Purification of beta-lactamases by affinity chromatography on phenylboronic acid-agarose.

Several beta-lactamases, enzymes that play an important part in antibiotic resistance, have been purified by affinity chromatography on boronic acid gels. The procedure is rapid, appears to be selective for beta-lactamases, and allows a one-step purification of large amounts of enzyme from crude cell extracts. We have found the method useful for any beta-lactamase that is inhibited by boronic acids. Two kinds of boronic acid column have been prepared, the more hydrophobic one being reserved for those beta-lactamases that bind boronic acids relatively weakly. beta-Lactamase I from Bacillus cereus, P99 beta-lactamase and K 1 beta-lactamase from Gram-negative bacteria are among the better-known beta-lactamases that have been purified by this method. The procedure has also been used to purify a novel beta-lactamase from Pseudomonas maltophilia in high yield; the enzyme has an exceptionally broad substrate profile and hydrolyses monocyclic beta-lactams such as azthreonam and desthiobenzylpenicillin.     

Molecular analysis of beta-lactamases from four species of Streptomyces: comparison of amino acid sequences with those of other beta-lactamases

Genes encoding extracellular beta-lactamases of Streptomyces badius, Streptomyces cacaoi, Streptomyces fradiae and Streptomyces lavendulae were cloned and mapped in Streptomyces lividans. DNA sequence analysis of the beta-lactamase genes revealed a high overall G + C content, ranging from 71 to 75 mol%, with a G + C content of 95 mol% at the third position of the codons for all four genes. The primary structure of the beta-lactamases including their signal peptides was deduced. The four beta-lactamases exhibited homology to each other and to class A beta-lactamases from other bacterial genera. We suggest that Streptomyces beta-lactamases are representatives of a superfamily of genes, from which class A beta-lactamases of Gram-negative bacteria may have evolved.     

Crystal structures of the Bacillus licheniformis BS3 class A beta-lactamase and of the acyl-enzyme adduct formed with cefoxitin

The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus beta-lactamase, many cephems are good substrates for a large number of beta-lactamases. However, the introduction of a 7alpha-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7alpha-methoxy group selectively reduces the hydrolytic action of many beta-lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 A resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the omega-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the alpha-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7beta-carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges.     

Characterization of a beta-lactamase produced by Pseudomonas paucimobilis.

A novel beta-lactamase enzyme produced by a strain of Pseudomonas paucimobilis is described. The enzyme differs from other recorded beta-lactamases from Gram-negative aerobic bacteria. It was constitutive, and had the characteristics of a penicillinase. One single band of beta-lactamase activity at pI 4.6 was seen on iso-electric focusing. The enzyme had a molecular mass of 30 kDa. The beta-lactamase was strongly inhibited by tazobactam, sulbactam and clavulanic acid but not by the thiol residue inhibitors p-chloromercuribenzoate and p-chloromercuriphenylsulphonic acid, or by metallo-enzyme inhibitors. Plasmid DNA was not demonstrable, suggesting that the enzyme was chromosomally encoded.     

Structural requirements for antibacterial activity and beta-lactamase stability of 7 beta-arylmalonylamino-7 alpha-methoxy-1-oxacephems

Replacement of a sulphur atom by an oxygen at the 1-position of the cephem nucleus generally resulted in fourfold to sixteenfold increase of antibacterial activity in each pair of the structural congeners. However, the increased antibacterial activity caused by the replacement was accompanied by instability to beta-lactamase to some extent, which was due presumably to the increased chemical reactivity of the beta-lactam ring system. The aim of the research effort is to confer beta-lactamase stability and expand the Gram-negative spectrum. Two types of substituents have been demonstrated to protect 1-oxacephem from enzymic hydrolysis and their protecting effects were specifically related to the types of beta-lactamases derived from Gram-negative bacteria: the 7 beta-malonylamino function is specific to cephalosporinase and the 7 alpha-methoxy group to penicillinase. The complementary effect of these substituents was clearly demonstrated. This line of studies led us to prepare the clinical candidate 6059-S, which possessed widely expanded antibacterial spectra against Gram-negative bacteria including indole-positive Proteus, Enterobacter, Serratia marcescens, Pseudomonas aeruginosa and Bacteroides fragilis.     

