Isolation and activity of Xenorhabdus antimicrobial compounds against the plant pathogens Erwinia amylovora and Phytophthora nicotianae.

AIMS: Broad-spectrum antibiotics produced by symbiotic bacteria [entomopathogenic bacterium (EPB)] of entomopathogenic nematodes keep monoxenic conditions in insect cadavers in soil. This study evaluated antibiotics produced by EPB for their potential to control plant pathogenic bacteria and oomycetes. METHODS AND RESULTS: Entomopathogenic bacterium produce antibiotics effective against the fire blight bacterium Erwinia amylovora, including streptomycin resistant strains, and were as effective in phytotron experiments as kasugamycin or streptomycin. Xenorhabdus budapestensis and X. szentirmaii antibiotics inhibited colony formation and mycelial growth of Phytophthora nicotianae. From X. budapestensis, an arginine-rich fraction (bicornutin) was adsorbed by Amberlite((R)) XAD 1180, and eluted with methanol : 1 n HCI (99 : 1). Bicornutin inactivated zoospores, and inhibited germination and colony formation of cystospores at <<25 ppm. An UV-active molecule (bicornutin-A, MW = 826), separated by HPLC and thin-layer chromatography, was identified as a novel hexa-peptide : RLRRRX. CONCLUSIONS: Xenorhabdus budapestensis produces metabolites with strong antibacterial and cytotoxic activity. Individual compounds can be isolated, identified and patented, but their full antimicrobial potential may be multiplied by synergic interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: Active compounds of two new Xenorhabdus species might control plant diseases caused by pathogens of great importance to agriculture such as Erw. amylovora and P. nicotianae.

Xenorhabdus antibiotics: a comparative analysis and potential utility for controlling mastitis caused by bacteria.

AIMS: The role of antibiotics produced by bacterial symbionts of entomopathogenic nematodes is to suppress growth of microbes in the soil environment. These antibiotics are active against Gram-positive and Gram-negative bacteria, and were tested against mastitis isolates from dairy cows. METHODS AND RESULTS: Two bioassays were adapted for Xenorhabdus antibiotics; an overlay method on agar plates, and serially diluted, cell-free, Xenorhabdus cultures. The antimicrobial activities of the liquid cultures of 13 strains from five Xenorhabdus species were further evaluated. Antimicrobial activities of the type strains of X. nematophila, X. budapestensis and X. szentirmaii were tested on mastitis isolates of Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae with both bioassays. A previously reported antibiotic from X. nematophila, nematophin, was synthesized in three steps from tryptamine and 4-methyl-2-oxovaleric acid sodium salt. CONCLUSIONS: The antibiotics of all three Xenorhabdus strains were powerful in either bioassay, but the sensitivity of the isolates differed from each other. While Kl. pneumoniae was the least susceptible, Staph. aureus had the highest sensitivity to each Xenorhabdus strain. Xenorhabdus szentirmaii and X. budapestensis were more potent antibiotic producers than X. nematophila, and raceme nematophin was ineffective against all mastitis isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that Xenorhabdus antibiotics are effective against mastitis isolates and should be further evaluated for their potential in mastitis control or prevention.

Cleaning validation procedure eased by using overpressured layer chromatography.

In the manufacturing plants of many pharmaceutical companies the reaction apparatus is suitable to produce different active pharmaceutical ingredients. After completing the production of a compound the equipment should be cleaned in order to avoid the cross contamination in the next lot of the other products. In the authors' laboratory several chromatographic methods were introduced to measure the amount of the residual substances remaining on the surface of the apparatus after the cleaning procedure. A sensitive and fairly rapid overpressured layer chromatographic (OPLC) procedure--suitable to separate and control five steroid hormone compounds (allylestrenol, estradiol, ethynodiol diacetate, levonorgestel, norethisterone) produced in the same equipment at different times--was developed and validated.

[Importance of professional proposals in the era of broadening antibiotic resistance]. / Egyes szakmai ajánlások jelentösége az antibiotikum-rezisztencia kiterjedésének korában.

A great degree of neglection has been portrayed on the consideration in two most important professional proposals in 6906 patients of 44 praxis in the basic health care in 1996: 1. the obviously viral respiratory diseases (including acute bronchitis, ordinarily) was treated in 56% of cases with antibacterial drugs (in 83% of all treatments with broad spectrum antibiotics), and 2. for patients with Streptococcal infections too broadened spectrum antibiotics for this purpose were undesirably prescribed (in 75.5% of treatments) by physicians. Taking into consideration the suitable efficient ("evidence based") anticausal therapy and professional proposals, as well, in 63.6% of all antibiotic therapy could be avoided by physicians. By all means, these prescribed legion of the aimless, unnecessary and simultaneously too broadened spectrum antibacterial drugs served for the spreading of antibiotic resistance only, and all of them were the results of the neglection of the most important professional proposals. These unnecessary treatments--excluding the increase of resistance or the improper, unsubstantiated raise in hopes of patients--cost too much: the sum of expenses were 49.5% of the cost for antibiotics in Hungary in 1996 (in our estimation). To measure and examine periodically the quality level of primary infectological care, new quality indicators were appointed by the authors: the attained quality levels of each medical praxis would have to be compared and approached to the desirable level of quality indicators (reflecting the "evidence based" infections care) for the abolition of the unreasonable, expensive and dangerous practice for patients, as well. It appears, that the wide application of the appropriate, "evidence based" anticausal therapy would be reached with control of offered quality (the offered values of the quality indicators) of physicians in primary care, only. The introduction of quality assurance in basic health care needs urgent comprehensive official action.

