A practical design of an elution system for preparative electrophoresis in polyacrylamide gel.

The previously applied direct spectrophotometric assay of β-lactamase activity toward cephalosporins was found readily applicable to penicillins too. The differential uv absorption spectra of various penicillins and their corresponding penicilloic acids were determined. The appropriate experimental conditions were examined and the spectrophotometric assay seems adequate for the study of several substrates in a mixture. Also this method was found highly suitable for computerized analysis of the kinetic data for the determination of Michaelis constants of the various penicillins. The use of the integrated form of the rate equation for the evaluation of the best estimates of Michaelis constants was found advantageous.     

Isolation and characterization of the acetyl-CoA synthetase from Penicillium chrysogenum. Involvement of this enzyme in the biosynthesis of penicillins.

Acetyl-CoA synthetase (ACS) of Penicillium chrysogenum was purified to homogeneity (745-fold) from fungal cultures grown in a chemically defined medium containing acetate as the main carbon source. The enzyme showed maximal rate of catalysis when incubated in 50 mM HCl-Tris buffer, pH 8.0, at 37 degrees C. Under these conditions, ACS showed hyperbolic behavior against acetate, CoA, and ATP; the Km values calculated for these substrates were 6.8, 0.18, and 17 mM, respectively. ACS recognized as substrates not only acetate but also several fatty acids ranging between C2 and C8 and some aromatic molecules (phenylacetic, 2-thiopheneacetic, and 3-thiopheneacetic acids). ATP can be replaced by ADP although, in this case, a lower activity was observed (37%). ACS in inhibited by some thiol reagents (5,5'-dithiobis(nitrobenzoic acid), N-ethylmaleimide, p-chloromercuribenzoate) and divalent cations (Zn2+, Cu2+, and Hg2+), whereas it was stimulated when the reaction mixtures contained 1 mM dithiothreitol, reduced glutathione, or 2-mercaptoethanol. The calculated molecular mass of ACS was 139 +/- 1 kDa, and the native enzyme is composed of two apparent identical subunits (70 kDa) in an alpha 2 oligomeric structure. ACS activity was regulated "in vivo" by carbon catabolite inactivation when glucose was taken up by cells in which the enzyme had been previously induced. This enzyme can be coupled "in vitro" to acyl-CoA:6-aminopenicillanic acid acyltransferase from P. chrysogenum, thus allowing the reconstitution of the functional enzymatic system which catalyzes the two latter reactions responsible for the biosynthesis of different penicillins. The ACS from Aspergillus nidulans can also be coupled to 6-aminopenicillanic acid acyltransferase to synthesize penicillins. These results strongly indicate that this enzyme can catalyze the activation (to their CoA thioesters) of some of the side-chain precursors required in these two fungi for the production of several penicillins. All these data are reported here for the first time.     

The Pharmacology of Semisynthetic Antibiotics

The chemical structures and reactions of penicillins and cephalosporins are reviewed in relation to their effects upon pharmacodynamic properties. The reactive betalactam ring is common to all penicillins and cephalosporin C analogues. This ring opens during acylation of the bacterial wall-building enzymes, but previous opening of the ring by acid or beta-lactamases destroys antibiotic activity.Semisynthetic substitutions may protect the ring by steric hindrance; this may actually inactivate certain penicillinases, so that resistant penicillins may potentiate penicillin G in some circumstances. However, the protective substitutions reduce the intrinsic activity of the synthetic penicillins themselves. Other properties which are affected include absorption, protein-binding, excretion, and possible allergenicity of the drugs. Effects on antibacterial spectrum may possibly be secondary to alteration of lipid solubility.     

