High-performance liquid chromatographic identification of beta-lactam antibiotics produced by dermatophytes

Thirteen of 48 dermatophyte isolates were found by bioassay to produce beta-lactam antibiotics and seven produced other antibiotics. Estimation and detection of specific beta-lactams in culture broths by derivatization and HPLC was only possible following concentration and extraction procedures. Analysis of the concentrated broths demonstrated the production of penicillin X and penicillin G by two Trichophyton mentagrophytes strains and by one Microsporum canis strain; one further T. mentagrophytes strain produced only penicillin X. Additions of the beta-lactam side-chain precursors, phenylacetic acid and phenoxyacetic acid, to fermentation media failed to increase the antibiotic titres.     

The streptococcolytic enzyme zoocin A is a penicillin-binding protein

Zoocin A is a streptococcolytic enzyme produced by Streptococcus equi subsp. zooepidemicus 4881 that has an unknown site of action on the peptidoglycans of susceptible organisms. Analysis of a mutant strain in which the genes for zoocin A and resistance to zoocin A were inactivated revealed that this strain was more susceptible to beta-lactam antibiotics than the parental organism. Purified zoocin A had weak beta-lactamase activity, bound radioactive penicillin covalently, and its streptococcolytic activity was inhibited by penicillin. Thus, zoocin A is a penicillin-binding protein and presumably is a D-alanyl endopeptidase.     

Replacement of the essential penicillin-binding protein 5 by high-molecular mass PBPs may explain vancomycin-β-lactam synergy in low-level vancomycin-resistant Enterococcus faecium D366

The mechanism of synergy between vancomycin and penicillin, as well as other beta-lactam antibiotics, was examined in a penicillin-resistant E. faecium (D366) expressing an inducible low-level resistance to vancomycin. It was demonstrated that penicillin per se was not able to reduce the inducible expression of the 39.5-kDa protein (VANB) or the carboxypeptidase activity which are involved in the mechanism of vancomycin resistance of this strain. Assays of competition between 3H-benzylpenicillin and diverse beta-lactam antibiotics suggested as the most likely explanation of the synergy that, once vancomycin resistance has been induced, the high-molecular mass penicillin-binding proteins (PBPs), and possibly PBP1 in particular, which have a high affinity for beta-lactam antibiotics, take over the role of the low-affinity PBP5 which is, in the non-induced strain, responsible for beta-lactam resistance.     

Formation of 6-Aminopenicillanic Acid, Penicillins, and Penicillin Acylase by Various Fungi

Several penicillin-producing fungi were examined for ability to produce 6-aminopenicillanic acid (6-APA) and penicillin acylase. 6-APA was found in corn steep liquor fermentations of Trichophyton mentagrophytes, Aspergillus ochraceous, and three strains of Penicillium sp. 6-APA was not detected in fermentations of Epidermophyton floccosum although penicillins were produced. 6-APA formed a large part of the total antibiotic production of T. mentagrophytes. The types of penicillins produced by various fungi were identified by paper chromatography, and it was found that all cultures produced benzylpenicillin. T. mentagrophytes and A. ochraceous showed increased yields of benzylpenicillin and the formation of phenoxymethylpenicillin in response to the addition to the fermentation medium of phenylacetic acid and phenoxyacetic acid, respectively. Washed mycelia of the three Penicillium spp. and two high penicillin-yielding strains of P. chrysogenum possessed penicillin acylase activity against phenoxymethylpenicillin. A. ochraceous, T. mentagrophytes, E. floccosum, and Cephalosporium sp. also had penicillin acylase activity against phenoxymethylpenicillin. Only two of the above fungi, T. mentagrophytes and E. floccosum, showed significant penicillin acylase activity against benzylpenicillin; in both cases it was very low. The acylase activity of A. ochraceous was considerably increased by culturing in the presence of phenoxyacetic acid. It is concluded that 6-APA frequently but not invariably accompanies the formation of penicillin, and that penicillin acylase activity against phenoxymethylpenicillin is present in all penicillin-producing fungi.     

The synthesis and in vitro testing of structurally novel antibiotics derived from acylnitroso Diels-Alder adducts

The structural similarity between beta-lactam antibiotics, such as penicillin, and isoxazolidine-3,5-dicarboxylic acids led to the hypothesis that isoxazolidine-3,5-dicarboxylic acids could be effective analogs of beta-lactam antibiotics. The syntheses of relevant isoxazolidine-3,5-dicarboxylic acids from acylnitroso Diels-Alder adducts and subsequent biological testing have shown that these first examples are inhibitors of Escherichia coli X580.     

