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In order to synthesize derivatives of galbonolide A and B with improved chemical stability and antifungal activity profiles, a panel of microorganisms consisting of various species of actinomycetes and fungi were screened. As a result, an organism, Streptomyces halstedii, was identified, which catalyzed the formation of two polar compounds, one from each of the galbonolides. The synthesis and the relative stability of these compounds were optimized by using washed cells, which had been prepared from the transforming organism, and reaction conditions, which included the usage of MES buffer with pH adjusted to 5.5 and incubation at 27°C. Under conditions thus established, two compounds were isolated and characterized by a combination of UV, mass, and NMR spectroscopic analysis. The data indicate the synthesis of 21-hydroxy derivatives of galbonolides A and B with reduced but still significant anti-fungal activity when compared to the parent galbonolides.

The mode of action of the known antifungal macrolides rustmicin (1) and galbonolide B (2) has been determined to be the inhibition of sphingolipid biosynthesis. A large scale fermentation and isolation process was developed for production of large quantities of rustmicin. New 21-hydroxy derivatives of both compounds were isolated from pilot scale fermentations and were also produced by biotransformation of rustmicin and galbonolide B.

A new metabolite, L-755,807, 1, was isolated from an endophytic Microsphaeropsis sp. in the course of searching for a bradykinin binding inhibitor. The structure of 1, including relative stereochemistry, was determined. 1 showed an IC50 of 71 μM in 3H-bradykinin binding to a cloned human B2 receptor.

A novel inositol mono-phosphatase inhibitor, L-671, 776 (1), was discovered from a culture of the hyphomycete, Memnoniella echinata (ATCC 20928). 1 has a molecular weight of 388 and a molecular formula of C23H32O5. The mode of inhibition is non-competitive, with a Ki of 450μM. It shows no inhibition of myo-inositol 1, 4-bisphosphate 1-phosphatase or myo-inositol 1, 4, 5-triphosphate 5-phosphatase, although it weakly inhibits myo-inositol 1, 4, 5-triphosphate 3-kinase (IC50 = 3 mM). It elevates inositol monophosphates in rat parotid slices (EC50 approximately 3 mM), but abolishes agonist effects. It also produces short-lived contraction of guinea pig trachea at 300 μM.

A novel HIV-1 protease inhibitor, L-696,474 (C 30 H 39 NO 4 , 477), was isolated from the fermentations of the fungus Hypoxylon fragiforme (ATCC 20995, MF5511) and purified by silica gel chromatography followed by crystallization. Spectroscopic studies have shown the competitive inhibitor L-696,474 to be a novel cytochalasin. Two related novel cytochalasins were also isolated and had no effect on the enzyme

Microbiological criteria provide the food industry and regulatory agencies with guidelines for control of food processing systems and are an underlying component of any critical control point that addresses a microbiological hazard in Hazard Analysis and Critical Control Points (HACCP) systems. Aerobic plate count (APC) or standard plate count (SPC) is commonly used to determine “total” numbers of microorganisms in a food product. Depending on the pathogen, low levels of the microorganism in the food product may or may not be of concern. Some microorganisms have such a low infective dose that their mere presence in a food presents a significant public health risk. For such microbes, the concern is not whether the pathogen is able to grow in the food but that the microorganism could survive for any length of time in the food. Food products frequently subject to contamination by harmful microorganisms, such as shellfish, may benefit from the application of microbiological criteria. Enterococci counts have few useful applications in microbiological criteria for food safety. Some microbiological criteria related to safety rely on tests for metabolites to indicate a potential hazard rather than direct tests for pathogenic or indicator microorganisms. The current acceptable limits are based on the criterion that there is an 80% probability that the plant is actually exceeding the target value if it exceeds the acceptable limit.

A novel illudane sesquiterpene antibiotic (1) with activity against methicillin-resistant Staphylococcus aureus (MRSA), has been isolated. Its MIC against MRSA was found to be 16 micrograms/mL. It also shows L-1210 cytotoxicity with an IC50 of 10-15 micrograms/mL.

