International Journal of Food Microbiology, 10 (1990) 73-90 73 Elsevier

FOOD 00298

Effect of polymyxin B nonapeptide and polymyxin B sulphate on trichothecene mycotoxin sensitivity

of yeasts using a conductimetric instrument

Peter Connolly and Janet E.L. Corry Food Science Dioision, Ministry of Agriculture, Fisheries and Food, London, U.K.

(Received 1 May 1989; accepted 17 August 1989)

The addition of polymyxin B sulphate (PBS), or an inactive by-product, polymyxin B nonapeptide (PBN) to a yeast bioassay system, increased its sensitivity to various toxic agents. The nil effect level (NEL) of T-2 toxin was reduced from 0.1 to 0.01 #g/ml for Kluyoeromyces fragilis GK 1005 in the presence of these agents when using a Malthus AT 192 conductimetric instrument. Other synergistic agents (DMSO, ethanol, cetyl trimethyl ammonium bromide and Triton X-100) gave poor results in the conductimetric system. PBN also increased sensitivity of K. fragilis GK 1005 towards cycloheximide in the Malthus system, and PBS reduced the NEL of T-2 toxin for K. fragilis GK 1005 in a disc diffusion assay from 0.2 to 0.04/~g per disc. No yeasts were found sensitive to the trichothecene deoxynivalenol (DON) even at a DON concentration of 10 ~g/ml, except in the presence of PBN and PBS. The minimal inhibitory concentration (mic) of DON in the presence of PBS was 2 #g/mi for K. fragilis GK 1005.

Key words: Conductance; Polymyxin B sulphate; Polymyxin B nonapeptide; Trichothecenes; T-2 Toxin; Deoxynivalenol; Yeast

Introduction

Trichothecene mycotoxins, produced ma in ly by F u s a r i u m spp., have been shown to be involved in m a n y toxicoses (Forgacs and Carll, 1962; Bamburg et al., 1969; Vesonder et al., 1973) and there is a need for rapid sensit ive assays to detect these toxins in foods and cultures. In s t rumen ta l methods for their detect ion inc lude high pressure l iquid chromatography, gas-liquid ch romatography and gas chromatogra- p h y / m a s s spectroscopy, often requir ing extensive c leanup procedures (Pathre and Mirocha, 1977). Enzyme- l inked i m m u n o s o r b e n t assays (ELISA) for t r ichothecenes have been developed (Zhang et al., 1986; G o o d b r a n d et al., 1987), bu t require separate ant ibodies for each studied toxin. Other suggested biochemical assays are inh ib i t ion of prote in synthesis (Ueno et al., 1973) a nd a g lu ta th ione epoxide

Correspondence to: J.E.L. Corry, J. Sainsbury plc, Scientific Services Division, Stamford House, Stamford Street, London, SE1 9LL, U.K.

0168-1605/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

74

transferase assay (Foster et al., 1975). In the presence of the enzyme glutathione reacts with the trichothecene epoxide moeity and residual glutathione is determined photometrically.

Bioassays used to detect trichothecenes include rodent skin tests, tissue culture techniques, chick embryo tests and pea germination inhibition (Watson and Lin- dsay, 1982). As bacteria have been reported to be insensitive to these toxins (Burmeister and Hesseltine, 1970) bioassays using micro-organisms have con- centrated on yeasts. Disc diffusion assays have used Rhodotorula rubra (Burmeister and Hesseltine, 1970; Stone et al., 1986) and Saccharomyces spp. and Kluy- veromyces fragilis (Schappert and Khachatourians, 1983; 1984b). More recent liquid culture methods involving impedimetric techniques used Hansenula fabianii and Pichia burtonii (Adak et al., 1987a,b).

Following the development of a medium suitable for monitoring the conductance changes produced by metabolically active yeast cultures (Connolly et al., 1988), work was carried out to assess the suitability of a conductimetric apparatus (the Malthus AT 192) as an analytical instrument to detect trichothecene mycotoxins.

