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THE ISOLATION AND PARTIAL CHARACTERIZATION
OF AN ENTEROTOXIN PRODUCED BY
AEROMONAS HYDROPHILA AND AEROMONAS SOBRIA

 

By

Dace Valduss

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1981

ABSTRACT
THE ISOLATION AND PARTIAL CHARACTERIZATION

OF AN ENTEROTOXIN PRODUCED BY
AEROMONAS HYDROPHILA AND AEROMONAS SOBRIA

By

Dace Valduss

The suckling mouse assay was used to test for enterotoxic activity
with human and environmental isolates of Aeromonas hydrophila and
Aeromonas sobria. Enterotoxic activity could be detected in 5 to 9 iso-
lates. Three of these isolates were Aeromonas sobria and 2 were
Aeromonas hydrophila. Negative strains remained negative for enterotoxic
activity even after ten-fold concentration of the supernatant. Cell free
filtrates tested in suckling mice produced a positive response indicating
that the enterotoxin is a cell-free exotoxin released into the growth
media. When heated at 60°C for 20 minutes, the filtrates lost enterotoxic
potential suggesting that the toxin is heat labile. Column chromatography
performed with concentrated enterotoxin indicated that the toxin is a
large molecular weight substance, perhaps protein in nature. Source of
isolation appears not to play a role in enterotoxin production since both

enrivonmental and human isolates were reactive in the assay.

DEDICATION

To my parents

Visvaldis and Ella Valduss

and my grandmother

Alma Rumba

Thank you for your encouragement, love, and understanding.

ii

ACKNOWLEDGMENTS

I would like to thank my research advisor, Dr. Robert J. Moon, for
his encouragement, guidance, and concern throughout this project.

I would also like to thank Dr. Elizabeth A. Werner, Dr. Maria J.
Patterson, Dr. Leland F. Velicer, Bob Leunk, Hassan Tavakoli, Bryon
Petschow, Lee Schwocho, Ellen Keitelman, Paul Rota, and Ruth Vrable for
their help and friendship throughout my graduate studies. Special thanks
to Barbara Dixon, Char Currie, and Cheryl Yonke for their patience and

friendship. Thank you all.

*******

iii

TABLE OF CONTENTS

 

 

 

Page
LIST OF TABLES ................................................ vi
LIST OF FIGURES ............................................... vii
INTRODUCTION .................................................. 1
LITERATURE REVIEW ............................................. 2
A Brief History of Aeromonas ............................. 2
Aeromonas Infection in Frogs ............................. 5
Aeromonas Infection in Humans ............................ 6
Enterotoxins of Gram Negative Bacteria ................... 7
Vibrio cholera Enterotoxin .......................... 7
Escherichia coli Enterotoxins ...... .... ............. 8
Salmonella Enterotoxin .............................. 11
Aeromonas Enterotoxin ............................... 12
Animai and Cell Models for Detection of Enterotoxin

Production .......................................... 16
MATERIALS AND METHODS ......................................... 18
Bacterial Strains ........................................ 18
Media and Maintenance of Cultures ........................ 18
Preparation of Cultural Supernatants ..................... 19
Filtration and Concentration ............................. 19
Mice ..................................................... 19
Suckling Mouse Assay ..................................... 20
Hemagglutination Assay ................................... 20
Column Chromatography .................................... 21
Statistics ............................................... 21
RESULTS ....................................................... 22
Enterotoxic Activity in Culture Supernatants ............. 22

Mannose Sensitive Hemagglutinating Activity of Aeromonas
Organisms ........................ . .................. 22

iv

Page

Enterotoxic Response in Suckling Mice with Aeromonas spp.

Following Filter Sterilization and Ultrafiltration... 25

Enterotoxic Activity Following Heating of Culture 25
Filtrates ..... .....................................

Passage of Concentrate Through G- 100 Sephadex Column ..... 29

DISCUSSION .................................................... 37

BIBLIOGRAPHY .................................................. 42

APPENDIX ...................................................... 50

LIST OF TABLES

TABLE

. Aeromonas Species and Source of Isolation ................
. Production of Enterotoxin-like Activity Assayed by the
Suckling Mouse Model in BHI Roller Tube Culture Super-
natants of Aeromonas ................... ..... .............
. Hemagglutinating Ability of Aeromonas Isolates ...........

. Effect of Filter Sterilization on the Enterotoxic Activity
of Aeromonas .............................................

. Reactivity of Filtrates Concentrated Ten-fold by Ultra-
filtration with a PM 30 Membrane Filter ..................

. Effect of Heating at 60°C on Enterotoxic Activity of
Aeromonas ................................... .............

Appendix 1: Comparison of Enterotoxic Activity with Other

Properties of the Genus Aeromonas .................... ....

vi

Page

23

24

26

27

28

3O

51

LIST OF FIGURES
FIGURE Page

1. Enterotoxin activity and protein concentration of fil-
trates from Aeromonas strain Em-56 following Sephadex
G-100 chromatography .................................... 32

2. Enterotoxin activity and protein concentration of fil-
trates from Aeromonas strain Cl-124 following Sephadex
G-100 chromatography .................................... 34

3. Enterotoxin activity and protein concentration of con-

centrated BHI broth following Sephadex G-100 chromatog-
raphy ................................ . .................. 36

vii

INTRODUCTION

In recent years, organisms previously considered saprophytic or
non-pathogenic for man have been implicated as the cause of human disease.
Many investigators call attention to the increasing numbers of infections

caused by Aeromonas hydrophila (20, 52, 60, 75, 85). The majority of

 

bacteremic infections have occurred in patients with chronic hepatic
disease or with an underlying neoplastic or hematologic disorder (60, 85,
88). Epidemiologically, Aeromonas has been isolated primarily from non-
fecally contaminated fresh water, city tap water and rural rivers, lakes
and canals (3, 4, 45). Aeromonads seem to thrive in water characterized
by a high content of non-fecal organic waste material. They have also
been isolated from fecal specimens from children and adults with diarrhea
of unexplained origin as well as from healthy carriers (3, 4, 24).

The primary objective of this study was to examine clinical and

environmental isolates of Aeromonas hydrophila and Aeromonas sobria for

 

enterotoxic activity in the suckling mouse assay. After isolating a
suckling mouse reactive "toxin", further studies were performed to define
the nature of this enterotoxic material with regard to filterability,
ability to be retained on ultrafiltration (PM30) membranes and stability
to heat. Attempts were also made to correlate other parameters such as
cytotoxic activity, piliation, motility and L050 with enterotoxic

activity.

LITERATURE REVIEW

A Brief History of Aeromonas

Bergey's Manual defines the genus Aeromonas as follows: "Short
(rarely more than 3 microns), rod shaped cells. Motile by means of
polar flagella, usually monotrichous: occasionally non-motile. Gram
negative, carbohydrates fermented with the production of H2, 02 and 2,3
butylene glycol. Methyl red negative. Slow or no fermentation of lactose.
The majority of the species thus far described are from water or are
known to be pathogenic to marine and fresh water animals such as fish
and amphibians." The manual lists four species: Aeromonas liguefaciens,
Aeromonas pyncatata, Aeromonas hydrophila and Aeromonas salmonicida.

Strains of bacteria which are now considered to belong to the genus
Aeromonas have been isolated and described in some detail during the past
70 years (26, 27, 71, 83). Many have undergone numerous name changes as
well as changes in grouping. Since much of the early literature is con-
fusing, a short description of each of the 4 main species of Aeromonas
is provided (based on Eddy's 1960 review of the literature and history of
Aeromonas [26]).

