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  • Vol. 55, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1989, p. 190-197 0099-2240/89/010190-08$02.00/0 Copyright © 1989, American Society for Microbiology

    Biotransformation and Detoxification of T-2 Toxin by Soil and Freshwater Bacteria

    STEVEN BEETON AND ALAN T. BULL*

    Biological Laboratory, University of Kent at Canterbury, Canterbury, Kent CT2 7NJ, United Kingdom

    Received 12 July 1988/Accepted 26 October 1988

    Bacterial communities isolated from 17 of 20 samples of soils and waters with widely diverse geographical origins utilized T-2 toxin as a sole source of carbon and energy for growth. These isolates readily detoxified T-2 toxin as assessed by a Rhodotorula rubra bioassay. The major degradation pathway of T-2 toxin in the majority of isolates involved side chain cleavage of acetyl moieties to produce HT-2 toxin and T-2 triol. A minor degradation pathway of T-2 toxin that involved conversion to neosolaniol and thence to 4-deacetyl neosolaniol was also detected. Some bacterial communities had the capacity to further degrade the T-2 triol or 4-deacetyl neosolaniol to T-2 tetraol. Two communities, TS4 and KS10, degraded the trichothecene nucleus within 24 to 48 h. These bacterial communities comprised 9 distinct species each. Community KS10 contained 3 primary transformers which were able to cleave acetate from T-2 toxin but which could not assimilate the side chain products, whereas community TS4 contained 3 primary transformers which were able to grow on the cleavage products, acetate and isovalerate. A third community, AS1, was much simpler in structure and contained only two bacterial species, one of which transformed T-2 toxin to T-2 triol in monoculture. In all cases, the complete communities were more active against T-2 toxin in terms of rates of degradation than any single bacterial component. Cometabolic interactions between species is suggested as a significant factor in T-2 toxin degradation.

    The trichothecenes are a group of toxic secondary metab- olites produced by several genera of fungi, such as Fusar- ium, Myrothecium, Trichothecium, and Stachybotrys (Table 1) (1, 15, 21). Ingestion of mouldy cereal crops contaminated by these toxigenic fungi has resulted in serious outbreaks of mycotoxicoses in humans and domesticated animals (4, 9). The toxicological effects of these mycotoxins include cyto- toxicity (7) and potent inhibition of eucaryotic protein syn- thesis (21).

    Studies on trichothecene transformations have centered on in vivo and in vitro mammalian systems, and in general, these studies have revealed the production of other less toxic trichothecenes (5, 13, 17, 23). Mammalian transformations of trichothecenes appear to be catalyzed by nonspecific car- boxylesterases located in the microsomal fraction of liver (5, 10). Early research also showed that a number of microor- ganisms were able to transform but not to degrade tricho- thecenes (3, 26, 27).

    Interest in microbial transformations was stimulated by the discovery of a detoxification pathway for T-2 toxin [4, - 15 - diacetoxy - 8a -(3 - methylbutyryl - oxy) - 12,13 - epoxy - trichothec-9-ene] and deoxynivalenol [3a,7a-15-trihydroxy- 12,13-epoxytrichothec-9-ene-8-oneJ, two naturally occurring trichothecenes found in mouldy cereals, to their respective nontoxic de-epoxy forms (11, 12). The possible role of soil bacteria in trichothecene transformation was suggested by Ueno et al. (22). Aerobic axenic cultures of a Curtobacte- rium species strain 114-2, isolated from soil, transformed T-2 toxin to T-2 triol [3a,43-15-trihydroxy-8-(3-methylbutyry- loxy)-12,13-epoxytrichothec-9-ene] via HT-2 toxin [15-ace- toxy - 8a - (3 - methylbutyryloxy) - 3a,4I3 - dihydroxy - 12,13 - epoxy-trichothec-9-ene], two trichothecenes belonging to the T-2 toxin series. T-2 toxin could be utilized by this bacterium as a sole source of carbon and energy, and the authors reported that prolonged incubation resulted in the

    * Corresponding author.

    disappearance of T-2 triol without the further production of any related trichothecene structures (16, 22). In common with in vitro transformations by mammalian systems (6, 10), the initial deacetylation reactions of the Curtobacterium isolate were catalyzed by soluble carboxylesterases. We have investigated the role of natural bacterial commu-

    nities and monocultures as agents for the detoxification and biodegradation of T-2 toxin and related trichothecenes. Bacterial communities capable of detoxification and biodeg- radation were enriched from soil and freshwater samples, and their detoxification capacities were screened by a re- cently described bioassay (19) based on the sensitivity of the yeast Rhodotorula rubra. The efficacy of bacterial commu- nities from widely diverse geographical locations in the detoxification of T-2 toxin is discussed, together with some features of the community structures.

