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Substantial concentrations of penicillin V potassium (PVK) have been found in livestock manure, soil, and wastewater effluents, which may pose potential threats to human health and contribute to the emergence of penicillin-resistant bacterial strains. In this study, bacterial strains capable of degrading PVK were isolated from sludge and characterized. Strain X-2 was selected for biodegradation of PVK. Based on morphological observations and 16S rRNA gene sequencing, strain X-2 was identified as an Ochrobactrum tritici strain. To enhance the PVK degradation ability of PVK, a whole-cell biodegradation process of Ochrobactrum tritici X-2 was established and optimized. In the whole-cell biodegradation process, the optimal temperature and pH were 30°C and 7.0, respectively. Under the optimized conditions, the degradation rate using 0.5 mg/ml PVK reached 100% within 3 h. During biodegradation, two major metabolites were detected: penicilloic acid and phenolic acid. The present study provides a novel method for the biodegradation of PVK using Ochrobactrum tritici strains, which represent promising candidates for the industrial biodegradation of PVK. IMPORTANCE Substantial concentrations of penicillin V potassium (PVK) have been found in the environment, which may pose potential threats to human health and contribute to the emergence of penicillin-resistant bacterial strains. In this study, antibiotic-degrading bacterial strains for PVK were isolated from sludge and characterized. Ochrobactrum tritici was selected for the biodegradation of PVK with high efficiency. To enhance its PVK degradation ability, a whole-cell biodegradation process was established and optimized using Ochrobactrum tritici The degradation rate with 0.5 mg/ml PVK reached 100% within 3 h. The potential biodegradation pathway was also investigated. To the best of our knowledge, the present study provides new insights into the biodegradation of PVK using an Ochrobactrum tritici strain, a promising candidate strain for the industrial biodegradation of β-lactam antibiotics., (Copyright © 2020 Wang et al.)

Penicillin V is a bacteriolytic β-lactam antibiotic drug. In the present work, we investigated the inhibitory effect of Penicillin V on the activity of mushroom tyrosinase for the first time. The molecular mechanism for the inhibition of tyrosinase by Penicillin V was investigated by means of kinetics analysis, fluorescence quenching and molecular docking techniques. The results showed that Penicillin V could inhibit both monophenolase and diphenolase activities with IC 50 of 16.6 ± 0.5 and 11.0 ± 0.2 mmol/L, respectively. The inhibitory type of Penicillin V on mushroom was mixed type, and the values of K I and K IS were 13.46 and 17.26 mmol/L, respectively. The fluorescence quenching and molecular docking showed that Penicillin V could form static interaction near the catalytic pocket of the enzyme to hinder the transportation of substrate to the active site, as well as reduce the copper plasticity for catalysis. Our results contributed to the usage of Penicillin V as a novel tyrosinase inhibitor with dual effect in field of antimicrobial and food preservation and could also provide guidance for the design of novel tyrosinase inhibitors.

(1) Background: Antibiotics are frequently used on farm animals, making animal husbandry a relatively large source of antibiotic pollution of the environment. The present study aims to isolate and acclimatize antibiotic-degrading bacterial strains for penicillin V potassium (PVK) from the contaminated soil of a pig farm. (2) Methods: Bacterial strains were isolated and acclimatized by continuous enrichment of cultures with PVK as the sole carbon source. The antibiotic susceptibility test, thiol mercury salt ultraviolet spectrophotometry (TMSUS), morphological observations, and 16S rDNA sequence analysis were used to identify and characterize the isolated strains. (3) Results: Four bacterial isolates (denoted as LM-1, LM-2, LM-3, LM-4) were obtained, and two of them (LM-1, LM-2) with the highest degradation rates were identified to belong to the same genera as Bacillus . These two isolates were found to be resistant to PVK antibiotic in an antibiotic sensitivity test. The TMSUS indicated that the strains LM-1 and LM-2 had good performance in PVK degradation (68% for LM-1, 66% for LM-2 in 48 h) when the initial PVK concentration was about 100 μg/mL. (4) Conclusions: Two bacterial strains isolated from the soil on a pig farm are effective in degrading PVK and can be potentially used for bioremediation of PVK antibiotic-contaminated soils.

The molecular recognition and binding interaction of beta-lactamase II from Bacillus cereus (Bc II) with penicillin V (PV) and sulbactam (Sul) at 277 K were studied by spectroscopic analysis and molecular docking. The results showed that a non-fluorescence static complex was separately formed between Bc II and two ligands, the molecular ratio of Bc II to PV or Sul was both 1:1 in the binding and the binding constants were 2.00 × 10 6 and 3.98 × 10 5 (L/mol), respectively. The negative free energy changes and apparent activation energies indicated that both the binding processes were spontaneous. Molecular docking showed that in the binding process, the whole Sul molecule entered into the binding pocket of Bc II while only part of the whole PV molecule entered into the pocket due to a long side chain, and electrostatic interactions were the major contribution to the binding processes. In addition, a weak conformational change of Bc II was also observed in the molecular recognition and binding process of Bc II with PV or Sul. This study may provide some valuable information for exploring the recognition and binding of proteins with ligands in the binding process and for the design of novel super-antibiotics., (Copyright © 2017 John Wiley & Sons, Ltd.)