State of the blood coagulating system in the treatment of puerperal infections using semisynthetic penicillins and cephaloridine]

The effect of some semi-synthetic penicillins, such as methicillin and oxacillin and cephalosporings, such as cephaloridin on the condition of the blood coagulation system in 85 patients with postnatal mastitis and endomyometritis was studied. It was shown that the above antibiotics had no significant effect on the parameters of the blood coagulating system. A decrease in the fibrinogen level was found in the mastitis patients treated with semi-synthetic penicillins and cephaloridin. An increase in the prothrombins index in the patients treated with methicillin oxacillin was shown. The use of the semi-synthetic penicillins and cephaloridin for the therapy of the puerpera with endomyometritis resulted in prolongation of the recalcification period. A decrease in the fibrinolytic activity most pronounced in therapy with cephaloridin was also found. Therefore, the above changes in the blood coagulating system of the puerpera with postnatal infections subjected to short-term treatment courses were insignificant and required no special correction.     

Correlation Between Growth Inhibition and the Binding of Various Penicillins and Cephalosporins to Staphylococcus aureus

The concentration of penicillin (or cephalosporin) required to achieve a given rate of binding to Staphylococcus aureus H correlates well with that required for inhibition of growth. This result suggests that the irreversible binding of penicillins and cephalosporins to cells is responsible for their biological activity.     

Antipseudomonal Activity of α-Sulfoaminopenicillins

A series of penicillins characterized by the presence of a sulfoamino or a modified sulfoamino group in the side chain was subjected to in vitro antimicrobial screening tests. Although the most potent members of the series were less active than benzylpenicillin against gram-positive bacteria and comparably active against most gram-negative bacteria, they were, on the average, 8 to 16 times more effective against strains of Pseudomonas aeruginosa. In other comparative laboratory tests against P. aeruginosa, these compounds were about as active as carbenicillin and four to eight times more active than ampicillin. An examination of structure-activity relationships indicated that maximal potency was obtained with penicillins having an alpha-(aromatic or heteroaromatic)-alpha-sulfoaminoacetamido side chain. The compound with an alpha-phenyl group was comparable in activity to those having an alpha-(2- or 3-thienyl) group, whereas any modification in position or structure of the alpha-sulfoamino group reduced activity. Results of studies with a cell-free P. aeruginosa beta-lactamase suggest that the marked inhibitory effects of alpha-sulfoamino penicillins for P. aeruginosa can be attributed, at least in part, to their high degree of resistance to this enzyme. Some derivatives, however, had weak antipseudomonal activity, despite possessing a high degree of beta-lactamase resistance.     

Beta-lactamase activity of bacteria of the genus Proteus]

beta-Lactamases of Proteus and their role in the mechanism of the microbe resistance to penicillins and ceporin were studied. It was found that the beta-lactamase of Proteus had low activity and were produced by both beta-lactamide resistant and sensitive clinical strains of Proteus. The resistant cultures of Proteus produced enzymes more frequently (3.4--5 times) than the sensitive ones. The synthesis of beta-lactamase in the clinical Proteus strains was inducable. The high induction coefficient was achieved only in the presence of high concentrations of the inductor. No significant dependence of the culture sensitivity level of ampicillin and ceporin on the induction level was observed. The most significant part of the constitutive enzyme in Proteus was intracellular, while that of the inducable enzyme was extracellular. No correlative dependence between the culture resistance levels to penicillins and ceporin and the enzyme activity was noted. The beta-lactamase activity was not found in the transconjugants with the in vitro acquired R-factor controlling the ampicillin and ceporin resistance, as well as in the resistant mutants selected on the media with increasing concentrations of the above antibiotics. Induction of beta-lactamase synthesis was not found in these strains either. The ability of Proteus to synthesize beta-lactamase can be lost on the strain storage under laboratory conditions which was not always accompanied by reduction of the culture sensitivity to ampicillin and ceporin. The enzymatic destruction of beta-lactamides was not the main mechanism of Proteus resistance to the above antibiotics.     

Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases.

