Vol. 14, No. 4 Printed in U.S.A.
INFECTION AND IMMUNITY, OCt. 1976, p. 1004-1010 Copyright © 1976 American Society for Microbiology
Assay ofEscherichia coli Enterotoxins by In Vivo Perfusion in the Rat Jejunum FREDERICK A. KLIPSTEIN,* CHUNG-SENG LEE, AND RICHARD F. ENGERT Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
Received for publication 10 February 1976
Assay of Escherichia coli enterotoxins by in vivo perfusion in rats was evaluated by examining the effects of variously prepared fractions of heat-labile (LT) and heat-stable (ST) toxins on water transport in this system. The assay was found to respond equally well, in a dose-related manner, to both LT and ST; it was sufficiently sensitive to detect the toxigenic effect of concentrations as small as 1 ng/ml. With the assay, it was found that LT is produced in cultures grown under aerobic, but not anaerobic, conditions; in contrast, ST is elaborated in stationary aerobic and anaerobic broth cultures but not in those grown under agitated aerobic conditions. Both toxins can be precipitated by either ammonium sulfate or acetone. The two toxin forms were completely separated from each other by sequential ultrafiltration. LT alone (thermolabile after exposure to 100°C for 30 min) was retained by a PM-30 membrane, and ST alone was present in UM-2 retentates; ST was retained more effectively by a UM-05 membrane, with a 1,000-fold increase in activity over that of the UM-2 retentate. Washed ultrafiltration retentates containing either LT or ST derived from the proper culture conditions all induced water secretion at concentrations of 100 ng or less per ml. These results indicate that in vivo perfusion in rats is a sensitive, duplicable assay for both the LT and ST forms of E. coli enterotoxin.
Enterotoxigenic strains of Escherichia coli produce two forms of enterotoxin: a low-molecular-weight, heat-stable toxin (ST) either alone or along with a large-molecular-weight, heatlabile toxin (LT) (11, 22, 32, 35). These two forms are generally considered to be separate, distinct entities based on dissimilarities in their physical (4, 14, 15, 23), antigenic (7, 11, 12, 35), and biological properties (3, 7, 8, 9). Whereas the techniques for producing LT alone are well recognized (13, 35), and sensitive, quantitative in vitro tests are available for its assay (3, 5, 9), such is not the case for ST. Cellfree broth filtrates of strains of E. coli that produce LT and ST contain both forms of toxin, and ST must be separated from LT by subsequent procedures based on their different physical properties such as heat lability (7, 24, 28). Assays for ST are confined to observing fluid accumulation within ligated loops of rabbits (7, 11, 27) or the whole intestinal tract of suckling mice (2, 31, 34). It seemed important, therefore, to determine whether clearly defined preparations of ST can be separated from LT and whether a more sensitive and quantitative assay system for ST could be developed. The toxigenic activity of enterotoxins of other coliform species has been assayed by the in vivo
marker perfusion technique in the rat jejunum (16, 17, 19). In the present study, we evaluated this system as an assay for E. coli entertoxins by determining the effect of LT and ST preparations, produced under various culture conditions and separated from each other by means of ultrafiltration (UF) procedures, on water transport in the rat jejunum.
MATERIALS AND METHODS Bacterial strains. Biotypes were determined by the API microtube 20 E profile system (Analytab Products, Inc., Plainview, N.Y.) (36). All three strains studied were jejunal isolates. (i) NTG. Nontoxigenic (NTG) strain BN (API biotype 5044572; serotype O1:H31) was isolated from a control subject in Puerto Rico; cell-free broth filtrates are inactive in rabbit ileal loop studies (18). (ii) ST only. Strain PR (API biotype 5044572; serotype O1:H31) was isolated from a Puerto Rican with tropical sprue (18); broth filtrates evoked in the rabbit ileal loop a positive fluid reaction that is unaltered by heating the preparation (21). (iii) LT/ST. Strain Haiti (API biotype 5044552; serotype 04:H32) was isolated from a Haitian with tropical sprue and shown by rat perfusion studies to produce both types of enterotoxin (20). Enterotoxin production. (i) By bacterial lysis. Sonic extracts were prepared by a modification of the method described by Gyles and Barnum (13). 1004
1005
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ASSAY OF E. COLI ENTEROTOXINS
The bacteria were grown either aerobically or anaerobically for 18 h at 37°C on Trypticase (BBL, Cockeysville, Md.) soy agar in a Roux bottle. The harvested confluent surface growth was sonically treated on a sonifier cell disrupter (Branson Sonic Power Co., Plainview, N.Y.) for 10 min; the suspension obtained was centrifuged for 30 min at 10,000 x g and 4°C, and the supernatant was passed through a 0.45-,.m membrane filter (Millipore Corp., Bedford, Mass.). The cell-free supernatant was then either lyophilized or passed through UF membranes. (ii) From broth cultures. For aerobic broth cultures, 0.1 ml of a 6-h subculture of bacteria was introduced into 250 ml of Trypticase soy broth in a 2,000 ml flask and incubated at 37°C for 18 h either under stationary conditions or with agitation at 200 shakes per minute. For anaerobic cultures, the same amount of bacteria was inoculated into 500 ml of the same medium in a 1-liter flask and incubated in an anaerobic chamber (Coy Manufacturing Co., Ann Arbor, Mich.) with 85% N2, 5% C02, and 10% H2 gas for 18 h at 37°C. After growth, the broth cultures were centrifuged for 45 min at 35,000 x g at 4°C, and the supernatant was passed through a 0.45-,um Millipore filter. The filtrate was then precipitated with either 8 volumes of acetone (1, 19) or 90% ammonium sulfate (AMS) (7). The acetone precipitates were solubilized in distilled water, the AMS precipitates were dissolved in 0.02 M ammonium bicarbonate (pH 7.8), and both solutions were then fractionated by UF. UF. Separation of the two forms of enterotoxin was based on the fact that LT has an apparent molecular weight of greater than 100,000 (4, 15, 23) and would be retained by a PM-30 UF membrane (molecular weight cutoff, 30,000), whereas ST, which has an apparent molecular weight in the range of 1,000 to 10,000 (1, 14), is partially retained by a UM-2 membrane (molecular weight cutoff, 1,000). The broth culture precipitates were passed sequentially through Amicon PM-30 and either UM2 or UM-05 (molecular weight cutoff, 500) membranes (Amicon Corp., Lexington, Mass.). Unless so specified, retentate fractions were taken to 10% of the original volume and lyophilized. Retentate fractions identified as "washed" were washed twice with two times the original volume, using 0.02 M ammonium bicarbonate, pH 7.80. Animal perfusion technique. Procedures for the in vivo evaluation of intestinal transport by means of marker perfusion studies in rats (30) and the results of perfusion studies of other enterotoxins examined in this animal system were described in detail elsewhere (16, 17, 19, 25, 37). Toxin fractions were tested in anesthetized, tracheostomized, Sprague-Dawley rats weighing from 150 to 250 g. Single 20-cm jejunal segments were perfused at a rate of 0.5 ml/min by using a peristaltic pump (model 1201, Harvard Apparatus Co., Millis, Mass.). Six 30-min test fractions were collected after a 30min steady-state period. The electrolyte solution described by Powell and Malawer (30) was used, except that the concentration of mannitol was reduced, when necessary, such that osmolality remained
isosmotic with rat plasma at 317 mosmol/kg (19, 30). Osmolalities were determined by measuring the freezing-point depression with an Advanced DigiMatic Osmometer (Advanced Instrument, Newton Heights, Mass.). Polyethylene glycol 4000 (PEG) concentrations were determined by the turbidimetric technique (26). Net transport of water, expressed in microliters per centimeter per 30 min, was calculated from changes in polyethylene glycol 4000 concentrations by the usual marker technique formula (30). Net lumen-to-blood transport is termed absorption and is signified by a plus sign, whereas a minus sign refers to net blood-to-lumen transport, or secretion. This laboratory (16, 17, 19) and others (10, 37) reported previously (and we so noted in the present study) that, whereas large concentrations of enterotoxin induce secretion throughout all six collection periods, smaller, submaximal concentrations evoke transient secretion during only several periods, usually the last few in the case of LT. Therefore, the mean value for all six periods may reflect absorption even though the toxin has evoked secretion at some time during the entire 210-min collection period. For this reason, we believe that the value for the 30-min collection period during which there is minimal absorption or maximal secretion (maximal effect) is a more sensitive and accurate reflection of toxin activity than the mean of all six periods. Values reported in this and previous studies (16, 17), therefore, are for the period of maximal effect. A positive enterotoxin effect is defined as the presence of secretion. The minimal effective dose (MED) of toxin is defined as the concentration (in dry weight per milliliter) that induces net secretion. The total amount of toxin perfused during the whole perfusion period is the concentration times 105 (the volume, in milliliters, perfused). Determinations reported are for assays in single rats except where noted otherwise.
