhat increased thermostability and a higher activation energy with cefazolin as substrate. These properties were expected for an enzyme produced by a thermophilic strain.     

Degradation of β-lactam antibiotics by polyacrylamide-entrapped β-lactamase-producingE. coli cells

Summary Cells of a -lactamase producingE. coli strain were immobilized with acrylamide and lyophilized. The gel particles containing the entrapped cells were used like an immobilized enzyme to study the inactivation of -lactam antibiotics. The substrate profile of the -lactamase was determined and the action of -lactamase inhibitors studied.     

The inhibition of β-lactamases from Gram-negative bacteria by clavulanic acid

The β-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme–clavulanate complex, a transiently stable species (t_½=4min at pH7.3 and 37°C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 β-lactamase and 1:1 for Klebsiella β-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 β-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella β-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t_½=40min at pH7.3 and 37°C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m⁻¹·s⁻¹) to produce a relatively stable complex (t_½=180min at pH7.3 and 37°C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate–enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH.     

A simple membrane reactor to obtain high cell density and plasmid encoded protein production from Escherichia coli

Summary A relatively simple system is described which yields high concentrations of cell mass and plasmid encoded protein. The maximum concentrations of cell mass and of a plasmid encoded protein, -lactamase, obtained were: 49 g/L dry weight and 1.4 g/L, respectively.     

Ascorbate as an induction inhibitor of β-lactamase in a strain of Enterobacter cloacae

H.A. SHOEB, H.I. AL-SHORA AND T. ABDEL-SALAM. 1995. The effect of ascorbate and anaerobiosis of β-lactamase content (constitutive and inducible) in relation to the susceptibility of a standard strain of Enterobacter cloacae to ampicillin was studied. Enterobacter cloacae ATCC 13047 showed increasing susceptibility to ampicillin when incubated anaerobically in the presence of increasing concentrations of ascorbic acid. The inducible β-lactamase activity in the cell-free extracts of Ent. cloacae decreased when the bacterium was grown aerobically in the presence of ascorbic acid. Under anaerobic growth conditions, however, ascorbic acid abrogated the induction of the enzyme completely. On the other hand, the constitutive enzymatic activity was markedly decreased as the bacterium was grown anaerobically. Thus under these growth conditions, ascorbate-anaerobiosis, the total β-lactamase level in the presence of ampicillin as inducer fell below the basal constitutive activity observed in the absence of ampicillin.     

Proteolytic inactivation of an extracellular (1 → 3)-β-glucanase from the fungus Acremonium persicinum is associated with growth at neutral or alkaline medium pH

Abstract The filamentous fungus Acremonium persicinum released high levels of proteolytic enzyme activity into the culture fluid during growth at pH 7 or above. Almost total inhibition of this crude activity by phenylmethylsulfonyl fluoride suggested that it was mainly due to the presence of a serine protease. This protease inactivated one of three extracellular (1 → 3)- β -glucanases produced by this fungus, although the activities of the remaining two (1 → 3)- β -glucanases did not appear to be affected. Growth of A. persicinum in acidic conditions resulted in the presence of much lower extracellular proteolytic activity and no apparent (1 → 3)- β -glucanase inactivation.     

The effect of long-chain alkyl betainates on the arylesterase-like activity of bovine serum albumin

The effect of a hydrolyzable cationic surfactant, tetradecyl betainate (tetradecyloxycarbonyl-N,N,N-trimethylmethanaminium chloride), on the arylesterase-like activity of bovine serum albumin (BSA) was investigated. The rate of hydrolysis of p-nitrophenyl hexanoate in the presence of BSA and varying concentrations of the surfactant was followed. The rate was found to be dependent on the concentration of the cationic surfactant and a maximum was found in the curve at ca. 3 m . The Michaelis-Menten constants (K_m/n, where n is the number of active sites) and the “catalytic” rate constants (k₂) were determined for the reactions, and were found to be 11 and 40 times larger, respectively, in the presence of the surfactant. The hydrolysis of radiolabeled tetradecyl betainate in the presence and absence of BSA was also followed. This study, together with a binding study based on gel permeation chromatography, showed that the surfactant binds to the protein, but that no hydrolysis of the betaine ester takes place while bound to BSA. It was thus concluded that the increased value of k₂ in the presence of a cationic surfactant was not, as has been previously suggested, due to an increased local hydroxide concentration resulting from the formation of a new pseudophase.     

