- Email: [email protected]
Production and properties of crude enterotoxin of Pseudomonas aeruginosa
International Journal of Food Microbiology, 10 (1990) 201-208 Elsevier
201
FOOD 00303
Production and properties of crude enterotoxin of Pseudomonas aeruginosa Sunita Grover, V.K. Batish and R.A. Srinivasan Dairy Microbiology Division, National Dairy Research Institute, Karnal, India
(Received 17 February 1989; revision received 30 August 1989; accepted 4 September 1989)
Pseudomonas aeruginosa CTM-3 was found to be the most potentially enterotoxigenic strain out of the 12 isolates recovered from milk, as a high fluid length ratio, i.e. F/L (1.1) in rabbit gut and a strong permeability response in rabbit skin (38.5 mm2 necrotic zone) was obtained with this culture. No clear-cut relationship between the two tests was observed. Six of the ethidium bromide (300 ~g/ml) cured variants of this culture completely lost their ability to produce enterotoxin indicating the possible involvement of a plasmid in enterotoxin synthesis. The crude enterotoxin from P. aeruginosa CTM-3 was completely inactivated in 15 s at 72°C. However, it was fairly stable at pH values in the range 4.5-7.5. Both pepsin and trypsin inactivated the enterotoxin activity at a concentration of 40 ~g/ml. Organic acids, formalin and hydrogen peroxide had no significant effect on the enterotoxin activity. The need for further investigations with purified preparations is emphasized.
Key words: P. aeruginosa CTM-3; Crude enterotoxin; Milk
Introduction P. aeruginosa produces a variety of extracellular toxins a n d enzymes that make it a p o t e n t i a l l y p a t h o g e n i c organism. The various extracellular p r o d u c t s p r o d u c e d i n c l u d e a m o n g others exotoxin A, enterotoxin, protease, lipase, lecithinase, elastase, D N a s e a n d h a e m o l y s i n (Liu, 1974). E n t e r o t o x i n induces fluid a c c u m u l a t i o n in the l u m e n of intestine a n d is distinct from the other toxic c o m p o u n d s a n d the lethal toxins ( K u b o t a a n d Liu, 1971; Liu, 1974). There have b e e n few reports o n the vascular p e r m e a b i l i t y factor a n d e n t e r o t o x i n p r o d u c t i o n in P. aeruginosa ( K a u s a m a a n d Suss, 1972; K a u s a m a , 1974; Shriniwas et al., 1979). The potential role of P. aeruginosa in gastro-intestinal infections appears to be overlooked. Moreover, the e n t e r o t o x i n has n o t yet b e e n fully characterized. Nevertheless, there are a few isolated reports i n d i c a t i n g the i n v o l v e m e n t of P. aeruginosa with gastroenteritis (Pande, 1976; Pottecher et al., 1979; H u t c h i n s et al., 1979). Hence, the present investigation i n t e n d s to study e n t e r o t o x i n p r o d u c t i o n b y P. aeruginosa strains isolated from milk a n d milk products a n d properties of the crude enterotoxin.
Correspondence address: S. Grover, Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001 (Haryana), India.
0168-1605/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
202 Materials and Methods
Cultures A total of 12 haemolytic strains of P. aeruginosa recovered from different milk and milk products as previously described (Grover and Srinivasan, 1988) were used in this investigation. In addition, the study also included a reference strain P. aeruginosa 46 isolated from gastroenteritis which was procured from Post-graduate Institute of Medical Sciences, Chandigarh. The cultures were maintained in the refrigerator on nutrient agar (Hi-media, India) slants with subculturing every 2 weeks. The cultures were thoroughly activated by incubation in Brain Heart Infusion broth (BHI, Difco) overnight at 37 o C before being subjected to enterotoxigenicity tests.
Enterotoxin production Enterotoxin was prepared from each test strain following the method of Kubota and Liu (1971) with some modifications. 1 ml of each activated culture was inoculated into 99 ml of BHI in 250-ml flasks to give a cell concentration of 106 cells/ml. After incubation, both at static and shake conditions (180 rpm) at 3 7 ° C for 18 h, the cells were harvested and the supernatant fluid passed through a membrane filter (Whatman, 0.45 g m ) to obtain a cell-free filtrate (CFF). The C F F was concentrated 8-10-fold by dialysis against polyethylene glycol 6000 (Hi-media, India) overnight at 4 o C.
Protein estimation Protein concentrations in the C F F were determined by the method of Lowry et al. (1951) using crystalline Bovine Serum Albumin (20 160 /~g/ml, Sigma) as standard.