Although trichothecenes are primarily protein synthesis inhibitors (Ueno et al., 1973), the degree of toxicity towards yeasts is believed to depend on the ability of the toxin to pass through the cell membrane (Schappert and Khachatourians, 1984a). In this study various membrane active agents (MAAs) were tested for their ability to enhance synergistically the toxicity of two trichothecenes, T-2 toxin and deoxynivalenol (DON). Several of these agents, i.e. cetyl trimethyl am- moniumbromide (CTAB), ethanol and Triton X-100, have been used before to enhance toxicity of trichothecenes towards yeasts (Schappert and Khachatourians, 1984a). Polymyxin B sulphate (PBS) and polymyxin B nonapeptide (PBN) have been used to increase toxicity of antibiotics towards yeasts (Boguslawski, 1985) and Gram-negative bacteria (Vaara and Vaara, 1983) and dimethyl sulphoxide (DMSO) has been used to increase sensitivity of bacteria to toxic agents and remove resistance towards antibiotics (Martin and Hanthal, 1974).

Preliminary work screening T-2 toxin and DON at 1 ~g /m l against over 180 yeasts confirmed the results of Schappert and Khachatourians (1983) and Adak et al. (1987a, b) that many strains were sensitive to T-2 toxin but none were sensitive to DON. Nine yeasts were further screened against 10/~g/ml DON and still found to be insensitive. Further studies were carried out using selected MAAs and assessing their effect in the presence of T-2 toxin and DON, using yeast strains considered most suitable. Most work was carried out using the Kluyverornyces fragilis strain of Schappert and Khachatourians (1984b) because it was the most sensitive to T-2 toxin.

Materials and Methods

Yeasts Yeasts used were Kluyveromyces fragilis G K 1005, courtesy of Professor G.

K_hachatourians, University of Saskatchewan, Canada; Candida albicans NCYC

75

1363, Hansenula canadensis NCYC 497, four strains of Saccharomyces cerevisiae (NCYC 177, NCYC 240, NCYC 244 and NCYC 975), Schizosaccharomyces pombe NCYC 380 and Torulopsis delbreukii NCYC 677 from the National Collection of Yeast Cultures, Norwich; and Hansenula fabianii CBS 5640 from the Centraal Bureau voor Schimmelcultures, Delft, Holland.

Preparation of yeast inocula Yeast inocula were prepared by inoculating into 9-ml volumes of CBAT (see

below) and incubating at 30 °C for 48 h. Cell concentrations were determined using haemocytometer slide counts and adjusted to 106 cel ls /ml in CBAT.

Media The medium used was carbon base ammonium tartrate (CBAT: Connolly et al.,

1988): carbon base (Difco) 22.0 g / l ; ammonium tartrate 3.0 g / l ; pH 5.5; auto- claved at 121°C for 15 min. The diluent was sterile quarter strength Ringers and 0.1% peptone.

Chemicals Trichothecenes T-2 toxin and deoxynivalenol were obtained from Sigma, Poole,

Dorset, U.K. Cetyl trimethyl ammoniumbromide, Triton X-100, cycloheximide and polymyxin B sulphate (PBS) were also obtained from Sigma. Polymyxin B non- apeptide (PBN) was obtained from Boehringer, Lewes, Sussex, U.K.

Preparation of polymyxin B sulphate (PBS) and polymyxin B nonapeptide (PBN) solutions

1 mg/ml and 3 mg /ml solutions of PBS and PBN respectively in CBAT were filter-sterilised and stored at 4 ° C until needed.

Growth analysis Medium conductance changes were monitored and recorded using a Malthus AT

192 (Malthus Instruments, Crawley, Surrey, U.K.) until the last sample reached the stationary conductance change phase unless otherwise stated.

Measurement parameters Peak response time (PRT) is the time at which the maximum rate of conductance

change occurs (Connolly et al., 1988).

Malthus assay procedure Assays using the Malthus AT 192 were carried out as follows: triplicate 1.6-ml

volumes of media for each condition to be studied were dispensed into sterile 2-ml Malthus cells. 0.4 ml of yeast inoculum were added and Malthus cells were sealed. After addition of the inoculum, cell concentration was 2 x 105 per ml. Incubation was carried out at 30 o C.

76

TABLE I

Concentration of membrane active agents (MAAs) and their effect at that concentration on the peak response time (PRT) 1 of Kluyoeromyces fragilis. Mean PRT in the absence of MAAs = 16.6 h

Membrane active Concentration Increase in PRT agent (MAA) (%)

Ethanol Dimethylsulphoxide (DMSO) Cetyl trimethyl ammonium bromide (CTAB) Triton X-100 Polymyxin B nonapeptide (PBN) Polymyxin B sulphate (PBS)