Pteroylpolyglutamate synthesis by lung- and culture-derived Pneumocystis carinii

Abstract Evaluation of four β-lactamase inhibitors in terms of their outer membrane permeability in Pseudomonas aeruginosa revealed that sulbactam and tazobactam diffused most efficiently and equally well. That of BRL42715 appeared to be a factor of ten lower than that of the above two, but it showed the strongest β-lactamase inhibitory activity. This is most likely due to its better β-lactamase inactivating activity. BRL42715 at 1.56 μg ml⁻¹ lowered the minimum inhibitory concentrations of ceftazidime and imipenem in a strain producing fully derepressed β-lactamase and an undetectable level of the outer membrane protein OprD2.     

Susceptibility of Gram-negative bacteria to β-lactam antibiotics and rapid characterization of β-lactamase activity

Various species of Gram-negative bacteria were tested for susceptibility to β-lactam antibiotics and for production of β-lactamase. The rapid cephalosporin 87/312 visual test was more sensitive than either the acidometric or the iodometric test; the iodometric test was least sensitive. Characteristic β-lactamase hydrolysis product patterns were obtained by scanning mixtures of β-lactamase-producing bacteria and cephalosporin substrates. β-Lactamase could not be detected on bacterial cells by fluorescent antibody techniques. The presence of β-lactamase can be correlated with minimal inhibitory concentrations of β-lactam antibiotics only in certain Gram-negative bacilli.     

Characterization of a novel SHV β-lactamase variant that resembles the SHV-5 enzyme

Abstract An SHV type β-lactamase frequently found in enterobacteria isolated in Greek hospitals was analyzed. The enzyme (SHV-5a) conferred resistance to ceftazidime and aztreonam. The DNA sequence of the structural gene was determined. The deduced amino acid sequence showed that positions 70–73 were occupied by the active site tetrad Ser-Thr-Phe-Lys. As in SHV-5, Ser-238 and Lys-240 were present. However, one deletion (Gly-54) and three substitutions (Arg-140 for Ala, Asn-192 for Lys and Val-193 for Leu) differentiate SHV-5a β-lactamase from SHV-5. Asn-192 and Val-193 have been reported to date only in the R974 plasmid-mediated SHV-1 β-lactamase. Hydrolysis studies with SHV-5a and SHV-5 showed that the enzymes behaved similarly. Additional evidence that they were functionally indistinguishable was provided by the similar MICs of β-lactams when the enzymes were expressed under isogenic conditions. The sequence differences, however, indicate that they are derived from different ancestors.     

Efficient release of thermostable β-galactosidase by glycine addition and thermal treatment of Escherichia coli cells

Efficient release of thermostable β-galactosidase from a recombinant Escherichia coli by the addition of glycine to the culture broth and subsequent thermal treatment was investigated. The enzyme release rate was strongly dependent on glycine concentration. The enzyme release rate was almost proportional to glycine concentrations up to 2% in phosphate buffer; however, inactivation of the enzyme was not observed following incubation for up to 3 h at 70°C even in the presence of 10% glycine. In a preliminary experiment, severe thermal inactivation was observed in the presence of polyethylene glycol (PEG), but glycine was able to suppress the inactivation. Thermal treatment of the cell suspension was effective for the improvement of the enzyme release rate. In the absence of glycine, the enzyme release rate was low at 37 and 45°C, even though the initial release rate was high at 0.5 h and 60°C. The combination of thermal treatment and addition of glycine to the cell suspension significantly improved the initial enzyme release rate and the amount of enzyme released to the extracellular fraction at 37 and 45°C was as high as that at 60°C during a 2-h incubation.     

