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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 89, Num. 2, 1994, pp. 209-210
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Mem. Inst. Oswaldo Cruz, Rio de Janeiro, Vol. 89(2):
209-210, apr./jun. 1994
RESEARCH NOTE
Conditions for the Production and Detection of Aeromonas
Enterotoxins
Mauro S Neves, Marly P Nunes (*)
Code Number:OC94042
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Departamento de Higiene Social e Analises Clinicas, Faculdade de
Farmacia * Departamento de Microbiologia Medica, Instituto de
Microbiologia, CCS, UFRJ, Ilha do Fundao, 21941-590 Rio de Janeiro,
RJ, Brasil
Key words: production - Aeromonas - enterotoxins
Aeromonas species have been associated with human infections
specially those from the gastrointestinal tract. Virulence factors
such as enterotoxins have been detected in some of these species (JM
Janda 1991 Clin Microbiol Rev 4: 397-410). These
enterotoxins, heat labile or heat stable, are products that
stimulate biochemical events in the intestine leading to diarrhoea
(MM Cahill 1990 J Appl Bacteriol 69: 1-16). The suckling
mouse test modified by V Burke et al. (1981 J Med Microbiol
14: 401-408) has been used as a model for the detection of
Aeromonas enterotoxins. The purpose of this study was to
evaluate the production of enterotoxins by Aeromonas cultured
in three different media as well as to evaluate the best time to
detect the action of enterotoxins by using the suckling-mouse test.
Ten Aeromonas (five A. hydrophila, three A.
caviae, and two A. sobria) recovered from clinical and
environmental sources were cultured in TSB-YE: Tryptone Soya Broth
(Oxoid) supplemented with 0.6% (w/v) of Yeast Extract (Oxoid), as
recommended by Burke et al. (loc. cit.). Aeromonas
were cultured into 5 ml of the broth dispensed in 25 ml Erlenmeyer
flasks incubated at 37 oC for 24 hr in shaker at 100 rpm. The
supernates were obtained after centrifugation of the growth cultures
at 10,000 X g for 20 min. Supernates containing only TSB-YE served
as negative controls and A. hydrophila (CIP7614) as a
positive control. The supernates (100 ul containing a drop of a 2%
Evans Blue solution) were inoculated with syringes intragastically
into three or more suckled Swiss albino mice (2-6 days old). After
inoculation the mice were maintained at 28 oC. In attempt to
evaluate the best time for the detection of the enterotoxins action,
groups of mice were killed at intervals of 1, 2 and 3 hr
respectively. Intestines were removed from the remaining bodies and
both intestinal weights (IW) and remaining body weights (BW) were
measured for each mice group and the ratios (IW/BW) calculated. We
considered as enterotoxigenic strains the ones that originated
ratios =/> 0.08 (Burke et al. loc. cit.) Culture
supernates from strains considered as toxigenic were heated at 56 oC
for 10 min in water bath and tested in mice as described earlier to
verify the effect of temperature in the enterotoxic activity.
After the preliminary experiments, it was established that the best
time to detect the enterotoxins action was 2 hr after the
inoculation of the mice, so, we have also decided to evaluate pathe
enterotoxins production in the medium proposed by CH Pai et al.
(1978 Infect Immun 19: 908-911) used for Yersinia spp.
and in the Evans medium for Escherichia coli (DJ Jr Evans
1973 Infect Immun 8: 725-730) under the same conditions
exposed above. Bacterial positive controls for these media included
Y. enterocolitica WA, serotype 8 (NCTC10938) and E.
coli (strain TR22/4, serotype 0128a, 128c:H12) as well as
negative controls consisting of non-inoculated media. All the
bacterial positive controls produced enterotoxins in their
respective media originating ratios (IW/BW) higher than 0.10 in the
suckling-mose assay. Supernates without bacteria, acting as negative
controls, originated always ratios (IW/BW0 under 0.06).
The results of all experiments are inserted in the Table. All
Aeromonas toxigenic supernates lost their activities after
heating at 56 oC-10 min.