The molecular recognition and interaction of beta-lactamase II from Bacillus cereus (Bc II) with penicillin V (PV) and sulbactam (Sul) especially conformational changes of Bc II in the binding process were studied through spectroscopy analysis in combination with molecular dynamics (MD) simulation. The results show that in the binding process, a new coordination bond is observed between the Zn 2 of Bc II and the carboxyl-O of PV or Sul by replacing His204. Electrostatic interaction between Zn 2 and the ligand provide main driving force for the binding affinity. Compared with apo Bc II, there are mainly four loops showing significant conformational changes in ligand-bound Bc II. A weak conformational transformation from β-sheets to random coils is observed in the loop2 of ligand-bound Bc II. The conformational transformation may depend on the functional group and binding pose of the ligand, giving the binding pocket greater flexibility and accordingly allowing for an induced fit of the enzyme-ligand binding site around the newly introduced ligand. The change in the loop2 of ligand-bound Bc II may lead to the opening of the binding pocket of Bc II. Therefore, loop2 can be considered a gate for control of ligand access in Bc II, hence its dynamic response should be considered in new drug design and development., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The degradation of penicillin G, penicillin V, and ampicillin in milk in the presence of β-lactamase was investigated by ultra-performance liquid chromatography coupled with electrospray ionization-time-of-flight mass spectrometry. Degradation products of the 3 penicillins in milk were identified based on the fact that the metabolites or degradation products contain a substructure of penicillin, and their degradation pathways in acidic milk in presence of β-lactamase were developed. The influence of factors on the degradation was investigated, including β-lactamase dosage, temperature, time, and acidity. The ratio of the 2 degradation products (penicilloic acid and penilloic acid) is different at different temperatures and pH. Penicilloic acid was the dominant species obtained at pH 6 under 40°C, but, being unstable, it could not be used as a standard for accurate analysis of penicilloic acid, and also could not be used as target for detection of penicillins in milk. Penilloic acid was the dominant species obtained at pH 2 above 40°C; it was stable and could be used as a standard for quantitative analysis and as target for detecting whether penicillins were used in milk., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Penicillin G and V have the same betalactam ring. Penicillin V (phenoxymethylpenicillin) results from the substitution of the phenyl acetic acid of benzylpenicillin by the phenoxy methyl side chain., Methods: Our patient was a 34-year-old man who experienced generalized urticaria after ingestion of Penicillin V. We performed skin prick tests and intradermal tests with a battery of betalactams including Penicillin V. We also determined specific IgE against penicillin V, penicillin G, amoxicillin, and ampicillin and performed a single-blind oral challenge with Penicillin V, amoxicillin, cefuroxime, and ceftazidime., Results: The results of skin prick and intradermal tests with the betalactams included were negative. Specific IgE with betalactams was < 0.10 IU/L. The result of a single-blind oral challenge with Penicillin V was positive: 40 minutes after receiving 125 mg of Penicillin V, the patient presented generalized pruritus with hives on his back and chest. He tolerated oral administration of amoxicillin, cefuroxime, and ceftazidime., Conclusion: We report an exceptional case of sensitization to Penicillin V with negative results in the allergy workup. Diagnosis was based on a positive single-blind oral challenge result. The patient tolerated other betalactams. We provide a brief summary of the most relevant recent patents.

In filamentous fungi, secondary metabolism is often linked with developmental processes such as conidiation. In this study we analyzed the link between secondary metabolism and conidiation in the main industrial producer of the β-lactam antibiotic penicillin, the ascomycete Penicillium chrysogenum. Therefore, we generated mutants defective in two central regulators of conidiation, the transcription factors BrlA and StuA. Inactivation of either brlA or stuA blocked conidiation and altered hyphal morphology during growth on solid media, as shown by light and scanning electron microscopy, but did not affect biomass production during liquid-submerged growth. Genome-wide transcriptional profiling identified a complex StuA- and BrlA-dependent regulatory network, including genes previously shown to be involved in development and secondary metabolism. Remarkably, inactivation of stuA, but not brlA, drastically downregulated expression of the penicillin biosynthetic gene cluster during solid and liquid-submerged growth. In agreement, penicillin V production was wild-type-like in brlA-deficient strains but 99% decreased in stuA-deficient strains during liquid-submerged growth, as shown by high-performance liquid chromatography (HPLC) analysis. Thus, among identified regulators of penicillin V production StuA has the most severe influence. Overexpression of stuA increased the transcript levels of brlA and abaA (another developmental regulator) and derepressed conidiation during liquid-submerged growth but did not affect penicillin V productivity. Taken together, these data demonstrate an intimate but not exclusive link between regulation of development and secondary metabolism in P. chrysogenum.

