![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Applied and Environmental Microbiology, December 1999, p. 5612-5614, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Hemolytic Activity and Siderophore Production in
Different Aeromonas Species Isolated from Fish
Jesús A.
Santos,*
César J.
González,
Andrés
Otero, and
María-Luisa
García-López
Department of Food Hygiene and Food
Technology, Veterinary Faculty, University of León, E-24071
León, Spain
Received 3 June 1999/Accepted 23 September 1999
 |
ABSTRACT |
The hemolytic activity and siderophore production of several
strains of motile aeromonads were determined. The hemolytic activity of
Aeromonas caviae and Aeromonas eucrenophila was
enhanced after trypsinization of the samples. The enhancement of
hemolysis was observed in strains that carried an aerolysin-like gene,
detected by a PCR procedure. Siderophore production was demonstrated in all but one strain of Aeromonas jandaei. No apparent
relationship was observed between the presence of plasmid DNA and
hemolysis or siderophore production.
| |
TEXT |
Members of the genus
Aeromonas have long been considered to be human pathogens.
They are implicated in a number of diarrheal and extraintestinal
infections, including septicemia, wound infections, burn-associated
sepsis, and respiratory tract infections (10, 11). However,
the ultimate mechanisms of pathogenicity are not clearly understood,
and several virulence factors have been proposed. Of these factors,
hemolytic molecules seem to be related to enterotoxigenicity. Aerolysin
(8) is the best-studied hemolysin, but Aeromonas strains can produce more than one hemolytic toxin with virulence properties (4, 22). In addition, production of siderophores has been reported for different species of Aeromonas
(1, 23) and might be related to the virulence properties.
Of the 14 species of Aeromonas currently described, only
five (A. hydrophila, A. veronii, A. caviae, A. jandaei, and A. schubertii) are
recognized as human pathogens, but other species, like A. trota and A. eucrenophila, caused fluid accumulation in
the rabbit ileal loop test after serial passages, indicating a
pathogenic potential (19, 20), and it has been demonstrated
that at least one strain of A. trota produces aerolysin
(12).
For the present article, we investigated the hemolytic activity of
strains of different species of Aeromonas isolated from freshwater fishes and the presence in them of the aerolysin gene (by
PCR), as well as the production of siderophores.
Bacterial strains.
Three strains of Aeromonas
caviae (AC 15, AC 16, and AC 17), two of A. eucrenophila (AE 1 and AE 2), one of A. jandaei (AJ 1),
and one of A. hydrophila (AH 154) were used throughout this work. The strains were isolated from samples of freshwater fish (pike,
[Esox lucius], brown trout [Salmo trutta],
and rainbow trout [Oncorhyncus mykiss]) and identified to
the species level by biochemical methods (5). Working
cultures of the microorganisms were kept on Trypticase soy agar (Oxoid)
slants at room temperature and routinely cultivated in Trypticase soy
broth (TSB) (Oxoid) at 28°C.
Determination of hemolytic activity.
The strains were tested
for beta-hemolytic activity on Columbia agar base (Oxoid) supplemented
with either 5% rabbit erythrocytes or 5% sheep erythrocytes with
incubation at 28°C for 48 h.
All of the strains were beta-hemolytic when assayed on rabbit blood
agar plates, and strains AC 17, AE 1, AE 2, and AH 154 were also
beta-hemolytic on sheep blood agar plates. However, strains AC 15 (A. caviae) and AJ 1 (A. jandaei) were
nonhemolytic, and strain AC 16 (A. caviae) was
alpha-hemolytic on sheep blood agar plates.
For quantification of hemolysin, bacteria were cultured in TSB at
28°C for 18 h, cells were pelleted by centrifugation at 10,000 × g for 10 min, and 500 µl of the supernatant
(filtered through 0.22-µm-pore-diameter filters) was added to 1 ml of
a 1% suspension of washed sheep or rabbit erythrocytes in
phosphate-buffered saline (PBS; Oxoid). For activation of hemolysin
precursor, the supernatants were treated with 50 µg of trypsin for 15 min at 37°C before adding the erythrocyte suspension. Both
nonactivated and activated reaction mixtures were incubated at 37°C
for 60 min and briefly centrifuged to remove unlysed erythrocytes, and A545 was measured to determine erythrocyte
lysis. Blanks were prepared by mixing the erythrocyte suspension with
sterile TSB (treated similarly to the samples), and the absorbance
corresponding to 100% of hemolysis was determined by adding a small
amount of sodium dodecyl sulfate to blank tubes.