Site-saturation mutagenesis was performed on the class A ROB-1 beta-lactamase at conserved Ser130, which is centrally located in the antibiotic binding site where it can participate in both protein-protein and protein-substrate hydrogen bonding. Mutation Thr130 gave a beta-lactamase hydrolysing penicillins and cephalosporins but which showed a 3-fold lower affinity (Km) for ampicillin and cephalexin, and a 30-fold lower hydrolytic (Vmax) activity for ampicillin. In contrast, the hydrolytic activity for cephalexin was similar to the wild-type for the Thr130 mutation. Mutation Gly130 gave a beta-lactamase hydrolysing only penicillins with an affinity and hydrolysis activity for these compounds approximately 15-fold lower than the wild-type, but no detectable activity against cephalosporins. Mutation Ala130 produced an enzyme capable of hydrolysing penicillins only at a low rate. Modelling the ROB-1 active site was done from the refined 2 A X-ray structure of the homologous Bacillus licheniformis beta-lactamase. Ampicillin and cephalexin were docked into the active site and were energy minimized with the CVFF empirical force field. Dockings were stable only when Ser70 was made anionic and Glu166 was made neutral. Interaction energies and distances were calculated for fully hydrated pre-acylation complexes with the Ser, Thr, Gly and Ala130 enzymes. The catalytic data from all mutations and the computed interactions from modelling confirmed that the Ser130 has a structural as well as a functional role in binding and hydrolysis of penicillins. This highly conserved residue also plays a substrate specificity role by hydrogen binding the carboxylic acid group of cephalosporins more tightly than penicillins.     

Effect of beta-lactam antibiotics on lipid bilayer membranes. II. Cephalosporin antibiotics

Interaction of cephalosporin antibiotics with flat bilayer lipid membranes (BLM) composed of vegetative or bacterial phospholipids was studied with respect to their effect on electroconductivity, capacity for binding to the bilayers and capacity for transmembrane transfer through the bilayers. By their effect on conductivity, the investigated cephalosporins could be divided into three groups: those having no effect on conductivity at the maximum concentrations (up to 1 mg/ml) i.e. cefoperazone, cefotaxime and ceftizoxime, those having an effect on conductivity at concentrations of 0.2 to 0.5 mg/ml i.e. cephalexin and cephalothin and those having a significant effect on conductivity at concentrations of 10 to 60 micrograms/ml i.e. N-acyl derivative of 7-ACA (substance 64) and 7-ACA derivative (substance 71). The cephalosporins had a capacity for binding to the bilayers and for incorporation into the bilayers. They penetrated to some extent through the bilayers composed of either vegetable or bacterial phospholipids. The lower rate of penetration through the BLM as compared to that of penicillins must be due to lower hydrophobicity of the cephalosporin molecules as compared to that of penicillins.     

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.     

Distribution of sulfanilamides and penicillins in the blood and organs of rats after their combined administration

The use of benzylpenicillin and ampicillin in combination with sulfalen or sulfadimethoxine increased the levels of the penicillins and sulfalen in some organs and tissues of rats. This was accompanied by a rise in the concentration gradients of the drugs. It is concluded that the combined use of the penicillins and sulfanilamides determines their increased penetration from the blood into other organs and tissues of the host.     

THE NEWER PENICILLINS

The newer penicillins give high promise of overcoming some of the few disadvantages of penicillin-G.They fall into three groups: The alpha-phenoxy-penicillins; the penicillinase resistant penicillins; and the penicillins with enhanced activity against gram-negative bacteria.The newer alpha-phenoxy-penicillins offer little over alpha-phenoxy methyl penicillin (penicillin-V). As the length of the side chain is increased, absorption and attainable serum concentration is also increased, but these are questionable benefits and probably not significant for therapeusis.The penicillinase-resistant penicillins have once more brought almost all severe staphylococcal infections within therapeutic range. One of them, methicillin, must be administered parenterally. It is the agent of choice for the treatment of severe, penicillin-G resistant staphylococcal infections, and this is its only clinical indication. Another, oxacillin, which may be administered orally, is partially resistant to gastric acid degradation, but must be given on an empty stomach. It is most useful as prolonged therapy following methicillin, in the treatment of mixed hemolytic streptococcal-penicillin-G resistant staphylococcal infections, and as primary therapy for moderately severe penicillin-G resistant staphylococcal infections.The third group is still mostly in the experimental stage, but some strains of Proteus, E. coli, Salmonella and Shigella are highly vulnerable to their action.Toxic and allergic reactions to the newer penicillins, and crossed allergic reactions with penicillin-G, present unsolved problems.     

