APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1999, p. 5293–5302 Vol. 65, No.

12
0099-2240/99/$04.00⫹0
Copyright © 1999, American Society for Microbiology. All Rights Reserved.

PCR Detection, Characterization, and Distribution of Virulence

Genes in Aeromonas spp.
CESAR ISIGIDI BIN KINGOMBE,1* GEERT HUYS,2 MAURO TONOLLA,3 M. JOHN ALBERT,4
JEAN SWINGS,2 RAFFAELE PEDUZZI,3 AND THOMAS JEMMI1
Laboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium2; Microbiology Section, Swiss Federal Veterinary Office,
Liebefeld-Bern,1 and Istituto Cantonale Batteriosierologico, Lugano,3 Switzerland; and International Centre for
Diarrhoeal Disease Research, Bangladesh (ICDDRB), Dhaka-2, Bangladesh4

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Received 20 May 1999/Accepted 22 September 1999

We found 73.1 to 96.9% similarity by aligning the cytolytic enterotoxin gene of Aeromonas hydrophila SSU
(AHCYTOEN; GenBank accession no. M84709) against aerolysin genes of Aeromonas spp., suggesting the
possibility of selecting common primers. Identities of 90 to 100% were found among the eight selected primers
from those genes. Amplicons obtained from Aeromonas sp. reference strains by using specific primers for each
gene or a cocktail of primers were 232 bp long. Of hybridization group 4/5A/5B (HG4/5A/5B), HG9, and HG12
or non-Aeromonas reference strains, none were positive. PCR-restriction fragment length polymorphism (PCR-
RFLP) with HpaII yielded three types of patterns. PCR-RFLP 1 contained two fragments (66 and 166 bp) found
in HG6, HG7, HG8, HG10, and HG11. PCR-RFLP 2 contained three fragments (18, 66, and 148 bp) found in
HG1, HG2, HG3, and HG11. PCR-RFLP 3, with four fragments (7, 20, 66, and 139 bp), was observed only in
HG13. PCR-amplicon sequence analysis (PCR-ASA) revealed three main types. PCR-ASA 1 had 76 to 78%
homology with AHCYTOEN and included strains in HG6, HG7, HG8, HG10, and HG11. PCR-ASA 2, with 82%
homology, was found only in HG13. PCR-ASA 3, with 91 to 99% homology, contained the strains in HG1, HG2,
HG3, and HG11. This method indicated that 37 (61%) of the 61 reference strains were positive with the primer
cocktail master mixture, and 34 (58%) of 59 environmental isolates, 93 (66%) of 141 food isolates, and 100
(67%) of 150 clinical isolates from around the world carried a virulence factor when primers AHCF1 and
AHCR1 were used. In conclusion, this PCR-based method is rapid, sensitive, and specific for the detection of
virulence factors of Aeromonas spp. It overcomes the handicap of time-consuming biochemical and other
DNA-based methods.