Characterization of beta-lactamase from Mycobacterium butyricum ATCC 19979

beta-lactamase has been purified to a homogeneous state from Mycobacterium butyricum ATCC 19979. The molecular weight (Mr = 29,000) and the isoelectric point (4,0) of the enzyme have been determined. The enzyme showed both penicillinase and cephalosporinase activity, but had relatively more of the former. With respect to substrate-profile the enzyme resembled the plasmid specified TEM-type beta-lactamases commonly encountered in Gram-negative bacteria. The enzyme was insensitive to p-chloromercuribenzoate, sodium chloride, or iodine inhibition.     

Sensitivity of Escherichia coli to various beta-lactams is determined by the interplay of outer membrane permeability and degradation by periplasmic beta-lactamases: a quantitative predictive treatment

In Gram-negative bacteria, beta-lactam antibiotics must overcome two barriers, the outer membrane and the periplasmic beta-lactamase, before they reach the targets of their action, penicillin-binding proteins. Although the barrier property of the outer membrane and catalytic property of the beta-lactamases have been studied and their significance in creating beta-lactam resistance emphasized, the interaction between these two barriers has not been treated quantitatively. Such treatment shows that the sensitivity, to a variety of beta-lactams, of the Escherichia coli K-12 cells containing very different levels of chromosomally coded AmpC beta-lactamase, or a plasmid-coded TEM-type beta-lactamase, can be predicted rather accurately from the penetration rate through the outer membrane and the hydrolysis rate in the periplasm. We further propose a new parameter, 'target access index', which is a quantitative expression of the result of interaction between the two barriers, and reflects the probability of success for the antibiotic to reach the targets.     

A search for beta-lactamase in chlamydiae, mycoplasmas, planctomycetes, and cyanelles: bacteria and bacterial descendants at different phylogenetic positions and stages of cell wall development

Bacteria from different phylogenetic positions such as chlamydiae, mycoplasmas, planctomycetes and also endosymbiotic murein-containing cyanelles were investigated for the production of beta-lactamases. No beta-lactamase activity was found in bacteria lacking murein such as Chlamydia pneumoniae, Mycoplasma pneumoniae, Pirellula marina and Planctomyces maris. In the murein-containing cyanelles of Cyanophora paradoxa no beta-lactamase activity could be detected.     

Co-existence of beta-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 beta-lactamase. A copper reducing assay, which was sensitive in the analytical range 2-20 micrograms/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 beta-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 beta-lactamases were constitutive enzymes in those organisms where both enzymes co-existed. Bacillus subtilis and B. cereus produced inducible and extracellular beta-lactamases. Acinetobacter calcoaceticus ATCC 21288 produced a constitutive beta-lactamase which was detected extracellularly.     

Guides for rational use of B-lactam antibiotics:resistance mechanism and clinical interpretation

beta-lactams are the antibiotic compounds most widely used against hospital and community acquired infections. However, resistance has emerged in both Gram-positive and Gram-negative bacteria, limiting their therapeutic efficacy. The choice of appropriate treatment depends on analysis of susceptibility data that indicates a specific mechanism of resistance. Correct interpretation of susceptibility tests permits a rational approach to the resistance problem and selection of alternatives for treatment. The laboratory must first be able to identify accurately microorganisms to the species level and then test a minimum of relevant antimicrobials. beta-lactam resistance in Enterobacteriaceae is mainly due to the production of plasmid or chromosomal encoded beta-lactamases. In Gram-negative non-fermenting bacteria, impermeability and efflux are relatively more important to the treatment selected. In Gram-positive bacteria, resistance mechanisms can involve changes in penicillin-binding proteins (PBPs), production of new PBPs or synthesis of beta-lactamases. The range of therapeutic options must be based on the current status of local resistance mechanisms.     

Unexpected advanced generation cephalosporinase activity of the M69F variant of SHV beta-lactamase

Infections with bacteria that contain hydrolytic beta-lactamase enzymes are becoming a serious problem in the United States. Mutations at Met-69, an amino acid proximal to the active site Ser-70 in the TEM-1 and SHV-1 beta-lactamases, have emerged as a puzzling cause of bacterial resistance to inhibitors of beta-lactamases. Site-saturation mutagenesis of the 69 position in SHV beta-lactamase was performed to determine how mutations of this non-catalytic residue play a role in increasing 50% inhibitory concentrations (IC(50) concentrations) for clinically important beta-lactamase enzyme inhibitors. Two distinct phenotypes are evident in the variant beta-lactamases studied: significantly increased minimum inhibitory concentrations (microg/ml) and IC(50) concentrations to clavulanic acid for the Met69Ile, Leu, and Val substitutions, and unanticipated increased minimum inhibitory concentrations and hydrolytic activity toward ceftazidime, an advanced generation cephalosporin antibiotic, for the Met69Lys, Tyr- and Phe-substituted enzymes. Molecular modeling studies emphasize the conserved structure of these substitutions despite great variation in substrate specificity. This study demonstrates the key role of Met-69 in defining substrate specificity of SHV beta-lactamases and alerts us to new phenotypes that may emerge clinically.     