We have identified a novel nonsteroidal ecdysteroid agonist. This compound was isolated from a methanol extract of Ajuga reptans L. (Lamiaceae) and the structure was identified by spectroscopic methods as 8- O -acetylharpagide. We have characterised this compound as an ecdysteroid agonist in a transactivation assay using β-galactosidase as the reporter gene regulated by ecdysteroid response elements. In this assay, 8- O -acetylharpagide has an EC 50 of 22 μM. The compound also competes with tritiated-ponasterone A for binding to the Drosophila ecdysteroid receptor. Finally, it induces differentiation of Drosophila Kc cells as would be expected of an ecdysteroid agonist. This iridoid glycoside is common to several plant species and may play a role in the natural defense mechanisms of plants.

Pneumocandin D0 (9), a new member of the echinocandin class of antifungal agents, has been isolated as a minor constituent from fermentation broths of the filamentous fungi Zalerion arboricola (ATCC 20957). The structure of 9 has been determined mainly on the basis of spectroscopic analysis and by comparison with published data for similar compounds. To date, pneumocandin D0 has been found to be the most potent inhibitor of Pneumocystis carinii development in vivo within the natural-occurring echinocandin family of antifungal agents.

Fifteen 13-ester, 13-carbanilate and 15-hydroxy derivatives of virginiamycin M 1 were synthesized and evaluated for their abilities to inhibit a) binding to the cholecystokinin receptor subtypes in guinea-pig brain (CCP-B) and rat pancreas (CCP-A), and b) microbial growth

Rubromycins, a class of quinone antibacterials, were discovered to selectively inhibit human immunodeficiency virus-1 (HIV-1) RNA-directed DNA polymerase (reverse transcriptase) (RT) activity more potently than cellular DNA polymerase alpha. beta- and gamma-rubromycin each inhibited equipotently HIV-1 RT and avian myeloblastosis virus RT, in a concentration-dependent manner, and were significantly weaker as inhibitors of calf thymus DNA polymerase alpha. These agents inhibited HIV-1 RT reversibly, were competitive with respect to template.primer, and were noncompetitive with respect to TTP. Dixon analyses yielded HIV RT Ki values of 0.27 +/- 0.014 and 0.13 +/- 0.012 microM for beta- and gamma-rubromycin, respectively. Similarly, using DNA polymerase alpha, the Ki values were 25.1 +/- 4.3 and 3.9 +/- 0.6 microM for beta- and gamma-rubromycin, respectively. Because these agents were toxic to noninfected human T lymphoid cells using concentrations at or above 6 microM, HIV-1 infectivity studies were carried out at 0.8-6 microM. At these concentrations, which are below the range expected to provide protection, no significant antiviral activity was observed. Although beta- and gamma-rubromycins did not possess sufficient HIV RT inhibitory potency or selectivity versus mammalian DNA polymerase to demonstrate antiviral activities, these studies support the hypothesis that specific molecules containing quinone functional groups can selectively inhibit viral polymerase activities over cellular polymerase activities. In addition, these studies suggest that rubromycins may be lead structures for the development of more potent and selective agents.

Thirty-one Kanagawa-positive and -negative Vibrio parahaemolyticus isolates from patient stools and seafood were examined for plasmid deoxyribonucleic acid content, culture characteristics, and indices of pathogenicity. No significant correlation was found between plasmid deoxyribonucleic acid contents and indices of pathogenicity for the strains tested.

Virulent strains of Yersinia enterocolitica and their plasmidless, avirulent derivatives were examined for their cell surface properties. Increased surface charge and hydrophobicity of Y. enterocolitica were found to be associated with the possession of a 40- to 48-megadalton plasmid. These surface properties were expressed, as were other plasmid-associated properties, at 37 but not at 22 degrees C. The concentration of calcium in the growth medium had a moderate effect on the expression of the cell surface properties. These cell surface properties were greatly reduced among plasmid-bearing cells grown on tryptic soy agarose regardless of growth temperatures. These properties were also associated with the ability of Y. enterocolitica to colonize the gastrointestinal tract of mice.