This paper investigates the accuracy of a matrix method proposed by other researchers to calculate time-varying flux control coefficients (dynamic FCCs) from elasticity coefficients by means of summation and connectivity theorems in the framework of metabolic control analysis. A mathematical model for the fed-batch penicillin V fermentation process is used as a case example for discussion. Calculated results reveal that this method produces significant calculation errors because the theorems are essentially valid only in steady state, although it may provide rough time-transient behaviors of FCCs. Strictly, therefore, dynamic FCCs should be directly calculated from the differential equations for metabolite concentrations and sensitivities., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Bulk of the penicillin produced is used as raw material for semi-synthetic penicillin (such as amoxicillin and ampicillin) and semi-synthetic cephalosporins (such as cephalexin and cefadroxil). In the present paper, an industrial penicillin V bioreactor train is optimized for multiple objectives simultaneously. An industrial train, comprising a bank of identical bioreactors, is run semi-continuously in a synchronous fashion. The fermentation taking place in a bioreactor is modeled using a morphologically structured mechanism. For multi-objective optimization for two and three objectives, the elitist non-dominated sorting genetic algorithm (NSGA-II) is chosen. Instead of a single optimum as in the traditional optimization, a wide range of optimal design and operating conditions depicting trade-offs of key performance indicators such as batch cycle time, yield, profit and penicillin concentration, is successfully obtained. The effects of design and operating variables on the optimal solutions are discussed in detail., (Copyright 2007 Wiley Periodicals, Inc.)

Uncertainty and variability affect economic and environmental performance in the production of biotechnology and pharmaceutical products. However, commercial process simulation software typically provides analysis that assumes deterministic rather than stochastic process parameters and thus is not capable of dealing with the complexities created by variance that arise in the decision-making process. Using the production of penicillin V as a case study, this article shows how uncertainty can be quantified and evaluated. The first step is construction of a process model, as well as analysis of its cost structure and environmental impact. The second step is identification of uncertain variables and determination of their probability distributions based on available process and literature data. Finally, Monte Carlo simulations are run to see how these uncertainties propagate through the model and affect key economic and environmental outcomes. Thus, the overall variation of these objective functions are quantified, the technical, supply chain, and market parameters that contribute most to the existing variance are identified and the differences between economic and ecological evaluation are analyzed. In our case study analysis, we show that final penicillin and biomass concentrations in the fermenter have the highest contribution to variance for both unit production cost and environmental impact. The penicillin selling price dominates return on investment variance as well as the variance for other revenue-dependent parameters., (Copyright 2004 Wiley Periodicals, Inc.)

The low penicillin-producing, single gene copy strain Wis54-1255 was used to study the effect of overexpressing the penicillin biosynthetic genes in Penicillium chrysogenum. Transformants of Wis54-1255 were obtained with the amdS expression-cassette using the four combinations: pcbAB, pcbC, pcbC-penDE, and pcbAB-pcbC-penDE of the three penicillin biosynthetic genes. Transformants showing an increased penicillin production were investigated during steady-state continuous cultivations with glucose as the growth-limiting substrate. The transformants were characterized with respect to specific penicillin productivity, the activity of the two pathway enzymes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthetase (IPNS) and the intracellular concentration of the metabolites: delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV), isopenicillin N (IPN), glutathione (GSH), and glutathione disulphide (GSSG). Transformants with the whole gene cluster amplified showed the largest increase in specific penicillin productivity (r(p))-124% and 176%, respectively, whereas transformation with the pcbC-penDE gene fragment resulted in a decrease in r(p) of 9% relative to Wis54-1255. A marked increase in r(p) is clearly correlated with a balanced amplification of both the ACVS and IPNS activity or a large amplification of either enzyme activity. The increased capacity of a single enzyme occurs surprisingly only in the transformants where all the three biosynthetic genes are overexpressed but is not found within the group of pcbAB or pcbC transformants. The indication of the pcbAB and pcbC genes being closely regulated in fungi might explain why high-yielding strains of P. chrysogenum have been found to contain amplifications of a large region including the whole penicillin gene cluster and not single gene amplifications. Measurements of the total ACV concentration showed a large span of variability, which reflected the individual status of enzyme overexpression and activity found in each strain. The ratio ACV:bisACV remained constant, also at high ACV concentrations, indicating no limitation in the capacity of the thioredoxin-thioredoxin reductase (TR) system, which is assumed to keep the pathway intermediate LLD-ACV in its reduced state. The total GSH pool was at a constant level of approx. 5.7 mM in all cultivations., (Copyright 2001 John Wiley & Sons, Inc.)

Late-exponential-phase Penicillium chrysogenum mycelia grown in a complex medium possessed an intracellular iron concentration of 650 mumol/L (2.2 +/- 0.6 mumol per g mycelial dry mass). This iron reserve was sufficient to ensure growth and antibiotic production after transferring mycelia into a defined low-iron minimal medium. Although the addition of Fe3+ to the Fe-limited cultures increased significantly the intracellular iron levels the surplus iron did not influence the production of penicillin V. Supplements of purified major P. chrysogenum siderophores (coprogen and ferrichrome) into the fermentation media did not affect the beta-lactam production and intracellular iron level. Neither 150 nor 300 mumol/L extracellular Fe3+ concentrations disturbed the glutathione metabolism of the fungus, and increased the oxidative stress caused by 700 mmol/L H2O2. Nevertheless, when iron was applied in the FeII oxidation state the oxidative cell injuries caused by the peroxide were significantly enhanced.