3.5% v/v 40 5.0% v/v 24 1/t g/ml 35 0.2% v/v 42

100 gg/ml 0 25 ~g/ml 0

1 PRT, peak response time - time at which maximum rate of conductance change occurs.

Production o f toxin dosed medium Por t ions of 1.0 m g / m l toxin s tock so lu t ions( in 95% d ich lo romethane , 5%

methano l ) were a d d e d to vo lumes of C B A T so that f inal toxin concen t r a t i on was 1.25 t imes grea ter than that requi red ( add i t i on of yeas t i nocu lum reduc ing the toxin concen t r a t i on b y a fac tor of 0.8). Con t ro l s ut i l i sed so lvent alone. M e d i u m was then hea ted with bo t t l e tops loosened at 6 0 ° C for 2 h to dr ive off solvents. This t r ea tmen t was def ined in p re l imina ry s tudies and it d id no t o therwise change m e d i a compos i t ions .

(i) T-2 toxin nil effect level (NEL) study. T-2 t ox in -dosed med ia were p r e p a r e d as above. Af t e r a d d i t i o n of the inoculum, final T-2 toxin concen t r a t i ons were 0.0, 0.05, 0.1, 0.4, 1.0 and 2.5 g g / m l .

(ii) T-2 toxin - cetyl trimethyl ammoniumbromide, ethanol and D M S O studies. T-2 tox in -dosed m e d i a were p r e p a r e d as above. Vo lumes of m e d i a were then d iv ided in to two equal por t ions . To the first was a d d e d one of the M A A s in Tab le I at 1.25 t imes the concen t r a t i on l isted in Tab le I ( to take in to account add i t i on of the inoculum) . To the second was a d d e d the same vo lume of steri le dis t i l led water . Af te r a d d i t i o n of the inoculum, final T-2 toxin concen t r a t i on was 0.0, 0.1, 0.4, 1.0 and 2.5 / z g /ml .

(iii) T-2 toxin - Triton X-IO0 studies. Tr i ton X-100 was a d d e d to C B A T to give concen t r a t ions of 0.0, 0.25, 0.75, 1.25, 2.5, 6.25 and 12.5% v / v . M e d i a were then d iv ided in to two al iquots. T-2 toxin was a d d e d to one and an equal vo lume of solvent was a d d e d to the other. Af te r a d d i t i o n of the inocu lum, the f inal T-2 toxin concen t r a t i on was 1.0 g g / m l and final concen t r a t i on of T r i t on X-100 was 0.0, 0.2, 0.6, 1.0, 2.0, 5.0 and 10.0% v / v .

(iv) T-2 toxin - polymyxin B nonapeptide (PBN) studies. T-2 tox in -dosed and con t ro l m e d i a (25 ml volumes) were d i spensed into s ter i le 1 oz universa l bo t t les to which 0.86 ml of the 3.0 m g / m l PBN so lu t ion was added . A f t e r a d d i t i o n of the inocu lum the f inal T-2 toxin concen t ra t ion was 0.0, 0.02, 0.05, 0.1 and 1 / ~ g / m l and the final P B N concen t ra t ion was 100 g g / m l .

(v) T-2 toxin - polymyxin B sulphate (PBS) studies. T-2 toxin dosed med ia were p r e p a r e d as above. 0.0625 g g / m l T-2 toxin m e d i u m was d i lu ted ( in to cont ro l

77

media) to T-2 toxin concentrations of 0.0025, 0.00625, 0.0125 and 0.025 /~g/ml. 25-ml volumes of media were dispensed into sterile 1 oz universal bottles to which 0.63 ml of the 1.0 m g / m l PBS solution were added. Final PBS concentration was 31.25 /~g/ml. After addition of the inoculum, the final T-2 toxin concentrations were 0.0, 0.002, 0.005, 0.01, 0.02, 0.05 and 0.1 /~g/ml. Conductance changes were monitored and recorded for 120 h.

(oi) Further T-2 toxin - Triton X-IO0 studies. Procedure was as in (iu) above except that (a) media contained 0.2% v / v Triton X-100 and (b) PBN was absent.

(uii) Cycloheximide studies. A solution of 1.25% w / v cycloheximide in CBAT was prepared and filter sterilised. Two 12.5-ml volumes were dispensed into sterile 1-oz universal bottles. Two 12.5-ml controls utilised filter-sterilised CBAT alone. To one volume of each (cycloheximide and control) 0.43 ml of PBN solution were added to give a PBN concentration of 125/~g/ml . To the other volumes 0.43 ml of filter-sterilised CBAT were added. After addition of the inoculum, final PBN concentration was 1 0 0 / t g / m l and final cycloheximide concentration was 1% w / v .