RESULTS Characteristics of the assay. (i) Onset of toxin action. Water transport during perfusion with the electrolyte solution alone (control) in 20 rats showed only slight variability during each of the six 30-min collection periods (Fig. 1). The mean (plus or minus standard error of the mean [± SEM]) value for minimum water absorption during any one of the six periods was +41 + 3. The onset of action of a preparation containing LT (the crude sonic extract of strain Haiti), perfused at a concentration of 2,000 ,g/ml, was not evident until the third perfusion period, or 90 min after introduction of the toxin, and the maximum effect was delayed until the sixth period. In contrast, the same dosage of a preparation containing ST (the UM2 retentate of a broth culture of strain Haiti) reduced water transport during the first test period. We were unable to determine whether water absorption was affected during the prior 30 min of exposure since reliable information
1006
INFECT. IMMUN.
KLIPSTEIN, LEE, AND ENGERT O CONTROL
E; LT
clear-cut relationship between dosage and effect on water transport for each preparation. Effect of specific LT and ST preparations. (i) Crude preparations of LT. Initially, the crude sonic extract and unwashed PM-30 reten-
O3 ST
Zt
rz,i
r-I
SZN 111;K
+60
i.. z
-tz
t
-ZZ
2
+40
.-. .: :
s+20-ttl1 1
2
3 4 PERIOD
5
6
FIG. 1. Effect on water transport of perfusing the electrolyte solution alone (control) in 20 rats and of LT and ST preparations, each tested in 5 rats at a concentration of 2,000 Mg/ml. Values are mean ± SEM for each 30-min period. LT is the crude sonic extract and ST is the UM-2 retentate of broth filtrate of strain Haiti.
concerning transport cannot be obtained during the preliminary 30-min steady state. (ii) Reproducibility of the assay. To determine whether toxin preparations that yield borderline or strongly positive water secretions can be differentiated clearly from NTG preparations and whether the result in a single rat is a reliable index of these differences, 12 different preparations were each perfused on multiple occasions at a concentration of 2,000 ,ug/ml (Fig. 2). The inactive material consisted of seven different preparations of strain BN (NTG) (the sonic extract and UF fractions of broth cultures grown under various conditions), each of which was tested in three rats. The five preparations from the toxigenic strains containing LT or ST, which yielded either a borderline or strongly active response, were each tested in five rats. Mean (+ SEM) values for water transport were absorption of +35 ± 2 for the NTG preparations, absorption of + 2 + 2 for the borderline active preparations, and secretion of -51 + 5 for the strongly active preparations. Values for the both borderline and strongly active preparations were significantly less (P < 0.001) than those of the inactive NTG preparations. The results obtained for each individual preparation in the various categories showed only slight variation when examined in more than one rat. (iii) Dosage-response relationship. The effect on water transport of perfusing differently prepared fractions of LT and ST of strain Haiti (LT/ST) at graded dosages of between 0.0001 and 2,000 Ag/ml is shown in Fig. 3. There was a
-60
J
2El00 2 BORDERLINE ACTIVE
-80-100FIG. 2. Results of repeated assays of individual preparations of variable toxigenic activity. Values are the means for three to five determinations (hatched area) and the range obtained in individual rats with each preAPIA (vertical barAJ. 'I
.
z
I
,
+40
1O
U@ -40
SONICATE
0 CRUDE 0 PM - 30 R(W)
-80 CONTROL
ST
.N
BROTH FILTRATE
OUM-2
R
EUM-05 RIW)
DOSAGE,
kg/ml
FIG. 3. Response of water transport to graded dosages of LT and ST preparations of strain Haiti. Values are mean ± SEM for the control solution and for assay in single rats for the toxin preparation6. R, Retentate fraction; W, washed fraction.
VOL. 14, 1976
ASSAY OF E. COLI ENTEROTOXINS
tate of broth filtrates, which had been grown under various culture conditions and precipitated with either AMS or acetone, were tested at a concentration of 2,000 ,ug/ml (Table 1). All preparations of strain PR (ST) were inactive. All preparations of strain Haiti (LT/ST) strain were active except for broth cultures grown
ST, cultures grown under anaerobic conditions
under anaerobic conditions. Exposure of the active preparations to 100°C for 30 min completely abolished their ability to induce water secretion. (ii) Crude preparations of ST. Unwashed UM-2 retentates of broth cultures grown under various conditions were initially tested at concentrations of 100 to 2,000 ,ug/ml. Retentates of cultures of both strains were active when the cultures were grown under stationary aerobic and anaerobic conditions, but the UF fractions of cultures grown under agitated aerobic conditions were inactive (Table 2). When AMS was used to precipitate ST, cultures grown under aerobic stationary conditions were more active than those grown anaerobically and, conversely, when acetone was used to precipitate
1007
were the most active. The toxigenic activity of these preparations was not affected by exposure to 100°C for 30 min, indicating that they consisted exclusively of ST. (iii) More-purified preparations of LT. Increased potency was achieved by passing the crude sonic extract through a PM-30 membrane and by washing the PM-30 retentates of both the sonic extract and broth filtrate. Passage of the Haiti (LT/ST) sonic extract through a PM30 membrane reduced the MED from 100 ,ug/ml for the crude sonic extract to 1 ,ug/ml for the PM-30 retentate; washing of the retentate resulted in a 1,000-fold increase in activity, with an MED of 0.001 ,g/ml (Table 3, Fig. 3). Washed PM-30 retentates of AMS and acetone precipitates of both the sonic extract and broth cultures of strain Haiti grown under agitated and stationary aerobic conditions, were all active when perfused at a concentration of 0.1 ,tg/ ml; however, none of the anaerobically grown preparations was active (Table 4). Perfusion of a similarly prepared sonic extract of strain PR
TABLE 1. LT: Effect of crude sonic extracts and unwashed PM-30 retentates of broth cultures on water transport Growth medium
Precipitate
Water transporta6,
Culture conditions
PR (ST)
Haiti (LT/ST)
Agar
(Sonic extract)
Aerobic
Stationary
+38
-87 (+34)
Broth Broth Broth
AMS AMS AMS
Aerobic Aerobic Anaerobic
Agitated Stationary Stationary
+30 +31 +35
-30 (+24) -14 (+15) +34
Broth Acetone Aerobic +31 -34 (+34) Agitated Broth 0 (+55) Acetone Aerobic +32 Stationary +42 Broth Acetone Anaerobic +22 Stationary a Results (microliters per centimeter per 30 min) are the average for assays in two rats for each preparation. Values in parentheses are for the same fraction after exposure to 100°C for 30 min. b All preparations were perfused at a concentration of 2,000 j±g/ml. TABLE 2. ST: Effect of unwashed UM-2 Retentates on water transport Water transporta Strain
PR (ST)
Culture conditons
Aerobic Aerobic Anaerobic
Agitated Stationary Stationary
Acetone precipitate
AMS precipitate 2,000b
1,000
100
+24 -41 (-37)
+25
+40
-37 -3
-7 +16
-30 (-27)
2,000b
+10
+4 -5 (-7)
1,000
+42 +22 +17
+5 +10 +55 Aerobic +18 Agitated +25 -44 (-68) -36 -5 Aerobic +4 Stationary +8 -52 (-76) -7 Anaerobic +7 -68 (-62) Stationary -11 a Results (microliters per centimeter per 30 min) are the average for assays in two rats for each preparation. Values in parentheses are for the same fraction after exposure to 100°C for 30 min. b Dosage in micrograms per milliliter.