The transport-enhancing effects of medium-chain fatty acids (caproic, caprylic, and capric acids) and their acylglycerols (mono-, di-, and triacylglycerols) were investigated by using Caco-2 cell monolayers as a model of the human intestinal epithelium. Penicillin V was used as a model for a hydrophilic bioactive compound. Among the fatty acids and acylglycerols tested, 1,2-dicaproin, monocaprin, monocaprylin, and capric acid sodium salt effectively enhanced the transport rate, whereas other substances enhanced the rate only slightly or not at all. With each of these four substances, the rate of enhancement was proportional to the concentration at low concentrations, but leveled off at high concentrations. The transport-enhancing effects were well correlated with the reduction in surface tension and with a physico-chemical parameter, denoted by the surface energy-lowering coefficient, characterizing the surface activity of a substance.

A phenoxymethylpenicillin amidohydrolase which hydrolyses phenoxymethylpenicillin to 6-aminopenicillanic acid (6-APA) has been isolated from two species of Penicillium chrysogenum. The amidohydrolase had a molecular mass of approx. 42 kDa. Its activity with benzylpenicillin as substrate was only 1.5% of that with phenoxymethylpenicillin and it was inhibited by its products. No penicillin formation from 6-APA and phenoxyacetyl or phenylacetyl coenzyme A was observed. The enzyme is thus distinct from the phenylacetyl coenzyme A:6-APA acyltransferase, which also has amidohydrolase activity and is involved in the final stages of the biosynthesis of penicillins.

A high-performance liquid chromatographic method has been developed for the determination of penicillin-V concentrations between 0.1 and 19 micrograms/ml in human plasma. Penicillin-V was isolated from plasma by solid-phase extraction on a C18/OH cartridge. The extracts were injected onto a reversed-phase HPLC system. A 125 x 4 mm C18 column was used to separate penicillin-V from its main metabolites, 5R- and 5S-penicilloic acid and endogenous compounds. The eluent consisted of 66% 0.02 M phosphoric acid buffer, to which tetrabutylammonium dihydrogenphosphate and 34% acetonitrile were added. The column effluent was monitored by ultraviolet spectrophotometry at 269 nm. Using this method, penicillin-V concentrations in plasma could be determined with an accuracy between -5.4 and 5.2% and a precision between 0.8 and 1.6%. The method has proved to be reliable and was used in bioavailability studies for the development of a new oral penicillin-V formulation.

Metabolites (both intra- and extracellular) involved in penicillin biosynthesis were measured during fed-batch cultivations with a high-yielding strain of Penicillium chrysogenum. The fed-batch cultivations were carried out on a complex medium containing corn steep liquor. Three distinct phases were observed: (a) a rapid growth phase where free amino acids present in the medium are metabolized, (b) a linear growth phase, and (c) a stationary phase. The specific penicillin production (rp) is initially high and, during the rapid growth phase, it increases slightly. During the linear growth phase rp is approximately constant [4-6 mg penicillin V (g dry weight)-1 h-1 depending on the operating conditions], whereas it decreases during the stationary phase. During the cultivations the tripeptide Aad-Cys-Val (the first metabolite in penicillin biosynthesis) and 8-hydroxypenillic acid (formed by carboxylation of 6-aminopenicillanic acid, 6-APA) were found to accumulate in the medium, whereas the concentrations of isopenicillin N and 6-APA were found to be approximately constant and low. About 3% of the Aad-Cys-Val formed in the first step of the penicillin biosynthetic pathway is lost to the medium and 4% of the isopenicillin N formed in the second step of the pathway is lost as extracellular isopenicillin N, 6-APA or 8-hydroxypenillic acid. Also the cyclic form of alpha-aminoadipic acid, 6-oxo-piperidine-2-carboxylic acid, was found to accumulate in the medium and it was found to be formed in an approximately constant ratio to penicillin V of 6 mol/100 mol.

The hydrolysis of penicillin-V to phenoxyacetic acid and 6-aminopenicillanic acid by the fungal enzyme penicillin-V amidase is of industrial importance since the 6-aminopenicillanic acid produced is an intermediate for semisynthetic penicillins. A rapid colorimetric assay of penicillin-V amidase was developed which uses 2-nitro-5-(phenoxyacetamido)-benzoic acid as a substrate. The released chromophore, 2-amino-5-nitrobenzoic acid, was detected at 405 nm. Using penicillin-V amidase from the fungus Fusarium oxysporum, the KM and Vmax for this substrate were 0.89 mM and 2.6 mumol/min/mg enzyme, respectively. Hydrolysis could be competitively inhibited by penicillin-V with a Ki of 4 mM. The change in the initial velocity of hydrolysis of 2-nitro-5-(phenoxyacetamido)-benzoic acid at 500 microM was linear over the range of 0.5 to 10 micrograms/ml enzyme. These results show that this new compound is useful in determining the presence and levels of penicillin-V amidase.

The addition of 0.1 M L-lysine to the fermentation medium reduced the production of penicillin by about 50% in Aspergillus nidulans. To analyse this effect at the molecular level, the expression of the penicillin biosynthesis genes acvA and ipnA, encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase and isopenicillin N synthetase, was studied by using translational fusions with different reporter genes (strain AXB4A, acvA-uidA, ipnA-lacZ fusions; AXB4B, acvA-lacZ, ipnA-uidA fusions) integrated in single copy at the chromosomal argB locus of Aspergillus nidulans. Irrespective of the reporter genes used the expression of acvA and ipnA fusion genes was repressed in L-lysine grown cultures. The expression of a fusion gene of an A. nidulans primary metabolism gene (oliC-lacZ) was not affected by L-lysine.