(oiii) Deoxynioalenol studies. To screen for susceptible yeasts deoxynivalenol (DON) dosed (12.5 ~ g / m l ) and control media were prepared as above. Volumes of PBS solution were added to give a concentration of 25 btg/ml PBS. After addition of the inoculum final D O N concentration was 10.0 ~tg/ml. The experiment was carried out in duplicate.

To carry out a minimal inhibitory concentration (MIC) study selected yeasts from above were studied with decreasing concentrations of D O N (with solvent controls). Final D O N concentrations studied were 0.0, 2.0, 4.0, 6.0 and 10.0 ktg/ml.

(ix) Disc diffusion assay. The disc diffusion assay of Schappert and Khachatourians (1984b) was repeated against T-2 toxin. A parallel experiment incorporating 25 # g / m l PBS into the agar instead of 0.5 /~g/ml cetyl trimethyl ammoniumbromide was included. The test was carried out in duplicate.

Results

Fig. 1 shows a typical conductance change profile for 2 x 105 ce l l s /ml of K. fragilis G K 1005 in CBAT at 30°C. Addition of T-2 toxin to the system is characterised by an increased time interval between the onset of the test and the time at which the maximum rate of conductance change, i.e. peak response time (PRT) is reached. The delay is proportional to the concentration of T-2 Toxin (Fig. 2).

Results here are expressed as a significant difference. That is, the difference between the PRT of the slowest reacting control and the fastest reacting toxin dosed sample of a test carried out in triplicate. A positive significant difference indicates no overlap of control and toxin dosed PRTs and a visible toxin effect should be seen using the Malthus instrument 's computer graphic functions. A negative significant difference is due to overlap of control and toxin dosed PRTs indicating no significant toxin effect.

78

E

v

o t.)

L L _ I II

~.i,.. 1 .0 3 0 480 ~', Time (h) ~::,,

I I 200 ~ ,

" - - - - . . . . . 41

- - 4 0 0

Fig. 1. Change of conductance vs. time for Kluveromyces fragilis GK 1005 in CBAT (Carbon base ammonium tartrate) at 30 ° C (inoculum of 2 x 105 cel ls /ml) .

Preliminary work (results not shown) studied the effect of the M A A s in the absence of toxin. Table I summarises their inhibitory effect on K. fragilis G K 1005. Fig. 3 indicates that 5% D M S O leads to a decrease in T-2 toxin sensitivity at low toxin concentrations but gives an increase in sensitivity at high concentrations.

Ethanol appears to have a similar but probably lesser effect to D M S O on T-2 toxin sensitivity (Fig. 4). Addition of cetyl trimethyl ammoniumbromide (CTAB) to

40-

3 0 "

rr a_ 20- .c "U D

10"

0 I 1 1 I 0.5 1.0 1.5 2.0 2.15

-0.~ %2 toxin (pg/ml)

Fig. 2. Effect of T-2 toxin on Kluyveromyces fragilis GK 1005. Mean and range of increase in peak response time (PRT) vs. T-2 toxin concentration for three experiments. Mean PRT of control with no

toxin = 1 6 . 0 h .

79

4 o . /

30.

_: 20 / /

10-

0 : I 0.5 1.0 1 5 2.0 2 5

-0.5 T-2 toxin (pg/ml)

Fig. 3. Effect of DMSO on T-2 toxin sensitivity of Kluyveromycesfragilis GK 1005. Control (no DMSO) ( I ) ; + 5% v / v DMSO (e). Mean control (no toxin, no DMSO) peak response time (PRT) = 17.3 h.

8 0 -

70-

/ /

6 0 - / / o-,/

5 0 -

.c_ 40- /

30 - /

20 - ,,

10-

I ! 0 015 110 1.5 2,0 2.5

-0 .5 T-2 toxin (p.g/ml)

Fig. 4. Effect of ethanol on T-2 toxin sensitivity of Kluyveromyces fragilis GK 1005. Control (no ethanol) ( I ) ; + 3.5% ethanol (e). Mean control (no toxin, no ethanol) peak response time (PRT) = 17.7 h.