Aerobacter liquefaciens (Aeromonas liquefaciens) was first isolated
by Beijerinck in 1900 from the ditches of the Delft. He proposed the
generic name Aerobacter and described the organism as having a single

polar flagellum and the ability to rapidly liquefy gelatin. In 1936

Kluyver and van Niel proposed that the name be changed to Aeromonas.
This species was included, for the first time, in the 7th edition of
Bergey's Manual as Aeromonas liquefaciens.

Bacillus punctatus (Aeromonas_punctata) was first isolated from
tap water by Zimmerman in 1890. In the first five editions of Bergey's

Manual, it was known as Achromobacter punctatum. In the 6th edition it

 

became Pseudomonas punctata and in the 7th edition Aeromonas punctata.

 

Some strains are very pathogenic to fish while others are nonpathogenic.

Bacterium salmonicida (Aeromonas salmonicida) was first isolated

 

by Emmerich and Neibel in 1894. It is non-motile and produces a pigment.
This organism differs from other Aeromonads by being Voges-Proskauer
negative and psycrophilic.

Bacillus hydrophilus fuscus (Aeromonas hydrophila). The earliest
strains were isolated from diseased frogs. The first person to investi-
gate septicemic diseases of frogs was Ernst, who in 1890 reported the
isolation of a bacterium from the blood of infected frogs. He named

the organism Bacillus ranicida. A year later, Sanarelli described an

 

organism which he isolated from the lymph and blood of frogs. He iso-
lated the same organism from the laboratory water supply. Sanarelli named

his organism Bacillus hydrophilus fuscus, since he did not believe it to

 

be identical to Bacillus ranicida. Sanarelli's bacteria was pathogenic

 

for frogs, toads, salamanders, lizards, sunfish and fresh water eels.
Injection of this organism into warm blooded animals such as guinea pigs,
rabbits, dogs, cats, mice, chickens and pigeons resulted in death within
a relatively short time. In 1893, Trambusti isolated the same species

from diseased frogs in his laboratory. His investigations were primarily

concerned with the isolation of toxic metabolic products from the organ-
ism. His experiments indicated that the toxic products were of two

kinds, one soluble and the other insoluble in alcohol. He attempted to
determine the physiologic action of pure cultures and toxins of the organ-
isms on experimental animals. Roger in 1893 described an epizootic
disease among frogs characterized by a distinct reddening of the legs.

He found that the viscera and blood of the frogs contained a small bacillus
in pure culture. The same organism was isolated from the water of the
aquarium which housed the infected frogs. Roger concluded that the
organism was the same organism isolated by Sanarelli. In the lst edition
of Bergey's Manual the organism was named Proteus hydrophilus and
described as having peritrichous flagella. Russel had previously shown
that the organism had a single polar flagellum. This error remained

until the 6th edition when the organism was placed in the genus

Pseudomonas. In 1943, Stanier proposed that the genus be changed to

 

Aeromonas. The first description of the morphological, cultural, bio-
chemical and pathogenic properties of Aeromonas hydrophila were described
by Russel in 1898. He confirmed much of the findings of Sanarelli and
Trambusti. He found the organism produced two potent toxins, one of
these resembling digitalis in its action and the other veratrin. Eddy in
1960 suggested revisions for the genus Aeromonas. He proposed combining
the species Aeromonas liquefaciens, Aeromonas hydrophila and Aeromonas
punctata into a single group, Aeromonas liquefaciens. Those organisms
which are polarly flagellated, methyl red positive, Voges-Proskauer nega-
tive and anaerogenic would be called Aeromonas formicans. He also

recommended that the name Aeromonas salmonicida be retained for the

 

non-motile, Voges-Proskauer negative and psycrophilic Aeromonads.

Ewing, Hugh and Johnson proposed a reorganization of the genus Aeromonas
that differed from Eddy (61). They proposed that the genus contain three
species: Aeromonas salmonicida, Aeromonas shigelloides, and Aeromonas

hydrophila (consisting of the genera Aeromonas liquefaciens and Aeromonas

 

 

Abydrophila). In 1962, after additional study, Eddy proposed further

 

revisions (27). He would include the species Aeromonas liquefaciens,

Aeromonas hydrophila, which he considered synonymous with Aeromonas

 

punctata, under the heading Aeromonas punctata. Aeromonas formicans
should be renamed Aeromonas caviae. Aeromonas salmonicida should be
retained.

Aeromonas sobria was first described by Popoff and Veron (71).

 

These organisms possess all the fundamental properties of the Aeromonads.
They differ from Aeromonas hydrophila in that they do not utilize
L-histidine, L-arabinose, L-arginine and salicin as a sole Carbon source.
They do not grow on KCN medium, hydrolyze esculin or ferment salicin.
These authors also proposed the retention of Aeromonas hydrophila as a
species. They believed that Aeromonas hydrophila could be divided into

two subspecies: anaerogenes and hydrophila, based on glucose fermenta-

 

tion and butanediol dehydrogenase production. Boulanger, Lallier and
Cousineau isolated Aeromonas sobria from healthy fish and confirmed the

biochemical differences described by Popoff and Veron (l3).

Aeromonas Infection in Frogs

Emerson and Norris first described the infection of frogs with

Aeromonas as "red-leg disease" for the hemorrhagic lesions found on the

abdomen and legs of diseased frogs. Infection can result from spontane-
ous or artificial infection of frogs with Aeromonas hydrophila (55).

The disease begins with a distinct sluggishness of the animal. Within a
few days, hemorrhagic areas are observed on the ventral surface of the
body. Extensive edema of the abdomen and thighs occurs several hours
before the frogs succumb to the disease. Post mortem examination reveals
edematous fluid beneath the skin of the abdomen and thighs. Small amounts
of blood tinged exudate can be found in the mouth along with small hemor-
rhagic areas on the surface of the tongue. Multiple petechial hemorrhages
are always seen on the surface of the thigh and abdominal musculature.

The peritoneal cavity of infected frogs contains hemolyzed bloody exudate
in which can be found large numbers of infecting organisms. The heart
muscle appears pale and flaccid. The blood vessels on the surface of the
stomach and intestines appear congested, while the organs appear dis-
tended. The liver is dark brown and mottled. The spleen becomes

enlarged while the kidneys show no abnormal changes. Aeromonas hydrophila
can be readily recovered from the organs, exudate and blood of infected
frogs. The portal of entry seems to be abraided skin.

Aeromonas also causes black rot in hen eggs (9). It is pathogenic
to a wide variety of fish (13). Aeromonas hydrophila has been isolated
from septicemias in snakes and salamanders (9, 19). In the case of snake
septicemias, the organism is transmitted from snake to snake by snake

mites (19).

Aeromonas Infection in Humans

In humans, Aeromonas has been associated with gastro-enteriris (6,

14, 19, 43, 57, 75) ce11u1itis (88), septicemia (1, 28, 52, 65, 73, 74, 85),

endocarditis (20), meningitis (72), wounds (45, 60, 90), and urinary
tract infections (39, 40). Aeromonads have also been isolated from the
stools of healthy carriers (3, 4, 24). Patients presenting with gastro-

intestinal disturbances caused by Aeromonas hydrophila usually have a

 

mild self limited diarrhea. Nausea, vomiting and abdominal pain have
also been observed (88). Those patients who present with septicemia
resemble septicemias caused by other gram negative organisms. These
patients usually have an underlying hematologic disorder, are immuno-
suppressed or have liver disease (1, 14, 28, 58, 65, 74, 79, 85, 87).
Septicemias caused by gram negative organisms in Laennec's cirrhosis are
due to blood invasion of intestinal bacteria that have crossed a damaged
intestinal mucosa, have bypassed the liver or have been insufficiently
filtered from the portal blood by the liver (88). It appears that the
gastrointestinal disturbances caused by Aeromonads may be due to an

enterotoxin or cytotoxin produced by the bacteria (14, 43, 57, 78).