By using whole-cell labeling assay with 125I-penicillin V, we observed a reduction in the binding of the radiolabeled beta-lactam to four or five penicillin-binding proteins (PBPs) in Haemophilus influenzae cells cultivated under specific conditions. PBPs 3A, 3B, 4, and 6 were altered after the growth of bacteria in diffusion chambers implanted in the peritoneal cavity of rats. PBP 2 was also modified when cells were cultivated in human cerebrospinal fluids. Because this observation may have important consequences on the efficacy of beta-lactams during antibiotic therapy, we characterized the physiological state of bacteria cultivated in animals in the hope of explaining how such important changes in cell properties develop in vivo. Since the development of natural genetic competence occurs at the stationary phase of growth in H. influenzae, we used a DNA transformation assay to evaluate the physiological state of bacteria grown in diffusion chambers implanted in rats. Chromosomal DNA isolated from an antibiotic-resistant donor strain was mixed with bacteria in diffusion chambers. At different times during a 5-h incubation period, recipient bacteria were collected from the chambers, CFU were determined by plate counting, and antibiotic-resistant transformants were isolated on selective plates. Genetic competence rapidly developed in cells grown in rats, and the frequency of transformation by test DNA was elevated. Electron microscopy revealed an irregular cell shape and blebs at the surface of bacteria cultivated in animals and in cerebrospinal fluids. In an attempt to induce a similar physiological state in vitro, we supplemented broth cultures with cyclic AMP or synchronized cultures by a nutritional upshift. No changes in PBPs were observed with supplemental cyclic AMP or during a single cell cycle. Finally, a reduction in the affinity of PBPs for 125I-penicillin V identical to that observed in bacteria grown in rats was observed in cells isolated from the stationary phase of growth in vitro. These results clearly indicate that H. influenzae cells grown in animals undergo a rapid change to a physiological state similar to that found in late-stationary-phase cultures in vitro. This observation indicates that the rational design of future and improved antibiotic therapy of H. influenzae infections should consider cell properties of slow-growing or latent bacteria.

Penicillin production with a high-producing strain Penicillium chrysogenum was investigated under well-controlled conditions in a stirred tank reactor with complex media containing lard oil and lactose on the one hand, and lactose on the other hand. With lard oil, cell growth and product formation rates were higher, and the production time was shorter by 40 h than without lard oil. On account of the longer production time without lard oil, the amount of beta-lactam compounds was higher (29.93 g l-1), but the mole fraction of the decomposed products (penicilloic acid and penilloic acid) was larger (0.282) than the amount of penicillin V (23.25 g l-1) and the decomposed mole fraction (0.0747) with lard oil. The final product concentrations were about the same (20.86 g l-1 or 35,462 IU ml-1 with lard oil, and 20.43 g l-1 or 34510 IU ml-1 without lard oil). The mole fractions of the by-product (p-OH-penicillin V) were 0.0365 and 0.066. The substitution of lard oil with lactose is possible without a considerable reduction of process performance.

The precise ultrastructural localization of penicillin-binding protein (PBP)-antibiotic complexes in Escherichia coli JM101, JM101 (pBS96), and JM101(pPH116) was investigated by high-resolution electron microscopy. We used mercury-penicillin V (Hg-pen V) as a heavy-metal-labeled, electron-dense probe for accurately localizing PBPs in situ in single bacterial cells grown to exponential growth phase. Biochemical data derived from susceptibility tests and bacteriolysis experiments revealed no significant differences between Hg-pen V and the parent compound, penicillin V, or between strains. Both antibiotics revealed differences in the binding affinities for PBPs of all strains. Deacylation rates for PBPs were slow despite the relatively low binding affinities of antibiotics. Cells bound most of the Hg-pen V added to cultures, and the antibiotic-PBP complex could readily be seen by electron microscopy of unstained whole mounts as distinct, randomly situated electron-dense particles. Fifty to 60% of the antibiotic was retained by cells during processing for conventional embedding so that thin sections could also be examined. These revealed similar electron-dense particles located predominantly on the plasma membrane and less frequently in the cytoplasm. Particles positioned on the plasma membranes were occasionally shown to protrude into the periplasmic space, thereby reflecting the high resolution of the Hg-pen V probe. Moreover, some particles were observed free in the periplasm, suggesting, for the first time, that a proportion of PBPs may not be restricted to the plasma membrane but may be tightly associated with the peptidoglycan for higher efficiency of peptidoglycan assembly. All controls were devoid of the electron-dense particles. The presence of electron-dense particles in cells of the wild-type JM101, demonstrated that our probe could identify PBPs in naturally occurring strains without inducing PBP overproduction.

The influence of stirrer speed in the third preculture on the performance of penicillin V production by Penicillium chrysogenum in complex medium in a 100-l air-lift tower loop reactor was investigated. The process performance in the main culture was improved by increasing the stirrer speed from 500 to 750 rpm: the pellet size was reduced to half, the cell growth was influenced only slightly, but the production phase was extended considerably, and the final penicillin concentration was increased from 5.1 g l-1 to 10.4 g l-1.