Penicillin Acylase Activity of Penicillium chrysogenum

The penicillin acylase activity of Penicillium chrysogenum was studied. Washed mycelial suspensions of a high penicillin-producing and a nonproducing strain were found to be similar in respect to relative acylase activity on benzylpenicillin, 2-pentenylpenicillin, heptylpenicillin, and phenoxymethylpenicillin. The relative rates for both strains, as determined by 6-aminopenicillanic acid formation, were approximately 1.0, 2.5, 3.5, and 6.0 on the penicillins in the order given. The high producing strain formed both 6-aminopenicillanic acid and "natural" penicillins in fermentations to which no side-chain precursor had been added. Therefore, its demonstrated ability to cleave the natural penicillins, 2-pentenylpenicillin and heptylpenicillin, suggests that at least some of the 6-aminopenicillanic acid produced during such fermentations arises from the hydrolysis of the natural penicillins. At pH 8.5, the mycelial acylase activity of the nonproducing strain was about three times that at pH 6.0; at 35 C, it was about 1.5 times as active as it was at 30 C. When tested on penicillin G or V, no differences in either total or specific penicillin acylase activity were observed among mycelia harvested from cultures of the nonproducer to which penicillin G, penicillin V, or no penicillin had been added. Acetone-dried mycelium from both strains displayed acylase activity, but considerably less than that shown by viable mycelium. Culture filtrates were essentially inactive, although a very low order of activity was detected when culture filtrate from the nonproducer was treated with acetone and the acetone-precipitated material was assayed in a minimal amount of buffer.     

Comparison of assay techniques for beta-lactamase activity

Two chemical, one physical and two microbiological techniques for the assay of β-lactamase activity have been compared using three partially purified β-lactamases of different specificities. Seven cephalosporins and three penicillins with a wide range of susceptibilities have been used as substrate. The different methods gave results that were in general agreement with the exception of the hydroxylamine assay. The cephalosporins were most rapidly assayed by the uv method. Its suitability for use in the assessment of new cephalosporin analogues was confirmed, but it could not be used for penicillins. The iodometric assay was rapid and reliable but required large amounts of substrate. Where the amount of substrate available was limited, the microbiological techniques were preferred. The results obtained for enzyme activities against two of the cephalosporins and against the penicillins were higher with the hydroxylamine assay than with the other methods and it had no apparent advantage.     

Generation of high antimycotic activity during degradation of β-lactam antibiotics

Cephalosporins generated antifungal principle(s) during their degradation in aqueous solution though their antibacterial property is lost progressively during this process. The degradation product(s) from cephalexin, cefadroxil, cephacetril and cefazolin showed high activity against the dermatophytes of the genus Trichophyton, Microsporum , and Epidermophyton. Growth of a plant pathogen Macrophomina phaseolina was also weakly inhibited. Unlike the cephalosporins none of the penicillins tested produced such an antifungally active substance in aqueous solution. The nature of antimycotic activity generated from cefazolin was different from that produced from cephalexin, cefadroxil and cephacetril.     

