Allergy to penicillin: fable or fact?

OBJECTIVE--To assess whether, on the basis of one blood test, penicillin allergy might be excluded sufficiently for general practitioners to give oral penicillin to patients claiming a history of penicillin allergy. DESIGN--Prospective study of patients referred by general practitioners. SETTING--Outpatient allergy clinic in a district general hospital. PATIENTS--175 referred patients who gave a history of immediate type reaction to penicillin, of whom 144 attended as requested and 132 completed the investigations. MAIN OUTCOME MEASURES--History and examination, serum radioallergosorbent test to phenoxymethylpenicillin and benzylpenicillin, and oral challenge with penicillin. RESULTS--Of 132 patients, four were confirmed to have penicillin allergy by the radioallergosorbent test and 128 had an oral penicillin challenge without ill effect. CONCLUSIONS--Most patients who gave a history of penicillin allergy are not so allergic, and their actual allergic state should be substantiated whenever feasible. For patients reporting minor or vague reactions negative findings with a radioallergosorbent test to phenoxymethylpenicillin and benzylpenicillin provide sufficient evidence to give oral penicillin safely.     

Why did the Fleming strain fail in penicillin industry?

Penicillin, discovered 75 years ago by Sir Alexander Fleming in Penicillium notatum, laid the foundations of modern antibiotic chemotherapy. Early work was carried out on the original Fleming strain, but it was later replaced by overproducing strains of Penicillium chrysogenum, which became the industrial penicillin producers. We show how a C(1357)-->T (A394V) change in the gene encoding PahA in P. chrysogenum may help to explain the drawback of P. notatum. PahA is a cytochrome P450 enzyme involved in the catabolism of phenylacetic acid (PA; a precursor of penicillin G). We expressed the pahA gene from P. notatum in P. chrysogenum obtaining transformants able to metabolize PA (P. chrysogenum does not), and observing penicillin production levels about fivefold lower than that of the parental strain. Our data thus show that a loss of function in P. chrysogenum PahA is directly related to penicillin overproduction, and support the historic choice of P. chrysogenum as the industrial producer of penicillin.     

A microplate assay for the coupled transglycosylase–transpeptidase activity of the penicillin binding proteins; a vancomycin-neutralizing tripeptide combination prevents penicillin inhibition of peptidoglycan synthesis

A microplate, scintillation proximity assay to measure the coupled transglycosylase–transpeptidase activity of the penicillin binding proteins in Escherichia coli membranes was developed. Membranes were incubated with the two peptidoglycan sugar precursors UDP-N-acetyl muramylpentapeptide (UDP-MurNAc(pp)) and UDP-[³H]N-acetylglucosamine in the presence of 40 μM vancomycin to allow in situ accumulation of lipid II. In a second step, vancomycin inhibition was relieved by addition of a tripeptide (Lys-d-ala-d-ala) or UDP-MurNAc(pp), resulting in conversion of lipid II to cross-linked peptidoglycan. Inhibitors of the transglycosylase or transpeptidase were added at step 2. Moenomycin, a transglycosylase inhibitor, had an IC₅₀ of 8 nM. Vancomycin and nisin also inhibited the assay. Surprisingly, the transpeptidase inhibitors penicillin and ampicillin showed no inhibition. In a pathway assay of peptidoglycan synthesis, starting from the UDP linked sugar precursors, inhibition by penicillin was reversed by a ‘neutral’ combination of vancomycin plus tripeptide, suggesting an interaction thus far unreported.     

Short chain α-pyrones capable of potentiating penicillin G against Pseudomonas aeruginosa

The dramatic increase in bacterial resistance over the past three decades has greatly reduced the effectiveness of nearly all clinical antibiotics, bringing infectious disease to the forefront as a dire threat to global health. To combat these infections, adjuvant therapies have emerged as a way to reactivate known antibiotics against resistant pathogens. Herein, we report the evaluation of simplified α-pyrone adjuvants capable of potentiating penicillin G against Pseudomonas aeruginosa, a Gram-negative pathogen whose multidrug-resistant strains have been labeled by the Centers for Disease Control and Prevention as a serious threat to public health.     