Physiological studies of the regulation of beta-lactamase expression in Pseudomonas maltophilia.

The kinetics of beta-lactamase induction in Pseudomonas maltophilia IID1275/873 were investigated. Upon induction with beta-lactam antibiotics, a correlation was seen between the increase in specific beta-lactamase activity and the generation time, as well as the concentration of inducer in the medium. The specific beta-lactamase activity increased slowly within the first 0.5 generation and then more rapidly; it decreased regularly after about 2 generations of growth in the presence of inducer. This decrease could presumably be attributed to the continuous breakdown of inducer by beta-lactamases in the culture medium. In a chemostat culture with continuous supply of fresh inducer-containing medium, the specific beta-lactamase activity could be stabilized at a high level over several generations. Removal of the beta-lactam after a certain induction time showed that a short exposure of the bacteria to inducer caused induction kinetics comparable to those resulting from continuous exposure of the cells to inducer. The two beta-lactamases of P. maltophilia, L1 and L2, were induced simultaneously under various experimental conditions.     

Relation of R Factor and Chromosomal β-Lactamase with the Periplasmic Space

The release of several R factor and chromosomal beta-lactamases by osmotic shock treatment was studied. It was found that those beta-lactamases with a molecular weight of about 20,000 were released, but those with a molecular weight of about 30,000 to 44,000 were not released during osmotic shock. This differential release did not depend on whether the structural genes were on the chromosome or on the genome of an R factor. The release or retention of the beta-lactamases appeared to be a characteristic of the enzyme rather than the host cell since the same results were obtained when the R factors were harbored by a variety of host bacteria. Studies with bacteria which produced more than one beta-lactamase showed that each enzyme reacted independently to the presence of other beta-lactamases produced by the host bacterium.     

Sensitivity of 5 beta-lactam antibiotics to beta-lactamase isolated from E. coli and its respective transconjugates

54 beta-lactamase producing E. coli were tested to observe their eventual capacity to transfer beta-lactamase production by conjugation to a receiving E. coli K12 C600 Na-. About 16% (9/54) of these strains transferred beta-lactamase producing capacity. MICs of five beta-lactam antibiotics (Ampicillin, Cephaloridine, Cephalexine, Cefuroxime, Cefotaxime) were performed against E. coli donors and E. coli K12 C600 transconjugates. It was observed a remarkable increase only of Ampicillin MICs against all transconjugates++. Beta-lactamases produced by donors and transconjugants were isolated and purified by sonication and high speed centrifugation. Sensitivity of the six antibiotics to these purified beta-lactamases was assessed by a spectrophotometric method that utilizes the velocity of cytochrome c reduction. beta-lactamases produced by transconjugants have identical substrate profile that beta-lactamases produced by donors.     

A new family of cyanobacterial penicillin-binding proteins. A missing link in the evolution of class A beta-lactamases

It is largely accepted that serine beta-lactamases evolved from some ancestral DD-peptidases involved in the biosynthesis and maintenance of the bacterial peptidoglycan. DD-peptidases are also called penicillin-binding proteins (PBPs), since they form stable acyl-enzymes with beta-lactam antibiotics, such as penicillins. On the other hand, beta-lactamases react similarly with these antibiotics, but the acyl-enzymes are unstable and rapidly hydrolyzed. Besides, all known PBPs and beta-lactamases share very low sequence similarities, thus rendering it difficult to understand how a PBP could evolve into a beta-lactamase. In this study, we identified a new family of cyanobacterial PBPs featuring the highest sequence similarity with the most widespread class A beta-lactamases. Interestingly, the Omega-loop, which, in the beta-lactamases, carries an essential glutamate involved in the deacylation process, is six amino acids shorter and does not contain any glutamate residue. From this new family of proteins, we characterized PBP-A from Thermosynechococcus elongatus and discovered hydrolytic activity with synthetic thiolesters that are usually good substrates of DD-peptidases. Penicillin degradation pathways as well as acylation and deacylation rates are characteristic of PBPs. In a first attempt to generate beta-lactamase activity, a 90-fold increase in deacylation rate was obtained by introducing a glutamate in the shorter Omega-loop.     