Enterotoxins of Gram Negative Bacteria

Vibrio cholera Enterotoxin

In 1884, Koch first suggested that cholera was a toxinosis. It is
now firmly established as the prototype of the enterotoxic enteropathies.
Toxin production in 1, cholera is coded for by a chromosomal gene (87).
The enterotoxin molecule has a molecular weight of 84,000 daltons and is
composed of 2 subunits, A and B (34). Subunit A has a molecular weight
of about 28,000 daltons and is responsible for the toxic activity of
choleragen (cholera toxin). Subunit A can be further separated into two

fragments: A1 with a molecular weight of 23,000 daltons and A2 with a

molecular weight of 2,500 daltons (67). Subunit B is the portion of the
protein which binds to Gm1 ganglioside in the outer membrane of intestinal
cells and is not toxigenic (33). The proposed structure of cholera toxin
is A85 or AB6. Cholera toxin spontaneously disassociates into cholera-
genoid, an aggregation of B subunits, with a molecular weight of 68,000
daltons. It is immunogenic but not toxigenic. Cholera toxin activates
membrane bound adenylate cyclase, which converts adenosine 5' triphosphate
(ATP) to cyclic AMP. Elevated levels of cyclic AMP result in a net secre-
tion of cholride and inhibition of sodium absorption resulting in loss of
water. Cholera toxin is heat labile. Activity can be measured in

suckling mice, rabbit ileal loops, skin permeability and Y1 adrenal cells

(7, 8, 12, 34, 69).

Escherichia coli Enterotoxins

Escherichia coli that produce diarrhea were formerly divided into

 

three groups: enteropathogenic, enterotoxigenic and enteroinvasive.
Enteropathogenic E. 9911 were identified by their somatic (0) and flagellar
(H) antigens (46). During the 1940's and 1950's certain strains of

E, coli identified by serotype were isolated from the stools of infants
with diarrhea during outbreaks in hospital nurseries (44). These 14
distinct antigenic types were designated as the classical enteropathogenic
serotypes. Since the 1950's, however, it was demonstrated that these
classical serotypes were found in stool specimens from healthy infants

as often as in stools from infants with diarrhea (51). Today there is con-
siderable doubt about the importance of the enteropathogenic E, coli.

Therefore, enteropathogenic E, coli are no longer considered to be a

separate group. The current literature distinguishes 52.2211 into two
groups--enterotoxigenic and enteroinvasive.

Enterotoxigenic E, £911 produce disease by elaboration of an exo-
toxin. Some strains of g. £911 produce labile toxin (LT), stable toxin
(ST) or both (44, 56, 82, 91). Toxin production is plasmid mediated.
Stable toxin has been shown to have a molecular weight of approximately
4,400 (2, 84). Stable toxin contains a large number of cystine residues
and has few hydrophobic amino acids (2). It is stable to heating at 100°
for 30 minutes (2, 36). It does not lose biological activity after
treatment with pronase, trypsin, proteinase K, deoxyribonuclease, ribonu-
clease and phospholipase C (2, 48). Acetone, phenol, chloroform and
methanol have no effect on the molecule. It is stable to acid even at
pH 1 but loses activity at pH greater than 9. Stable toxin can be
detected in suckling mice and rabbit ileal loops (30, 36, 68). The
onset of fluid accumulation in rabbit ileal loops is rapid with maximum
volume being produced between 4-6 hours after inoculation (30). ST does
not stimulate adenylate cyclase but appears to activate increased levels
of cyclic guanosine 3', 5' monophosphate (GNP) in intestinal tissue by
activating guanylate cyclase (56).

Labile toxin has a molecular weight of 73,000 daltons (56). It
has two components with molecular weights of 44,000 and 30,000 (56).

Its activity is increased by trypsin and completely destroyed by pronase
and proteinase K (56). This toxin is labile to heat and acid but is stable
to alkaline conditions. Its mechanism of action is similar to cholera
toxin (45). LT also appears to be antigenically related to cholera

toxin (15, l6, l7). Anti-cholera toxin neutralizes the toxic activity of

10

cholera toxin as well as labile toxin jg_yjyg_(15, 16, 17). Cholera
toxin also shows immunologic relatedness with LT in immunodiffusion
tests. Anti-LT sera produce a precipitin band with both LT and CT and
neutralize both toxins. Klipstein et al. reported weak neutralization
of stable toxin with anti-LT and anti-CT (53). Labile toxin can be
assayed in rabbit ileal loops, skin permeability tests, Y.I adrenal cells,
CHO cells, and pigeon erythrocyte lysates. The suckling mouse in non-
responsive (21, 48). Onset of fluid response in rabbit ileal loops is
immediate at high doses but delayed at low doses (30). The maximum volume
is produced in no less than 10 hours. The ability of the organisms to
attach to intestinal epithelium may be a prerequisite for enteric infec-
tion. Colonization of the small intestine is followed by enterotoxin
production. Intestinal colonization is mediated by pili antigens desig-
nated as K 88 in swine (49), K 99 in cows and sheep (47), and coloniza-
tion factor antigen (CFA) in man (31).

Enteroinvasive E, 2911 have the ability to produce a dysentery
like illness similar to that caused by Shigella. Invasive E, 9911 invade
the epithelial cells of the colon. The organisms cause destruction of
the brush border and are engulfed by an envagination of the cell membrane
(70). The organisms are contained within vacuoles in epithelial cells
from which they invade and kill adjacent cells. This results in inflamma-
tion and erosion or ulceration of the epithelium with the production of

a dysentery like diseases manifested by bloody mucoid stools.

11

Salmonella Enterotoxin

Salmonella enterotoxin is less well defined. Gianella studied

 

invasive and noninvasive strains of Salmonella (37). He found that non-
invasive strains of Salmonella did not invoke a fluid response while 3

of 6 invasive strains did elicit a positive response. Whole organisms
were necessary for fluid accumulation. Sterile filtrates elicited no
response. Sedlock, Koupal and Deibel on the other hand reported activity
in suckling mice with culture filtrates (81). Sedlock and Deibel reported
that responses in rabbit ileal loops could be enhanced if prior to admin-
istration of culture supernatant the intestinal lumen is washed with a
mucolytic agent. Fluid secretion could also be observed in untreated
intestinal loops if enterotoxin was administered with a live, invasive

Salmonella strain which did not evoke a secretory response (81). Koupal

 

and Deibel found that Salmonella toxin was heat stable, pH stable,
pronase sensitive and shared some properties with E, 9211 labile and
stable toxins (54). Data from our laboratory indicate that live viable
organisms are essential for toxic activity. Culture filtrates, concen-
trates of filtrates as well as sonicates and heat inactivated culture
supernatants did not produce activity. It seems that the suckling mouse
system is not assaying for a true enterotoxin such as CT or LT with

Salmonella. Enterotoxin activity with Salmonella can be assayed in

 

rabbit ileal loops, skin permeability assays and the suckling mouse (54,
77, 80). The exact mechanism of suckling mouse reactivity with

Salmonella is not yet understood since Salmonella enterotoxin does not

 

appear to activate adenylate cyclase by the same mechanism as y, cholera

toxin (38).