[The possibilities of GC/MS and HPLC in the analysis of sugars and acids in natural matrices]. / A GC/MS és a HPLC lehetóségei a természetes mátrixok karbonsav és szacharidtartalmának analitikájában.

A GC/MS procedure is described for the simultaneous quantitation of the minor and major constituents of several natural matrices, including fruits, vegetables, mushrooms, drug/industrial plants and honeys, as their trimethylsilyl derivatives, from one solution, by one injection. Selected minor components have been determined on the basis of their characteristic fragment ions, in the presence of extremely high excess of saccharides of various degree of polymerization. Selective fragmentation of these minor compounds in the ion trap detector provided possibilities for distinguishing them. In the case of honeys the method permitted the simultaneous quantitation of o-phosphoric, malic, shikimic, citric/isocitric, quinic, margaric, oleic and stearic acids, 5-hydroxymethylfurfurol and proline with the extremely high sugar contents of honeys (fructose, glucose, galacturonic acid, inositol, sucrose, trehalose, turanose, maltose, gentiobiose, isomaltose, raffinose, erlose, melezitose, maltotriose, panose, isomaltotriose) and allowed the fast evaluation of sugar and acid constituents of fifteen honeys from various floral and geological origin. Results revealed that (i) the minor components varied in the concentration range of 0.0001 to 0.43%, and, (ii) together with the saccharides of honeys made up the total of identified and determined constituents from 87.8% to 98.5%. Quantitative evaluation of the minor constituents was performed on the basis of their selective fragment ion values with an average reproducibility of 6.7% (RSD).

[How to proceed in the age of increasing antibiotic resistance?]. / Hogyan tovább az antibiotikumrezisztencia kiterjedésének korában?

The authors examined the infection control activity, the quality of basic health care of 6906 patients in 44 praxis. The mass of accomplished treatments was compared with the virtual mass of desirable treatments according to up-to-date considerations. The presumptive purposivity and expectable efficiency of the realised treatments didn't satisfy the justly expected professional claims. The decrease in quality was basically due to the cognitive dissonance (deficiencies of cognition and/or erroneous adoptation of theoretical and practical knowledge) of physicians. The unnecessarily prescribed (polypragmatic) and simultaneously purposeless broad spectrum of antibiotic treatments ran to two-third of the total--mainly in the cases or viral and beta-hemolytic Streptococcal infections; the number of antibacterial treatments was three times more than needed. Better compromise could be reached by the introduction of quality control in the field of infection treatments in the basic health care system. Spreading of the antibiotic resistance cannot be prevented without of quality assurance in the field of infection control and without the wide use of a more reasonable kind of treatment practice.

The cerebroside sulfate activator from pig kidney: derivitization, cerebroside sulfate binding, and metabolic correction.

Highly purified cerebroside sulfate activator from pig kidneys was characterized by a number of chemical and biological procedures. Methods for chemical modifications were evaluated in an attempt to obtain biologically active derivatives. Iodination, dabsylation, and to a lesser degree reductive methylation provided useful products with good retention of cerebroside sulfate activator activity. Other procedures resulted in largely inactive derivatives or losses in both protein and biological activities. Attempts at renaturation of cerebroside sulfate activator subjected to various denaturing conditions appeared to be successful in many instances, but it was uncertain if the protein structure had actually been disrupted. The binding of cerebroside sulfate by activator was estimated by gel filtration under conditions similar to those of its assay. The formation of a relatively stable 1:1 complex was observed, collaborating results with the human protein. The complex was stable enough to be isolated and shown to be an efficient substrate for arylsulfatase A. The effectiveness of the pig kidney cerebroside sulfate activator for correcting the metabolic defect in activator-deficient human fibroblasts was compared with human materials. The pig kidney protein was taken up more efficiently by the cells and resulted in a better metabolic correction than material from human liver, but was somewhat less effective than a preparation from human urine.

The cerebroside sulfate activator from pig kidney: purification and molecular structure.

The activator protein for hydrolysis of cerebroside sulfate by arylsulfatase A was purified from pig kidney in high yield. This protein, also known as sphingolipid activator protein-1 and saposin-B, was particularly rich in pig kidney. Purification was achieved by a simple procedure involving homogenation and heat treatment followed by affinity, ion exchange, and gel filtration chromatographies. The final product was better than 90% pure by gel electrophoresis and HPLC. It was possible to sequence more than 60 amino acids from the N-terminus with only a few uncertain residues. The sequence differed from that predicted for the human protein by about 10%, with most amino acid variations being conservative. There appeared to be a residual glycosyl substituent on asparagine 21, but the sugar content was low and the protein failed to bind to concanavalin A. The cerebroside sulfate activator proved to be exceptionally resistant to denaturation or protease digestion. The apparent molecular mass was approximately 20,000 Da on preparative gel-filtration columns, but was variable when estimated by HPLC gel filtration. Values ranging from 30,000 to over 100,000 Da were observed in neutral buffers, while values around 15,000-16,000 Da were seen in acidic buffers such as those used for assay of the biological activity. This was further decreased to a putative subunit of 7000-8000 Da under severe denaturing conditions. Pig kidney is a convenient source for the large-scale preparation of this interesting protein which has heretofore been obtained from human sources.

International registration of clinical engineers.

This paper outlines the moves being made by the International Federation for Medical and Biological Engineering to establish an International Clinical Engineering Board. The purpose of this board is to stimulate, through the establishment of National Examining Authorities, international agreement on the minimum requirements needed for the registration or certification of professional clinical engineers.