Characterization of eight beta-lactamases of Gram-negative bacteria

Eight kinds of beta-lactamases produced by gram-negative bacteria were characterized by the following properties: molecular weight, isoelectric point, pH optimum, molecular activity, immunochemical reactivity, and kinetic parameters with respect to twelve kinds of common beta-lactam antibiotics. These beta-lactamases included two types of penicillinases mediated by R plasmids and six kinds of species-specific cephalosporinases. To determine a reliable value of the kinetic parameter, Km, we introduced a continuous and acidimetric assay method of beta-lactamase activity with a pH stat.     

In vitro activity of beta-lactam antibiotics and their sensitivity to beta-lactamase

beta-lactamase production was evaluated by chromogenic cephalosporin 87/312 in 116 E. coli isolated from clinical sources. Such test revealed beta-lactamase production in 54 strains out of 116 (46%): MICs of eight beta-lactam antibiotics (Ampicillin, Piperacillin, Cefazoline, Cephaloridine, Cephalexine, Cefuroxime, Cefotaxime, Cefotaxime) were determined using a miniaturized dilution broth method. Cefotaxime and Ceftriaxome and Ceftriaxone showed the highest antibacterial activity. All beta-lactamases produced by E. coli strains under examination were isolated and purified by ultrasonic disruption and high speed centrifugation. Sensitivity of the eight antibiotics to purified beta-lactamases was assessed by a spectrophotometric method that utilizes the velocity of cytochrome c reduction. The sensitivity to beta-lactamases was reflected in the in vitro activity of the antibiotics as assessed by the determination of the MICs.     

Types of beta-lactamase determined by plasmids in gram-negative bacteria.

Two species of beta-lactamase determined by plasmids in enteric bacteria that show some resemblance to TEM enzymes are described. Both are distinct from all other plasmid-mediated beta-lactamases and differ from the TEM beta-lactamases in ability to hydrolyze some substrates, in isoelectric point, in immunological specificity, and in susceptibility to inhibition. One of the enzyme species, mediated by plasmid p453, has been briefly described previously. We have discovered that this beta-lactamase, designated SHV-1, is unique in its response to inhibition by the sulfhydryl group reagent p-chloromercuribenzoate, because the hydrolysis of cephaloridine but not that of benzylpenicillin is affected. This enzyme is found in a variety of plasmid types which were transferred from several bacterial species collected from a wide geographic range. The other enzyme species is novel; only a single plasmid determining this kind of beta-lactamase (designated HMS-1) has been detected.     

Molecular characterization of TEM-type beta-lactamases identified in cold-seep sediments of Edison Seamount (south of Lihir Island, Papua New Guinea)

To determine the prevalence and genotypes of beta-lactamases among clones of a metagenomic library from the cold-seep sediments of Edison seamount (10,000 years old), we performed pulse-field gel electrophoresis, antibiotic susceptibility testing, pI determination, and DNA sequencing analysis. Among the 8,823 clones of the library, thirty clones produced beta-lactamases and had high levels of genetic diversity. Consistent with minimum inhibitory concentration patterns, we found that five (16.7%) of thirty clones produced an extended-spectrum beta-lactamase. 837- and 259-bp fragments specific to blaTEM genes were amplified, as determined by banding patterns of PCR amplification with designed primers. TEM-1 was the most prevalent beta-lactamase and conferred resistance to ampicillin, piperacillin, and cephalothin. TEM-116 had a spectrum that was extended to ceftazidime, cefotaxime, and aztreonam. The resistance levels conferred by the pre-antibiotic era alleles of TEM-type beta-lactamases were essentially the same as the resistance levels conferred by the TEM-type alleles which had been isolated from clinically resistant strains of bacteria of the antibiotic era. Our first report on TEM-type beta-lactamases of the pre-antibiotic era indicates that TEM-type beta-lactamases paint a picture in which most of the diversity of the enzymes may not be the result of recent evolution, but that of ancient evolution.