80

60-

5o

40 / ~ , ~ % /

c 30

~ ,

10

O~ ~. 0.5/~ 0.5 1.0 1.5 2.0 2.5

T-2 toxin (p.g/ml) Fig. 5. Effect of cetyl trimethyl ammoninmbromide (CTAB) on T-2 toxin sensitivity of Kluyverornyces fragilis GK 1005. Control (no CTAB) II; + 1 # g / m l CTAB e. Mean control (no toxin, no DMSO) peak

response time (PRT) = 17.2 h.

25

~~ 2 0 : ~

-615. tm

10-

5-

,% I I I u

1 2 3 7 , , , ,

4 5 6 ~ I r 10 Triton x-lO0 (%v/v)

-05

Fig. 6. Effect of Triton X-100 on the toxicity of 1 # g / m l T-2 toxin on Kluyveromycesfragilis GK 1005. Mean significant difference between control and toxin dosed triplicates in the presence of varying amounts of Triton X-100 for three experiments. Mean control (no toxin, no Triton X-100) peak response

time (PRT) = 16.6 h.

251 /

! " J 20- /

/ /

/

~" 15- / / a. /

~- 7 / " D /

J 10- / /

J / J

J J

/ j 5- / . /

/ /

J /

g l

0.5J ~ O. 1 0.2 0.3 0.4 05 0.6 0.7 0.8 O. 9 1.0

T-2 toxin (I.tg/ml)

Fig. 7. Effect of Tr i ton X-100 on T-2 toxin sens i t iv i ty of Kluyveromycesfragilis G K 1005 in the presence (O) and absence (U) of 0.2% Tr i ton X-100. Mean cont ro l (no toxin, no Tr i ton X-100) peak response t ime

(PRT) = 16.8 h.

25-

_ / / / /

.c 15- / / E ~ .c_ / /

10- / D /

5- / / /

/ /

/ Control 0 / ,_~._______-a

002 004 0.06 0.08 01 -0.5 T-2 toxin (gg/ml)

Fig. 8. Effect of T-2 toxin on Kluyveromycesfragilis G K 1005 in the presence (O) and absence (m) of 100 # g / m l po lymyxin B nonapept ide (PBN). Mean con t ro l (no toxin, no PBN) peak response t ime

(PRT) = 17.3 h.

82

20-

15-

o_ lO- c

a

5-

/" / ~ ' / - [

/

/

i # / /#

J

OOl

Control

o i ' ' i

0.02 003 0.04 005 - 0 5 T-2 toxin (~.g/ml)

Fig. 9. Effect of T-2 toxin on Klvyvesomyces fragilis OK 1005 in the presence (e ) and absence ( I ) of 25 ~ g / m l polymyxin B sulphate (PBS). Mean control (no toxin, no PBS) peak response time (PRT) = 16.9 h.

the system led to a large increase in sensitivity, with no growth occurring at T-2 toxin concentrations higher than 1 /~g/ml. Unfortunately, CTAB also gave an unpredictable response (Fig. 5). Fig. 6 emphasises that, except at levels of 0.2% or below, Triton X-100 considerably reduced T-2 toxin sensitivity. In view of this, the effect of 0.2% Triton X-100 was studied and was found to reduce the T-2 toxin MIC from 0.1 to about 0.05 /~g/ml (Fig. 7).

Figs. 8 and 9 show that both PBN and PBS considerably increase the sensitivity of K. fragilis to T-2 toxin. After these results had been obtained with PBN, lower T-2 toxin concentrations were studied with PBS to determine the new MIC. With both agents T-2 toxin concentrations greater than 0.1 /~g/ml allowed no growth response within 80 h of commencing the test whereas the PRT was reached in approximately 16 h in their absence. S. cerevisiae N C Y C 975, normally relatively insep.fitive to T-2 toxin, N E L > 2.5 f fg /ml (unpublished results) also showed increased sensitivity in the presence of PBN (Table II). Table III shows that PBN also increases the sensitivity of K. fragilis GK 1005 to cycloheximide.