Aeromonas spp. comprise mesophilic motile and psychro-
philic nonmotile gram-negative ubiquitous bacteria. World-
wide studies have demonstrated that Aeromonas spp. are uni-
versally distributed and widely isolated from clinical (38),
environmental (29, 38, 44), and food samples (2, 5), where they
may grow even at low temperatures (46). They are an example
of emerging bacterial pathogens. Even though they have been
recognized as primary fish pathogens for a long time, their
status as primary human pathogens was not clear until recently
(18, 42). It is estimated that aeromonads may cause up to 13%
of the reported gastroenteritis cases in the United States (10).
Aeromonas spp. are opportunistic pathogens that are at the
same time infectious (4, 33) and enterotoxigenic (12, 16).
Routine detection of pathogenic Aeromonas spp. was not
efficient until now due to the diversity of the hybridization
groups (HGs), and also, 17% of isolates could not be grouped
into any of the known HGs with biochemical tests (19).
By definition, the detection of a pathogen requires rapid and
specific methods for isolation, identification, and enumeration.
Such procedures may assist in the control of potentially patho-
genic microorganisms from environmental and food samples,
which are mostly regarded as the main transport vectors to
human populations.
According to the International Commission on Microbiolog-
ical Specifications for Food (30), many classical microbial pro-
cedures for the detection of Aeromonas spp. are laborious and
* Corresponding author. Mailing address: Microbiology Section,
Swiss Federal Veterinary Office, Schwarzenburgstrasse 161, CH-3003
Liebefeld-Bern, Switzerland. Phone: 41 31 322 22 63. Fax: 41 31 323 85
70. E-mail: cesar.kingombe@bvet.admin.ch.
time consuming or do not allow quantitative assessment of
these organisms. A complete review of methodologies for the
isolation, identification, and enumeration of Aeromonas spp.
from clinical, environmental, and food samples has been done
by Joseph and coworkers (34). Since then, other isolation
methods have been evaluated and compared for the ability to
isolate or detect Aeromonas spp. in food and environmental
items (20, 48, 51) and this indicates the need for a reliable,
universal, and standard method for the detection of these
pathogens in clinical, environmental, and food samples.
The identification of mesophilic Aeromonas spp. remains
difficult in routine surveys due to the complex classification of
Aeromonas bacteria into phenotypically defined phenospecies
and into genomospecies delineated on the basis of DNA-DNA
hybridization studies. At least 15 genospecies or HGs related
to 14 phenospecies have been validated or proposed (13).
Biochemical allocation of unknown aeromonads into HGs usu-
ally involves a large series of tests, ranging from 9 (1) to 136
(35) in number. Well-equipped laboratories can perform cel-
lular fatty acid methyl ester (FAME) composition analysis
(27), DNA fingerprinting by amplified fragment length poly-
morphism analysis (28), multilocus enzyme electrophoresis
(MEE), or ribotyping (50) to identify Aeromonas spp. to the
genomospecies level. Nevertheless, the above-mentioned
methods cannot reveal the pathogenic or nonpathogenic char-
acter of unknown Aeromonas sp. isolates (39).
An interesting approach for the direct detection of poten-
tially pathogenic Aeromonas sp. isolates is the use of virulence
determinants as genetic markers. A significant number of Aero-
monas sp. virulence genes have been described, including the
aerolysin genes (15, 24, 25, 26); hemolysin genes from A. hy-