12

Aeromonas Enterotoxin

Much of the early literature on Aeromonas dealt with classification
of the organism or its pathogenesis for fish and frogs (26, 27, 71, 83).
Only recently has Aeromonas been considered a possible human pathogen
with the ability to produce an enterotoxin (3, 4, 13, 43). Annapurna and
Sanyal tested 50 strains of Aeromonas hydrophila isolated from diarrheic
and healthy human feces, from domestic animals (cow, buffalo, goat and
chickens), from drinking water, sewage and the river Ganges (4). Live
cultures and cell-free filtrates of 47 and the 50 strains gave positive
responses in rabbit ileal loops. The three negative strains became posi-
tive after two passages in ileal loops of rabbits. The amount of fluid
produced was comparable to that of a toxigenic strain of Vibrio cholera.
The enterotoxin was inactivated after heating at 60°C for 20 minutes
and 65° for 10 minutes. The enterotoxin could be precipitated with
ammonium sulphate and was non-dialysable. They believed that the toxin
was protein. They also could find no histopathologic changes in ileal
loops other than the depletion of mucus from goblet cells. Dubey and
Sanyal also found that cell-free culture filtrates and crude enterotoxin
preparations caused fluid accumulation in rabtit ileal loops (24).
The activity was lost after heating at 60° for 20 minutes or 56°C for
30 minutes. The toxin was acid labile and maximum activity was produced
at a pH range of 8-10.

Boulanger, Lallier and Cousineau isolated Aeromonas sobria from
healthy fish and Aeromonas hydrophila from both healthy and moribund

fish (13). Six of the eight Aeromonas sobria strains produced one type

 

of hemolysis on 5% bovine blood agar while 12 of 13 strains of

13

Aeromonas hydrophila produced two types of hemolysis suggesting that

Aeromonas hydrophila was more hemolytic than Aeromonas sobria. They also

 

found that the Aeromonads were proteolytic and produced a cytotoxin.

All of the Aeromonas sobria isolates reacted positively in the suckling

 

mouse and rabbit ileal loops. Four of the Aeromonas hydrophila strains

 

were positive in both the suckling mouse and rabbit ileal loop while
seven strains were positive in the suckling mouse only. These studies
seemed to suggest that Aeromonas might produce two types of toxin; the
first, a toxin that produces an immediate response and is detectable in
suckling mice and the second a toxin that produces a delayed response and
is detectable in rabbit ileal loops. This would indicate that
Aeromonas enterotoxin has properties in common with both the stable and
labile toxins of E, £911, These investigators could find no antigenic
relationship between the toxins from Aeromonas and E, £911,

Sanyal, Singh and Sen compared the enterotoxicity of Aeromonas

hydrophila isolated from healthy and diarrheagenic adults and children

 

with isolates of Plesiomonas shigelloides (78). They found that, regard-

 

less of source, 12 of 14 strains of Aeromonas hydrophila produced a posi-
tive response in rabbit ileal loops while very few of the Plesiomonas
shigelloides gave a positive response.

Ljungh, Popoff and WadStrdm reported isolating an enterotoxin from
eleven human isolates of Aeromonas (57). Enterotoxin was assayed in
rabbit intestinal loops, rabbit skin and Y1 adrenal cells. These investi-
gators reported interference in all three assay systems by hemolysins and
a cytotoxic protein produced by the Aeromonads. This interference could

be inactivated by heating at 56° for 10 minutes or by a specific

14

antihemolysin. They also found that neither cholera antitoxin nor anti-
serum to E, £911 enterotoxin could neutralize the activity. The action
of Aeromonas enterotoxin may be similar to cholera toxin since both cause
the release of corticosteroids from Y1 adrenal cells. These investigators
also reported that Plesiomonas shigelloides produced only low levels of
enterotoxin.

Gurwith, Bourque, Cameron, Forrest and Green reported isolation of

Aeromonas hydrophila from a 67 year old Indian patient with a cholera-like

 

illness (43). Culture filtrates were cytotoxic in mouse adrenal cells,
HeLa cells and human foreskin fibroblasts. The toxin was heat and acid
labile had an estimated molecular weight of 45,000 daltons. When tested
in suckling mice, live cultures and sterile filtrates produced less than
positive results but death occurred in all mice in 4 hours. Intraperi-
toneal injection into adult mice caused death within 24 hours. Fluid
accumulation could be demonstrated in rabbit ileal loops.

Cumberbatch et a1. studied 96 isolates of Aeromonas hydrophila for
cytotoxin and hemolysin production (18). Sixty-six isolates were both
cytotoxic and hemolytic while the others produced neither cytotoxin nor
hemolysin. Cytotoxic activity correlated with enterotoxic activity as
well as with a positive lysine decarboxylase phenotype or a positive
Voges-Proskauer phenotype. Enterotoxic activity could not be correlated
with the presence or absence of plasmids. They also isolated in low titer
a substance that causes rounding and detachment of MAT cells and HeLa
cells. They believed that this rounding factor does not resemble the
cytotoxic enterotoxins of E, ggli_and Vibrio cholera, since these entero-

toxins have no effect on HeLa cells. These authors believe the cytotoxin

15

to be a proteolytic substance produced by the Aeromonads. Whether it
contributes to the enteropathogenicity of Aeromonas cannot be determined
at the present time.
Wadstrdm, Ljungh and Wretlind also found enterotoxin activity in
Aeromonas using rabbit ileal loops, rabbit skin tests and Y.l adrenal
cells (89). They noticed that the skin test could be affected by the
hemolytic and cytotoxic factors produced by the organism. Heating the
test samples at 56° for 10 minutes inactivated the cytotoxic and hemolytic
factors. Hemolysins also lysed the adrenal cells but heating inhibited
this activity. Addition of specific anti-hemolysins and incubation at
20° for 30 minutes also inhibited the hemolytic activity and allowed detec-
tion of enterotoxin in skin tests and adrenal cell tests. Heating of
crude enterotoxin samples at 80° for 10 minutes showed that the toxin is
heat labile like the enterotoxins of Vibrio cholera and E, 2911.
Bernheimer and Avigad have isolated an extracellular hemolytic
toxin which they have called "aerolysin" (10). They estimated its molecu-
lar weight to be 50,000 daltons. Aerolysin is heat labile and apparently
protein. Wretlind, Mdllby and Wadstrdm isolated two hemolysins from

Aeromonas hydrophila which were cytotoxic for HeLa cells and human fibro-

 

blasts and produced dermonecrosis in rabbits (92, 93). These hemolysins
were unstable to heating and were inactivated by proteolytic enzymes.
Donta and Haddow also reported cytotoxic activity for Aeromonas

hydrophila (23). Their cytotoxin also was heat labile. They could not

 

find enterotoxic activity either by rabbit ileal loop assay or Y1 adrenal

cell assay.

16

Animal and Cell Models for Detection
of Enterotoxin Production

 

 

A variety of animal and cell models have been utilized to study
enterotoxin activity (21, 29, 32, 42, 79, 82). The rabbit ileal loop
was one of the first systems utilized (30, 37, 44). With this procedure,
rabbits are fasted for 48 hours prior to use. Under local anesthetic,
the small bowel is flushed with phosphate buffered saline and ligated
segments averaging 4 to 5 centimeters in length are prepared. Each seg-
ment receives a 1.0 m1 intraluminal injection of enterotoxic material, PBS
(negative control) or cholera toxin (positive control). All samples are
injected at least in duplicate and in random sequence. After 18 hours,
the ileal sections are excised and measurements are taken of the volume
of fluid accumulated with the segment.