In order to analyze the mutual displacement effects on the protein binding of beta-lactam antibiotics, binding experiments with the human serum albumin (HSA) were performed for cephalothin (CET) and phenoxymethylpenicillin (PCV) by using the centrifugal ultrafiltration method. The numbers of primary and secondary binding sites, n1 and n2, and the affinity constants for the primary and secondary binding sites, K1 and K2 were determined for CET to be 1.00 +/- 0.06 (mean +/- S.D.) and 4.54 +/- 0.12 and 2.59 x 10(3) +/- 0.10 x 10(3) (M-1) and 2.59 x 10(2) +/- 0.16 x 10(2) (M-1), respectively, and for PCV to be 0.94 +/- 0.10 and 5.41 +/- 0.40 and 3.52 x 10(3) +/- 0.25 x 10(3) (M-1) and 4.07 x 10(2) +/- 0.54 x 10(2) (M-1), respectively. Using the predicted optimum unbound concentration of PCV, i.e., 4.6 x 10(-4) M, the displacement effect of PCV to the binding of CET at the primary site has been demonstrated, while no significant effect was observed at the secondary binding site. Moreover, a competitive displacement effect of CET was also demonstrated for the binding of PCV to HSA at the primary binding site, suggesting that CET and PCV bound to HSA at the same primary binding site.

The regularities of biosynthesis of 6-aminopenicillanic acid (6-APA), benzylpenicillin (BP) and phenoxymethylpenicillin (PMP) by the strains under the investigation did not significantly differ. In the absence of the precursor both the strains mainly synthesized 6-APA. Phenylacetic acid (PAA) and phenoxyacetic acid (POAA) provided directed biosynthesis: the fungus synthesized BP or PMP depending on the precursor nature. When the amount of the precursors was not sufficient, 6-APA was synthesized along with the penicillins. PAA proved to be a more active precursor than POAA. When both precursors were present in the fermentation broth, only BR was synthesized. An important distinction of strain 316A was its increased sensitivity to PAA especially in the initial period. After an increase in the PAA concentration the growth rate of strain 316A lowered to a greater extent than that of strain 284A. This was likely to determine the higher levels of penicillin production by strain 316A in the presence of POAA, a nontoxic precursor. A procedure for supplying the precursors was developed. Under the laboratory conditions it provided high levels of the penicillin production.

In 45 patients hospitalized with febrile erysipelas that had been treated with oral penicillin, punch biopsies of infected skin were performed and analysed for phenoxymethylpenicillin (pcV) concentrations. The curves for the concurrent serum and tissue levels followed the same course, indicating a rapid diffusion of pcV from serum into tissue. Penicillin concentrations in infected skin exceeded the minimal inhibitory concentrations (MIC) of the streptococci isolated for the first 4 h after tablet ingestion. Venous insufficiency was highly prevalent and pcV concentrations in affected patients were slightly but not significantly higher. A theoretical basis for the successful treatment of erysipelas with oral penicillin is therefore at hand.

Penicillin V (phenoxymethyl penicillin) is produced by industrial strains of Penicillium chrysogenum in the presence of phenoxyacetic acid (POAc), a side-chain precursor for the penicillin V molecule. The wild-type strain of P. chrysogenum produces an undesirable penicillin byproduct, para-hydroxypenicillin V (p-OH penicillin V), in addition to penicillin V, via para-hydroxylation of POAc and subsequent incorporation of the p-OH phenoxyacetic acid into the penicillin molecule. Most of the p-OH penicillin V is produced late in cycle when the POAc concentration in the medium is nearly depleted. The level of p-OH penicillin V produced by the control strain ranges up to 10-15% of the total penicillins produced. 3-Phenoxypropionic acid and p-bromophenylacetic acid partially inhibit the formation of p-OH penicillin V with a minimal effect on penicillin V productivity. Mutants deficient in their ability to hydroxylate POAc were found to produce lower levels of p-OH penicillin V. Multi-step mutation and screening, starting with the wild-type strain, have culminated in isolation of mutants which produce p-OH penicillin V as 1% of the total penicillins with no adverse effect on penicillin V productivity.

The penDE gene from Penicillium chrysogenum has been isolated; the gene is located in close vicinity of the pcbC gene. Amplification of the pcbC-penDE gene cluster in Penicillium chrysogenum Wis54-1255 leads to a significant increase in penicillin production. In selected transformants an increase of up to 40% is observed.

Wild-type strains of Penicillium chrysogenum produce lower penicillin V titers in media containing excess glucose. Two mutant strains were isolated and shown to produce normal penicillin V titers in the presence of excess glucose. These strains, designated as glucose-repression insensitive (GRI) mutants, produced higher penicillin V titers than the wild-type strain in media containing lactose as the main carbohydrate source. In lactose-based media, the production of penicillin V was depressed to a much lesser extent by in-cycle additions of glucose with the GRI mutants when compared to the wild-type strain. In short-term biosynthesis experiments using washed cells in a medium containing glucose as the sole carbon source, the GRI mutants produced penicillin V at a faster rate than the wild-type strain. In fed-batch fermentations in 14-liter fermentors, where glucose was fed continuously and pH controlled, both GRI mutants produced more than 10% higher penicillin V titers than the wild-type strain. These results suggest that isolation of GRI mutants is an effective way to select for higher producing strains and that the synthesis of penicillin synthesizing enzymes in GRI mutants may be less repressed by glucose than in wild-type strains.