5293
5294 BIN KINGOMBE ET AL.
drophila and A. sobria (22), A. salmonicida (23), and A. caviae
(52); an extracellular lipase gene from A. hydrophila (7); a
cytolytic enterotoxin gene of A. hydrophila (16); and a hemo-
lytic toxin gene of A. trota (36). The first study using the PCR
technique for specific detection of an A. hydrophila virulence
gene, the hole-forming toxin gene (25), was accomplished in
Canada by Pollard and coworkers (47). Using a pair of primers
designed on the basis of this known gene sequence, the authors
were able to detect beta-hemolysin-positive A. hydrophila
strains from patients with diarrhea, whereas control strains of
hemolytic A. sobria, nonhemolytic Aeromonas spp., and A.
caviae strains, even those producing cytotoxin or enterotoxin,
were negative with these primers. A Swedish study (8) using
the same primers confirmed the Canadian findings (47) when
screening for the presence of the aerolysin gene in water, fish,
and foods isolates. Another PCR technique for the detection
of two hemolysin genes of A. sobria was conducted in Japan
(49), where none of the virulence factors found with other
Aeromonas spp. were detected.
The present report describes the development of a new PCR
method that detects cytolytic enterotoxin and aerolysin genes
in Aeromonas spp. by using one pair of primers for a specific
gene or a cocktail of primers for the most known virulence
genes of Aeromonas spp. In combination with PCR-restriction
fragment length polymorphism (PCR-RFLP) and/or PCR-am-
plicon sequence analysis (PCR-ASA), the described PCR assay
also allows partial determination of the HG of a potentially
virulent isolate from a clinical, environmental, or food sample.
MATERIALS AND METHODS
Bacterial strains and cultural media. (i) Bacterial strains. For the reference
strains and the clinical, environmental, and food isolates used in this investiga-
tion, see Tables 2 and 3. All strains were identified by either FAME, MEE, or
ribotyping as described previously (6, 27, 39, 50) or by serotyping with a unpub-
lished serology test developed at the National Veterinary Laboratory, Aarhus,
Denmark.
(ii) Isolate preservation. All strains were purified on Columbia sheep blood
agar (bio-Merieux, Geneva, Switzerland) upon receipt in our laboratory. Subse-
quently, a typical colony was grown in Tryptone soya broth (Oxoid CM 129;
Fakola AG, Basel, Switzerland) at 28°C for 16 h. For preservation purposes,
stock cultures were prepared in 30% sterilized glycerol (Glycerol G-5150; Sigma,
Buchs, Switzerland) and maintained at ⫺70°C.
Bacterial DNA extraction for PCR. Approximately 100 ␮l of a Tryptone soya
broth (Oxoid) culture grown for 16 h at 28°C was used for DNA extraction using
the InstaGene matrix (Bio-Rad Laboratories AG, Glattbrugg, Switzerland) in
accordance with the manufacturer’s instructions. Subsequently, 5 ␮l of the DNA
solution was used as a template for PCR amplification.
Strategies for primer design. An A. hydrophila cytolytic enterotoxin gene
(AHCYTOEN) was described (16) as a multivirulence gene including lethality in
mice, hemolysis, cytotoxicity, and enterotoxigenicity. Some of those activities are
part of the virulence factors of other Aeromonas spp. For this reason, the
AHCYTOEN gene was chosen as the reference gene in this investigation.
First, we aligned the AHCYTOEN gene with other aerolysin genes, i.e.,
GenBank accession no. M16495 (24), U40711 (52), and AF064068 (36) and
EMBL accession no. X65043, X65044, X65045, X65046 (21), X65048 (22), and
Y00559 (26), to find the related regions. This alignment was performed with the
Bestfit program of the GCG software (Genetics Computer Group, Inc., Madison,
Wisc.). Finally, unique and specific primers were selected from open reading
frame 2 (ORF2) of the AHCYTOEN gene by using Primer Designer 3 software
(Scientific & Educational Software, Durham, N.C.). The selected primers were
then simultaneously compared to other sequences in the GenBank database to
verify their identity and similarity to Aeromonas sp. genes and those of other
species.
Master mixture preparation and PCR condition optimization. As previewed
by the Primer Designer 3 software, the eight selected primers (AHCF1, -2, and
-3 and AHCR1, -2, -3, -4, and -5 [see Table 1]) constitute five combinations of
primers or a cocktail of all of the primers that produce an amplicon of 232 bp
under identical amplification conditions. The annealing temperature indicated
by the software was increased by 10°C in order to increase the stringency of
primer annealing and to avoid nonspecific binding. Of the five possible primer
combinations, only the combination of AHCF1 and AHCR1 was used for clin-
ical, environmental, and food isolates because they displayed 100% homology
with the counterpart AHCYTOEN gene and an extracellular hemolysin gene
(EMBL accession no. X65045) (21), which represented the two main groups of
APPL. ENVIRON. MICROBIOL.