Another animal model that has been utilized is the suckling mouse
(50, 62, 66). Dean et a1. developed the system to detect ST production
by E, £911 (21). The mice used in the assay were 1-4 days old. He in-
jected the test material through the body wall directly into the milk—
filled stomachs of the mice. After 4 hours, the mice were sacrificed,
the intestines removed and examined for fluid accumulation. Giannella
modified the assay by suggesting the incubation time be changed to 3
hours, the growth medium for the bacteria be Casamino acids--yeast extract
broth, and that roller cultures be used to obtain better toxin produc-
tion (36). The assay was further investigated by Moon et al. who
reported optimal results when mice used in the assay were less than 6
days old (62). False negatives were produced if incubation was performed

at 37°C and older mice were used.

17

The rabbit skin permeability assay was another system developed to
detect enterotoxic activity (29, 32). Culture filtrates of enterotoxic
organisms produce erythema, induration and increased capillary perme-
ability when injected into the skin of rabbits. Injection of Evan's blue
dye several hours after the culture filtrates enhanced the reading of
the test. A rapid blanching reaction was observed with E, gglj_and

Salmonella if the dye was injected 1 hour after the filtrates. This

 

factor was heat stable in contrast to delayed permeability factor which
was heat labile (77).

_I_r1 M cell systems such as Y1 adrenal cells and Chinese hamster
ovary cells (CHO) have also been frequently used (22, 41, 43, 86).
In the Y1 assay, enterotoxic activity is detected by morphological round-
ing and steroidogenesis in the monolayer cultures. The CHO cells respond
by activation of adenylate cyclase with accumulation of cAmp and cell
elongation. CHO cells are 100 to 10,000 times more sensitive for cholera
toxin and 5 to 100 times more sensitive for LT than either the rabbit

ileal loop or the rabbit skin permeability tests.

MATERIALS AND METHODS

Bacterial Strains

Aeromonas hydrophila strains Em-56, E-3, E-16, E-14 and E-2 were

 

of environmental origins. Strain 01-7 was isolated from a diver leg

wound. Aeromonas sobria strains Cl-124 and Cl-125 were isolated from

 

blood cultures while strain 54 was of environmental origin. All strain
were kindly provided by Dr. Richard Walker of the Naval Medical Research

Command, Bethesda, MD. Escherichia coli H10407 was obtained from

 

J. W. Peterson, University of Texas, Galveston. Salmonella typhimurium
E9288 was obtained from E. V. Morse, School of Veterinary Medicine,

Purdue University, Lafayette, Indiana.

Media and Maintenance of Cultures

All cultures were maintained on agar slants consisting of 2.0%
peptone (Difco Laboratories, Detroit, Michigan) 0.5% NaCl and 2.0% agar
(32) at 4° with transfers made every 6-10 weeks. For permanent storage,
all strains were cultured overnight in 10 ml of Brain Heart Infusion
(BHI) broth (Difco Laboratories). The cultures were centrifuged at
12,100 x g for 20 minutes in a Sorvall RC-5 Superspeed Refrigerated
Centrifuge (Ivan Sorvall Inc., Norwalk, Connecticut). The bacterial cell
pellets were washed two times with 0.85% NaCl. Five m1 of glycerol
(Mallinckrodt, St. Louis, Missouri) were added and the suspension was
maintained at -20°C.

18

19

Preparation of Cultural Supernatants

Organisms were inoculated from agar slants into 5 ml of BHI broth
in 150 x 16 mm tubes. The tubes were placed in a Wheaton Roller Culture
Apparatus (Wheaton Scientific, Millville, New Jersey), set at I rev/min
and incubated at 37°C for 18-24 hours. The cultures were then centrifuged
in a Sorvall RC-5 Superspeed Refrigerated Centrifuge at 12,100 x g for
20 minutes. The resultant broth supernatants were used to inoculate

suckling mice, except where filtration is specified.

Filtration and Concentration

Culture supernatants were filtered through 0.45 um Swinnex l3 mem-
brane filters (Millipore Corporation, Bedford, Massachusetts). The fil-
trates were assayed for enterotoxic activity. Filtrates were concentrated
ten-fold with an Amicon ultrafiltration unit fitted with a PM 30 membrane
(Amicon Corporation, Lexington, Massachusetts). Retentates and filtrates

were both tested in suckling mice.

Mice

Newborn HA/ICR suckling mice (Harlan Industries, Incorporated,
Cumberland, Indiana) 3 to 5 days old were used in the suckling mouse assay.
Mice were separated from their mothers immediately before use. The

average of three mice was used to determine each experimental point.

20

Suckling Mouse Assay

 

The procedures of Dean et al. were performed with the following
modification. Each of three mice were injected intragastrically
(percutaneously with a 30% gauge needle) with either 0.1 ml of culture
supernatant, filtrate or concentrate containing two drops of 2% Evans
blue dye/ml. The mice were incubated for 2; hours at room temperature
and then sacrificed by cervical dislocation. The abdomen was opened and
the entire intestine, excluding the stomach was removed with forceps and
weighed. The ratio of gut weight to remaining carcass weight was calcu-
lated and an average value obtained for each group of 3 mice. Activity
is expressed as the ratio of intestinal weight to remaining body weight
(IW/BW). Values greater than or equal to .086 are considered positive,
values between .080 and .085 are indeterminate and values less than or
equal to .079 are negative. Each assay was performed at least in dupli-

cate utilizing three mice per assay.

Hemagglutination Assay

 

Hemagglutination tests were performed according to the methods of
Deguid et a1. (25) with the following alterations. Citrated guinea pig,
sheep or human red blood cells were washed three times in saline and made
up to a 3% suspension in saline. Alternatively, red cells were washed
and resuspended in saline containing 0.5% mannose to test for suscepti-
bility of hemagglutination to mannose. Sedimented bacterial cells from
BHI roller tubes, tryptose slants, peptone slants, 1% peptone roller

tubes and human blood agar plates were suspended in 0.25 ml saline.

21

Two drops of bacterial suspension were tested with 2 drops of red cell

suspension with or without mannose.

Column Chromatography

Following ultrafiltration, selected positive filtrates were
chromatographed on a 25 x 2.6 cm. G-lOO Sephadex column (Pharmacia Fine
Chemicals Inc., Piscataway, New Jersey) equilibrated with 0.1 N tris-HCl
buffer at pH 7.0. Chromatography was carried out at 5°C. One ml frac-
tions were collected. Protein determinations were carried out on these
fractions by the method of Lowry (59). Fractions were pooled into groups

of 10 and tested in suckling mice.

Statistics

 

The mean of IW/BW ratios i 1 standard error of the mean for each
sample tested was calculated. The student's t-test was used to evaluate

results when indicated (11).

RESULTS

Enterotoxic Activity in Culture Supernatants

Culture supernatants from all strains of Aeromonas sobria showed
positive activity in the suckling mouse (IW/BW‘3_.086). Of those, two
were of human origin (Cl-124 and C1-125 both isolated from blood cultures)
and one (strain 54) of environmental origin. Positive responses were
also seen with two strains of Aeromonas_hydrgphila (Em-56 of environmental
origin and 01-7 isolated from a diver's leg wound). Three strains of

Aeromonas hydrophila (E-16, E-14 and E-2) were negative (IW/BW.§_.O79).