    MATERIALS AND METHODS Chemicals. T-2 toxin, deoxynivalenol, and neosolaniol

    [4,-15-diacetoxy-3co,8a-dihydroxy-12,13-epoxytrichothec-9- ene] were gifts from the Chemical Defence Establishment, Porton Down, Wiltshire, England. Standard trichothecene kits containing T-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol [3a,4,B,8a - 15 - tetrahydroxy - 12,13 -epoxytrichothec - 9 - ene], verrucarol [4,B-15-dihydroxy-12,13-epoxytrichothec-9-ene], and diacetoxyscirpenol [3-hydroxy-4,-15-diacetoxy-12,13- epoxytrichothec-9-ene] were purchased from Sigma London Chemical Company, Pool, Dorset, England. Tetraethylene pentamine, 4-dimethylamino pyridine, 3-nitrobenzoyl chlo- ride, and 4-nitrobenzyl pyridine were purchased from Fluka, Buch, Switzerland. All other chemicals or solvents were either chemically pure or of analytical reagent grade.

    Environmental samples. Environmental samples for en- richment isolation of T-2 toxin-detoxifying microorganisms were collected from the following sites (bacterial community designations in parentheses): leaf litter and soils, Canter- bury, Kent, England (KS); the River Stour, Kent, England

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  • TRANSFORMATION OF T-2 TOXIN 191

    TABLE 1. Chemical structures and chromatographic data for T-2 toxin and other trichothecenes

    1 2 3 A

    T-2trolOH H0129.0 2.1

    CH2R 2~~

    4-Deactheneosolanio OH OAOH N LN

    Retenionime (m) o p-ntrobetuoedervatves

    (TS);hoteffuncaermsrenea pape manufacture HPChin

    result' 1 2 3 A B

    T-2 toxin OAc OAc V 0.53 0.59 9.45 HT-2 toxin OH OAc V 0.23 0.21 18.40 Neosolaniol OAc OAc OH 0.32 0.30 8.35 T-2 tdiol OH OH V 0.12 0.09 26.17 4-Deacetylneosolaniol OH OAc OH NDod ND ND T-2 tetraol OH OH OH 0.05 0.02 20.54

    a See diagram above. OAc, Acetate; V, isovalerate. lDetermined by TLC (see Materials and Methods). oRetention time (min) of p-nitrobenzoyl derivatives. reND, Not determined.

    (KW); soils from deciduous forest, Chiang Mai, Thailand (TS); effluent water from screen paper manufacture, Chiang Mai, Thailand (TW1); soils from Signy Island, Antarctica (AS); the River Rhine, Cologne, Federal Republic of Ger- many (GW and GU). Soil samples (ca. 1.0 g) were shaken with distilled water (5.0 ml), and water samples (1.0 ml) were used directly as inocula for enrichment cultures. Enrichment isolation. Soil and water inocula were used to

    set up enrichument cultures in a basal medium (8) supple- mented with the following trace elements (grams liter-L): H3BO3, 0.05; MnSO4 -4H20, 0.04; (NH4)6Mo7024, 0-02; KI, 0.01; CUS04 - H2O, 0.004; CoCl6 -6H20, 0.00. The initial pH of the medium was adjusted to 7.0 with NaOH solution (1 M). Initial experiments were based on a T-2 toxin concentration of 1 mg ml-a, but at this concentration, T-2 toxin was insoluble, and therefore the concentration was reduced to 1 mM (0.046 mgml-t), at which T-2 toxin was soluble. Incubations (10 ml) were made in 100-ml Erlen- meyer flasks at 30C and 200 irpm. Isolates AS1 and AS2 were incubated at 4°C without shaking. After 7 days of incubation, samples (0.1 ml) of the enrichment cultures were removed aseptically and inoculated into 10 ml of fresh T-2 toxin medium. Daily samples (0.46 ml) were removed asep- tically for analysis. The course of T-2 toxin biotransforma- tion was followed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) of culture supernatants. Supernatants were shaken three times with equal volumes of ethyl acetate (0.46 ml), and the organic phase was analyzed. The efficiency of this extraction proce- dure has been demonstrated by Yagen et al. (25), who reported recoveries of T-2 toxin and HT-2 toxin from plasma of 95 and 102%, respectively, by using a single ethyl acetate extraction. We have obtained similar recoveries from culture supernatants. The culture supernatants were also subjected to a bioassay to determine residual trichothecene toxicity. Bacteriological analyses were made as described below.

    Assimilation of other trichothecenes by bacterial communi- ties. To basal medium (10 ml), separate additions of the

    following trichothecenes were made: HT-2 toxin, 0.02 mM; neosolaniol, 1.0 mM; diacetoxyscirpenol, 1.0 mM; deoxyni- valenol, 1.0 mM; roridin A, 0.05 mM; verrucarin A, 0.5 mM; verrucarol, 0.1 mM. Incubations, sampling, and analyses were made as for T-2 toxin enrichment experiments. Chemical analysis of trichothecenes. In TLC analysis, two

    solvent systems were used: chloroform-acetone (3:2, vol/ vol) (system A) and chloroform-methanol (95:5, vol/vol) (system B). T-2 toxin and trichothecene metabolites were visualized either by spraying with 10% H2SO4 and then heating or by treatment with 4-(p-nitrobenzyl) pyridine and then with tetraethylenepentamine (20, 21). Photographic negatives of de