A quick and simple method was developed to distinguish hydrophobic from hydrophilic cells. The latex particle agglutination test is based on the hydrophobic interactions between cells and latex particles which result in the agglutination of the suspension mixture. There was a direct correlation between the expression of plasmid-associated cell surface properties and latex particle agglutination by Yersinia enterocolitica. Multivalent cation-induced agglutination of suspensions of washed cells of virulent Y. enterocolitica and latex particles is indicative of their amphipathic character. Electrostatic interaction may also play a role in the latex particle agglutination reaction.

SUMMARY Brucella abortus biotype 1 was isolated from the blood of an experimentally-infected cow five weeks post exposure and from vaginal swabs on the day of abortion. Antibody activity at a positive level was detected in the sera from this cow by an ELISA method three weeks post exposure to Brucella abortus and between the fifth and twelfth week post exposure by conventional methods, i.e. card, Rivanol (Riv), standard agglutination tube (SAT) and complement fixation (CFT). A regression of antibody activity was then observed until all sera collected in week 23 after exposure and weekly to week 49 were negative by conventional methods. Antibody activity at positive levels was again detected by all conventional methods one week before this cow aborted. However, antibody activity was detectable by enzyme-linked immunosorbent assay (ELISA) in all sera collected from week 3 to week 52 post exposure. These data indicate that an ELISA method is capable of identifying B. abortus-infected cows when results of conventional serologic methods are negative.

Asperlicins B (1, C31H29N5O5), C (2, C25H18N4O2), D (3, C25H18N4O2), and E (4, C25H18N4O3) are novel cholecystokinin antagonists produced by Aspergillus alliaceus. The structures of these compounds have been determined by 1H NMR and MS analysis.

Yersinia enterocolitica counts of inoculated beef and pork as determined from confirmed isolates picked from trypticase soy agar plates were similar to the counts of confirmed enamel-black colonies on bismuth sulfite agar plates. With three strains of Y. enterocolitica increases in count occurred on raw beef held over a 10-day period at 0–1°C. When inoculated raw or cooked beef and pork were stored at 7°C (0–10 days) or at 25°C (0–24 hr) large increases in Y. enterocolitica count occurred. At 25°C the increases in Y. enterocolitica counts were somewhat greater on cooked than on raw products. These differences in count may have been caused by (a) differences in the physicochemical characteristics of the meat (raw vs cooked) and/or (b) differences in the level and type of microbial flora that developed on these products. In addition to Y. enterocolitica, Staphylococcus and Micrococcus spp. often were dominant on cooked products, Pseudomonas and Microbacterium spp. on raw meats.

Characteristics are presented for Yersiniu enterocoliticu-like organisms isolated from vacuum-packaged beef and lamb stored for 21-35 days at l-3°C. Isolation of this organism was more frequent after 28 days of storage under vacuum conditions than under nonvacuum conditions (leaker packages). A higher incidence of isolates was obtained from cuts packaged under high vacuum conditions.

Seventeen Yersinia enterocolitica-like isolates from vacuum-packaged beef and lamb are described. A majority of the isolates were motile at 25 degrees C but not at 36 degrees C, utilized citrate at 25 degrees C (delayed or negative at 36 degrees C) and produced acid from rhamnose, raffinose and melibiose. Increases in count of Y. enterocolitica occurred on raw beef stored at 0--1 degrees C over a 10-day period. No viable Y. enterocolitica were detected in beef roasts which were inoculated with about 3 x 10(6) cells/g and subsequently heated to a final internal temperature of 60--62 degrees C. Y. enterocolitica counts of inoculated beef steaks stored at 1--5 degrees C for 21--35 days were consistently higher in the more oxygen-permeable films than in vacuum packages. Colonies of Y. enterocolitica and related bacteria from meats produced shiny, enamel-black colonies on bismuth sulfite agar with plate incubation at 25 degrees C for 3--5 days.