TABLE II

Effect of 100 / tg /ml polymyxin B nonapeptide (PBN) on sensitivity of Saccharomyces cereuisiae N C Y C 975 to T-2 toxin (Nil effect level for this organism is > 2.5/.tg/ml T-2 toxin)

T-2 toxin PRT 1 Mean PRT+__ range Significant concentration (h) (h) difference 2 ( ~ g /ml) (h)

0.0 13.2; 12.8; 13.6 13.2+0,4 0.1 12.4; 12.4; 13.2 12.7 +_ 0,4 - 1.3 1.0 19.2; 16.8; 20.8 18.9+2,0 3.3

1 PRT: see Table I. 2 Significant difference: see Results.

83

T A B L E I l l

Effect of 100 ~ g / r n l p o l y m y x i n B nonapep t i de (PBN) on sens i t iv i ty of Kluyveromyces fragilis G K 1005

to 1% ( w / v ) cyc lohex imide

P R T (h) * M e a n Signif icant PRT_+ range (h) d i f ference (h) 2

Con t ro l I 16.4; 16.4; 15.6 16.1 + 0 . 4

(No cycloheximide , no PBN) 1% Cyc lohex imide 32.0; 34.8; 36.0 34.3 + 2.0

(No PBN) Con t ro l II 17.2; 16.4; 17.6 17.1 + 0 . 6

( + PBN no cycloheximide) 1% Cyc lohex imide 40.8; 41.6; 40.0 40.7 -+ 0.8

( + PBN)

15.8

22.2

1 P R T : see Tab le I. 2 S igni f icant difference: see Results .

All yeasts listed in Materials and Methods were tested against 10 /~g /ml D O N in the presence of PBS. Table IV demonstrates that seven out of nine of the test yeasts became sensitive to D O N although in the absence of PBS or PBN they had previously been insensitive (data not included). K. fragilis G K 1005 and S. cerevisiae NCYC 975 were picked for further studies and NELs of < 2.0 and 6.0 /zg/ml D O N were determined, respectively (Fig. 10). Although T. delbreukii N C Y C 677 appears from Table IV to be the most D O N sensitive yeast, this yeast was unsuitable for further study due to its long and often unreproducible peak response time.

T A B L E IV

Effect of deoxyn iva leno l (10 b tg /ml ) on P R T (h) 1 in the p resence of 25 ~ g / m l p o l y m y x i n B su lpha te on

test yeas ts

Yeas t 1st Expt 2 2nd Exp t 2 M e a n 3 Sensi t iv i ty 4

T-2 tox in D O N

C. albicans N C Y C 1363 - 1 . 2 0.2 - 0 . 5 + 0 . 7 - - H. canadensis N C Y C 497 - 1 . 0 0.2 - 0 . 4 ± 0 . 6 - -

H. fabianii CBS 5640 4.4 6.6 5.5 + 1.1 + + + K. fragilis G K 1005 7.2 12.5 9.9 + 2.7 + + + + + + Sacch. cerevisiae N C Y C 240 3.8 5.1 4 . 5 + 0 . 7 + + +

Sacch. cerevisiae N C Y C 244 6.0 5.5 5.8 + 0.3 + + + + Saeeh. cerevisiae N C Y C 975 4.8 3.1 4.0___0.9 - + + Sehiz. pombe N C Y C 380 0.4 0.2 0.3 ___ 0.1 + + + T. delbreukii N C Y C 766 16.1 25.1 20.8 + 4 . 7 - + + +

1 PRT: see Tab le I. 2 Expressed as s igni f icant difference, see Resul ts . 3 M e a n + range. 4 _ to + + + denotes inc reas ing toxin sensi t ivi ty.

84

t4-

12-

10-

.=

r-',

6 -

4.

2-

2 4 6 ~ - 8 " ~ 10

D O N (p.g lml)

Fig. 10. Effect of increasing levels of deoxynivalenol (DON) on peak response time (PRT) of Kluy- veromyces fragilis G K 1005 ( I ) and Saccharomyces cerevisiae N C Y C 975 (e). Mean control (no toxin)

peak response time (PRT) = 16.5 and 12.3 h, respectively.

Fig. 11 shows that when the assay of Schappert and Khachatourians (1984b) was duplicated the same limit of detection for T-2 toxin was found, although smaller zones of inhibition were found than in the original work. Substituting PBS for CTAB increased sensitivity such that the limit of detection was decreased from 0.2 to 0.04 fig T-2 toxin. In no instance was the range of measurements greater than 1 mm for any toxin concentration studied.

Discussion

Yeasts Previous work in this laboratory (results not shown) indicated that of over 180

yeasts studied K. fragilis GK 1005 was by far the most T-2 toxin sensitive. S. cerevisiae NCYC 975 was found to be relatively insensitive towards T-2 toxin at concentrations up to 2.5 #g /ml . The yeasts in Table IV were of intermediate sensitivity towards T-2 toxin, sensitivity decreasing apparently in the order S. cerevisiae N C Y C 244, S. pombe NCYC 380, S. cerevisiae NCYC 240, and H. fabianii CBS 5640. C. albicans NCYC 1363, H. canadensis NCYC 497, S. cerevisiae NCYC 975 and T. delbreukii were not sensitive to T-2 toxin at a concentration of 1.0/zg/ml.