Haiti (LT/ST)
1008
INFZCT. IMMUN.
KLIPSTEIN, LEE, AND ENGERT
(ST), grown aerobically, at a concentration of 100 ug/ml yielded absorption of +53. (iv) More-purified preparation of ST. To determine whether a UM-05 membrane retains ST more effectively than a UM-2 membrane, the acetone precipitates of anaerobically grown broth cultures of BN (NTG), PR (ST), and Haiti (LT/ST) were passed sequentially through PM30 and UM-05 membranes (Table 3, Fig. 3). The activity in the unwashed UM-05 retentate of material from strains PR and Haiti had an MED of 0.1 ,ug/ml, which represented a 1,000fold increase in activity over that of the unwashed UM-2 retentates. Washing the UM-05 retentate increased the potency of material from strain Haiti by 10-fold, yielding an MED 0.01 ,ug/ml, and that of strain PR by 100-fold, resulting in an MED of 0.001 ,ug/ml. The toxigenic activity of the washed preparations of these two strains was not reduced when the preparations were perfused, at their MED, after exposure to 100°C for 30 min. All of the washed UM-05 retentates of cultures of strain Haiti that were grown under stationary aerobic and anaerobic (but not agitated aerobic) conditions were found to be active when perfused at a concentration of 0.1 ,g/ml (Table 4). In contrast, the washed UM-05 retentate of strain BN
(NTG) gave absorption of +37 when perfused at concentration of 1 ug/ml. DISCUSSION Previous in vivo marker perfusion studies concerned with the effect of E. coli enterotoxins on water transport in rabbits and dogs employed 50- to 2,500-,tg/ml dosages of relatively crude material containing both LT and ST (10, 29, 33). Bywater found that UF fractions containing only ST also stimulate water secretion in calves (1). In vivo perfusion in rats was shown previously to be a sensitive, quantitative assay for the heat-stable enterotoxins elaborated by Klebsiella pneumoniae and Enterobacter cloacae (16, 17, 19), and the results of the present study indicate that this assay system responds equally well to both the LT and ST forms of E. coli enterotoxin. The assay system is reproducible, such that perfusion in a single rat yields reliable information; it responds in a dose-related fashion to both LT and ST fractions; and it is sufficiently sensitive to detect toxigenic activity at concentrations as low as 1 ng/ml. In previous studies concerned with the onset of action of the two forms ofE. coli enterotoxin, the preparations used contained both ST and a
TABLE 3. Effect of PM-30 and UM-05 retentates, before and after washing, on water transport Water transport (p1/cm per 30 min) Strain Toxin Prep Fraction Washed la
0.1
0.01
0.001
0.0001
Haiti Haiti
LT LT
Sonic extract Sonic extract
PM-30 PM-30
No Yes
-45
+36 -107
-23
-3
+29
Haiti Haiti
ST ST
Brothb Brothb
UM-05 UM-05
No Yes
-21 -107
-15 -33
0 -8
+15 +18
PR ST Brothb No UM-05 -58 -13 +32 PR ST Brothb Yes UM-05 -120 -53 -11 a Dosage in micrograms per milliliter. b Broth cultures were grown under anaerobic conditions and precipitated with acetone.
+21
TABLE 4. Effect of washed PM-30 and UM-05 retentates of strain Haiti on water transport Water transporta Toxin
LT LT LT LT LT
Prepn
Sonic extract Sonic extract Sonic extract Broth Broth
UF
PM-30 PM-30 PM-30 PM-30 PM-30
Ppt
AMS Acetone AMS Acetone
ST Broth AMS UM-05 ST Broth Acetone UM-05 a All preparations were perfused at a concentration of 0.1 ug/ml.
(p1/cm per 30 min)
Aerobic
Anaerobic
Agitated
Stationary
-22 -39
-107 -84 -50 -29 -11
+9 +12
+8 +7
-11 -1
-23 -33
+7
VOL. 14, 1976
ASSAY O E. COLJ ENTEROTOXINS
LT, and the respective effect of each form was separated by testing the preparations before and after heat treatment (7, 28). Evans and her colleagues found that their thermostable preparation resulted in prompt fluid accumulation within ligated rabbit ileal loops even at the lowest dosage, whereas the onset of fluid accumulation in response to the heat-labile material was rapid at a high dose but delayed at low doses (7). Determining serial fluxes of water in canine jejunal loops, Nalin and his co-workers showed that their thermostable toxin induced prompt secretion, whereas the effect of their thermolabile preparation was delayed until 3 h after exposure to the toxin (28). When preparations containing exclusively ST or LT were perfused in the present study, a clear-cut difference between their onset of action was apparent: ST altered water transport during the earliest period of testing, but the effect of LT was not manifest until after exposure to the toxin for 90 min. Different culture conditions were found to favor maximum production of LT and ST in the present study: LT was produced under aerobic but not anaerobic conditions, whereas ST was elaborated in both stationary aerobic and anaerobic cultures but not in those maximally aerated by agitation. LT alone can be recovered from whole-cell lysates of growth on agar slants (6, 24, 27, 35) or in broth cultures (13, 22), where maximum aeration enhances its production (6, 8, 27); it can also be recovered from cell-free broth filtrates and precipitated by AMS (7, 22, 27, 29), but these preparations are only partially heat labile, indicating the presence of both LT and ST (7, 27). We found LT activity to be present in AMS and acetone precipitates of both sonic extracts of cells grown on agar and of cell-free broth filtrates. ST can be recovered from cell-free filtrates of aerobic cultures on soft agar (1, 2, 24, 35) or in broth (11, 14, 27, 29), and this form of toxin can be precipitated by either AMS or acetone (1, 7, 28, 35). In the present study, stationary aerobic culture conditions yielded the most active material precipitated by AMS, whereas anaerobic culture conditions yielded maximum output of ST when acetone was employed. Thermostable toxin preparations elaborated by K. pneumoniae and E. cloacae have been reported to have similar optimal culture conditions (16, 17). Due to the difference in their molecular weights, E. coli LT and ST can be separated by passage through graded polymeric membranes. LT, which has a molecular weight of 102,000 (4), is retained by both XM-50 and XM-100 UF membranes (4, 15, 23), whereas ST, which has an apparent molecular weight of between 1,000
and 10,000, passes through both the XM-50 membrane and the smaller pore sized PM-10 membrane (1, 14, 23). In the present study, LT and ST were completely separated from each other by sequential passage of broth filtrates through graded membranes: preparations in the PM-30 retentate were completely heat labile and those in the UM-2 retentate, after sequential passage through a PM-30 membrane, retained their toxigenic activity after exposure to 100°C for 30 min. Jacks and Wu reported that E. coli ST is completely retained by a UM-2 membrane (14); on the other hand, Bywater detected ST activity in both the retentate and filtrate fractions of this membrane (1), and the same has been reported to hold true for K. pneumoniae and E. cloacae ST preparations (16, 17). We therefore tried out the effect of the smaller pore sized UM-05 membrane and found it to retain E. coli ST more effectively, yielding fractions with 1,000-fold more activity than the UM-2 retentate. Washing the membranes markedly enhanced toxigenic activity; this was probably due to removal of inactive, small-molecular-weight products of growth through the PM-30 membrane and to more complete desalting of the retentate fraction in the case of the UM-05 membrane.