Escherichia coli Mutants Tolerant to Beta-Lactam Antibiotics

Two types of Escherichia coli mutants tolerant to beta-lactam antibiotics were isolated. One is E. coli chi2452, which showed a tolerant response against beta-lactam antibiotics when grown at 42 degrees C, and the others are the mutants C-80 and C-254, selected from mutagenized E. coli chi1776 by cycles of exposure to ampicillin, cephaloridine, and starvation of the nutritionally required diaminopimelic acid. Beta-lactam antibiotics caused rapid loss of viability and lysis in cultures of chi1776 or in chi2452 grown at 32 degrees C. In contrast, the same antibiotics caused only a reversible inhibition of growth in mutants C-80 and C-254 or in cultures of chi2452 grown at 42 degrees C. Beta-lactam antibiotics that show high affinity for penicillin-binding proteins 2 or 3 (mecillinam and cephalexin, respectively) induced similar morphological effects (ovoid cell formation and filament formation) in both parent and mutant strains. In contrast, beta-lactam antibiotics which have a high affinity for penicillin-binding protein 1 (e.g., cephaloridine or cefoxitin), which cause rapid lysis in the parental strains, caused cell elongation in the tolerant bacteria. In contrast to the parental cells, autolytic cell wall degradation was not triggered by beta-lactam treatment of chi2452 cells grown at 42 degrees C or in mutants C-80 and C-254. The total autolytic activity of mutants C-80 and C-254 was less than 30% that of the parent strain. However, virtually identical autolytic activities were found in cells of chi2452 grown either at 42 or 32 degrees C. Possible mechanisms for the penicillin tolerance of E. coli are considered on the basis of these findings.     

Enzymatic synthesis of beta-lactam antibiotics via direct condensation

In this paper, the feasibility of precipitation driven synthesis of acidic and zwitterionic beta-lactam antibiotics is studied. As an example of the first type, penicillin G was produced in good yield (160 mmol kg(-1)) directly from the free acid and amine aqueous substrate suspension, where the synthesis product precipitated. Such a precipitation driven synthesis via direct reversal of the hydrolytic reaction is thermodynamically unfavourable for zwitterionic beta-lactam antibiotics, such as amoxicillin. In this paper, a novel method is suggested to help favour precipitation of (poorly soluble) product salts by deliberate addition of certain counter-ions. After screening a number of different counter-ions, it was found that the amoxicillin anion forms a poorly soluble salt with Zn(2+). Despite increased beta-lactam degradation due to the presence of zinc ions, in a synthetic reaction with 0.1 M ZnSO(4) present the synthetic yield could be increased at least 30-fold.     

Overproduction of a single protein, Pc-Pex11p, results in 2-fold enhanced penicillin production by Penicillium chrysogenum

Current industrial production of beta-lactam antibiotics, using the filamentous fungus Penicillium chrysogenum, is the result of many years of strain improvement by classical mutagenesis. More efficient production strains showed significant increases in the number and volume fraction of microbodies in their cells, organelles that harbor key enzymes involved in the biosynthesis of beta-lactam antibiotics. We have isolated the P. chrysogenum cDNA encoding Pc-Pex11p, a peroxin that is involved in microbody abundance. We demonstrate that overproduction of Pc-Pex11p in P. chrysogenum results in massive proliferation of tubular-shaped microbodies and a 2- to 2.5-fold increase in the level of penicillin in the culture medium. Notably, Pc-Pex11p-overproduction did not affect the levels of the enzymes of the penicillin biosynthetic pathway. Our results suggest that the stimulating effect of enhanced organelle numbers may reflect an increase in the fluxes of penicillin and/or its precursors across the now much enlarged microbody membrane.     

Industrial production of beta-lactam antibiotics

The industrial production of beta-lactam antibiotics by fermentation over the past 50 years is one of the outstanding examples of biotechnology. Today, the beta-lactam antibiotics, particularly penicillins and cephalosporins, represent the world's major biotechnology products with worldwide dosage form sales of approximately 15 billion US dollars or approximately 65% of the total world market for antibiotics. Over the past five decades, major improvements in the productivity of the producer organisms, Penicillium chrysogenum and Acremonium chrysogenum (syn. Cephalosporium acremonium) and improved fermentation technology have culminated in enhanced productivity and substantial cost reduction. Major fermentation producers are now estimated to record harvest titers of 40-50 g/l for penicillin and 20-25 g/l for cephalosporin C. Recovery yields for penicillin G or penicillin V are now >90%. Chemical and enzymatic hydrolysis process technology for 6-aminopenicillanic acid or 7-aminocephalosporanic acid is also highly efficient (approximately 80-90%) with new enzyme technology leading to major cost reductions over the past decade. Europe remains the dominant manufacturing area for both penicillins and cephalosporins. However, due to ever increasing labor, energy and raw material costs, more bulk manufacturing is moving to the Far East, with China, Korea and India becoming major production countries with dosage form filling becoming more dominant in Puerto Rico and in Ireland.     