TABLE
Ratios (intestinal weights/remaining body weights) obtained
after inoculation of mice with Aeromonas supernates in
relation to time and culture media
===================================================================
Ratios obtained from supernates
cultured in the medium proposed
Strains Origin by Burke et al. in relation to
time (hr) (a)
--------------------------------
1 2 3
A. hydrophila (AHPA) water 0.076 0.070 0.055
A. hydrophila (AHU) urine 0.080 0.080 0.097
A. hydrophila (AH1031-2) feces 0.086 0.090 0.087
A. hydrophila (AH0712-1) feces 0.075 0.080 0,097
A. hydrophila (AHScut) cutaneous 0.060 0.066 0.077
infection
A. caviae (ACU) urine 0.070 0.060 0.055
A. caviae (ACO121 - 1) feces 0.078 0.067 0.056
A. caviae (ACO32-2) feces 0.098 0.086 0.100
A. sobria (ASO42-1) feces 0.070 0.090 0.070
A. sobria (AS4831-2) feces 0.090 0.086 0.075
---------------------------------------------------------------------
Ratios obtained in mice
inoculated 2 hr with
Strains Origin supernates cultured in
two media (a)
----------------------
CH Pai Evans
A. hydrophila (AHPA) water 0.075 0.067
A. hydrophila (AHU) urine 0.067 0.086
A. hydrophila (AH1031-2) feces 0.080 0.070
A. hydrophila (AH0712-1) feces 0.090 0.066
A. hydrophila (AHScut) cutaneous 0.070 0.078
infection
A. caviae (ACU) urine 0.067 0.064
A. caviae (ACO121 - 1) feces 0.067 0.070
A. caviae (ACO32-2) feces 0.097 0.082
A. sobria (ASO42-1) feces 0.080 0.072
A. sobria (AS4831-2) feces 0.083 0.076
=====================================================================
a: strains considered as toxigenic originated ratios =/> 0.08.
We concluded that the best time to detect the action produced by the
enterotoxins in the suckling-mouse assay was 2 hr since 60% of the
Aeromonas strains showed positivity. Burke et al. (loc.
cit.) considered possible to identify known positive and
negative Aeromonas strains correctly by as early as 2 hr when
the suckling-mouse test was made at 28 oC, however, they recommend 3
hr because they have found a better discrimination at this time. If
our suckling-mouse assays were observed only 3 hr after inoculation,
only 40% of our strains would be positive since the enterotoxins
would not be detected in the two A. sobria species (species
that were not tested by Burke et al. (loc. cit.) that showed
positivity only after 2 hr. Those investigators also observed that
enterotoxins lost their activities at 56 oC-10 min. AG Dean et al.
(1972 J Infect Dis 125: 407-411) described the suckling-mouse
test to detect E. coli heat-stable enterotoxin. In
relation to Aeromonas enterotoxins, those detected by this
assay are heat-labile, although, a second enterotoxin heat-stable
(100 oC-30 min) was discovered and detected in the rat perfusion
system (CW Houston et al. 1991 Experientia 47: 424-426).
Like in the experiments of Burke et al. (loc. cit.),
we also concluded that TSB-YE was the best medium for the production
of enterotoxins since 60% of the Aeromonas strains were
positive if compared to the other media analyzed. Our findings
clearly show that the incubation for the detection of enterotoxins
in the suckling-mouse assay proposed by Burke et al. (loc.
cit.) would give negative results specially for A. sobria
strains that showed positivity in 2 hr. Based on these results we
recommend TSB-YE for the production of Aeromonas enterotoxins
and 2 hr as the time necessary to detect them in the suckling-mouse
assay.
Acknowledgments: to Dr Maria Regina F Toledo for supplying
some Aeromonas strains (AH1031-2, AHO712-1, ACO121-1,
ACO32-2, ASO42-1 and AS4831-2).
Copyright 1994 Fundacao Oswaldo Cruz - FIOCRUZ
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