Assay for enterotoxic activity The enterotoxic activity of the crude enterotoxin preparation was determined in ligated ileal loops of rabbits and vascular permeability response in rabbit skin following the methods of De and Chatterjee (1953) and Craig (1965), respectively. The extent of the enterotoxin response in rabbit gut was quantitated by determining F / L ratio, i.e., the ratio of the fluid accumulated (ml) in the ligated ileal loop and the length (cm) of the loop.
Effect of curing agents P. aeruginosa CTM-3 was grown separately in the presence of different concentrations (0-300 g g / m l ) of acridine orange (Sigma), acriflavine (Sigma) and ethidium bromide (Sigma) and sub-lethal concentrations were determined. Twentyfour colonies surviving such concentrations were randomly picked and screened for enterotoxin production and haemolytic activity on rabbit blood agar (Cruickshank et al., 1975).
Properties of crude enterotoxin Effect of temperature. The stability of the enterotoxin prepared as described above was determined by subjecting 10-ml volumes in 30-ml tubes to different
203 combinations of time and temperature, i.e., 7 ° C for 7 days (refrigerator), 30 ° C for 24 h, 4 0 ° C for 1 h, 6 3 ° C for 30 rain and 7 2 ° C for 15 s (Water bath with circulation, Tempo, India). The temperatures in the water bath were controlled by monitoring the temperature in tubes containing the same volume of uninoculated broth. Compensation was made for come up times. The samples were tested for residual enterotoxic activity by the rabbit ileal loop assay. Effect ofpH. The method of Mundell et al. (1976) was followed to study the effect of p H on crude enterotoxin. The p H of the enterotoxin samples was adjusted to 4.5, 5.5, 6.5 and 7.5 using 1 N HCI or 1 N N a O H . The acid or base was added in very small increments with rapid mixing as a precaution against toxin inactivation. After p H adjustment samples were membrane-filtered and kept at 37 ° C for 5 h and tested by the rabbit ileal loop assay. Effect of pepsin and trypsin. Pepsin (800 u n i t s / m g protein, Sigma) and trypsin (10000 u n i t s / m g protein, Sigma) were added to the concentrated C F F in levels of 0 - 5 0 / ~ g / m l . After 5 h at 37 ° C the treated C F F was tested for enterotoxic activity in rabbit gut. Prior to enzyme treatment the p H of the samples was adjusted to 7.0. Effect of organic acids. Potassium sorbate (Analar, BDH), propionic acid (Analar, BDH) and butyric (Analar, BDH) were added separately to the crude enterotoxin samples at levels of 0.1, 0.5, 1.0 and 1.5%. The lactic acid (Analar, B D H ) concentrations used in this study were 0.15, 0.25, 0.35 and 0.45%. The samples were kept for 5 h at 37 ° C before subjecting them to ileal loop assay. Effect of formalin. Formalin (formaldehyde solution, 37.41% w / v , Analar, BDH) was added to a level of 0.1, 0.5, 1.0 and 1.5% to the crude enterotoxin and incubated at 37 ° C for 5 h whereafter toxin activity was tested by rabbit ileal loop assay.
Minimum effective dose (MED) The minimum effective dose was determined by injecting different concentrations of the concentrated crude enterotoxin into ileal loops of rabbits. A dose-response curve was plotted (Fig. 3). The minimum effective dose (MED) was determined by locating the mid point of the linear portion of the curve (Burrows and Musteikis, 1966).
Results and Discussion
Screening of P. aeruginosa strains for enterotoxin production The data presented in Table I show that seven out of 12 P. aeruginosa isolates examined during this study exhibited a positive response in the ligated rabbit gut and vascular permeability reaction in rabbit skin. The most potential enterotoxigenic isolate was CTM-3 as this culture produced a high F / L ratio (1.1) in the rabbit gut and hence this culture was selected for further studies. Two types of accumulated fluids were seen in the positive ligated ileal loops of rabbits, one being reddish brown and the other clear. The accumulation of reddish brown fluid in the rabbit gut may be due to extensive haemorrhage and necrosis brought about by the crude enterotoxin of P. aeruginosa in the ileal loops. Our findings in this regard are
204 TABLE I Screening of P. aeruginosa isolates for enterotoxin production R a b b i t ileal loop assay
R a b b i t skin p e r m e a b i l i t y test
F / L ratio 1
Response
A r e a of blueing ( m m 2)
Response
Control C-9
0.091 0.695
+
0.00 28.28
+
C-10 CTM-3 CMN-3 CMS-3
0.839 1.100 0.845 0.822
+ + + +
31.18 38.50 24.76 25.97
+ + + +
P-7 P-8 IC-6 IC-7 B-1 B-2 PTM-1
0.857 0.900 0.913 0.482 0.247 0.345 1.017 1.140
+ + + -
28.28 0.00 0.00 38.50 24.76 33.19 40.91 50.28
+ + + + + +
Strain
P. aeruginosa 46 F/L
+ +
ratio is the ratio of fluid (ml) a c c u m u l a t e d a n d the length ( c m ) of the ligated ileal loop.