1009
ACKNOWLEDGMENTS This work was supported by grants from the WilliamsWaterman Fund of the Research Corporation, New York City, N.Y., and the Hillsdale Fund, Greensboro, N.C. LITERATURE CITED 1. Bywater, R. J. 1972. Dialysis and ultrafiltration of a heat-stable enterotoxin from Escherichia coli. J. Med. Microbiol. 5:337-343. 2. Dean, A. G., Y. C. Ching, R. G. Williams, and L. B. Harden. 1972. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J. Infect. Dis. 125:407-411. 3. Donta, S. T. 1974. Detection of heat-labile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture. Science 183:334-336. 4. Dorner, F. 1975. Escherichia coli enterotoxin. Purification and partial characterization. J. Biol. Chem. 250:8712-8719. 5. Dorner, F., and P. Mayer. 1975. Escherichia coli enterotoxin: stimulation of adenylate cyclase in broken-cell preparations. Infect. Immun. 11:429. 6. Etkin, S., and S. L. Gorbach. 1971. Studies on enterotoxin from Escherichia coli associated with acute diarrhea in man. J. Lab. Clin. Med. 78:81-87. 7. Evans, D. G., D. J. Evans, and N. F. Pierce. 1973. Differences in the response of rabbit small intestine to heat-labile and heat-stable enterotoxins of Escherichia coli. Infect. Immun. 7:873-880. 8. Evans, D. J., D. G. Evans, and S. L. Gorbach. 1973. Production of vascular permeability factor by enterotoxigenic Escherichia coli isolated from man. Infect. Immun. 8:725-730. 9. Guerrant, R. L., L. L. Brunton, T. C. Schnaitman, L. I. Rebhun, and A. G. Gilman. 1974. Cyclic adenosine monophosphate and alteration of Chinese hamster
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cell morphology: a rapid, sensitive in vitro for the enterotoxins of Vibrio cholerae and Escherichia coli. Infect. Immun. 10:320-327. Guerrant, R. L., U. Ganguly, A. G. T. Casper, E. J. Moore, N. F. Pierce, and C. C. J. Carpenter. 1973. Effect of Escherichia coli on fluid transport across canine small bowel. Mechanism and time-course with enterotoxin and whole bacterial cells. J. Clin. Invest. 52:1707-1714. Gyles, C. L. 1971. Heat-labile and heat-stable forms of the enterotoxin from E. coli strains enteropathogenic for pigs. Ann. N.Y. Acad. Sci. 179:314-322. Gyles, C. L. 1974. Immunological study of the heatlabile enterotoxins of Escherichia coli and Vibrio cholerae. Infect. Immun. 9:564-570. Gyles, C. L., and D. A. Barnum. 1969. A heat-labile enterotoxin from strains of Escherichia coli enteropathogenic for pigs. J. Infect. Dis. 120:419-429. Jacks, T. M., and B. J. Wu. 1974. Biochemical properties of Escherichia coli low-molecular-weight, heatstable enterotoxin. Infect. Immun. 9:342-347. Jacks, T. M., B. J. Wu, A. C. Braemer, and D. E. Bidlack. 1973. Properties of the enterotoxic component in Escherichia coli enteropathogenic for swine. Infect. Immun. 7:178-189. Klipstein, F. A., and R. F. Engert. 1976. Purification and properties of Klebsiella pneumoniae heat-stable enterotoxin. Infect. Immun. 13:373-381. Klipstein, F. A., and R. F. Engert. 1976. Partial purification and properties of Enterobacter cloacae heatstable enterotoxin. Infect. Immun. 13:1307-1314. Klipstein, F. A., L. V. Holdeman, J. J. Corcino, and W. E. C. Moore. 1973. Enterotoxigenic intestinal bacteria in tropical sprue. Ann. Intern. Med. 79:632-641. Klipstein, F. A., I. R. Horowitz, R. F. Engert, and E. A. Schenk. 1975. Effect of Klebsiella pneumoniae enterotoxin on intestinal transport in the rat. J. Clin. Invest. 56:799-807. Klipstein, F. A., H. B. Short, R. F. Engert, L. Jean, and G. A. Weaver. 1976. Contamination of the small intestine by enterotoxigenic coliform bacteria among the rural population of Haiti. Gastroenterology 70:1034-1041. Klipstein, F. A., and E. A. Schenk. 1975. Enterotoxigenic intestinal bacteria in tropical sprue. II. Effect of the bacteria and their enterotoxins on intestinal structure. Gastroenterology 68:642-655. Kohler, E. M. 1971. Observations on enterotoxins produced by enteropathogenic Escherichia coli. Ann. N.Y. Acad. Sci. 176:212-219. Lariviere, S., and C. L. Gyles. 1973. Preliminary charovary assay
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25. 26. 27. 28.
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35.
36.
37.
