United States Agency for International Development

Four hundred and seventy-five strains, which included 394 type cultures of Streptomyces and representatives of 14 other actinomycete genera, were studied. Overall similarities of these strains for 139 unit characters were determined by the SSM and SJ coefficients and clustering by the UPGMA algorithm. Test error and overlap between the phena defined were within acceptable limits. Cluster-groups were defined by the SSM coefficient at the 70.1% similarity (S) level and by the SJ coefficient at the 50% S-level. Clusters were distinguished at the 77.5% SSM and 63% SJ S-levels. Groupings obtained with the two coefficients were generally similar, but there were some changes in the definition and membership of cluster-groups and clusters. The phenetic data obtained, together with those from previous diverse studies, indicated that the genera Actinopycnidium, Actinosporangium, Chainia, Elytrosporangium, Kitasatoa and Microellobosporia should be reduced to synonyms of Streptomyces, while Intrasporangium, Nocardioides and Streptoverticillium remained as distinct genera in the family Streptomycetaceae. Nocardiopsis dassonvillei also showed strong phenetic affinity to Streptomyces, despite its chemotaxonomic differences. Actinomadura sensu stricto was phenetically distinguishable from Streptomyces and 'Nocardia' mediterranea was recognized as a taxon distinct from both these genera and from Nocardia sensu stricto. Most of the Streptomyces type cultures fell into one large cluster-group. At the 77.5% SSM S-level, they were recovered in 19 major and 40 minor clusters, with 18 strains recovered as single member clusters. The status of the latter as species was therefore confirmed. Most of the minor clusters, consisting of two to five strains, can also be regarded as species. The major clusters varied in size (from 6 to 71 strains) and in there homogeneity. Therefore, it is suggested that they be regarded as species-groups until further information is available. The results provide a basis for the reduction of the large number of Streptomyces species which have been described. They also demonstrate that the previous use of a limited number of subjectively chosen characters to define species-groups or species has resulted in artificial classifications.

Numerical Classification of Streptomyces and Related Genera

In the past, many investigations involving the selective isolation of Streptomycetes from soil have been carried out. However, there seems little agreement on which media are most suitable for this purpose. A brief survey of the literature revealed a total of 21 recommended media.

Selection of Media for Isolation of Streptomycetes

Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B1 complexes formed with 12 bacterial strains in both viable and nonviable (heat- or acid-treated) forms was assessed by repetitive aqueous extraction. By the fifth extraction, up to 71% of the total aflatoxin B1 remained bound. Nonviable bacteria retained the highest amount of aflatoxin B1. Lactobacillus rhamnosus strain GG (ATCC 53103) and L. rhamnosus strain LC-705 (DSM 7061) removed aflatoxin B1 from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B1 to an antibody in an indirect competitive inhibition enzyme-linked immunosorbent assay suggests that surface components of these bacteria are involved in binding. Further evidence is the recovery of around 90% of the bound aflatoxin from the bacteria by solvent extraction. Autoclaving and sonication did not release any detectable aflatoxin B1. Variation in temperature (4 to 37°C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B1 released. Binding of aflatoxin B1 appears to be predominantly extracellular for viable and heat-treated bacteria. Acid treatment may permit intracellular binding. In all cases, binding is of a reversible nature, but the stability of the complexes formed depends on strain, treatment, and environmental conditions. Food contaminants entering the body through the oral route are directly exposed to the action of gut microflora. Normal healthy intestinal microflora contains many strains of lactic acid bacteria (LAB), some of which have been isolated, ascribed health benefits, and termed probiotic strains (22). The protective effect of LAB against food mutagens such as heterocyclic amines, N-nitroso compounds, and aflatoxins has been reported (8, 12, 19, 24, 27). Many of these studies have involved Lactobacillus strains, and physical binding has been proposed as one mechanism of mutagen removal. This study focuses on the nature of the binding of aflatoxin B1 (AFB1) by 12 LAB strains. The potent mycotoxin AFB1 is a secondary metabolite of Aspergillus fungi that grow on a variety of food and feed commodities at any stage during growth, harvest, storage, and transportation. The occurrence of aflatoxin contamination is global, with severe problems especially prevalent in developing countries (11). Aflatoxins are of great concern because of their detrimental effects on the health of humans and animals, including carcinogenic, mutagenic, teratogenic, and immunosuppressive effects (3). Aflatoxins are also of industrial importance due to the economic losses resulting from condemnation of contaminated crops, cheese defects, and impaired growth and feed efficiency of animals fed contaminated feeds. Consequently there is a great demand for

Surface binding of aflatoxin B(1) by lactic acid bacteria.