in agreement with those of Kubota and Liu (1971) and Shriniwas et al. (1979) who observed that P. aeruginosa had the ability to cause fluid accumulation in the rabbit gut. Shriniwas et al. (1979) also reported two distinct groups of P. aeruginosa strains producing clear and bloody fluids respectively in the rabbit gut. During the present investigation, no clear relationship between the enterotoxin production in rabbit gut and skin permeability response in rabbit skin was observed as some of the enterotoxin-negative strains evoked a positive response in rabbit skin and vice-versa. Contrary to our findings, Shriniwas et al. (1979) observed that strains capable of producing enterotoxin also exhibited a positive skin response, although the reverse was not true. This discrepancy may be due to variation in the biological response to the permeability factor produced by P. aeruginosa depending upon the type of animal used and its resistance. Regarding the effect of cultural conditions on enterotoxin production, our findings indicate that though there was a small increase in growth of P. aeruginosa when cultured under agitated (aerated) conditions, there was no difference in enterotoxin production under either stationary or agitated conditions of growth (data not shown). The results of the present study concerning the effect of curing agents on growth of P. aeruginosa CTM-3 shows that this culture was fairly resistant to ethidium bromide, acriflavine and acridine orange even at 300 /~g/ml (data not shown). Earlier, Albert et al. (1945) and Holloway and Fragie (1960) reported that P. aeruginosa was naturally resistant to acridine dyes. Ethidium bromide appeared to be the most effective curing agent in this study as six randomly picked colonies surviving ethidium bromide treatment lost the ability to produce enterotoxin and one became deficient in haemolytic activity. Contrary to this,
205 Watanabe and Fukasawa (1961) found acriflavine more effective in eliminating the drug resistance in P. aeruginosa. The loss of enterotoxin production in six of the cured variants examined in this study suggests the possible involvement of a plasmid for controlling enterotoxin synthesis in P. aeruginosa CTM-3. However, to substantiate these findings, more extensive work on the plasmid biology of this culture needs to be carried out.
Properties of the crude enterotoxin The results pertaining to the effect of different temperatures on the crude enterotoxin of P. aeruginosa CTM-3 is illustrated in Fig. 1. From the data, it is quite clear that the crude toxin was heat labile as evidenced by the apparent complete loss of activity at 7 2 ° C for 15 s. At 63°C, there was 57% decrease in activity after 30 min. Kubota and Liu (1971) reported that enterotoxin from P. aeruginosa was destroyed at 60 ° C after 30 min. The enterotoxin preparation appeared to be quite stable at a wide range of pH, i.e., 4.5 to 7.5 (data not shown). There was no loss of activity at these values indicating a wide p H spectrum for the enterotoxin activity. The effect of proteolytic enzymes on the crude enterotoxin of the test culture is shown in Fig. 2. Both pepsin and trypsin inactivated the enterotoxic activity completely at the level of 4 0 / ~ g / m l . The loss of enterotoxic activity on digestion with the proteolytic enzymes suggests that the enterotoxin is protein in nature. Similar observations were made by K u b o t a and Liu (1971).
C--CONTROL TC--TOXIN
(BHI) CONTROL
1"1 1.0-
T
0"g0-8-
O p-
0.70-6-
.e. la.
7d
0.50"4-
rain 0.30.2-
~
0.1 -
C
TC
7
30
4 0 63
TEMPERATURE
w..15 sec.
72
(°C)
Fig. 1. Effect of t e m p e r a t u r e o n activity of crude e n t e r o t o x i n ( F / L ratio = r a t i o of fluid (ml) accum u l a t e d a n d l e n g t h (cm) of the ligated ileal loop).
206 PEPSIN
2p -
<:
0-9
0.9 ~
0-8
0"8
0-7
0
0.6
0-7 0'6
(Z:
IX
.~
0-5 "~
TRYPSIN
1-0
0"5
0"4 u.. 0.3 0"4 I 0.2
0-3
0-2
0-1
0-1 0
I
110 210 310 40
I
10 20
I
I
30 4 0
50
,~/m t
pg/mt
Fig. 2. Effect of proteolytic enzymes on crude activity of crude enterotoxin (For explanation of F / L ratio see legend to Fig. 1).
The data presented in Table II show that there was no appreciable loss in activity of the crude enterotoxin on treatment with some c o m m o n food preservatives. The enterotoxin was quite resistant to formalin and hydrogen peroxide upto 1.5% concentration. Hence, our studies indicate that the commonly used food preservatives and sterilants used in food plants have little effect on the preformed enterotoxin of P. aeruginosa. Fig. 3 shows the minimum effective dose (MED) of the crude enterotoxin preparation of P. aeruginosa CTM-3. As is evident form this figure, the M E D was fairly high, i.e., 10 m g / m l (Burrows and Musteikis, 1966). The high M E D of the anterotoxin obtained in this study could be attributed to the crude nature of the preparation.