 

All three were of environmental origin. Strain E+3 of Aeromonas

hydrophila was indeterminate in the suckling mouse. All the Aeromonads

 

were found to be motile indicating that motility does not seem to corre-

late with enterotoxic activity (Table 2).

Mannose Sensitive Hemagglutinating Activity
of Aeromonas Organisms
Organisms were grown in various media (BHI broth, tryptose slants,
1% peptone broth and slants, and on human blood agar plates) and tested
for hemagglutinating activity with guinea pig red cells, sheep red cells
and human 0 red cells with and without mannose. Only strains 54, E-16

and E-l4 possessed mannose sensitive hemagglutinating activity with

22

23

Table l. Aeromonas Species and Source of Isolation

 

 

 

 

 

 

 

 

 

 

Number Species Source
Cl-124 Aeromonas sobria Blood culture
Em-56 Aeromonas hydrophila Environmental
Cl-125 Aeromonas sobria Blood culture

54 Aeromonas sobria Environmental
Cl-7 Aeromonas hydrophila Diver's leg wound
E-3 Aeromonas hydrophila Environmental
E-l6 Aeromonas hydrophila Environmental
E-l4 Aeromonas hydrophila Environmental

 

E-2 Aeromonas hydrophila Environmental

 

 

24

Table 2. Production of Enterotoxin-like Activity Assayed by the
Suckling Mouse Model in BHI Roller Tube Culture Supernatants
of Aeromonasa

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Number IW/BW Ratio Organism Motility
C1-124 .103 i .008 Aeromonas sobria +
9
Em-56 .102 i 2008 Aeromonas hydrophila +
11
Cl-125 .097 i .004 Aeromonas sobria +
(11)
54 .090 i .002 Aeromonas sobria +
(10)
Cl-7 .086 i .003 Aeromonas hydrophila +
9
E-3 .085 i .002 Aeromonas hydrophila +
8
E-l6 .079 i .005 Aeromonas hydrophila +
9 .
E-l4 .077 i .00? Aeromonas hydrophila +
8
E-2 .074 i .003 Aeromonas hydrophila +
6
E-9288 .105 i .004 Salmonella typhimurium ND
9
H10407 .130 i .015 Escherichia coli ND
6
BHI broth .075 i .003
(7)

 

aFigures represent mean i 1 standard error of the mean.
Numbers in parentheses indicate the number of separate tests performed.

25

guinea pig red cells (Table 3). Growth on slants or in roller tube cul-

tures did not affect the hemagglutinating ability of the strains.

Enterotoxic Response in Suckling Mice with
Aeromonas spp. Following Filter
Sterilization andTUltrafiltration

To test for the presence of a cell-free exotoxin, culture super-
natants were filter sterilized through a 0.45 micron milipore filter.
Results in Table 4 show that enterotoxic activity was not lost by passage
through a filter. The five positive strains of Aeromonas remained posi-
tive, the three negative strains remained negative and the indeterminate
strain remained indeterminate.

The possibility that small amounts of enterotoxin were being pro-
duced by the negative strains was also investigated. Culture filtrates
were concentrated ten-fold by ultrafiltration with a PM 30 membrane.

The retentates and filtrates from the Amicon were tested in suckling
mice. Results in Table 5 show that ten-fold concentration did not
increase suckling mouse reactivity.

When the positive strains were concentrated ten-fold, enterotoxic
activity could only be found in the retentate. This suggests that the
toxic molecule is of 30,000 or greater molecular weight. Uninoculated

broth concentrated ten-fold was negative.

Enterotoxic Activity Following_Heating of
Culture Filtrates

 

Culture filtrates of Aeromonas sobria (Cl-124, Cl-125 and 54) and

 

Aeromonas hydrophila (Em-56) were heated in a 60° waterbath for 10, 15,

 

26

Table 3. Hemagglutinating Ability of Aeromonas Isolates

 

 

 

Guinea Pig Sheep and
Number RBC's Human RBC's Enterotoxicity
C1-124 BHI broth a - - +
other media - - ND
Em-56 BHI broth - - +
other media - - ND
Cl-125 BHI broth - - +
other media - - ND
53" BHI broth + - +
other media + - ND
C1-7 BHI broth - - +
other media - - ND
E;§_ BHI broth - - indeterm
other media - - ND
E-16b BHI broth + - -
other media + - ND
E-l b BHI broth + - -
other media + - ND

 

aOther media include BHI broth, tryptose slants, human blood agar plates,
1% peptone broth and slants.

bCultures were positive when tested with guinea pig red cells and grown
on all media except 1% peptone broth and slants.

27

Table 4. Effect of Filter Sterilization on the Enterotoxic Activity of

 

 

 

Aeromonasa

Unfiltered IW/BW Filtered IW/BW
Number Ratio Ratio
01-124 .103 i .008 (9) .104 i .004 (7)
Em-56 .102 i .008 (11) .096 i .008 (8)
C1-125 .097 i .004 (11) .103 i .006 (7)
54 .090 i .002 (10) .091 i .005 (5)
C1-7 .086 i .003 (9) .084 i .002 (4)
E-3 .085 i .002 (8) .083 i .002 (3)
E-16 .079 i .005 (9) .075 i .004 (3)
E-14 .077 i .005 (8) .080 i .007 (3)
E-2 .074 i .003 (6) .073 i .001 (5)
E-9288 .105 i .004 (9) .076 i .007 (6)
(Salmonella)

 

aFigures represent mean i 1 standard error of the mean.
Numbers in parentheses indicate the number of separate tests performed.

28

 

 

 

 

Table 5. Reactivity of Filtrates Concentrated Ten-fold by Ultrafiltra-
tion with a PM 30 Membrane Filter
IW/BWa
Culture Filtrate

Number Supernatant Filtrate 10X Concentrate (Amicon)
Cl-124 .103 i .008 .104 i .004 .092 i .005 .078 1 .002

(9) (7) (5) (3)
Em-56 .102 i .008 .096 i .008 .102 i .004 .082 i .002

(ll) (8) (2) (3)
Cl-125 .097 i .004 .103 i .006 .094 i .002 .076 i .001

(ll) (7) (3) (3)
54 .090 i .002 .091 i .005 .092 t .010 .073 i .004

(10) (5) (3) (3)
C1-7 .086 i .003 .084 i .002 .086 i .000 .078 i .002

(9) (4) (3) (3)
E-3 .085 i .002 .083 i .002 .080 i .003 .079 i .001

(8) (3) (3) (3)
E-16 .079 i .005 .075 i .004 .079 i .003 .077.i .004

(9) (3) (3) (3)
E-l4 .077 i .005 .080 i .007 .077 i .001 .079 i .001

(8) (3) (3) (2)
E-2 .074 i .003 .073 i .001 .074 i .002 .075 i .003

(6) (5) (3) (3)
Broth .075 i .003 - .084 i .003 -

(7) (3)

 

aFigures represent mean i 1 standard error of the mean.

Numbers in parentheses indicate the number of separate tests performed.

29

and 20 minutes respectively, cooled to room temperature and injected

into suckling mice. Table 6 shows a gradual loss of enterotoxic activity
with prolonged heat treatment. E, £911 enterotoxin treated in a similar
fashion did not show a loss of activity. Heating for 20 minutes or longer

destroys all enterotoxic activity in Aeromonads.