Electron microscopic examination of virulence plasmid-bearing cells of Yersinia enterocolitica revealed the formation of a fibril structure when grown at 37 degrees C but not at 22 degrees C. Plasmidless cells did not exhibit any surface matrix. The formation of this surface component was associated with increased cell surface charge and hydrophobicity, formation of long chains of cells, and autoagglutination. This structure is distinct from the rigid appendages (fimbriae) which are elaborated by certain Yersinia strains.

Feeding of 14C-labeled amino acids to resting cells of Aspergillus alliaceus strongly supported the intuitive hypothesis that asperlicin is biosynthesized from tryptophan, anthranilate and leucine. The resting cell system was used also to prepare 25 asperlicin analogs via directed biosynthesis in presence of analogs of tryptophan and leucine.

An enzyme-linked immunosorbent assay (ELISA) method was evaluated for the detection of antibodies to Brucella abortus in cows milk. Milk samples from seropositive or -negative cows were sed to determine the distribution of absorbance values to classify milk as ELISA positive or ELISA negative. Brucella abortus was isolated from milk samples from 10 (45%) of the 22 cows whose milk and serum were ELISA positive. The ELISA was evaluated and determined to be an appropriate method for detecting antibodies to B abortus in bovine milk.

The relationship of subtherapeutic feeding and parenteral injection of antibiotics to the presence of antibiotic resistant strains of Escherichia coli in the fecal microflora of commercial turkeys has been investigated. Cloacal swabs collected from 137 commercial turkeys were examined for E coli resistant to gentamicin. Gentamicin-resistant E coli organisms were isolated and tested for resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, and tetracycline. Strains of E coli resistant to gentamicin were identified in 118 of 137 (86.1%) specimens evaluated. There were 5 different antibiotic resistance patterns exhibited by the gentamicin-resistant strains of E coli. All strains showed a common antibiotic resistance pattern of gentamicin, kanamycin, and streptomycin. The results of the antibiotic susceptibility tests were compared to the known history of antibiotic usage in each flock. There was no significant correlation between the use of subtherapeutic concentrations of antibiotics and the frequency of gentamicin resistant E coli. However, the frequency of gentamicin-resistant E coli was closely related to the age of the bird, with birds less than 12 weeks of age being most likely to harbor E coli resistant to gentamicin. This age-dependent frequency of gentamicin-resistant E coli was associated with the common practice of dipping eggs in gentamicin and injecting newly hatched poults with gentamicin, but not with the feeding of subtherapeutic concentrations of antibiotics.

Inoculation of live Corynebacterium pseudotuberculosis, culture supernatant, ammonium sulfate-fractionated crude exotoxin, or chromatographically purified exotoxin preparations into gnotobiotic small ruminants (n = 13) caused death of the ruminants within 48 hours. Characteristic changes observed in animals living greater than or equal to 2 hours after inoculation included hemorrhage and edema at the site of injection, severe hemolytic anemia and hemoglobinuria, dark red fluid in body cavities, lung edema, and icterus. The crude exotoxin preparation caused a syndrome of acute shock in 2 lambs that died within 15 minutes after inoculation. Clinical and pathologic responses of animals inoculated with culture supernatant and purified toxin were similar. Histopathologic evidence indicated that the exotoxin caused necrotic changes in the proximal convoluted tubules of the kidneys. Inoculation with live organisms caused multiple foci of suppurative inflammation in skeletal muscle and adjacent adipose tissue, whereas such changes were not observed in animals administered exotoxin preparations. Although C pseudotuberculosis exotoxin induced a hemolytic anemia in the experimental animals, it did not lead to in vitro lysis of ovine, caprine, or bovine erythrocytes, unless they had been sensitized with Rhodococcus (Corynebacterium) equi filtrate. The toxic sphingomyelin-specific phospholipase D from C pseudotuberculosis had a molecular weight of 31,000 daltons and an isoelectric point of approximately 9.6. The elution profile of exotoxin on a carboxymethyl Sephadex column was studied and the majority of the enzymatic activity was eluted by a NaCl gradient (0.25M to 0.7M) with a maximum at 0.35M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)