TABLE II Effect of organic acids/salts, formalin and hydrogen peroxide on the activity of crude enterotoxin Additives Propionic acid Butyric acid Potassium sorbate Formalin Hydrogen peroxide
Enterotoxic activity ( F / L ratio) 1 at a concentration (%) of 0.00
0.10
0.50
1.00
1.50
1.20 1.20 1.00 1.00 1.00
1.20 1.22 0.90 1.00 1.00
1.19 1.20 0.80 0.90 1.00
1.10 1.00 0.75 0.79 0.80
1.08 1.20 0.72 0.80 0.71
Enterotoxic activity ( F / L ratio) 1 at a concentration (%) of
Lactic acid
0.00
0.15
0.25
0.35
0.45
1.20
1.15
1.12
1.13
0.97
1 For explanation of F / L ratio see footnote to Table I.
207
1.1 1,0 1.9 0-8
o
o
°'t: . . . . . J
=
/i
,
F 0.2
01
~ 0
_
_
4 8 12 16 20 24. TOXIN CONC. ( m 9 / r n l )
Fig. 3. Minimum effective dose curve of P. aeruginosa crude enterotoxin (For explanation of F / L ratio see legend to Fig. 1).
I t is c o n c l u d e d t h a t P. aeruginosa is c a p a b l e o f p r o d u c i n g e n t e r o t o x i n s u n d e r defined laboratory conditions. However, in order to understand the significance of this enterotoxin, its nature and mode of action needs to be fully ascertained by investigations with complete or partial purified preparations of the enterotoxin.
References
Albert, A., Rubbor, S.D., Goldacre, R.J., Davey, M.E. and Stone, J.D. (1945) The influence of chemical constitution on antibacterial activity. II. A general survey of the acridine series. Br. J. Exptl. Pathol. 26, 160. Burrows, W. and Musteikis, G.M. (1966) Cholera infection and toxin in the rabbit ileal loop. J. Infect. Dis. 116, 183. Craig, J.P. (1965) A permeability factor (toxin) found in cholera stools and culture filtrates and its neutralization by convalescent cholera sera. Nature 207, 614. Cruickshank, R., Duguid, J.P., Marmion, B.P. and Swain, R.H.A. (1975) Medical Microbiology, Vol. II, 12th edn. Churchill Livingstone, London. De, S.N. and Chatterjee, D.N. (1953) An experimental study of mechanism of action of Vibrio cholerae on the intestinal mucous membrane. J. Pathol. Bacteriol. 66, 559. Grover, S. and Srinivasan, R.A. (1988) Isolation and characterization of Pseudomonas aeruginosa from milk and milk products. Indian J. Dairy Sci. 41,326. Holloway, B.W. and Fragie, B. (1960) Fertility factors and genetic linkage in Pseudomonas aeruginosa. J. Bacteriol. 80, 362. Hutchins, P., Hindocha, P., Phillips, A. and Walker-Smith, J. (1979) Traveller's diarrhoea with a vergeance. Lancet ii, 1373. Kausama, H. (1974) Presence of co-factor for the vascular permeability factor. Infect. Immun. 10, 1185. Kausama, H. and Suss, R.H. (1972) Vascular permeability factor of Pseudomonas aeruginosa. Infect. Immun. 5, 363. Kubota, Y. and Liu, P.V. (1971) An enterotoxin of Pseudomonas aeruginosa J. Infect. Dis. 123, 97.
208 Liu, P.V. (1974) Extracellular toxins of Pseudomonas aeruginosa. J. Infect. Dis. 130 (S), 94. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265. Mundell, D.H., Anselmo, C.R. and Wishnow, R.M. (1976) Factors influencing heat labile Escherichia coil enterotoxin activity. Infect. lmmun. 14, 383. Pande, R.C. (1976) Bacteriology of infantile diarrhoea and gastro-enteritis in Allahabad. Indian J. Pathol. Microbiol. 19, 169. Pottecher, B., Goetz, M.L., Jacquemarie, M.A., Reed, E. and Lavillaureix, J. (1979) Infectious enterocolitis in patients receiving intensive care and fed via a nasogastric drip. Ann. Anesthesiol. Fr. 20, 595. Shriniwas, Menon, U. and Bhujwala, R.A. (1979) Production of permeability factor and enterotoxin by Pseudomonas aeruginosa. Indian J. Med. Res. 70, 380. Watanabe, T. and Fukasawa, T. (1961) Episome mediated transfer of drug resistance in enterobacteriaceae. II. Elimination of resistance factors with acridine dyes. J. Bacteriol. 81,679.