Passage of Concentrate Through G-lOO
Sephadex Column

 

Culture filtrates of strains Em-56, Cl-124 and broth were concen-
trated ten-fold with an Amicon PM 30. TWO mls of this concentrate was
layered onto a Sephadex G-100 column. One ml fractions were collected.
These fractions were tested for protein content by the method of Lowry
and then pooled in groups of 10 and tested in suckling mice. Figures 1,
2, and 3 represent the graphs of protein concentration of each of the
fractions. Enterotoxic activity is represented by bars. The greatest
protein concentration can be found in tubes 40-50 of both the broth and
culture fractions. Enterotoxic activity can be seen in pooled fractions
11-19 and 20—29 for the Aeromonas strains. No enterotoxic activity could
be detected in the fractions of sterile broth. The large concentration
of protein in tubes 40-50 can best be explained as unused constituents

of the BHI broth.

30

Table 6. Effect of Heating at 60°C on Enterotoxic Activity of Aeromonas

 

 

 

IW/BWa
culture

Number Supernatant 60° - 10 min 60° - 15 min 60° - 20 min
Cl-124 .103 i .008 .090 i .003 .004 i .002 .078 i .003
(9) (3) (3) (4)

Em-56 .102 i .008 .089 i .006 .089 i .003 .076 i .000
(ll) (3) (2) (5)

C1-125 .097 i .004 .094 i .000 .084 i .001 .075 i .003
(11) (2) (2) (5)

54 .090 i .002 .090 i .004 .079 i .005 .076 i .002
(10) (2) (3) (5)

 

aFigures represent mean i 1 standard error of the mean.
Numbers in parentheses indicate the number of separate tests performed.

31

Figure l. Enterotoxin activity (bars) and protein concentration
(line) of filtrates from Aeromonas strain Em-56 follow-
ing Sephadex G-100 chromatography. Numbers in parenthe-
ses indicate the number of separate tests performed.

32

Enterotoxic Activity (IW/BW)

_ mcampu

consaz mash

 

.ou mo om .mm om .me cc .mm on .mm om .mH .oH
No.0.
«o.¢
mo.o
ANV A
. - . NV
Hoe + mac . - . Amy -
coo + bee floo.+ mac.
mo. mo: mm: Amy .
ooo.+ mac. Lev
.eLooou=_ moo.“ omo. Amv -
won aoo.+mmo.
won
oH.o

 

 

 

 

 

 

  

   

 

 

     

 

 

 

 

 

 

 

~.H

¢.H

o.H

m.H

o.N

~.~

milligrams protein

33

Figure 2. Enterotoxin activity (bars) and protein concentration
(line) of filtrates from Aeromonas strain Cl-124
following Sephadex G-100 chromatography. Number in
parentheses indicate the number of separate tests
performed.

34
Enterotoxic Activity (IW/BW)

. N mcamwm

consaz mash

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

on mm on mm om me oe mm on mm om ma 0H m
P H b) b D b n
No.04
eo.o.
no.0,
amv
.. .. . E -
v00 + “NO A¢V g. +ONO Amv I .
mo.o. o: Hoe. M was. .(V mm: coo + Hmo Aev u . u w.H
.5885 [Snag - 89.38.
. moo. + awo. won
t Jcnr 1 o N
3.0l . N.~

 

 

milligrams protein

35

Figure 3. Enterotoxin activity (bars) and protein concentration
(line) of concentrated BHI broth following Sephadex
G-100 chromatography. Numbers in parentheses indicate
the number of separate tests performed.

36
Enterotoxic Activity (IW/BW)

m mczmwm

Longsz mash

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2 8 8 mm 8 3 S .mm on .3 8 .3 .3 .m
a N.
/
8.? :.
two
8.? .m.
I 00H
8.? . N;
“WW 1 ~50. Amv A v F v
.+ I . m
Hoo +~No hmv - m - . . - . .
goo. + 45o. Coo. +¢~o woo + See Amv , a H
mm: amqll .
mm: mm: mm: moo.+-o._
8.? .\ 3.. e. 1.: b4
4
T a;
3.? . .om
r

w.~

milligrams protein

DISCUSSION

Many organisms previously considered to be nonpathogenic for humans
have recently been implicated in diarrhea outbreaks. These same organ-
isms cause fluid accumulation in the rabbit ileal loops (8, 43). Among
these, Aeromonas spp. have been associated with diarrhea and many investi-
gators have studied the enterotoxic potential of organisms in this genus
(3, 4, 43). Several studies have examined the enterotoxic activity of
Aeromonas in rabbit ileal loops, the rabbit skin test, the adrenal cell
assay and the suckling mouse (3, 4, 13, 24, 43, 71). In this study, 5 of
the 9 strains obtained from researchers at the Naval Medical Research
Institute were shown to be enterotoxic in the suckling mouse assay while
1 was indeterminate and 3 were negative. All of the strains isolated
from humans were positive.

True enterotoxins are exotoxins which are released into the culture
medium during growth of an organism. Cell-free preparations of cultures
of E, 9911 and E. cholera can cause fluid accumulation in both rabbit
ileal loops and suckling mice (7, 8, 21, 64). Salmonella, according to
Gianella, only induce a positive response when whole organisms are
present (37). Yet, Sedlock, Koupal and Deibel report activity in
suckling mice with culture filtrates (81). Annapurna, Sanyal and other
investigators have reported positive responses in suckling mice and
rabbit ileal loop assays with cell-free preparations of Aeromonas cul-

tures (3, 4, 24, 57, 79). In this study, culture supernatants which were

37

38

filter sterilized by passage through a 0.45 milipore filter still
retained the ability to induce a positive response in suckling mice.
It appears, therefore, that the Aeromonas enterotoxin, like the E. oli

and Virbrio cholera enterotoxin, is a cell-free exotoxin, released into

 

the medium by the growing organism.

Since some investigators found that toxic material can be retained
by a PM 30 membrane, positive strains were concentrated in the Amicon
unit (77, 81). In this study, only the retentates from the PM 30 showed
positive activity. Filtrates from the PM 30 were negative in suckling
mice. This would indicate that the toxin has a molecular weight of
30,000 or greater. Since the Aeromonads showing a negative or indetermin-
ate response might be producing toxin at a low level, attempts were also
made to concentrate the filtrates with an Amicon PM 30 ultrafiltration
unit. After ten-fold concentration, no increase in suckling mouse
reactivity was found.

Annapurna and Sanyal have reported loss of enterotoxic activity in
rabbit ileal loops of Aeromonas culture filtrates after heating (3, 4).
It was desired to know if this phenomenon could also be seen in the
suckling mouse assay. A gradual loss of enterotoxic activity could be
seen with prolonged heat treatment. No enterotoxic activity remained
after heating culture filtrates for 20 minutes at 60°C. This indicates
that Aeromonas enterotoxin may be similar to E. 9911 labile toxin and
Vibrio cholera enterotoxin.

Since it was believed that the enterotoxic molecule may be protein
in nature, column chromatography was performed with concominant protein

analysis of the resulting fractions. Although individual peaks of

39

protein could not be isolated (possibly due to the protein rich BHI broth
used for growth of the Aeromonads), enterotoxic activity could be found
only in 2 pools of the fractions. Since these pools were eluted very
early from the column, it can be postUlated that the toxin is a relatively
large molecule with a molecular weight ranging from 30,000 to 150,000
daltons. It is not clear at this point whether the protein present in
these fractions is due to the enterotoxin or to constituents of the growth
media.