Aflatoxin B1: A review on metabolism, toxicity, occurrence in food, occupational exposure, and detoxification methods.

Yeasts, molds, bacteria, actinomycetes, algae, and fungal spores were screened for their ability to degrade aflatoxin. Some molds and mold spores partially transformed aflatoxin B1 to new fluorescing compounds. Only one of the bacteria, Flavobacterium (aurantiacum?) NRRL B-184, removed aflatoxin from solution. Both growing and resting cells of B-184 took up toxin irreversibly. Toxin-contaminated milk, oil, peanut butter, peanuts, and corn were completely detoxified, and contaminated soybean was partially detoxified by addition of B-184. Duckling assays showed that detoxification of aflatoxin solutions by B-184 was complete, with no new toxic products being formed.

Microbial detoxification of aflatoxin.

A method has been developed for the production of aflatoxin by growing Aspergillus flavus NRRL 3145 on solid substrate wheat. Optimal yields of 900 mug of aflatoxin G(1) and 900 mug of aflatoxin B(1) per g of substrate were obtained in 4 to 5 days at 28 C. A study of aflatoxin production on hulls and groats of oats and on whole oats by A. flavus strains NRRL 2999, NRRL 3000, and NRRL 3145 revealed that aflatoxin was produced on all three substrates, although production was very slight on hulls. Strain NRRL 3145 grown on solid substrate groats produced the largest amounts of aflatoxin: 580 mug of B(1) and 450 mug of G(1) per g of substrate. A densitometric method for reading thin-layer chromatographic plates is described; this is more objective and more accurate than the visual methods previously used for the determination of all four aflatoxins.

Production of aflatoxin on wheat and oats: measurement with a recording densitometer.

Yields of from 200 to 300 mg per liter of aflatoxins B1 and G1 were produced by two strains of Aspergillus flavus in 20-liter fermentors under proper conditions of inoculum (well-dispersed growth) and aeration (0.5 volume per volume per min of air, 300 rev/min, 30 psi back pressure, baffles). Peak yields were usually attained in 72 hr, after which the aflatoxin concentration declined rapidly. Degradation of aflatoxin depended primarily on mycelial lysis and high-aeration conditions. Cultures previously reported not to degrade aflatoxin could be induced to do so under these conditions. The percentage and rate of toxin degradation were independent of toxin concentration, and appeared to be nonenzymatic and nonspecific. Degradation simulating that occurring in the fermentor was achieved by reacting aflatoxin with peroxidized methyl esters of vegetable oil; initial degradation was rapid and appeared to involve a complex series of reactions.

Aflatoxin production and degradation by Aspergillus flavus in 20-liter fermentors.

Removal of aflatoxin B(1) from liquid cultures by resting and growing cells of Flavobacterium aurantiacum NRRL B-184 was studied. Spectrophotometic and thin-layer techniques served as aflatoxin assays. Cells grown in the presence of 5 ppm or higher levels of aflatoxin developed aberrant morphological forms. These toxin concentrations partially inhibited growth, and the nature of the inhibition suggested that aflatoxin interfered with cell wall synthesis. Incubation of 1.0 x 10(11) resting cells per milliliter with 7.0 mug/ml of aflatoxin B(1) during a 4-hr period facilitated complete toxin removal from a buffered aqueous medium. Autoclaved cells and cell wall preparations could remove a fraction of the aflatoxin of a test system. However, the toxin removed by autoclaved cells and cell walls could be extracted by washing with water but the aflatoxin B(1) removed by intact cells could not be extracted into the liquid phase. The uptake of aflatoxin B(1) by resting cells was sensitive to temperature and pH. Ruptured preparations of F. aurantiacum were not able to remove or modify the aflatoxin in an aqueous solution.

H. H. Hall

Papers

Microbial detoxification of aflatoxin.

Production of aflatoxin on wheat and oats: measurement with a recording densitometer.

Aflatoxin production and degradation by Aspergillus flavus in 20-liter fermentors.

Aflatoxin B1 Uptake by Flavobacterium aurantiacum and Resulting Toxic Effects

Further studies in the evaluation of carbohydrate utilization tests as aids in the differentiation of species of Streptomyces.