virulence factors in the genus Aeromonas, i.e., enterotoxins and hemolysins. The
cocktail master mixture containing all eight primers was used only for the ref-
erence strains, which were found to be negative for the AHCF1-AHCR1 primer
combination master mixture. All primers were purchased from MWG-Biotech
GmbH (Ebersberg, Germany).
Optimization of the PCR protocol was performed by using 50 ␮l of a PCR
mixture containing 5 ␮l of template DNA, 8 ␮l of a mixture containing each
deoxynucleoside triphosphate at 0.2 mM (Roche Diagnostics AG, Rotkreuz,
Switzerland), 5 ␮l of GeneAmp 10⫻ PCR buffer II (Perkin-Elmer, Rotkreuz,
Switzerland), 5 ␮l of a 25 mM MgCl2 solution (3.0 mM final concentration;
Perkin-Elmer), 0.25 ␮l of a 200 ␮M solution of each primer (1 ␮M final con-
centration), 0.10 ␮l of Tween 20 (2% final concentration) (Sigma), 0.25 ␮l of
AmpliTaq Gold at 5 U/␮l (1.25-U final concentration) (Perkin-Elmer), and 26.15
␮l of sterile double-distilled water, to make the final volume 50 ␮l. The PCR
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mixture was made in a sterile, laminar-airflow environment in a large volume to
produce approximately 100 tubes of 45 ␮l each, combining all of the ingredients
except the template DNA, and stored at ⫺20°C. PCR amplification was per-
formed with a GeneAmp 9600 PCR system (Perkin-Elmer) by using the follow-
ing temperature program: 1 cycle of denaturation for 10 min at 95°C; 25 cycles
of melting at 95°C for 15 s, annealing at 66°C for 30 s, and elongation at 72°C for
30 s; and a final extension round at 72°C for 10 min. The PCR amplicons were
separated electrophoretically by loading a total amplicon volume of 20 ␮l in-
cluding 6 ␮l of stop solution buffer onto a 2.5% agarose gel (pulsed-field-certified
agarose; Bio-Rad Laboratories AG). Electrophoresis was performed in 0.5⫻
Tris-borate-EDTA buffer–double-distilled water (Roche Diagnostics AG) for 1 h
at 100 V. Amplicons were visualized with UV light after the agarose gel had been
soaked for 15 min in an ethidium bromide solution (0.5 g/ml; Bio-Rad Labora-
tories AG).
Characterization of amplicons. The PCR products were purified by using the
QIAquick PCR Purification Kit (QIAGEN AG, Basel, Switzerland) and quan-
tified by using the GeneQuant instrument (Pharmacia-Biotech Europe GmbH,
Dübendorf, Switzerland) in accordance with the instructions provided by the
manufacturers. PCR-RFLP analysis using the endonuclease HpaII as indicated
by the Clone Manager software (Scientific & Educational Software) and PCR-
ASA were used to characterize the 135 amplicons available for this purpose.
The following procedure was used for PCR-RFLP analysis. A 10-␮g sample of
the purified PCR product was digested for 16 h at 37°C with endonuclease HpaII
(Roche Diagnostics AG) in accordance with the manufacturer’s instructions. The
fragments were separated by electrophoresis of 20 ␮l of the digested product in
3.5% agarose gel (pulsed-field-certified agarose; Bio-Rad) and subsequently
visualized with ethidium bromide fluorescence.
For PCR-ASA, the cycle sequencing reaction mixture was prepared with the
Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosys-
tems, Rotkreuz, Switzerland). Excess terminators were removed by the Centri-
Sep Spin Column Purification System (PE Applied Biosystems) before prepara-
tion and loading of the samples onto the ABI PRISM 310 Genetic Analyzer
System (PE Applied Biosystems) as specified by the manufacturer’s protocol.
The obtained sequences were aligned with the amplicon of the AHCYTOEN
gene predicted by the Primer Designer 3 software by using the Bestfit program
of the GCG software.
RESULTS
Sequence alignment and primer design strategies. Results
of sequence alignment and the primer design strategies are
detailed in Table 1. Alignment of the AHCYTOEN gene with
the aerolysin genes of Aeromonas spp. revealed high sequence
similarities ranging from 73.1 to 96.9% (Table 1). The most
commonly shared region of the AHCYTOEN gene was ORF2,
with its 1,515-bp length. The search for primers within this
ORF provided two 22-bp primers unique to the AHCYTOEN
gene, i.e., AHCF1 (5⬘-GAG AAG GTG ACC ACC AAG
AAC A-3⬘) and AHCR1 (5⬘-AAC TGA CAT CGG CCT TGA
ACT C-3⬘). Comparison of these primers to sequences in the
GenBank database generated six more primers, including for-
ward primers AHCF2 and AHCF3 and reverse primers
AHCR2, AHCR3, AHCR4, and AHCR5 (Table 1), from the
other aerolysin genes with high sequence identities to AHCF1
and AHCR1 ranging from 90 to 100%. More base mismatches
in the generated primers were found with AHCR1 than with
AHCF1.
PCR amplification. Results of PCR amplification using well-
characterized reference strains and wild-type isolates are re-
ported in Tables 2 and 3. For the reference strains, PCR
amplification using a specific combination of primers for a
specific gene or using the cocktail master mixture produced an
VOL. 65, 1999 PCR DETECTION OF AEROMONAS VIRULENCE GENES 5295