201
FOOD 00303
Production and properties of crude enterotoxin of Pseudomonas aeruginosa Sunita Grover, V.K. Batish and R.A. Srinivasan Dairy Microbiology Division, National Dairy Research Institute, Karnal, India
(Received 17 February 1989; revision received 30 August 1989; accepted 4 September 1989)
Pseudomonas aeruginosa CTM-3 was found to be the most potentially enterotoxigenic strain out of the 12 isolates recovered from milk, as a high fluid length ratio, i.e. F/L (1.1) in rabbit gut and a strong permeability response in rabbit skin (38.5 mm2 necrotic zone) was obtained with this culture. No clear-cut relationship between the two tests was observed. Six of the ethidium bromide (300 ~g/ml) cured variants of this culture completely lost their ability to produce enterotoxin indicating the possible involvement of a plasmid in enterotoxin synthesis. The crude enterotoxin from P. aeruginosa CTM-3 was completely inactivated in 15 s at 72°C. However, it was fairly stable at pH values in the range 4.5-7.5. Both pepsin and trypsin inactivated the enterotoxin activity at a concentration of 40 ~g/ml. Organic acids, formalin and hydrogen peroxide had no significant effect on the enterotoxin activity. The need for further investigations with purified preparations is emphasized.
Key words: P. aeruginosa CTM-3; Crude enterotoxin; Milk
Introduction P. aeruginosa produces a variety of extracellular toxins a n d enzymes that make it a p o t e n t i a l l y p a t h o g e n i c organism. The various extracellular p r o d u c t s p r o d u c e d i n c l u d e a m o n g others exotoxin A, enterotoxin, protease, lipase, lecithinase, elastase, D N a s e a n d h a e m o l y s i n (Liu, 1974). E n t e r o t o x i n induces fluid a c c u m u l a t i o n in the l u m e n of intestine a n d is distinct from the other toxic c o m p o u n d s a n d the lethal toxins ( K u b o t a a n d Liu, 1971; Liu, 1974). There have b e e n few reports o n the vascular p e r m e a b i l i t y factor a n d e n t e r o t o x i n p r o d u c t i o n in P. aeruginosa ( K a u s a m a a n d Suss, 1972; K a u s a m a , 1974; Shriniwas et al., 1979). The potential role of P. aeruginosa in gastro-intestinal infections appears to be overlooked. Moreover, the e n t e r o t o x i n has n o t yet b e e n fully characterized. Nevertheless, there are a few isolated reports i n d i c a t i n g the i n v o l v e m e n t of P. aeruginosa with gastroenteritis (Pande, 1976; Pottecher et al., 1979; H u t c h i n s et al., 1979). Hence, the present investigation i n t e n d s to study e n t e r o t o x i n p r o d u c t i o n b y P. aeruginosa strains isolated from milk a n d milk products a n d properties of the crude enterotoxin.
Correspondence address: S. Grover, Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001 (Haryana), India.
0168-1605/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
202 Materials and Methods
Cultures A total of 12 haemolytic strains of P. aeruginosa recovered from different milk and milk products as previously described (Grover and Srinivasan, 1988) were used in this investigation. In addition, the study also included a reference strain P. aeruginosa 46 isolated from gastroenteritis which was procured from Post-graduate Institute of Medical Sciences, Chandigarh. The cultures were maintained in the refrigerator on nutrient agar (Hi-media, India) slants with subculturing every 2 weeks. The cultures were thoroughly activated by incubation in Brain Heart Infusion broth (BHI, Difco) overnight at 37 o C before being subjected to enterotoxigenicity tests.
Enterotoxin production Enterotoxin was prepared from each test strain following the method of Kubota and Liu (1971) with some modifications. 1 ml of each activated culture was inoculated into 99 ml of BHI in 250-ml flasks to give a cell concentration of 106 cells/ml. After incubation, both at static and shake conditions (180 rpm) at 3 7 ° C for 18 h, the cells were harvested and the supernatant fluid passed through a membrane filter (Whatman, 0.45 g m ) to obtain a cell-free filtrate (CFF). The C F F was concentrated 8-10-fold by dialysis against polyethylene glycol 6000 (Hi-media, India) overnight at 4 o C.
Protein estimation Protein concentrations in the C F F were determined by the method of Lowry et al. (1951) using crystalline Bovine Serum Albumin (20 160 /~g/ml, Sigma) as standard.
Assay for enterotoxic activity The enterotoxic activity of the crude enterotoxin preparation was determined in ligated ileal loops of rabbits and vascular permeability response in rabbit skin following the methods of De and Chatterjee (1953) and Craig (1965), respectively. The extent of the enterotoxin response in rabbit gut was quantitated by determining F / L ratio, i.e., the ratio of the fluid accumulated (ml) in the ligated ileal loop and the length (cm) of the loop.