"In vitro" synthesis of benzylpenicillin and phenoxymethylpenicillin has been carried out by direct N-acylation of 6-aminopenicillanic acid (6-APA) with S-phenylacetyl- and (S-phenoxyacetyl)glutathione. The reactions were catalyzed by the enzyme acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum and in both cases the synthesis of antibiotics was enhanced by CoA. Penicillin K, a natural penicillin, was also synthesized "in vitro" by incubating (S-octanoyl)glutathione, 6-APA and AT, but in this case the formation of antibiotic required the presence of CoA. Furthermore, benzylpenicillin was obtained from (S-phenylacetyl)cysteinylglycine and 6-APA, suggesting that some intermediates of the gamma-glutamyl cycle are directly involved in the biosynthesis of penicillins. To explain "in vivo" formation of this beta-lactam antibiotic, a biosynthetic pathway which includes several glutathione-S-derivatives and a non-enzymatic reaction, is proposed.

The bioavailability of 11 brands of phenoxymethylpenicillin was investigated in a cross-over-study in healthy volunteers. There were found some differences in the velocity of absorption, in the peaks and in the blood levels after 4 hours. The absorption rate was evaluated by comparison of area under the curve and urine-recovery after oral administration of different brands. It is suggested to control each brand of oral antibiotic preparations on bioavailability.

The absorption rate and serum level curves of two commercial phenoxymethylpenicillin mixture preparations were compared in adult volunteers. Both the potassium salt (Primcillin) and the benzathine salt (V-Pen ped forte) of V-penicillin were rapidly absorbed and the mean peak serum levels were obtained with both preparations within the first hour after single dose administration. The mean peak level obtained with the potassium salt was several-fold higher (p less than 0.001) and was also reached within a shorter time than that of the benzathine preparation. These results confirm the earlier evidence, obtained with other formulae, for the superior bioavailability of the potassium salt of V-penicillin as compared to most other derivatives used in V-penicillin mixtures.

The penicillin concentration in the nasopharyngeal secretion after penicillin administration was studied in 33 patients undergoing tonsillectomy. Benzylpenicillin was either administrated by the intravenous or the intramuscular route in doses of 0.6-1.2 g. Phenoxymethylpenicillin was given perorally in doses of 0.5-1.0 g. The secretion from the nasopharynx was collected prior to the operation both in a disposable collector and on a filter paper disc. A serum sample was also drawn prior to and immediately after the operation. The serum penicillin concentration reached levels of 16.0 mg/l, 5.8 mg/l and 3.6 mg/l (mean values) when given by the intravenous, the intramuscular and the peroral route, respectively. The mean penicillin concentration in nasopharyngeal secretion was 1.6 mg/l, 0.3 mg/l and 0.3 mg/l for these three administration modes. In the majority of cases the concentration of penicillin in the nasopharyngeal secretion exceeded the minimum inhibitory concentration for Streptococcus pneumoniae (0.006-0.12 mg/l).

Fourty patients undergoing tonsillectomy for recurrent tonsillitis were administered penicillin V, amoxicillin, cefaclor or josamycin. Antibiotic concentrations in serum and tonsillar tissues were determined by microbiological assay. Cefaclor demonstrated a superior diffusion than penicillin V and amoxicillin, but nevertheless inferior to that of josamycin.

1.2 mega U Penicillin V potassium was given to 20 patients 2 hours (group 1) and another 19 patients 3 hours (group 2) before tonsillectomy. After the operation biological determinations of the levels of active principle were made from tonsillar tissue and serum with statistical evaluation of the results (Spearman's rank correlation). There was a significant connection between the serum and tonsillar tissue concentrations (in children and adults) in both groups. The level of the active principle in group 2 was only slightly lower than in group 1. Many of the micro-organisms responsible for tonsillitis can be influenced by the concentrations found here, so that application of Penicillin V can often be expected to have a good therapeutic effect.

The gastrointestinal absorption of penicillin V (pc-V) was investigated in 6 children, 6-12 months old, with suspected coeliac disease. The diagnosis was set after small bowel biopsy and absorption tests of vitamin A and d-xylose. As control groups served 7 children with diarrhoea but with normal small bowel biopsy and/or absorption tests and a group of 9 children with upper respiratory tract infection of the same ages as the children in the test group. The absorption of calcium pc-V in oil suspension (Penicals) was impaired in the patients with suspected coeliac disease compared to that of the control groups. There was no significantly different absorption of pc-V between the control children with diarrhoea and those with upper respiratory tract infection. After 6-8 months of gluten free diet in the children with suspected coeliac disease their absorptive ability of oral calcium pc-V in suspension form was equal with that of a control group.

Different salts of penicillin V (pc-V): potassium pc-V (Calciopen and Kåvepenin), calcium pc-V (Penicals) and benzathine pc-V (Meropenin) were given to 37 children (age 2 months to 4 years) with upper respiratory infections. The gastro-intestinal absorption of the drug given in a mixture was followed for three hours after administration by determination of the serum levels from capillary samples. Administration of the mixtures containing the potassium pc-V resulted in a more rapid absorption and in significantly higher plasma concentrations at 30 min than did administration of the preparations containing the calcium and benzathine salts. In four children with coeliac disease, verified by intestinal biopsy, the absorption of potassium pc-V (Calciopen) was compared with that of calcium pc-V (Penicals). A decreased absorption was found and this was most pronounced when the calcium salt was given.