The quantification of the extracellular hemolytic activity (Fig.
1) indicated that rabbit erythrocytes are
more sensitive to the action of the hemolysin(s). Apart from the strain
of A. hydrophila (AH 154), none of the strains showed an
important extracellular hemolytic activity towards sheep erythrocytes,
but treatment of the supernatants with trypsin resulted in significant
increments of this activity in two strains of A. caviae (AC
15 and AC 16) and in both A. eucrenophila strains, being
particularly important in AE 1 (Fig. 1A).
View larger version (40K):
[in this window]
[in a new window]
|
FIG. 1.
Extracellular hemolytic activity of the strains of
Aeromonas towards sheep (A) and rabbit (B) erythrocytes.
 , untreated samples;  , trypsin-treated samples.
|
|
The extracellular activity against rabbit erythrocytes was stronger,
and again, trypsin activation significantly increased the activity of
strains AC 17 and AJ 1 (Fig. 1B).
The production of hemolytic toxins has been regarded as strong evidence
of pathogenic potential in aeromonads. However, nonhemolytic aeromonads
have been implicated as human pathogens (16). Regarding this, the routine use of sheep blood agar appears to be inappropriate, considering that sheep erythrocytes are less sensitive than
erythrocytes from other mammals (3, 13). In our study, we
observed stronger activity against rabbit blood, both on agar plates
and in quantification assays.
Production of hemolysin as a precursor activatable by trypsin has been
reported in several strains of Aeromonas (8, 13). In our case, trypsinization enhanced hemolytic activity in some of the
strains tested (even though all of the strains were highly proteolytic)
(5), especially when less-sensitive sheep erythrocytes were
used. The hemolytic activity present before trypsinization in some
strains might be due to hemolysin(s) other than aerolysin. In a recent
report, Wong et al. (22) demonstrated the presence of two
unrelated hemolytic genes in one strain of A. hydrophila, both involved in virulence, but not the only genes responsible for pathogenesis.
PCR detection of aerolysin genes.
DNA was isolated from the
strains of Aeromonas by a common procedure (14).
The published sequences of aerolysin-like genes of several
Aeromonas species (7-9, 12, 21) were screened for conserved regions, and a pair of oligonucleotide primers were synthesized. A forward primer, 5'-GC(A/T) GA(A/G) CCC (A/G)TC TAT
CC(A/T) G (positions 69 to 87 of the aerolysin gene
[8]), was designed to target a conserved region in all
of the available aerolysin sequences. The reverse primer 5'-TTT CTC CGG
TAA CAG GAT TG (positions 321 to 302) was the Aero1b primer described by Pollard et al. (17).
PCR was performed on a Mastercycler Personal apparatus (Eppendorf).
Initial denaturation was done at 94°C for 3 min followed by 30 cycles
with denaturation at 92°C for 30 s, annealing at 55°C for
30 s, and extension at 72°C for 1 min. A final extension step
was done at 72°C for 1.5 min. Aliquots from each reaction were
analyzed by 1% agarose gel electrophoresis and viewed, after ethidium
bromide staining, under UV light.
An amplification product of the expected size (252 bp) was observed
with DNA template from strains AC 15, AC 16, AE 1, and AH 154 (Fig.
2). The amplification of the aerolysin
gene was observed in strains that also showed enhancement of the
hemolytic activity by trypsinization, suggesting that some A. caviae and A. eucrenophila strains can produce an
aerolysin-like gene activable under certain conditions.
View larger version (78K):
[in this window]
[in a new window]
|
FIG. 2.
Detection of the aerolysin gene by PCR in different
species of Aeromonas. Lanes: M, 123-bp ladder; 1, AC 15; 2, AC 16; 3, AC 17; 4, AE 1; 5, AE 2; 6, AJ 1; 7, AH 154.
|
|
Siderophore detection.
The chrome azurol S siderophore
detection agar (CAS agar) (18), prepared as described by
Barghouthi et al. (1), was used to detect siderophore
production. Single colonies of each isolate were transferred to this
medium, and the plates were incubated at 30°C for 48 h. The
presence of an orange halo around the colonies was recorded as positive
for siderophore production. Production of siderophore was detected in
all but one isolate. Only one A. jandaei strain (AJ 1)
showed no orange halo on CAS agar plates.
The production of siderophore for the isolates of Aeromonas
is not remarkable, because it is a well-documented trait for this genus. More interesting is the lack of siderophore activity by a strain
of A. jandaei, a species previously reported as siderophore producing (23). Naidu and Yadav (15) found that
environmental isolates of A. hydrophila were less efficient
than clinical strains for production of siderophores.