Effect of curing agents P. aeruginosa CTM-3 was grown separately in the presence of different concentrations (0-300 g g / m l ) of acridine orange (Sigma), acriflavine (Sigma) and ethidium bromide (Sigma) and sub-lethal concentrations were determined. Twentyfour colonies surviving such concentrations were randomly picked and screened for enterotoxin production and haemolytic activity on rabbit blood agar (Cruickshank et al., 1975).
Properties of crude enterotoxin Effect of temperature. The stability of the enterotoxin prepared as described above was determined by subjecting 10-ml volumes in 30-ml tubes to different
203 combinations of time and temperature, i.e., 7 ° C for 7 days (refrigerator), 30 ° C for 24 h, 4 0 ° C for 1 h, 6 3 ° C for 30 rain and 7 2 ° C for 15 s (Water bath with circulation, Tempo, India). The temperatures in the water bath were controlled by monitoring the temperature in tubes containing the same volume of uninoculated broth. Compensation was made for come up times. The samples were tested for residual enterotoxic activity by the rabbit ileal loop assay. Effect ofpH. The method of Mundell et al. (1976) was followed to study the effect of p H on crude enterotoxin. The p H of the enterotoxin samples was adjusted to 4.5, 5.5, 6.5 and 7.5 using 1 N HCI or 1 N N a O H . The acid or base was added in very small increments with rapid mixing as a precaution against toxin inactivation. After p H adjustment samples were membrane-filtered and kept at 37 ° C for 5 h and tested by the rabbit ileal loop assay. Effect of pepsin and trypsin. Pepsin (800 u n i t s / m g protein, Sigma) and trypsin (10000 u n i t s / m g protein, Sigma) were added to the concentrated C F F in levels of 0 - 5 0 / ~ g / m l . After 5 h at 37 ° C the treated C F F was tested for enterotoxic activity in rabbit gut. Prior to enzyme treatment the p H of the samples was adjusted to 7.0. Effect of organic acids. Potassium sorbate (Analar, BDH), propionic acid (Analar, BDH) and butyric (Analar, BDH) were added separately to the crude enterotoxin samples at levels of 0.1, 0.5, 1.0 and 1.5%. The lactic acid (Analar, B D H ) concentrations used in this study were 0.15, 0.25, 0.35 and 0.45%. The samples were kept for 5 h at 37 ° C before subjecting them to ileal loop assay. Effect of formalin. Formalin (formaldehyde solution, 37.41% w / v , Analar, BDH) was added to a level of 0.1, 0.5, 1.0 and 1.5% to the crude enterotoxin and incubated at 37 ° C for 5 h whereafter toxin activity was tested by rabbit ileal loop assay.
Minimum effective dose (MED) The minimum effective dose was determined by injecting different concentrations of the concentrated crude enterotoxin into ileal loops of rabbits. A dose-response curve was plotted (Fig. 3). The minimum effective dose (MED) was determined by locating the mid point of the linear portion of the curve (Burrows and Musteikis, 1966).
Results and Discussion
Screening of P. aeruginosa strains for enterotoxin production The data presented in Table I show that seven out of 12 P. aeruginosa isolates examined during this study exhibited a positive response in the ligated rabbit gut and vascular permeability reaction in rabbit skin. The most potential enterotoxigenic isolate was CTM-3 as this culture produced a high F / L ratio (1.1) in the rabbit gut and hence this culture was selected for further studies. Two types of accumulated fluids were seen in the positive ligated ileal loops of rabbits, one being reddish brown and the other clear. The accumulation of reddish brown fluid in the rabbit gut may be due to extensive haemorrhage and necrosis brought about by the crude enterotoxin of P. aeruginosa in the ileal loops. Our findings in this regard are
204 TABLE I Screening of P. aeruginosa isolates for enterotoxin production R a b b i t ileal loop assay
R a b b i t skin p e r m e a b i l i t y test
F / L ratio 1
Response
A r e a of blueing ( m m 2)
Response
Control C-9
0.091 0.695
+
0.00 28.28
+
C-10 CTM-3 CMN-3 CMS-3
0.839 1.100 0.845 0.822
+ + + +
31.18 38.50 24.76 25.97
+ + + +
P-7 P-8 IC-6 IC-7 B-1 B-2 PTM-1
0.857 0.900 0.913 0.482 0.247 0.345 1.017 1.140
+ + + -
28.28 0.00 0.00 38.50 24.76 33.19 40.91 50.28
+ + + + + +
Strain
P. aeruginosa 46 F/L
+ +
ratio is the ratio of fluid (ml) a c c u m u l a t e d a n d the length ( c m ) of the ligated ileal loop.