acterization of the heat-labile enterotoxin of Escherichia coli Fll (P155). J. Infect. Dis. 128:312-320. Lariviere, S., C. L. Gyles, and D. A. Barnum. 1972. A comparative study of the rabbit and pig gut loop systems for the assay of Escherichia coli enterotoxin. Can. J. Comp. Med. 36:319-328. McDonel, J. L. 1974. In vivo effects of Clostridium perfringens enteropathogenic factors on the rat ileum. Infect. Immun. 10:1156-1167. Malawer, S. J., and D. W. Powell. 1967. An improved turbidometric analysis of polyethylene glycol utilizing an emulsifier. Gastroenterology 53:250-256. Moon, H. W., and S. C. Whipp. 1971. Systems for testing the enteropathogenicity of Escherichia coli. Ann. N.Y. Acad. Sci. 176:197-211. Nalin, D. R., A. K. Bhattacharjee, and S. H. Richardson. 1974. Cholera-like toxic effect of culture filtrates of Escherichia coli. J. Infect. Dis. 136:595-601. Pierce, N. F., and C. K. Wallace. 1972. Stimulation of jejunal secretion by a crude Escherichia coli enterotoxin. Gastroenterology 63:439-448. Powell, D. W., and S. J. Malawer. 1968. Relationship between water and solute transport from isosmotic solutions by rat intestine in vivo. Am. J. Physiol. 215:49-55. Sack, D. A., J. G. Wells, M. H. Merson, R. B. Sack, and G. K. Morris. 1975. Diarrhoea associated with heatstable enterotoxin-producing strains of Escherichia coli. Lancet 2:239-241. Sack, R. B. 1975. Human diarrheal disease caused by enterotoxigenic Escherichia coli. Annu. Rev. Microbiol. 29:333-353. Sherr, H. P., J. G. Banwell, A. Rothfeld, and T. R. Hendrix. 1973. Pathophysiological response of rabbit jejunum to Escherichia coli enterotoxin. Gastroenterology 65:895-901. Shore, E. G., A. G. Dean, K. J. Holik, and B. R. Davis. 1974. Enterotoxin-producing Escherichia coli and diarrheal disease in adult travelers: a prospective study. J. Infect. Dis. 129:577-582. Smith, H. W., and C. L. Gyles. 1970. The relationship between two apparently different enterotoxins produced by enteropathogenic strains of Escherichia coli of porcine origin. J. Med. Microbiol. 3:387-401. Smith, P. B., K. M. Tomfohrde, D. L. Rhoden, and A. Balows. 1972. API system: a multitube micromethod for identification of Enterobacteriaceae. Appl. Microbiol. 24:449-452. Sullivan, R., and T. Asano. 1971. Effects of staphylococcal enterotoxin B on intestinal transport in the rat. Am. J. Physiol. 220:1793-1797.
INFECTION AND IMMUNITY, OCt. 1976, p. 1004-1010 Copyright © 1976 American Society for Microbiology
Assay ofEscherichia coli Enterotoxins by In Vivo Perfusion in the Rat Jejunum FREDERICK A. KLIPSTEIN,* CHUNG-SENG LEE, AND RICHARD F. ENGERT Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
Received for publication 10 February 1976
Assay of Escherichia coli enterotoxins by in vivo perfusion in rats was evaluated by examining the effects of variously prepared fractions of heat-labile (LT) and heat-stable (ST) toxins on water transport in this system. The assay was found to respond equally well, in a dose-related manner, to both LT and ST; it was sufficiently sensitive to detect the toxigenic effect of concentrations as small as 1 ng/ml. With the assay, it was found that LT is produced in cultures grown under aerobic, but not anaerobic, conditions; in contrast, ST is elaborated in stationary aerobic and anaerobic broth cultures but not in those grown under agitated aerobic conditions. Both toxins can be precipitated by either ammonium sulfate or acetone. The two toxin forms were completely separated from each other by sequential ultrafiltration. LT alone (thermolabile after exposure to 100°C for 30 min) was retained by a PM-30 membrane, and ST alone was present in UM-2 retentates; ST was retained more effectively by a UM-05 membrane, with a 1,000-fold increase in activity over that of the UM-2 retentate. Washed ultrafiltration retentates containing either LT or ST derived from the proper culture conditions all induced water secretion at concentrations of 100 ng or less per ml. These results indicate that in vivo perfusion in rats is a sensitive, duplicable assay for both the LT and ST forms of E. coli enterotoxin.
Enterotoxigenic strains of Escherichia coli produce two forms of enterotoxin: a low-molecular-weight, heat-stable toxin (ST) either alone or along with a large-molecular-weight, heatlabile toxin (LT) (11, 22, 32, 35). These two forms are generally considered to be separate, distinct entities based on dissimilarities in their physical (4, 14, 15, 23), antigenic (7, 11, 12, 35), and biological properties (3, 7, 8, 9). Whereas the techniques for producing LT alone are well recognized (13, 35), and sensitive, quantitative in vitro tests are available for its assay (3, 5, 9), such is not the case for ST. Cellfree broth filtrates of strains of E. coli that produce LT and ST contain both forms of toxin, and ST must be separated from LT by subsequent procedures based on their different physical properties such as heat lability (7, 24, 28). Assays for ST are confined to observing fluid accumulation within ligated loops of rabbits (7, 11, 27) or the whole intestinal tract of suckling mice (2, 31, 34). It seemed important, therefore, to determine whether clearly defined preparations of ST can be separated from LT and whether a more sensitive and quantitative assay system for ST could be developed. The toxigenic activity of enterotoxins of other coliform species has been assayed by the in vivo
marker perfusion technique in the rat jejunum (16, 17, 19). In the present study, we evaluated this system as an assay for E. coli entertoxins by determining the effect of LT and ST preparations, produced under various culture conditions and separated from each other by means of ultrafiltration (UF) procedures, on water transport in the rat jejunum.
MATERIALS AND METHODS Bacterial strains. Biotypes were determined by the API microtube 20 E profile system (Analytab Products, Inc., Plainview, N.Y.) (36). All three strains studied were jejunal isolates. (i) NTG. Nontoxigenic (NTG) strain BN (API biotype 5044572; serotype O1:H31) was isolated from a control subject in Puerto Rico; cell-free broth filtrates are inactive in rabbit ileal loop studies (18). (ii) ST only. Strain PR (API biotype 5044572; serotype O1:H31) was isolated from a Puerto Rican with tropical sprue (18); broth filtrates evoked in the rabbit ileal loop a positive fluid reaction that is unaltered by heating the preparation (21). (iii) LT/ST. Strain Haiti (API biotype 5044552; serotype 04:H32) was isolated from a Haitian with tropical sprue and shown by rat perfusion studies to produce both types of enterotoxin (20). Enterotoxin production. (i) By bacterial lysis. Sonic extracts were prepared by a modification of the method described by Gyles and Barnum (13). 1004
1005
VOL. 14, 1976
ASSAY OF E. COLI ENTEROTOXINS