Serum penicillin concentrations and urine excretion rates of oral benzathine penicillin G and phenoxymethyl penicillin potassium were evaluated in children. Mean peak serum concentration of 0.134 microgram/ml of benzathine penicillin G and 1.018 microgram/ml of phenoxymethyl penicillin potassium were measured at 125 and 35 min. The mean half-life times were 1.36 and 0.74 hr, and for area under the curve, the values were 0.34 and 1.68 microgram . hr/ml for benzathine penicillin G and phenoxymethyl penicillin potassium groups, respectively. Phenoxymethyl penicillin potassium had more reliable pharmacokinetic properties and would be the preferred forms of oral penicillin. However, all patients receiving penicillin G had penicillinemia, which was enough to inhibit most strains of Streptococcus pyogenes for a longer period than phenoxymethyl penicillin potassium. Palatability of phenoxymethyl penicillin is less than for benzathine penicillin G; the latter may be used for children in whom compliance may be a problem.

Amino acids have been coupled to the carboxyl group of penicillin V and cephalothin by methods that keep the beta-lactam ring intact. Derivatives were successfully obtained with both neutral (Leu, Val, Ala, Ile, Trp, Tyr, Gly) and one acidic (Glu) amino acids. The new compounds were inactive in vitro against Staphylococcus aureus or Micrococcus luteus. Incubation in the presence of purified carboxypeptidases (A, B), soluble lysosomal fractions from liver, or cellular homogenates from liver, kidney, fibroblasts, and macrophages did not allow recovery of the antibacterial activity. Injection in mice also failed to cause liberation of microbiologically active compounds. HPLC studies confirmed that the amide linkage between the antibiotic and the amino acid was not hydrolyzed in the presence of soluble lysosomal fractions from liver. However, conversion of cephalothin and cephalothin-leucine to desacetyl derivatives was observed in the presence of soluble lysosomal fractions and extracts from liver and semipurified orange peel acetylesterase(s). It is concluded that amino acid derivatives of beta-lactam antibiotics do not offer potential chemotherapeutic use as prodrugs.

Antibacterial Concentrations in Serum and Dental Tissues after Preoperative Application of Propicillin. After resecting radicular and follicular cysts in the jaws, the serum, bone, capsule and fluid of the cysts from 40 patients were studied for their propicillin concentration. The patients received 1 Mega unit (0.7 g) propicillin orally as oblong tablets one or two hours before surgery. One or two hours after the application of propicillin, the distribution coefficients between serum and the three tissues were 0.78 and 0.87 for the spongious bone, 0.07 and 0.06 for the cyst capsule and 0.14 and 0.08 for the cyst fluid. The mean concentrations in all three tissues were distinctly above the MICs of the bacteria most frequently isolated from the mouth for at least two hours.

Sixty-one patients, aged six months to ten years, with acute purulent otitis media were treated with penicillin V for ten days. The drug was given twice a day in a total daily dose of 50 mg/kg body weight. The concentration of penicillin was determined in serum and in middle ear exudate on one of the first six days of treatment, 60, 120 or 180 minutes after administration of the drug. Bacterial cultures were taken from the nasopharynx before and during treatment and from the middle ear exudate at the time of myringotomy. The serum level of penicillin decreased during treatment. There was also a decrease in penicillin concentration of the middle ear exudate during treatment. This decrease was most pronounced during the first two days. In a few cases Haemophilus influenzae was found in the middle ear exudate during treatment. In these cases the concentration of penicillin in the exudate was found to be higher than the mean concentration at corresponding times. The rapid decrease in the exudate concentration of the drug two to three days after the beginning of therapy may suggest that the patients responded to the treatment and that it is the initial concentration that decides whether an infection will respond to the therapy. High exudate concentrations later on seem to be due mainly to a continuation of the inflammatory reaction and had slight or no clinical effect. Thus, the present investigation produced no strong evidence for a ten-day treatment compared with, for example, a five-day treatment.

The pharmacokinetics of phenoxymethylpenicillin in tonsillar tissue was studied in 33 patients who underwent tonsillectomy, mainly because of repeated tonsillitis or peritonsillitis. The patients were operated on 30-240 min after an oral penicillin dose of 12.5 mg/kg body weight. The mean serum concentration was 2.8 micrograms/ml for 10 patients operated upon 80-95 min after drug administration. The mean tissue concentration for these patients at the same time was 0.6 micrograms/g. The mean concentration of penicillin in tissue after 240 min was 0.05 micrograms/g. 19% of the serum concentration was recovered in the tonsillar tissue. No significant difference between penicillin concentration in the centre or surface of the tonsils was found. The tissue concentration was calculated to be above 0.03 micrograms/ml for at least 4-5 h. 30% of the patients harboured penicillinase producing bacteria on their tonsils, mainly Staphylococcus aureus. These patients had a lower tissue concentration than those lacking the bacteria, but the difference was not statistically significant (p = 0.095).