in agreement with those of Kubota and Liu (1971) and Shriniwas et al. (1979) who observed that P. aeruginosa had the ability to cause fluid accumulation in the rabbit gut. Shriniwas et al. (1979) also reported two distinct groups of P. aeruginosa strains producing clear and bloody fluids respectively in the rabbit gut. During the present investigation, no clear relationship between the enterotoxin production in rabbit gut and skin permeability response in rabbit skin was observed as some of the enterotoxin-negative strains evoked a positive response in rabbit skin and vice-versa. Contrary to our findings, Shriniwas et al. (1979) observed that strains capable of producing enterotoxin also exhibited a positive skin response, although the reverse was not true. This discrepancy may be due to variation in the biological response to the permeability factor produced by P. aeruginosa depending upon the type of animal used and its resistance. Regarding the effect of cultural conditions on enterotoxin production, our findings indicate that though there was a small increase in growth of P. aeruginosa when cultured under agitated (aerated) conditions, there was no difference in enterotoxin production under either stationary or agitated conditions of growth (data not shown). The results of the present study concerning the effect of curing agents on growth of P. aeruginosa CTM-3 shows that this culture was fairly resistant to ethidium bromide, acriflavine and acridine orange even at 300 /~g/ml (data not shown). Earlier, Albert et al. (1945) and Holloway and Fragie (1960) reported that P. aeruginosa was naturally resistant to acridine dyes. Ethidium bromide appeared to be the most effective curing agent in this study as six randomly picked colonies surviving ethidium bromide treatment lost the ability to produce enterotoxin and one became deficient in haemolytic activity. Contrary to this,
205 Watanabe and Fukasawa (1961) found acriflavine more effective in eliminating the drug resistance in P. aeruginosa. The loss of enterotoxin production in six of the cured variants examined in this study suggests the possible involvement of a plasmid for controlling enterotoxin synthesis in P. aeruginosa CTM-3. However, to substantiate these findings, more extensive work on the plasmid biology of this culture needs to be carried out.
Properties of the crude enterotoxin The results pertaining to the effect of different temperatures on the crude enterotoxin of P. aeruginosa CTM-3 is illustrated in Fig. 1. From the data, it is quite clear that the crude toxin was heat labile as evidenced by the apparent complete loss of activity at 7 2 ° C for 15 s. At 63°C, there was 57% decrease in activity after 30 min. Kubota and Liu (1971) reported that enterotoxin from P. aeruginosa was destroyed at 60 ° C after 30 min. The enterotoxin preparation appeared to be quite stable at a wide range of pH, i.e., 4.5 to 7.5 (data not shown). There was no loss of activity at these values indicating a wide p H spectrum for the enterotoxin activity. The effect of proteolytic enzymes on the crude enterotoxin of the test culture is shown in Fig. 2. Both pepsin and trypsin inactivated the enterotoxic activity completely at the level of 4 0 / ~ g / m l . The loss of enterotoxic activity on digestion with the proteolytic enzymes suggests that the enterotoxin is protein in nature. Similar observations were made by K u b o t a and Liu (1971).
C--CONTROL TC--TOXIN
(BHI) CONTROL
1"1 1.0-
T
0"g0-8-
O p-
0.70-6-
.e. la.
7d
0.50"4-
rain 0.30.2-
~
0.1 -
C
TC
7
30
4 0 63
TEMPERATURE
w..15 sec.
72
(°C)
Fig. 1. Effect of t e m p e r a t u r e o n activity of crude e n t e r o t o x i n ( F / L ratio = r a t i o of fluid (ml) accum u l a t e d a n d l e n g t h (cm) of the ligated ileal loop).
206 PEPSIN
2p -
<:
0-9
0.9 ~
0-8
0"8
0-7
0
0.6
0-7 0'6
(Z:
IX
.~
0-5 "~
TRYPSIN
1-0
0"5
0"4 u.. 0.3 0"4 I 0.2
0-3
0-2
0-1
0-1 0
I
110 210 310 40
I
10 20
I
I
30 4 0
50
,~/m t
pg/mt
Fig. 2. Effect of proteolytic enzymes on crude activity of crude enterotoxin (For explanation of F / L ratio see legend to Fig. 1).
The data presented in Table II show that there was no appreciable loss in activity of the crude enterotoxin on treatment with some c o m m o n food preservatives. The enterotoxin was quite resistant to formalin and hydrogen peroxide upto 1.5% concentration. Hence, our studies indicate that the commonly used food preservatives and sterilants used in food plants have little effect on the preformed enterotoxin of P. aeruginosa. Fig. 3 shows the minimum effective dose (MED) of the crude enterotoxin preparation of P. aeruginosa CTM-3. As is evident form this figure, the M E D was fairly high, i.e., 10 m g / m l (Burrows and Musteikis, 1966). The high M E D of the anterotoxin obtained in this study could be attributed to the crude nature of the preparation.