The bacteria were grown either aerobically or anaerobically for 18 h at 37°C on Trypticase (BBL, Cockeysville, Md.) soy agar in a Roux bottle. The harvested confluent surface growth was sonically treated on a sonifier cell disrupter (Branson Sonic Power Co., Plainview, N.Y.) for 10 min; the suspension obtained was centrifuged for 30 min at 10,000 x g and 4°C, and the supernatant was passed through a 0.45-,.m membrane filter (Millipore Corp., Bedford, Mass.). The cell-free supernatant was then either lyophilized or passed through UF membranes. (ii) From broth cultures. For aerobic broth cultures, 0.1 ml of a 6-h subculture of bacteria was introduced into 250 ml of Trypticase soy broth in a 2,000 ml flask and incubated at 37°C for 18 h either under stationary conditions or with agitation at 200 shakes per minute. For anaerobic cultures, the same amount of bacteria was inoculated into 500 ml of the same medium in a 1-liter flask and incubated in an anaerobic chamber (Coy Manufacturing Co., Ann Arbor, Mich.) with 85% N2, 5% C02, and 10% H2 gas for 18 h at 37°C. After growth, the broth cultures were centrifuged for 45 min at 35,000 x g at 4°C, and the supernatant was passed through a 0.45-,um Millipore filter. The filtrate was then precipitated with either 8 volumes of acetone (1, 19) or 90% ammonium sulfate (AMS) (7). The acetone precipitates were solubilized in distilled water, the AMS precipitates were dissolved in 0.02 M ammonium bicarbonate (pH 7.8), and both solutions were then fractionated by UF. UF. Separation of the two forms of enterotoxin was based on the fact that LT has an apparent molecular weight of greater than 100,000 (4, 15, 23) and would be retained by a PM-30 UF membrane (molecular weight cutoff, 30,000), whereas ST, which has an apparent molecular weight in the range of 1,000 to 10,000 (1, 14), is partially retained by a UM-2 membrane (molecular weight cutoff, 1,000). The broth culture precipitates were passed sequentially through Amicon PM-30 and either UM2 or UM-05 (molecular weight cutoff, 500) membranes (Amicon Corp., Lexington, Mass.). Unless so specified, retentate fractions were taken to 10% of the original volume and lyophilized. Retentate fractions identified as "washed" were washed twice with two times the original volume, using 0.02 M ammonium bicarbonate, pH 7.80. Animal perfusion technique. Procedures for the in vivo evaluation of intestinal transport by means of marker perfusion studies in rats (30) and the results of perfusion studies of other enterotoxins examined in this animal system were described in detail elsewhere (16, 17, 19, 25, 37). Toxin fractions were tested in anesthetized, tracheostomized, Sprague-Dawley rats weighing from 150 to 250 g. Single 20-cm jejunal segments were perfused at a rate of 0.5 ml/min by using a peristaltic pump (model 1201, Harvard Apparatus Co., Millis, Mass.). Six 30-min test fractions were collected after a 30min steady-state period. The electrolyte solution described by Powell and Malawer (30) was used, except that the concentration of mannitol was reduced, when necessary, such that osmolality remained
isosmotic with rat plasma at 317 mosmol/kg (19, 30). Osmolalities were determined by measuring the freezing-point depression with an Advanced DigiMatic Osmometer (Advanced Instrument, Newton Heights, Mass.). Polyethylene glycol 4000 (PEG) concentrations were determined by the turbidimetric technique (26). Net transport of water, expressed in microliters per centimeter per 30 min, was calculated from changes in polyethylene glycol 4000 concentrations by the usual marker technique formula (30). Net lumen-to-blood transport is termed absorption and is signified by a plus sign, whereas a minus sign refers to net blood-to-lumen transport, or secretion. This laboratory (16, 17, 19) and others (10, 37) reported previously (and we so noted in the present study) that, whereas large concentrations of enterotoxin induce secretion throughout all six collection periods, smaller, submaximal concentrations evoke transient secretion during only several periods, usually the last few in the case of LT. Therefore, the mean value for all six periods may reflect absorption even though the toxin has evoked secretion at some time during the entire 210-min collection period. For this reason, we believe that the value for the 30-min collection period during which there is minimal absorption or maximal secretion (maximal effect) is a more sensitive and accurate reflection of toxin activity than the mean of all six periods. Values reported in this and previous studies (16, 17), therefore, are for the period of maximal effect. A positive enterotoxin effect is defined as the presence of secretion. The minimal effective dose (MED) of toxin is defined as the concentration (in dry weight per milliliter) that induces net secretion. The total amount of toxin perfused during the whole perfusion period is the concentration times 105 (the volume, in milliliters, perfused). Determinations reported are for assays in single rats except where noted otherwise.
RESULTS Characteristics of the assay. (i) Onset of toxin action. Water transport during perfusion with the electrolyte solution alone (control) in 20 rats showed only slight variability during each of the six 30-min collection periods (Fig. 1). The mean (plus or minus standard error of the mean [± SEM]) value for minimum water absorption during any one of the six periods was +41 + 3. The onset of action of a preparation containing LT (the crude sonic extract of strain Haiti), perfused at a concentration of 2,000 ,g/ml, was not evident until the third perfusion period, or 90 min after introduction of the toxin, and the maximum effect was delayed until the sixth period. In contrast, the same dosage of a preparation containing ST (the UM2 retentate of a broth culture of strain Haiti) reduced water transport during the first test period. We were unable to determine whether water absorption was affected during the prior 30 min of exposure since reliable information
1006
INFECT. IMMUN.
KLIPSTEIN, LEE, AND ENGERT O CONTROL
E; LT
clear-cut relationship between dosage and effect on water transport for each preparation. Effect of specific LT and ST preparations. (i) Crude preparations of LT. Initially, the crude sonic extract and unwashed PM-30 reten-
O3 ST
Zt
rz,i
r-I
SZN 111;K
+60
i.. z
-tz
t
-ZZ
2
+40
.-. .: :
s+20-ttl1 1
2
3 4 PERIOD
5
6
FIG. 1. Effect on water transport of perfusing the electrolyte solution alone (control) in 20 rats and of LT and ST preparations, each tested in 5 rats at a concentration of 2,000 Mg/ml. Values are mean ± SEM for each 30-min period. LT is the crude sonic extract and ST is the UM-2 retentate of broth filtrate of strain Haiti.
concerning transport cannot be obtained during the preliminary 30-min steady state. (ii) Reproducibility of the assay. To determine whether toxin preparations that yield borderline or strongly positive water secretions can be differentiated clearly from NTG preparations and whether the result in a single rat is a reliable index of these differences, 12 different preparations were each perfused on multiple occasions at a concentration of 2,000 ,ug/ml (Fig. 2). The inactive material consisted of seven different preparations of strain BN (NTG) (the sonic extract and UF fractions of broth cultures grown under various conditions), each of which was tested in three rats. The five preparations from the toxigenic strains containing LT or ST, which yielded either a borderline or strongly active response, were each tested in five rats. Mean (+ SEM) values for water transport were absorption of +35 ± 2 for the NTG preparations, absorption of + 2 + 2 for the borderline active preparations, and secretion of -51 + 5 for the strongly active preparations. Values for the both borderline and strongly active preparations were significantly less (P < 0.001) than those of the inactive NTG preparations. The results obtained for each individual preparation in the various categories showed only slight variation when examined in more than one rat. (iii) Dosage-response relationship. The effect on water transport of perfusing differently prepared fractions of LT and ST of strain Haiti (LT/ST) at graded dosages of between 0.0001 and 2,000 Ag/ml is shown in Fig. 3. There was a
-60
J
2El00 2 BORDERLINE ACTIVE
-80-100FIG. 2. Results of repeated assays of individual preparations of variable toxigenic activity. Values are the means for three to five determinations (hatched area) and the range obtained in individual rats with each preAPIA (vertical barAJ. 'I
.
z
I
,
+40
1O
U@ -40
SONICATE
0 CRUDE 0 PM - 30 R(W)
-80 CONTROL
ST
.N
BROTH FILTRATE
OUM-2
R
EUM-05 RIW)
DOSAGE,
kg/ml
FIG. 3. Response of water transport to graded dosages of LT and ST preparations of strain Haiti. Values are mean ± SEM for the control solution and for assay in single rats for the toxin preparation6. R, Retentate fraction; W, washed fraction.