β-Lactamase Formation and Resistance of Proteus morganii to Various Penicillins and Cephalosporins

Ten strains of Proteus morganii were selected and the production of β-lactamase was studied. The cellular level of β-lactamase from P. morganii was regulated by the concentration of the inducer in the growth media, and the enzyme activity appeared within 10 min while its highest value was obtained 2 hr after the addition of benzylpenicillin as the inducer. The resistance level to cephaloridine was generally related to the amount of β-lactamase activity among the ten strains of P. morganii, but no relation was found between the ampicillin susceptibilities and the amounts of β-lactamase in their cell-free preparations. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by a crude enzyme was five times higher than that of benzylpenicillin. Semisynthetic penicillins were resistant to hydrolysis and exhibited competitive inhibition. Among the semisynthetic penicillins, dicloxacillin and carbenicillin were powerful competitive inhibitors of the β-lactamase from P. morganii.     

Activity of Selected Penicillins In Vitro and in Experimental Bacterial Infections in Mice

The minimal inhibitory concentration and the 50% mouse protective dose for five key penicillins are presented. Such data provide a useful guide in evaluating the activity of new penicillins, in comparison with those already commercially available, and establishes the amounts of antibiotic necessary to obtain a PD(50) for several experimental model infections in mice.     

Interaction of zwitterionic penicillins with the OmpF channel facilitates their translocation

To study translocation of beta-lactam antibiotics of different size and charge across the outer bacterial membrane, we combine an analysis of ion currents through single trimeric outer membrane protein F (OmpF) porins in planar lipid bilayers with molecular dynamics simulations. Because the size of penicillin molecules is close to the size of the narrowest part of the OmpF pore, penicillins occlude the pore during their translocation. Favorably interacting penicillins cause time-resolvable transient blockages of the small-ion current through the channel and thereby provide information about their dynamics within the pore. Analyzing these random fluctuations, we find that ampicillin and amoxicillin have a relatively high affinity for OmpF. In contrast, no or only a weak interaction is detected for carbenicillin, azlocillin, and piperacillin. Molecular dynamics simulations suggest a possible pathway of these drugs through the OmpF channel and rationalize our experimental findings. For zwitterionic ampicillin and amoxicillin, we identify a region of binding sites near the narrowest part of the channel pore. Interactions with these sites partially compensate for the entropic cost of drug confinement by the channel. Whereas azlocillin and piperacillin are clearly too big to pass through the channel constriction, dianionic carbenicillin does not find an efficient binding region in the constriction zone. Carbenicillin's favorable interactions are limited to the extracellular vestibule. These observations confirm our earlier suggestion that a set of high-affinity sites at the narrowest part of the OmpF channel improves a drug's ability to cross the membrane via the pore.     

Patterns of clometacillin circulation in the body of laboratory animals]

Distribution of clometacillin in mice, rats and rabbits was studied in comparison with some other penicillins. It was found that clometacillin was superior to all other penicillins used for comparison with respect to the circulation time in the blood after intravenous, intramusclar or intrastomach administration. As for the capacity for penetrating into the tissues from the blood, clometacillin was not inferior to ampicillin and benzylpenicillin and was superior to propicillin, though it was bound by the blood serum proteins to a greater extent than the other penicillins used for comparison.     

Achievements and problems from the view of a physician

Penicillin made possible the cure of many common, and also the most serious, infections, such as meningococcal meningitis and bacterial endocarditis, often with few or no sequelae. Endocarditis had been invariably fatal. Semisynthetic penicillins added new dimensions of convenience of administration and a broader spectrum in the presence of many beta-lactamases. A quantum step forward was permitted by the derivatives of cephalosporin C. Specific clinical advances were (1) the opportunity to use these in some penicillin-allergic patients, (2) activity against wider range of Gram-negative bacilli, (3) activity against Bacteroides fragilis (cefoxitin), (4) more complete renal excretion after oral cephalosporins than with oral penicillins, and (5) delayed renal excretion. Major remaining problems limiting beta-lactam use are (1) allergy, (2) resistant organisms, (3) relatively poor entry into the cerebrospinal fluid (especially of cephalosporins, (4) some nephrotoxicity, (5) local irritation of veins and tissues during administration, and (6) poor results in patients with agranulocytosis.