A boronic-acid-based probe for fluorescence polarization assays with penicillin binding proteins and β-lactamases

Penicillin binding proteins (PBPs) and β-lactamases are involved in interactions with β-lactam antibiotics connected with both antibacterial activity and mediation of bacterial β-lactam resistance. Current methods for identifying inhibitors of PBPs and β-lactamases can be inefficient and are often not suitable for studying weakly and/or reversibly binding compounds. Therefore, improved ligand binding assays for PBPs and β-lactamases are needed. We report the development of a fluorescence polarization (FP) assay for PBPs and "serine" β-lactamases using a boronic-acid-based, reversibly binding "tracer." The tracer was designed based on a crystal structure of a covalent complex between a boronic acid and PBP1b from Streptococcus pneumoniae. The tracer bound to three different PBPs with modest affinity (K(d)=4-12 μM) and more tightly to the TEM1 serine β-lactamase (K(d)=109 nM). β-Lactams and other boronic acids were able to displace the tracer in competition assays. These results indicate that fluorescent boronic acids are suited to serve as reversibly binding tracers in FP-based assays with PBPs and β-lactamases and potentially with other related enzymes.     

5.5 Å crystallographic structure of penicillin β-lactamase and radius of gyration in solution

An electron density map of crystalline R-TEM Escherichia coli β-lactamase (penicillinase) has been calculated from X-ray diffraction data at 5.5 Å resolution with protein phases based on Friedel mates from a high-quality samarium derivative. The mean figure of merit for 854 independent reflections is 0.75. The monomeric molecule is slightly ellipsoidal and contains one and possibly two regions of α-helix which are 25 Å long. The Crystallographic search for the substrate binding site has so far been inconclusive. The radius of gyration of the enzyme in solution at pH 7 is 17.1 ± 1.0 Å from small-angle X-ray scattering measurements. This compares with 18.6 å calculated from the low-resolution electron density map of the molecule in the crystal.     

Different roads to discovery; Prontosil (hence sulfa drugs) and penicillin (hence β-lactams)

The important chemotherapeutic agents, Prontosil and pentenylpenicillin (penicillin F), were investigated initially by two men, Domagk and Fleming, who had been influenced by the horrendous wound infections of World War I. The very different pathways leading to their development and to that of the successor antibacterials (sulfa drugs, further penicillins, semi-synthetic penicillins), including the role played by patents, are discussed.     

Use of α-aminoadipic acid for the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp

1. l-alpha-Amino[6-(14)C]adipic acid has been prepared from the dl-amino acid by oxidation of the l-isomer with l-amino acid oxidase to alpha-oxo[6-(14)C]adipic acid and by transamination of the latter with l-glutamic acid in an extract of a Cephalosporium sp. prepared by ultrasonic treatment of the mycelium. 2. The optical configuration of small amounts of (14)C-labelled alpha-aminoadipic acid from the mycelium of the Cephalosporium sp. has been determined by treatment with l-amino acid oxidase and measurement of the proportion of radioactivity subsequently retained on a column of a strong cation-exchange resin. 3. alpha-Aminoadipic acid which had been labelled in the mycelium from [1-(14)C]acetate appeared to contain more than 99% of the l-isomer. 4. l-alpha-Amino[(14)C]adipic acid (sodium salt) was taken up much more rapidly than the d-isomer, or alpha-oxo[6-(14)C]adipic acid, by suspensions of washed mycelium of the Cephalosporium sp. in water. The pool of intracellular alpha-aminoadipic acid was expandable. 5. Intracellular products found to be labelled with (14)C from l-alpha-amino[(14)C]adipic acid were delta-aminovaleric acid, saccharopine, lysine, protein, compounds which behaved like penicillin N, cephalosporin C and deacetylcephalosporin C respectively on paper chromatography and electrophoresis, and a peptide whose amino acid residues include alpha-aminoadipic acid, cysteine and valine. 6. l-alpha-Amino[(14)C]adipic acid acted as a precursor of the delta-(d-alpha-aminoadipoyl) side chains of extracellular penicillin N and cephalosporin C. 7. (14)C from d-alpha-amino[(14)C]adipic acid was incorporated into penicillin N and cephalosporin C, but the incorporation was accompanied by a relatively high dilution of specific radioactivity and some l-alpha-amino[(14)C]adipic acid was found in the intracellular pool. 8. These findings are discussed in relation to the origin of the d- configuration of the alpha-aminoadipoyl side chain of the antibiotics.     