VOL. 14, 1976
ASSAY OF E. COLI ENTEROTOXINS
tate of broth filtrates, which had been grown under various culture conditions and precipitated with either AMS or acetone, were tested at a concentration of 2,000 ,ug/ml (Table 1). All preparations of strain PR (ST) were inactive. All preparations of strain Haiti (LT/ST) strain were active except for broth cultures grown
ST, cultures grown under anaerobic conditions
under anaerobic conditions. Exposure of the active preparations to 100°C for 30 min completely abolished their ability to induce water secretion. (ii) Crude preparations of ST. Unwashed UM-2 retentates of broth cultures grown under various conditions were initially tested at concentrations of 100 to 2,000 ,ug/ml. Retentates of cultures of both strains were active when the cultures were grown under stationary aerobic and anaerobic conditions, but the UF fractions of cultures grown under agitated aerobic conditions were inactive (Table 2). When AMS was used to precipitate ST, cultures grown under aerobic stationary conditions were more active than those grown anaerobically and, conversely, when acetone was used to precipitate
1007
were the most active. The toxigenic activity of these preparations was not affected by exposure to 100°C for 30 min, indicating that they consisted exclusively of ST. (iii) More-purified preparations of LT. Increased potency was achieved by passing the crude sonic extract through a PM-30 membrane and by washing the PM-30 retentates of both the sonic extract and broth filtrate. Passage of the Haiti (LT/ST) sonic extract through a PM30 membrane reduced the MED from 100 ,ug/ml for the crude sonic extract to 1 ,ug/ml for the PM-30 retentate; washing of the retentate resulted in a 1,000-fold increase in activity, with an MED of 0.001 ,g/ml (Table 3, Fig. 3). Washed PM-30 retentates of AMS and acetone precipitates of both the sonic extract and broth cultures of strain Haiti grown under agitated and stationary aerobic conditions, were all active when perfused at a concentration of 0.1 ,tg/ ml; however, none of the anaerobically grown preparations was active (Table 4). Perfusion of a similarly prepared sonic extract of strain PR
TABLE 1. LT: Effect of crude sonic extracts and unwashed PM-30 retentates of broth cultures on water transport Growth medium
Precipitate
Water transporta6,
Culture conditions
PR (ST)
Haiti (LT/ST)
Agar
(Sonic extract)
Aerobic
Stationary
+38
-87 (+34)
Broth Broth Broth
AMS AMS AMS
Aerobic Aerobic Anaerobic
Agitated Stationary Stationary
+30 +31 +35
-30 (+24) -14 (+15) +34
Broth Acetone Aerobic +31 -34 (+34) Agitated Broth 0 (+55) Acetone Aerobic +32 Stationary +42 Broth Acetone Anaerobic +22 Stationary a Results (microliters per centimeter per 30 min) are the average for assays in two rats for each preparation. Values in parentheses are for the same fraction after exposure to 100°C for 30 min. b All preparations were perfused at a concentration of 2,000 j±g/ml. TABLE 2. ST: Effect of unwashed UM-2 Retentates on water transport Water transporta Strain
PR (ST)
Culture conditons
Aerobic Aerobic Anaerobic
Agitated Stationary Stationary
Acetone precipitate
AMS precipitate 2,000b
1,000
100
+24 -41 (-37)
+25
+40
-37 -3
-7 +16
-30 (-27)
2,000b
+10
+4 -5 (-7)
1,000
+42 +22 +17
+5 +10 +55 Aerobic +18 Agitated +25 -44 (-68) -36 -5 Aerobic +4 Stationary +8 -52 (-76) -7 Anaerobic +7 -68 (-62) Stationary -11 a Results (microliters per centimeter per 30 min) are the average for assays in two rats for each preparation. Values in parentheses are for the same fraction after exposure to 100°C for 30 min. b Dosage in micrograms per milliliter.
Haiti (LT/ST)
1008
INFZCT. IMMUN.
KLIPSTEIN, LEE, AND ENGERT
(ST), grown aerobically, at a concentration of 100 ug/ml yielded absorption of +53. (iv) More-purified preparation of ST. To determine whether a UM-05 membrane retains ST more effectively than a UM-2 membrane, the acetone precipitates of anaerobically grown broth cultures of BN (NTG), PR (ST), and Haiti (LT/ST) were passed sequentially through PM30 and UM-05 membranes (Table 3, Fig. 3). The activity in the unwashed UM-05 retentate of material from strains PR and Haiti had an MED of 0.1 ,ug/ml, which represented a 1,000fold increase in activity over that of the unwashed UM-2 retentates. Washing the UM-05 retentate increased the potency of material from strain Haiti by 10-fold, yielding an MED 0.01 ,ug/ml, and that of strain PR by 100-fold, resulting in an MED of 0.001 ,ug/ml. The toxigenic activity of the washed preparations of these two strains was not reduced when the preparations were perfused, at their MED, after exposure to 100°C for 30 min. All of the washed UM-05 retentates of cultures of strain Haiti that were grown under stationary aerobic and anaerobic (but not agitated aerobic) conditions were found to be active when perfused at a concentration of 0.1 ,g/ml (Table 4). In contrast, the washed UM-05 retentate of strain BN
(NTG) gave absorption of +37 when perfused at concentration of 1 ug/ml. DISCUSSION Previous in vivo marker perfusion studies concerned with the effect of E. coli enterotoxins on water transport in rabbits and dogs employed 50- to 2,500-,tg/ml dosages of relatively crude material containing both LT and ST (10, 29, 33). Bywater found that UF fractions containing only ST also stimulate water secretion in calves (1). In vivo perfusion in rats was shown previously to be a sensitive, quantitative assay for the heat-stable enterotoxins elaborated by Klebsiella pneumoniae and Enterobacter cloacae (16, 17, 19), and the results of the present study indicate that this assay system responds equally well to both the LT and ST forms of E. coli enterotoxin. The assay system is reproducible, such that perfusion in a single rat yields reliable information; it responds in a dose-related fashion to both LT and ST fractions; and it is sufficiently sensitive to detect toxigenic activity at concentrations as low as 1 ng/ml. In previous studies concerned with the onset of action of the two forms ofE. coli enterotoxin, the preparations used contained both ST and a
TABLE 3. Effect of PM-30 and UM-05 retentates, before and after washing, on water transport Water transport (p1/cm per 30 min) Strain Toxin Prep Fraction Washed la
0.1
0.01
0.001
0.0001
Haiti Haiti
LT LT
Sonic extract Sonic extract
PM-30 PM-30
No Yes
-45
+36 -107
-23
-3
+29
Haiti Haiti
ST ST
Brothb Brothb
UM-05 UM-05
No Yes
-21 -107
-15 -33
0 -8
+15 +18
PR ST Brothb No UM-05 -58 -13 +32 PR ST Brothb Yes UM-05 -120 -53 -11 a Dosage in micrograms per milliliter. b Broth cultures were grown under anaerobic conditions and precipitated with acetone.
+21
TABLE 4. Effect of washed PM-30 and UM-05 retentates of strain Haiti on water transport Water transporta Toxin
LT LT LT LT LT
Prepn
Sonic extract Sonic extract Sonic extract Broth Broth
UF
PM-30 PM-30 PM-30 PM-30 PM-30
Ppt
AMS Acetone AMS Acetone
ST Broth AMS UM-05 ST Broth Acetone UM-05 a All preparations were perfused at a concentration of 0.1 ug/ml.