The effects of βbT-lactams on the isoelectric focusing of βbT-lactamases

A range of concentrations of ceftazidime (4–64 mg I^-1) was shown to cause no induction of the TEM-1 and TEM-5 β-lactamases produced by Escherichia coli Nb. Increasing the concentration of ceftazidime in cultures of E. coli Nb caused a concomitant increase in the intensity of a satellite band of pI 5.2. The same increase in this satellite band was observed when ceftazidime was added to cell-free β-lactamase peparations from E. coli Nb and the separate addition of 11 different β-lactams to TEM-1 showed that each compound produced its own unique pattern of satellite bands. In addition, the mixing of ceftazidime with TEM-1 and 13 other TEM-derived β-lactamases caused a similar satellite band to be observed but ceftazidime did not have the same effect on PSE or SHV β-lactamases. Consequently, the addition of ceftazidime to a β-lactamase preparation prior to isoelectric focusing (IEF) may help to verify if a particular β-lactamase is TEM-derived. Purification of the satellite bands by electrodialysis and their subsequent re-focusing demonstrated that the ceftazidime-induced satellite bands can revert to a protein which has a pI similar to the parent band, illustrating the possible reversibility and dynamic nature of β-lactamase satellite bands on IEF. These results enable a better interpretation to be made of β-lactamase satellite bands observed on IEF.     

Starvation-independent sporulation in Myxococcus xanthus involves the pathway for β-lactamase induction and provides a mechanism for competitive cell survival

Myxococcus xanthus is a Gram-negative, soil-dwelling bacterium with a complex life cycle which includes fruiting body formation and sporulation in response to starvation. This developmental process is slow, requiring a minimum of 24–48 h, and requires cells to be at high cell density on a solid surface. It is known that, in the absence of starvation, vegetatively growing cell suspensions can form 'glycerol spores' when exposed to high levels of glycerol, usually 0.5 M. The cells differentiate from rods to resistant spheres rapidly (2–4 h) and synchronously. We have found that the chromosomally encoded β-lactamase of M. xanthus can be induced by numerous β-lactam antibiotics as well as by non-specific inducers including glycine and many D -amino acids. In addition, D -cycloserine, phosphomycin, and hen egg-white lysozyme also induce β-lactamase in this bacterium. Unexpectedly, agents which induce β-lactamase can induce 'glycerol spores'; all of the agents tested which induce glycerol spores (glycerol, DMSO, ethylene glycol) also induce β-lactamase. During the induction of sporulation, β-lactamase activity increases, reaching a peak during the morphological transition from rod-shaped cells to spherical spores. These spores are viable and resistant to many treatments which disrupt vegetatively growing rods but are not as resistant as fruiting body spores. The concomitant induction of β-lactamase and starvation-independent sporulation suggests that these processes share a common signal-transduction pathway. These results also suggest that starvation-independent sporulation may be an adaptation of cells in order to resist agents that damage peptidoglycan structure and therefore threaten cell survival.     

Induction studies showing evidence of the similarities between an inducible intracellular and extracellular β-d-glucosidase produced by a species of Monilia

Abstract A Monilia sp. produced an inducible intracellular β- d -glucosidase (IG-2) which is the nascent form of the extracellular enzyme (EG-1) prior to its secretion into the extracellular medium. The other intracellular β- d -glucosidase (IG-1) produced was a constitutive enzyme. Highest yields of the inducible β- d -glucosidase resulted when Monilia sp. was grown on insoluble cellulose. Cellobiose and d -glucose appeared to repress β- d -glucosidase formation at high substrate levels and synthesis occurred only once the levels of these sugars in the medium were nearly depleted.