The global regulator LaeA controls penicillin biosynthesis, pigmentation and sporulation, but not roquefortine C synthesis in Penicillium chrysogenum

The biosynthesis of the beta-lactam antibiotic penicillin is an excellent model for the study of secondary metabolites produced by filamentous fungi due to the good background knowledge on the biochemistry and molecular genetics of the beta-lactam producing microorganisms. The three genes (pcbAB, pcbC, penDE) encoding enzymes of the penicillin pathway in Penicillium chrysogenum are clustered, but no penicillin pathway-specific regulators have been found in the genome region that contains the penicillin gene cluster. The biosynthesis of this beta-lactam is controlled by global regulators of secondary metabolism rather than by a pathway-specific regulator. In this work we have identified the gene encoding the secondary metabolism global regulator LaeA in P. chrysogenum (PcLaeA), a nuclear protein with a methyltransferase domain. The PclaeA gene is present as a single copy in the genome of low and high-penicillin producing strains and is not located in the 56.8-kb amplified region occurring in high-penicillin producing strains. Overexpression of the PclaeA gene gave rise to a 25% increase in penicillin production. PclaeA knock-down mutants exhibited drastically reduced levels of penicillin gene expression and antibiotic production and showed pigmentation and sporulation defects, but the levels of roquefortine C produced and the expression of the dmaW involved in roquefortine biosynthesis remained similar to those observed in the wild-type parental strain. The lack of effect on the synthesis of roquefortine is probably related to the chromatin arrangement in the low expression roquefortine promoters as compared to the bidirectional pbcAB-pcbC promoter region involved in penicillin biosynthesis. These results evidence that PcLaeA not only controls some secondary metabolism gene clusters, but also asexual differentiation in P. chrysogenum.     

Production and release of peptidoglycan and wall teichoic acid polymers in pneumococci treated with beta-lactam antibiotics

Autolysin-defective pneumococci treated with inhibitory concentrations of penicillin and other beta-lactam antibiotics continued to produce non-cross-linked peptidoglycan and cell wall teichoic acid polymers, the majority of which were released into the surrounding medium. The released cell wall polymers were those synthesized by the pneumococci after the addition of the antibiotics. The peptidoglycan and wall teichoic acid chains released were not linked to one another; they could be separated by affinity chromatography on an agarose-linked phosphorylcholine-specific myeloma protein column. Omission of choline, a nutritional requirement and component of the pneumococcal teichoic acid, from the medium inhibited both teichoic acid and peptidoglycan synthesis and release. These observations are discussed in terms of plausible mechanisms for the coordination between the biosynthesis of peptidoglycan and cell wall teichoic acids.     

Involvement of O8-antigen in altering beta-lactam antibiotic susceptibilities in Escherichia coli.

In spite of being dispensable, O-antigens are believed to facilitate various cellular processes and alter antibiotic sensitivities. Escherichia coli K-12 (CS109) strains are lacking in O-antigens and are reported to be sensitive to antibiotics. To our surprise, E. coli 2443 (expressing O8-antigen) manifested two- to fourfold higher sensitivities toward penicillin and its derivatives than strain CS109. However, sensitivities toward other structurally unrelated antibiotics remained unchanged. To understand the rationale behind such observations, we replaced the rfb locus of strain 2443 with that of E. coli K-12. The beta-lactam sensitivities of 2443 cells with replaced rfb locus appeared to be identical to those for CS109. Therefore, it is quite reasonable to hypothesize the possible involvement of O8-antigen in beta-lactam sensitization.     

Interactions between beta-lactam antibiotics and isolated membranes of Streptococcus faecalis ATCC 9790.

The DD-carboxypeptidase-exchange membrane-bound enzyme in Streptococcus faecalis ATCC 9790 reacts with beta-lactam antibiotics to form complexes with rather long half-lives. Depending upon the antibiotic, the second-order rate constants for complex formation range from 0.75-560 M-1 S-1 (at 37 degrees C and in water) and the first-order rate constants for complex breakdown range from 1.3 to 26 x 10(-5) s-1 (at 37 degrees C and in 5 mM phosphate buffer pH 7.5). There are about 30 pmol of DD-carboxypeptidase-exchange enzyme per mg of membrane protein. The degradation products arising from benzylpenicillin are phenylacetylglycine and probably N-formyl-D-penicillamine. Isolated membranes also contain other penicillin binding sites (about 70 pmol/mg membrane protein). That part of benzylpenicillin which reacts with at least some of these latter sites is slowly degraded into penicilloic acid. Normal functioning of the DD-carboxypeptidase-exchange membrane-bound enzyme is important, if not essential, for cell growth. With the beta-lactam antibiotics tested inhibition of cell growth is mainly related to the rates of formation of the inactive enzyme-antibiotic complexes. The relationship, however, is not a direct one probably due to the competitive effect exerted by the other penicillin binding sites.     