(p1/cm per 30 min)
Aerobic
Anaerobic
Agitated
Stationary
-22 -39
-107 -84 -50 -29 -11
+9 +12
+8 +7
-11 -1
-23 -33
+7
VOL. 14, 1976
ASSAY O E. COLJ ENTEROTOXINS
LT, and the respective effect of each form was separated by testing the preparations before and after heat treatment (7, 28). Evans and her colleagues found that their thermostable preparation resulted in prompt fluid accumulation within ligated rabbit ileal loops even at the lowest dosage, whereas the onset of fluid accumulation in response to the heat-labile material was rapid at a high dose but delayed at low doses (7). Determining serial fluxes of water in canine jejunal loops, Nalin and his co-workers showed that their thermostable toxin induced prompt secretion, whereas the effect of their thermolabile preparation was delayed until 3 h after exposure to the toxin (28). When preparations containing exclusively ST or LT were perfused in the present study, a clear-cut difference between their onset of action was apparent: ST altered water transport during the earliest period of testing, but the effect of LT was not manifest until after exposure to the toxin for 90 min. Different culture conditions were found to favor maximum production of LT and ST in the present study: LT was produced under aerobic but not anaerobic conditions, whereas ST was elaborated in both stationary aerobic and anaerobic cultures but not in those maximally aerated by agitation. LT alone can be recovered from whole-cell lysates of growth on agar slants (6, 24, 27, 35) or in broth cultures (13, 22), where maximum aeration enhances its production (6, 8, 27); it can also be recovered from cell-free broth filtrates and precipitated by AMS (7, 22, 27, 29), but these preparations are only partially heat labile, indicating the presence of both LT and ST (7, 27). We found LT activity to be present in AMS and acetone precipitates of both sonic extracts of cells grown on agar and of cell-free broth filtrates. ST can be recovered from cell-free filtrates of aerobic cultures on soft agar (1, 2, 24, 35) or in broth (11, 14, 27, 29), and this form of toxin can be precipitated by either AMS or acetone (1, 7, 28, 35). In the present study, stationary aerobic culture conditions yielded the most active material precipitated by AMS, whereas anaerobic culture conditions yielded maximum output of ST when acetone was employed. Thermostable toxin preparations elaborated by K. pneumoniae and E. cloacae have been reported to have similar optimal culture conditions (16, 17). Due to the difference in their molecular weights, E. coli LT and ST can be separated by passage through graded polymeric membranes. LT, which has a molecular weight of 102,000 (4), is retained by both XM-50 and XM-100 UF membranes (4, 15, 23), whereas ST, which has an apparent molecular weight of between 1,000
and 10,000, passes through both the XM-50 membrane and the smaller pore sized PM-10 membrane (1, 14, 23). In the present study, LT and ST were completely separated from each other by sequential passage of broth filtrates through graded membranes: preparations in the PM-30 retentate were completely heat labile and those in the UM-2 retentate, after sequential passage through a PM-30 membrane, retained their toxigenic activity after exposure to 100°C for 30 min. Jacks and Wu reported that E. coli ST is completely retained by a UM-2 membrane (14); on the other hand, Bywater detected ST activity in both the retentate and filtrate fractions of this membrane (1), and the same has been reported to hold true for K. pneumoniae and E. cloacae ST preparations (16, 17). We therefore tried out the effect of the smaller pore sized UM-05 membrane and found it to retain E. coli ST more effectively, yielding fractions with 1,000-fold more activity than the UM-2 retentate. Washing the membranes markedly enhanced toxigenic activity; this was probably due to removal of inactive, small-molecular-weight products of growth through the PM-30 membrane and to more complete desalting of the retentate fraction in the case of the UM-05 membrane.
1009
ACKNOWLEDGMENTS This work was supported by grants from the WilliamsWaterman Fund of the Research Corporation, New York City, N.Y., and the Hillsdale Fund, Greensboro, N.C. LITERATURE CITED 1. Bywater, R. J. 1972. Dialysis and ultrafiltration of a heat-stable enterotoxin from Escherichia coli. J. Med. Microbiol. 5:337-343. 2. Dean, A. G., Y. C. Ching, R. G. Williams, and L. B. Harden. 1972. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J. Infect. Dis. 125:407-411. 3. Donta, S. T. 1974. Detection of heat-labile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture. Science 183:334-336. 4. Dorner, F. 1975. Escherichia coli enterotoxin. Purification and partial characterization. J. Biol. Chem. 250:8712-8719. 5. Dorner, F., and P. Mayer. 1975. Escherichia coli enterotoxin: stimulation of adenylate cyclase in broken-cell preparations. Infect. Immun. 11:429. 6. Etkin, S., and S. L. Gorbach. 1971. Studies on enterotoxin from Escherichia coli associated with acute diarrhea in man. J. Lab. Clin. Med. 78:81-87. 7. Evans, D. G., D. J. Evans, and N. F. Pierce. 1973. Differences in the response of rabbit small intestine to heat-labile and heat-stable enterotoxins of Escherichia coli. Infect. Immun. 7:873-880. 8. Evans, D. J., D. G. Evans, and S. L. Gorbach. 1973. Production of vascular permeability factor by enterotoxigenic Escherichia coli isolated from man. Infect. Immun. 8:725-730. 9. Guerrant, R. L., L. L. Brunton, T. C. Schnaitman, L. I. Rebhun, and A. G. Gilman. 1974. Cyclic adenosine monophosphate and alteration of Chinese hamster
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cell morphology: a rapid, sensitive in vitro for the enterotoxins of Vibrio cholerae and Escherichia coli. Infect. Immun. 10:320-327. Guerrant, R. L., U. Ganguly, A. G. T. Casper, E. J. Moore, N. F. Pierce, and C. C. J. Carpenter. 1973. Effect of Escherichia coli on fluid transport across canine small bowel. Mechanism and time-course with enterotoxin and whole bacterial cells. J. Clin. Invest. 52:1707-1714. Gyles, C. L. 1971. Heat-labile and heat-stable forms of the enterotoxin from E. coli strains enteropathogenic for pigs. Ann. N.Y. Acad. Sci. 179:314-322. Gyles, C. L. 1974. Immunological study of the heatlabile enterotoxins of Escherichia coli and Vibrio cholerae. Infect. Immun. 9:564-570. Gyles, C. L., and D. A. Barnum. 1969. A heat-labile enterotoxin from strains of Escherichia coli enteropathogenic for pigs. J. Infect. Dis. 120:419-429. Jacks, T. M., and B. J. Wu. 1974. Biochemical properties of Escherichia coli low-molecular-weight, heatstable enterotoxin. Infect. Immun. 9:342-347. Jacks, T. M., B. J. Wu, A. C. Braemer, and D. E. Bidlack. 1973. Properties of the enterotoxic component in Escherichia coli enteropathogenic for swine. Infect. Immun. 7:178-189. Klipstein, F. A., and R. F. Engert. 1976. Purification and properties of Klebsiella pneumoniae heat-stable enterotoxin. Infect. Immun. 13:373-381. Klipstein, F. A., and R. F. Engert. 1976. Partial purification and properties of Enterobacter cloacae heatstable enterotoxin. Infect. Immun. 13:1307-1314. Klipstein, F. A., L. V. Holdeman, J. J. Corcino, and W. E. C. Moore. 1973. Enterotoxigenic intestinal bacteria in tropical sprue. Ann. Intern. Med. 79:632-641. Klipstein, F. A., I. R. Horowitz, R. F. Engert, and E. A. Schenk. 1975. Effect of Klebsiella pneumoniae enterotoxin on intestinal transport in the rat. J. Clin. Invest. 56:799-807. Klipstein, F. A., H. B. Short, R. F. Engert, L. Jean, and G. A. Weaver. 1976. Contamination of the small intestine by enterotoxigenic coliform bacteria among the rural population of Haiti. Gastroenterology 70:1034-1041. Klipstein, F. A., and E. A. Schenk. 1975. Enterotoxigenic intestinal bacteria in tropical sprue. II. Effect of the bacteria and their enterotoxins on intestinal structure. Gastroenterology 68:642-655. Kohler, E. M. 1971. Observations on enterotoxins produced by enteropathogenic Escherichia coli. Ann. N.Y. Acad. Sci. 176:212-219. Lariviere, S., and C. L. Gyles. 1973. Preliminary charovary assay
10.
11.
12. 13. 14.
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16. 17. 18. 19.
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