Plasmid analysis and curing.
The strains of
Aeromonas were cultured in TSB at 28°C for 14 to 16 h, and plasmid DNA was isolated by the procedure of Birnboim and Doly
(2). Plasmids were separated by electrophoresis in 0.8%
agarose gels, stained with ethidium bromide, and visualized under UV light.
Two strains of A. caviae (AC 15 and AC 16) carried several
plasmids, whereas no plasmid DNA was isolated from the remaining strains of Aeromonas under the conditions used in this
study. The strains AC 15 and AC 16 were selected for plasmid curing
experiments. The strains were cultured in TSB with ethidium bromide (10 to 300 µg/ml) at 28°C for 48 h, and the cultures were diluted
and plated onto Trypticase soy agar. Some colonies were picked from the
plates, and plasmid DNA was isolated and analyzed as described above.
Hemolytic activity and siderophore production of cured strains were
tested in agar plates as described above. No phenotypic changes in
hemolytic activity or siderophore production were observed in the cured strains.
Hanes and Chandler (6) found that an R plasmid of A. hydrophila regulated attachment and hemolytic activities and a
cured strain showed greater activities than the parental strain. In our
experiments, no differences were found in hemolytic activities and
siderophore production in A. caviae, indicating that these activities are not directly regulated by plasmid DNA.
In conclusion, the presence of an aerolysin-related gene was
demonstrated by PCR in some strains of A. caviae and
A. eucrenophila of environmental origin. The presence of
this gene is related to the hemolysis of sheep erythrocytes after
activation of the hemolytic protein with trypsin. We are conducting
further studies to elucidate the nature of the hemolytic activities
found in our strains.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the Spanish CICYT (Project
ALI 97-0601).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Higiene y Tecnología de los Alimentos, Facultad de Veterinaria,
Universidad de León, E-24071 León, Spain. Phone:
34/987/291286. Fax: 34/987/291284. E-mail:
dhtjsb{at}unileon.es.
| |
REFERENCES |
| 1.
|
Barghouthi, S.,
R. Young,
M. O. J. Olson,
J. E. L. Arceneaux,
L. W. Clem, and B. R. Byers.
1989.
Amonabactin, a novel tryptophan- or phenylalanine-containing phenolate siderophore in Aeromonas hydrophila.
J. Bacteriol.
171:1811-1816[Abstract/Free Full Text].
|
| 2.
|
Birnboim, H. C., and J. Doly.
1979.
A rapid alkaline extraction procedure for screening recombinant plasmid DNA.
Nucleic Acids Res.
7:1513-1523[Abstract/Free Full Text].
|
| 3.
|
Brenden, R., and J. M. Janda.
1987.
Detection, quantitation and stability of the beta hemolysin of Aeromonas spp.
J. Med. Microbiol.
24:247-251[Abstract].
|
| 4.
|
Chopra, A. K.,
C. W. Houston, and A. Kurosky.
1991.
Genetic variation in related cytolytic toxins produced by different species of Aeromonas.
FEMS Microbiol. Lett.
78:231-237.
|
| 5.
|
González-Serrano, C. J.
1996.
Ph.D. thesis.
Universidad de León, Leon, Spain
|
| 6.
|
Hanes, D. E., and D. K. F. Chandler.
1993.
The role of a 40-megadaltons plasmid in the adherence and hemolytic properties of Aeromonas hydrophila.
Microb. Pathog.
15:313-317[Medline].
|
| 7.
|
Hirono, I.,
T. Aoki,
T. Asao, and S. Kozaki.
1992.
Nucleotide sequences and characterization of haemolysin genes from Aeromonas hydrophila and Aeromonas sobria.
Microb. Pathog.
13:433-446[Medline].
|
| 8.
|
Howard, S. P.,
W. J. Garland,
M. J. Green, and J. T. Buckley.
1987.
Nucleotide sequence of the gene for the hole-forming toxin aerolysin of Aeromonas hydrophila.
J. Bacteriol.
169:2869-2871[Abstract/Free Full Text].
|
| 9.
|
Husslein, V.,
B. Huhle,
T. Jarchau,
R. Lurz,
W. Goebel, and T. Chakraborty.
1988.
Nucleotide sequence and transcriptional analysis of the aerC aerA region of Aeromonas sobria encoding aerolysin and its regulatory regions.