For enterotoxigenic organisms to be pathogenic, the organisms must
first be able to adhere to the intestinal mucosa. Many investigators
have studied properties of the organisms that might be responsible for
adherence. With E, cholera, adhesion has been associated with motility

(35). Studies performed with the Aeromonads (see Table 2) showed that

 

all the isolates, including those strains negative in the suckling mouse,
were motile indicating that the same mechanism might not be operating
here. In enterotoxigenic E. 9911, adherence is determined by the
presence of K antigens in animals and colonization factor antigens in
humans. These antigens were non-flagellar, surface appendages believed
to be pili. If these antigens are lost, the organisms lose their ability
to produce disease even though they still retain their enterotoxi-
genicity (61). Coliform bacteria also have the ability to agglutinate
red blood cells and this property has been attributed to the presence of
pili (76). Hemagglutination attributed to Type 1 pili can be inhibited
by low concentrations of mannose (5). Studies were undertaken to deter-
mine if piliation, the ability to attach to buccal epithelial cells or

the hemagglutinating ability of the Aeromonads had any bearing on

 

40

enterotoxic activity and disease production. Transmission electron
microscopy studies performed at the Naval Medical Research Institute

showed that all of the Aeromonads possessed pili. The Aeromonads also

 

showed variability in regard to attachment to buccal epithelial cells

(see Appendix 1). From these studies, no correlation could be established
for these parameters with enterotoxic activity or disease production.
Hemagglutination studies performed in our laboratory with guinea pig red
cells showed that three strains possessed mannose sensitive hemagglutin-
ating pili, but that only one of these strains was enterotoxic. Since some
investigators believe that pili production is enhanced with the use of
enriched media (human blood agar, peptone broth, tryptose agar), further
agglutination studies were performed. The bacteria were grown in BHI
broth, 1% peptone slants and broth, tryptose slants and on human blood
agar plates. In addition, human group 0 red cells and sheep red blood
cells both with and without mannose were employed. Variations in temper-
ature were also tested (data not shown). In each case, only the 3

strains that showed hemagglutinating ability with the guinea pig red

cells showed hemagglutinating ability under variations in growth condi-
tions. Agglutination was seen only with guinea pig red cells. Sheep

and human 0 cells did not agglutinate. Possession of hemagglutinating
pili appears not to be correlated with enterotoxin production.

Some investigators report a correlation of enterotoxic activity
with the production of a cytotoxin (18, 89). In this study, all strains
that produced an enterotoxin were also cytotoxic for Y1 adrenal cells
(see Appendix 1). It is not clear at the present time if this represents
the same toxic activity. Suckling mouse reactivity was also correlated

with a lower LD50 for adult mice.

SUMMARY

Aeromonads, like Vibrios, cause little change in the gastric

 

epithelium when producing disease indicating that a toxin is responsible
for disease production. From these studies, it can be concluded that

Aeromonads have the ability to produce an enterotoxin. The toxin, like

 

1, cholera toxin, is filterable and heat labile. Both toxins can be
assayed in the same animal models. Like cholera toxin, Aeromonas toxin
induces steroidogenesis in Y1 adrenal cells. Enterotoxic activity in

Aeromonads is correlated with cytotoxic activity and a low L050.

 

Motility, the possession of hemagglutinating pili and the ability to
adhere to buccal epithelial cells do not seem to play a role in entero-
toxic potential. Cholera toxin is a large molecular weight protein.
Aeromonas toxin also appears to be of a relatively large molecular weight

(30,000-150,000 daltons). The exact chemical composition is not known.

41

10.

11.

BIBLIOGRAPHY

. Abrams, E., C. H. Zierdt and J. A. Brown. 1971. Observations on

Aeromonas h dro hila Septicaemia in a Patient with Leukemia.
J. Clinical Pathology. 24: 491-492.

. Alderete, J. F. and D. C. Robertson. 1978. Purification and

Chemical Characterization of the Heat-Stable Enterotoxin Produced
by Porcine Strains of Enterotoxigenic Escherichia coli. Infect.
Immun. 19: 1021-1030.

 

. Annapurna, E., and S. C. Sanyal. 1975. Studies on the Enteropatho-

genicity of Aeromonas hydrophila in an Experimental Model.
Ind. Jour. Prev. Soc. Med. 6: 234-237.

 

. Annapurna, E., and S. C. Sanyal. 1977. Enterotoxicity of

Aeromonas hydrophila. J. Med. Micro. 10: 317-323.

 

. Atkinson, H. M., and T. J. Trust. 1980. Hemagglutination Properties

and Adherence Ability of Aeromonas hydrophila. Infect. Inmun.
27: 938-946.

. Baman, S. I. 1980. Aeromonas hydrophila as the Etiologic Agent in

Severe Gastroenteritis: Report of a Case. Amer. Jour. of Med.
Tech. 43: 179-181.

. Banwell, J. G., and H. Sherr M.D. 1973. Effect of Bacterial Entero-

toxins on the Gastrointestinal Tract. Gastroenterology. 65:
467-497.

. Baselski, V., R. Briggs, and C. Parker. 1977. Intestinal Fluid

Accumulation Induced by Oral Challenge with Vibrio cholera or
Cholera Toxin in Infant Mice. Infect. Immun. l5: 704-712.

. Bergey's Manual of Determinative Bacteriology, 7th Edition.

The Williams and Wilkins Co., Baltimore, N.Y., 1957.

Bernheimer, A. W. and L. S. Avigad. 1974. Partial Characterization
of Aerolysin, a Lytic Exotoxin from Aeromonas hydrophila.
Infect. Immun. 9: 1016-1021.

Bhattachanjya, G. K., and R. A. Jackson. 1977. Inferences About

a Population, pp. 233-243. In: Statistical Concepts and Methods.
New York: John Wiley and Sons, Inc.

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APPENDIX

Further tests on the strains performed by researchers at the Naval
Medical Research Institute yielded the following data. Piliation studies,
performed with transmission electron microscopy, showed that all the
strains possessed pili with densities from +/- to 4+. Cytotoxicity
studies done using Y1 adrenal cells indicate that 6 of the 9 isolates pro-
duced a cytotoxin. Attachment studies of Aeromonads to buccal epithelial
cells produced variable results. LD50 studies performed in adult mice
showed great variation among the strains. Suckling mouse assays performed
in our laboratory detected enterotoxic activity with the strains of
Aeromonas which were cytotoxic and had a low LD50 for adult mice
(Appendix 1). Like the enterotoxic activity, cytotoxic activity was heat
labile (data not shown). Apparently, piliation and attachment do not

seem to play a role in enterotoxin production. The exact role of cyto-

toxic potential with regard to enterotoxicity has not been established.

50

51

Appendix 1. Comparison of Enterotoxic Activity with Other Properties of
the Genus Aeromonas

 

 

 

Enterotoxic b b Pilia-
Number Responsea Cytotox. LD50 Attachment t10n
01-124 positive + 3.7 5.1 3+
Em-56 positive + 6.5 1.6 1+
C1-125 positive + 6.6 7.2 2+
54 positive + 4.5 1.2 1+
Cl-7 positive + 4.5 10.2 4+
E-3 indeterminate + 5.4 4.1 4+
E-16 negative - 8.2 1.2 +/-
E-14 negative - 7.8 1.1 1+
E-2 negative - 8.4 4.5 1+

 

aData obtained at Michigan State University, East Lansing, Michigan.

bData obtained at the Naval Medical Research Institute, Bethesda, Md.

 
   

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