Immobilization of permeabilized whole cell penicillin G acylase from Alcaligenes faecalis using pore matrix crosslinked with glutaraldehyde

The activity of penicillin G acylase from Alcaligenes faecalis increased 7.5-fold when cells were permeabilized with 0.3% (w/v) CTAB. The treated cells were entrapped by polyvinyl alcohol crosslinked with boric acid, and crosslinked with 2% (v/v) glutaraldehyde to increase the stability. The conversion yield of penicillin G to 6-aminopenicillanic acid was 75% by immobilized system in batch reaction. No activity was lost after 15 cycles and about 65% enzyme activity was retained at the end of the 31th cycle.     

Changing glycine 21 for glutamic acid in the β-subunit of penicillin G acylase from Kluyvera citrophila prevents protein maturation

Summary Active penicillin acylase from Kluyvera citrophila strain ATCC 21 285 consists of two different -and -subunits derived from a single precursor by post-translational processing. Using the chemical mutagen hydroxylamine we have treated plasmid pYKD59 containing the active penicillin acylase gene (pga) from K. citrophila and have generated different point mutant penicillin acylase genes, one producing a muturation deficient precursor. This point mutation has changed the gylcine 310 residue of the precursor for a glutamic acid (residue number 21 of the mature -subunit). The introduction of a charged residue in this position did not prevent translocation of the precursor to the periplasm but the resultant molecule was not able to undergo subsequent post-translational modification to yield the active protein.     

Partial purification of penicillin acylase from Escherichia coli in poly(ethylene glycol)–sodium citrate aqueous two-phase systems

Studies on the partition and purification of penicillin acylase from Escherichia coli osmotic shock extract were performed in poly(ethylene glycol)–sodium citrate systems. Partition coefficient behavior of the enzyme and total protein are similar to those described in other reports, increasing with pH and tie line length and decreasing with PEG molecular weight. However, some selectivity could be attained with PEG 1000 systems and long tie line at pH 6.9. Under these conditions 2.6-fold purification with 83% yield were achieved. Influence of pH on partition shows that is the composition of the system and not the net charge of the enzyme that determines the behaviour in these conditions. Addition of NaCl to PEG 3350 systems significantly increases the partition of the enzyme. Although protein partition also increased, purification conditions were possible with 1.5 M NaCl where 5.7-fold purification and 85% yield was obtained. This was possible due to the higher hydrophobicity of the enzyme compared to that of most contaminants proteins.     

Influence of mass transfer limitations on the enzymatic synthesis of β-lactam antibiotics catalyzed by penicillin G acylase immobilized on glioxil-agarose

Mass transfer effects were investigated for the synthesis of ampicillin and amoxicillin, at pH 6.5 and 25 degrees C, catalyzed by penicillin G acylase immobilized on agarose. The influence of external mass transfer was analysed using different stirring rates, ranging form 200 to 800 rpm. Above 400 rpm, the film resistance may be neglected. Intra-particle diffusion limitation was investigated using biocatalysts prepared with different enzyme loads and agarose with different mean pore diameters. When agarose with 6, 8 and 10% of crosslinking were used, for the same enzyme load, substrates and products concentration profiles presented no expressive differences, suggesting pore diameter is not important parameter. An increase on enzyme load showed that when more than 90 IU of enzyme activity were used per mL of support, the system was influenced by intra-particle mass transfer. A reactive-diffusive model was used to estimate effective diffusivities of substrates and products.     

Synthesis of the penicillin precursor, δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV), by an immobilized enzyme preparation

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase activity has been partially-purified from cell-free extracts of Streptomyces clavuligerus by ammonium sulfate precipitation. The salt precipitated enzyme was immobilized on an anion exchange resin and synthesis of ACV was observed by exposing the immobilized enzyme preparation to a reaction mixture containing l--aminoadipic acid, l-valine and l-cysteine in the presence of appropriate cofactors. Reaction mixtures containing l--aminobutyric acid(aB) in place of l-valine synthesized the ACV analog ACaB. Immobilized ACV synthetase can be reused, and after six cycles of reaction, 28.9% of original activity remains.     

Enzymatic analysis of the effect of naturally occurring Leu138Pro mutation identified in SHV β-lactamase on hydrolysis of penicillin and ampicillin

Background  

The aim of this study was to analyze the significance of leucine to proline substitution at position 138(Leu138Pro) on the hydrolysis of penicillin and ampicillin that we identified in the bla _SHV gene of clinical Escherichia coli swine isolate.