85

~ ~ PBS'

15- ._~ / s " K"_.o

/ ...o . . . . .

10- /

/ ' ' ' ', 'o 0.2 0.4 0.6 0 8 1.

T-2 toxin (p.g/ml)

Fig. 11. Effect of T-2 toxin on zone of no growth for Kluyverornycesfragilis GK 1005 in a disc diffusion assay. S&K (111), Schappert and Kachatourians (1984b); S&K ~l (©) and S&K #2 ( i ) , Schappert and Kachatourians assay reproduced here; PBS #1 (z~) and PBS ~2 (A), 25 #.ig/ml PBS instead of 0.5% cetyl

trimethyl ammoniumbromide , as used by Schappert and Kachatourians.

Effect of membrane actiue agents Although DMSO has been used to increase toxin sensitivity of bacteria (Mart in

and Hanthal, 1974) no such effects have been observed with yeasts, and in one instance DMSO was reported to decrease the sensitivity of a yeast to a toxin (Danilenko et al., 1982) as happened here at low T-2 toxin concentrations (Fig. 3). We observed increased sensitivity to T-2 toxin in the presence of 5% v / v DMSO at toxin concentrations greater than 1 #~g/ml.

In the present work ethanol increased sensitivity of K. fragilis to higher levels of T-2 toxin but failed to decrease the N E L (Fig. 4).

Alcohols have been shown to change membrane conformation (Ingrains, 1976) and may therefore lead to altered membrane permeabil i ty characteristics. Cetyl trimethyl ammoniumbromide (CTAB) gave an unpredictable response and at con- centrations of T-2 toxin greater than 1 /xg/ml no growth was seen within 120 h of commencing the test (Fig. 5). Previous workers (Schappert and Khachatourians, 1984a) found a decrease in the N E L of T-2 toxin on Saccharomyces carlsbergensis when using 1 #xg/ml CTAB or 5% v / v ethanol, which they found to be the optimal

86

concentration for T-2 toxin sensitivity. Results from previous work (not shown) indicate that 5% ethanol led to a greater increase in T-2 toxin sensitivity than 3.5% ethanol, but this concentration of ethanol was inhibitory per se. Ethanol inhibits ammonium uptake by yeasts (Leao and Van Uden, 1983) which might explain the decreased growth response in the presence of ethanol in the carbon base ammonium tartrate medium used in the present studies. Schappert and Khachatourians (1984a) used a yeast (S. carlsbergensis) relatively insensitive to T-2 toxin in comparison to K. fragilis G K 1005 and lowered the MIC from 2.0 to 0 .5 / zg /ml when using CTAB, ethanol and Triton X-100,

Different levels of Triton X-100 had a profound effect on T-2 toxin sensitivity of K. fragilis G K 1005. Low levels increased sensitivity while high levels (up to 10%) decreased toxin response so that the toxic effect was eliminated (Fig. 6). Similar results have been found previously (Schappert and Khachatourians, 1984a). Unlike ethanol, Triton X-100 (0.2% v / v ) decreased the NEL of K. fragilis G K 1005 from 0.1 to 0.05 btg/ml T-2 toxin (Fig. 7).

Effect of polyrnyxin B nonapeptide (PBN) and polymyxin B sulphate (PBS) PBN and PBS were found to be the most effective membrane active agents. Figs.

8 and 9 indicate a large increase in sensitivity of K. fragilis G K 1005 towards T-2 toxin due to PBN and PBS, reducing the N E L of T-2 toxin to 0.005 / tg /ml . With both these agents no growth or conductivity response was evident after 120 h of commencing the test when the T-2 toxin concentration was greater than 0.1 ~ g / m l . Unlike the other agents used in this study, PBN and PBS at concentrations of 100 /~g/ml and 25 ~tg/ml respectively had no detectable effect upon the growth of K. fragilis G K 1005 (Table I),

S. cerevisiae N C Y C 975, a yeast relatively insensitive towards T-2 toxin is also susceptible to the synergistic action of PBN (Table II) although it is still less sensitive than K. fragilis G K 1005 in the presence of PBN. Table I I I indicates that synergism is not specific to T-2 toxin, although the increase in sensitivity is less marked with cycloheximide.