Mol. Microbiol.
2:507-517[Medline].
|
| 10.
|
Janda, J. M.
1991.
Recent advances in the study of the taxonomy, pathogenicity, and infectious syndromes associated with the genus Aeromonas.
Clin. Microbiol. Rev.
4:397-410[Abstract/Free Full Text].
|
| 11.
|
Janda, J. M., and S. L. Abbot.
1998.
Evolving concepts regarding the genus Aeromonas: an expanding panorama of species, disease presentations, and unanswered questions.
Clin. Infect. Dis.
27:332-344[Medline].
|
| 12.
|
Khan, A. A.,
E. Kim, and C. E. Cerniglia.
1998.
Molecular cloning, nucleotide sequence, and expression in Escherichia coli of a hemolytic toxin (aerolysin) gene from Aeromonas trota.
Appl. Environ. Microbiol.
64:2473-2478[Abstract/Free Full Text].
|
| 13.
|
Kozaki, S.,
T. Asao,
Y. Kamata, and G. Sakaguchi.
1989.
Characterization of Aeromonas sobria hemolysin by use of monoclonal antibodies against Aeromonas hydrophila hemolysins.
J. Clin. Microbiol.
27:1782-1786[Abstract/Free Full Text].
|
| 14.
|
Lawson, P.,
S. E. Gharbia,
H. N. Shah, and D. R. Clark.
1989.
Recognition of Fusobacterium nucleatum subgroups Fn-1, Fn-2 and Fn-3 by ribosomal RNA gene restriction patterns.
FEMS Microbiol. Lett.
53:41-45[Medline].
|
| 15.
|
Naidu, A. J., and M. Yadav.
1997.
Influence of iron, growth temperature and plasmids on siderophore production in Aeromonas hydrophila.
J. Med. Microbiol.
46:833-838[Abstract].
|
| 16.
|
Namdari, H., and E. J. Bottone.
1990.
Microbiologic evidence supporting the role of Aeromonas caviae as a pediatric enteric pathogen.
J. Clin. Microbiol.
28:837-840[Abstract/Free Full Text].
|
| 17.
|
Pollard, D. R.,
W. M. Johnson,
H. Lior,
S. D. Tyler, and K. R. Rozee.
1990.
Detection of the aerolysin gene in Aeromonas hydrophila by the polymerase chain reaction.
J. Clin. Microbiol.
28:2477-2481[Abstract/Free Full Text].
|
| 18.
|
Schwyn, B., and J. B. Neilands.
1987.
Universal chemical assay for the detection and determination of siderophores.
Anal. Biochem.
160:47-56[Medline].
|
| 19.
|
Singh, D. V., and S. C. Sanyal.
1997.
Enterotoxicity, haemolytic activity and antibiotic susceptibility of Aeromonas eucrenophila strains isolated from water and infected fish.
Indian J. Exp. Biol.
35:144-147[Medline].
|
| 20.
|
Singh, D. V., and S. C. Sanyal.
1997.
Enteropathogenicity of Aeromonas jandaei and A. trota.
FEMS Immunol. Med. Microbiol.
17:243-250[Medline].
|
| 21.
|
Wang, G.,
K. D. Tyler,
C. K. Munro, and W. M. Johnson.
1996.
Characterization of cytotoxic, hemolytic Aeromonas caviae clinical isolates and their identification by determining presence of a unique hemolysin gene.
J. Clin. Microbiol.
34:3203-3205[Abstract].
|
| 22.
|
Wong, C. Y. F.,
M. W. Heuzenroeder, and R. L. P. Flower.
1998.
Inactivation of two haemolytic toxin genes in Aeromonas hydrophila attenuates virulence in a suckling mouse model.
Microbiology
144:291-298[Abstract].
|
| 23.
|
Zywno, S. R.,
J. E. L. Arceneaux,
M. Altwegg, and B. R. Byers.
1992.
Siderophore production and DNA hybridization groups of Aeromonas spp.
J. Clin. Microbiol.
30:619-622[Abstract/Free Full Text].
|
Applied and Environmental Microbiology, December 1999, p. 5612-5614, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Vivas, J., Carracedo, B., Riano, J., Razquin, B. E., Lopez-Fierro, P., Acosta, F., Naharro, G., Villena, A. J.
(2004). Behavior of an Aeromonas hydrophila aroA Live Vaccine in Water Microcosms. Appl. Environ. Microbiol.
70: 2702-2708
[Abstract]
[Full Text]
Top
Abstract
Text