Microbial isopenicillin N synthase genes: structure, function, diversity and evolution.

Clinically and economically, penicillins and cephalosporins are the most important class of the beta-lactam antibiotics. They are produced by a wide variety of microorganisms including numerous species of Streptomyces, some unicellular bacteria and several filamentous fungi. A key step common to their biosynthetic pathways is the conversion of a linear, cysteine-containing tripeptide to a bicyclic beta-lactam antibiotic by isopenicillin N synthase. Recent successes in the cloning and expression of isopenicillin N synthase genes now permit production of a plentiful supply of this enzyme, which may be used for structural and mechanistic studies, or for biotechnological applications in the creation of novel beta-lactam compounds from peptide analogues. New ideas concerning the evolution and prevalence of the penicillin and cephalosporin biosynthetic genes have emerged from studies of isopenicillin N synthase genes.     

Evaluation of a simple carrier molecule to enhance drug penetration of dermal layers by utilizing multivariate methods, structure property correlations, and continuous system modeling

Nicotinic acid is shown to be comparable to dihydropyridine in its capacity to facilitate penetration of an attached antibacterial drug through dermal layers. Antibacterial drugs examined with nicotinic acid or dihydropyridine carriers were beta-lactam antibiotics: methicillin, oxacillin, benzylpenicillin, penicillin F, penicillin dihydro F, propicillin, carbenicillin, penicillin K, penicillin X, and ampicillin. An oxymethyl (-O-CH2-) group is inserted as the linker between the antibiotic and the carrier group. Structure Property Correlations and multivariate methods such as regression analysis, cluster analysis, principal component analysis, discriminate analysis, self-organizing tree algorithm, and factor analysis clearly showed that nicotinic acid performs as an effective carrier drug and is comparable to dihydropyridine. The skin penetration constant Kp was calculated for all 10 antibiotics having either dihydropyridine or nicotinic acid as carrier, and was found to have a mean of 5.13E-05 cm/hour and 1.83E-05 cm/hour, respectively. The standard deviation for each group showed the numerical values overlap as did the 90% confidence interval for each group. A hierarchical tree organization of skin shows the overlapped dermal layers as they exist in normal skin and for the model utilized in this work. A Deming-Regression analysis also shows the nicotinic acid and dihydropyridine structures to have similar and correlated water solubility. Plotting Kp of the dihydropyridine structures as independent variable versus Kp of the nicotinic acid structures show good correlation (Pearson correlation r = 0.6606) and no significant departure from linearity. Connected box plots showed the majority of Kp values for each group of modified antibiotics to exist in a tight cluster. Polar graph of the Log Kow values showed the two groups of modified antibiotics to be correlated and numerically adjacent in trend. ChemSketch property calculations and modeling demonstrates the affects of structural oxygens, nitrogens, carbonyl groups, amide groups, and aromatic rings that are important in understanding the pervasiveness through dermal layers. Continuous model analysis by acslXtreme is utilized and demonstrates three models of the dispersion of drugs through dermal layers based on diffusivity constant (D), Log Kp from Log Kow and formula weight, and Kp as a function of time.     

Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion

A new beta-lactam-inducible penicillin-binding protein (PBP) that has extremely low affinity to penicillin and most other beta-lactam antibiotics has been widely found in highly beta-lactam(methicillin)-resistant Staphylococcus aureus (MRSA). The gene for this protein was sequenced and the nucleotide sequence in its promoter and close upstream area was found to show close similarity with that of staphylococcal penicillinase, while the amino acid sequence over a wide range of the molecule was found to be similar to those of two PBPs of Escherichia coli, the shape-determining protein (PBP 2) and septum-forming one (PBP 3). Probably the MRSA PBP (Mr 76462) evolved by recombination of two genes: an inducible type I penicillinase gene and a PBP gene of a bacterium, causing the formation of a beta-lactam-inducible MRSA PBP.     

Mutants of Caulobacter crescentus resistant to beta-lactam antibiotics

A number of mutants of Caulobacter crescentus CB15 resistant ot ampicillin were isolated. The mutants differred in their resistance to several beta-lactam antibiotics. No differences in composition of the penicillin-binding proteins of the mutants compared to the parental strain, or in the affinity of these proteins to penicillin or ampicillin were found. The mutants were found to differ from the parent and also in many cases from each other in outer membrane protein composition.