TABLE II Effect of organic acids/salts, formalin and hydrogen peroxide on the activity of crude enterotoxin Additives Propionic acid Butyric acid Potassium sorbate Formalin Hydrogen peroxide
Enterotoxic activity ( F / L ratio) 1 at a concentration (%) of 0.00
0.10
0.50
1.00
1.50
1.20 1.20 1.00 1.00 1.00
1.20 1.22 0.90 1.00 1.00
1.19 1.20 0.80 0.90 1.00
1.10 1.00 0.75 0.79 0.80
1.08 1.20 0.72 0.80 0.71
Enterotoxic activity ( F / L ratio) 1 at a concentration (%) of
Lactic acid
0.00
0.15
0.25
0.35
0.45
1.20
1.15
1.12
1.13
0.97
1 For explanation of F / L ratio see footnote to Table I.
207
1.1 1,0 1.9 0-8
o
o
°'t: . . . . . J
=
/i
,
F 0.2
01
~ 0
_
_
4 8 12 16 20 24. TOXIN CONC. ( m 9 / r n l )
Fig. 3. Minimum effective dose curve of P. aeruginosa crude enterotoxin (For explanation of F / L ratio see legend to Fig. 1).
I t is c o n c l u d e d t h a t P. aeruginosa is c a p a b l e o f p r o d u c i n g e n t e r o t o x i n s u n d e r defined laboratory conditions. However, in order to understand the significance of this enterotoxin, its nature and mode of action needs to be fully ascertained by investigations with complete or partial purified preparations of the enterotoxin.
References
Albert, A., Rubbor, S.D., Goldacre, R.J., Davey, M.E. and Stone, J.D. (1945) The influence of chemical constitution on antibacterial activity. II. A general survey of the acridine series. Br. J. Exptl. Pathol. 26, 160. Burrows, W. and Musteikis, G.M. (1966) Cholera infection and toxin in the rabbit ileal loop. J. Infect. Dis. 116, 183. Craig, J.P. (1965) A permeability factor (toxin) found in cholera stools and culture filtrates and its neutralization by convalescent cholera sera. Nature 207, 614. Cruickshank, R., Duguid, J.P., Marmion, B.P. and Swain, R.H.A. (1975) Medical Microbiology, Vol. II, 12th edn. Churchill Livingstone, London. De, S.N. and Chatterjee, D.N. (1953) An experimental study of mechanism of action of Vibrio cholerae on the intestinal mucous membrane. J. Pathol. Bacteriol. 66, 559. Grover, S. and Srinivasan, R.A. (1988) Isolation and characterization of Pseudomonas aeruginosa from milk and milk products. Indian J. Dairy Sci. 41,326. Holloway, B.W. and Fragie, B. (1960) Fertility factors and genetic linkage in Pseudomonas aeruginosa. J. Bacteriol. 80, 362. Hutchins, P., Hindocha, P., Phillips, A. and Walker-Smith, J. (1979) Traveller's diarrhoea with a vergeance. Lancet ii, 1373. Kausama, H. (1974) Presence of co-factor for the vascular permeability factor. Infect. Immun. 10, 1185. Kausama, H. and Suss, R.H. (1972) Vascular permeability factor of Pseudomonas aeruginosa. Infect. Immun. 5, 363. Kubota, Y. and Liu, P.V. (1971) An enterotoxin of Pseudomonas aeruginosa J. Infect. Dis. 123, 97.
208 Liu, P.V. (1974) Extracellular toxins of Pseudomonas aeruginosa. J. Infect. Dis. 130 (S), 94. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265. Mundell, D.H., Anselmo, C.R. and Wishnow, R.M. (1976) Factors influencing heat labile Escherichia coil enterotoxin activity. Infect. lmmun. 14, 383. Pande, R.C. (1976) Bacteriology of infantile diarrhoea and gastro-enteritis in Allahabad. Indian J. Pathol. Microbiol. 19, 169. Pottecher, B., Goetz, M.L., Jacquemarie, M.A., Reed, E. and Lavillaureix, J. (1979) Infectious enterocolitis in patients receiving intensive care and fed via a nasogastric drip. Ann. Anesthesiol. Fr. 20, 595. Shriniwas, Menon, U. and Bhujwala, R.A. (1979) Production of permeability factor and enterotoxin by Pseudomonas aeruginosa. Indian J. Med. Res. 70, 380. Watanabe, T. and Fukasawa, T. (1961) Episome mediated transfer of drug resistance in enterobacteriaceae. II. Elimination of resistance factors with acridine dyes. J. Bacteriol. 81,679.