Effect of PBS on the toxicity of deoxynivalenol Table IV shows for the first time sensitivity of yeasts towards DON, albeit at a

concentration three magnitudes higher than that needed for T-2 toxin to show a response. Seven of the nine yeasts were found to be sensitive to DON. Five of these seven were found to be sensitive to 1 / t g / m l T-2 toxin, while two yeasts were sensitive to neither T-2 nor DON. Using PBS, the lower limit of toxicity of D O N towards K. fragilis G K 1005 was less than 2 ~ g / m l . Although only a few yeasts have been tested using this toxin K. fragilis G K 1005 was found to be the most sensitive yeast to D O N as well as T-2 toxin, except for T. delbreukii N C Y C 677. Reproducibility of growth response of T. delbreukii was so poor that less than 6 ~ g / m l D O N could not be detected (results not shown). Results indicate that PBS can remove resistance of S. cerevisiae N C Y C 975 to D O N (Table IV).

Of the 60 or so trichothecenes presently known D O N has a relatively low toxicity, but its frequent occurrence in cereal crops necessitates its detection (Pathre and Mirocha, 1978; Elliot, 1982; Tanaka et al., 1986). Prior to this study, no

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microorganisms sensitive to DON had been reported, and with the exception of skin necrosis tests few data exist on DON in bioassays (Watson and Lindsay, 1982).

Polymyxin B is produced by Bacillus polymyxa and is toxic towards bacterial, fungal and animal cells. It is composed of a cyclic peptide portion joined to a fatty acyl moeity (Glasby, 1979). Proteolytic cleavage of the fatty acyl portion removes the antimicrobial activity of both portions but the non-toxic product, polymyxin B nonapeptide (PBN), increases sensitivity towards toxic agents (Chihara et al., 1973; Vaara and Vaara, 1983; Boguslawski, 1985).

Polymyxin B acts by binding to membranes, destroying osmotic properties and causing leakage of metabolites from within the cell (Storm et al., 1977). It is believed that phosphatidyl ethanolamine is the target molecule in the cell membrane, forming two bonds with suitably protonated amino residues in polymyxin: an electrostatic bond with the phosphate group and a bond involving proton exchange with the amino group of the ethanolamine. Stabilisation of the peptide to allow this bonding to take place comes from the fatty acyl portion of the molecule which inserts into the hydrophobic interior of the cell membrane (Storm et al., 1977). Because polymyxin B nonapeptide lacks the hydrophobic anchor, interactions of the peptide and membrane are of a more transient nature, explaining firstly the need for higher concentrations of PBN than PBS and secondly the former's relative non- toxicity. Its non-toxicity to yeasts means PBN may prove an ideal synergistic agent for mycotoxin studies.

The MIC of T-2 toxin for K. fragilis is 0.01 /~g/ml (0.02 #g per assay) in the presence of PBN or PBS. This figure compares with 0.2/~g for the same yeast using a disc diffusion assay and the synergistic agent cetyl trimethyl ammoniumbromide (Schappert and Khachatourians, 1984b), 0.019 #g per assay for Rhodutorula rubra UV mutants (Stone et al., 1986) and 0.024, 0.036 and 0.8 ~tg per assay for Hansenula fabianii, Pichia burtonii and Candida albicans, respectively (Adak et al., 1987b). We have tested R. rubra mutants (Stone et al., 1986) against T-2 toxin in the Malthus instrument (results not shown) and although very sensitive, the rate of growth of the yeast was too slow to warrant further study. Stone et al. (1986) suggest that an assay time of greater than 48 h is required for the most T-2 toxin sensitive UV mutant. Using K. fragilis G K 1005 in the present work the peak response time was reached in approximately 16 h and visual inspection of conductance change curves indicated presence of T-2 toxin as little as 6 h after commencing the test. Increasing the assay temperature to 35°C increases the speed of response of the yeast (results not published).

The most important practical result described here is the reduction of T-2 toxin NEL in the disc diffusion assay. Sensitivity towards D O N is too slight to be detected by disc assay and few laboratories possess a conductimetric instrument such as the Malthus AT. The present work however indicates that for a bioassay the Malthus instrument is both faster and more sensitive than conventional methods. Further work will be carried out in an attempt to improve sensitivity to DON in both the disc assay and conductimetric instrument. The response of the assay system to a wider range of mycotoxins as well as extracts from mycotoxin-con- taminated foods and feedstuffs will also be investigated.

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