TABLE 1. Sequence alignment and primer design for the detection of Aeromonas sp. virulence genes
Nucleotide sequence % % Similarity by No. of PCR-RFLP
Organism Primer combinationb Site locations
accession no. Similaritya PCR-ASAc fragmentsd

A. hydrophila GBe M84709 100.0 C1, AHCF1-AHCR1 1661–1682, 1871–1892 100.0g 3

A. hydrophila EMh X65045 96.9 C1, AHCF1-AHCR1 919–940, 1871–1892 100.0 3
A. hydrophila GB M16495 86.9 C2, AHCF1-AHCR2 1309–1330, 1519–1540 92.2 3
A. hydrophila EM X65043 84.7 C2, AHCF1-AHCR2 1203–1224, 1413–1433 90.5 3
A. hydrophila EM X65044 84.7 C2, AHCF1-AHCR2 1202–1223, 1413–1433 90.1 3
A. sobria EM X65046 74.4 C3, AHCF1-AHCR3 1117–1138, 1327–1348 76.7 2
A. salmonicida EM X65048 74.7 C3, AHCF1-AHCR3 1119–1140, 1329–1350 76.7 2
A. caviae GB U40711 77.5 C4, AHCF2-AHCR4 985–1006, 1195–1216 84.1 2

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A. sobria EM Y00559 73.6 C5, AHCF3-AHCR5 1639–1660, 1849–1870 81.5 4
A. trota GB AF064068 73.1 C5, AHCF3-AHCR5 1174–1195, 1383–1405 81.9 4
a
Compared to the 2,528 bp of AHCYTOEN (GenBank nucleotide sequence accession no. M84709).
b
Primer sequences with mismatched bases underlined: AHCF1, 5⬘-GAGAAGGTGACCACCAAGAACA-3⬘; AHCR1, 5⬘-AACTGACATCGGCCTTGAACTC-3⬘;
AHCF2, 5⬘-GAGAAGGTCTCCACCAAGAACA-3⬘; AHCR2, 5⬘ AGCTGACATCGGCCTTGAACTC-3⬘; AHCF3, 5⬘-GAGAAGGTCTCGACCAAGAACA-3⬘;
AHCR3, 5⬘-AATTGACCTCGGCCTTGAACTC-3⬘; AHCR4, 5⬘ AGCTGACATCCGCCTTGAACTC-3⬘; AHCR5, 5⬘ AGCTCATATCCGCTTTGAACTC-3⬘.
c
Compared to the 232-bp amplicon sequence of the AHCYTOEN gene.
d
PCR-RFLP of 232 bp using HpaII.
e
GB, GenBank.
f
Primer combination number.
g
Value of 100.0% ⫽ 232-bp amplicon size.
h
EM, European Molecular Biology Laboratory.

amplicon of 232 bp, as predicted by the Primer Designer 3
software. Of the 61 Aeromonas reference strains tested, the 30
(49.2%) that were positive for the primer combination
AHCF1-AHCR1 master mixture were found in HG1 (A. hy-
drophila), HG2 (A. bestiarum), HG3 (A. hydrophila and A.
salmonicida), HG6 (A. eucrenophila), HG7 (A. sobria), and
HG8 (A. veronii biogroup sobria). When the cocktail master
mixture was used, 37 (61%) of the 61 reference strains were
positive. Some of the reference strains negative with the
AHCF1-AHCR1 mixture, e.g., in HG6 (A. eucrenophila),
HG10 (A. veronii biogroup veronii), HG11 (A. encheleia), and
HG13 (A. trota), turned out to be positive with the primer
cocktail master mixture. No representatives of HG4/5A/5B (A.
caviae-A. media complex), HG9 (A. jandaei), or HG12 (A.
schubertii) were found to be positive for the targeted genes.
Two positive representative reference strains of each HG were
tested with all five combinations, including the cocktail master
mixture. Some of them produced more than one amplicon as
evidence that they possess more than one virulence factor, e.g.,
strain ATCC 43979T (HG7) (results not shown). The applica-
tion of this PCR procedure to isolates from clinical, environ-
mental, and food isolates when using the primer combination
AHCF1-AHCR1 showed that 227 (65%) of 351 strains were
positive for the targeted virulence markers. Use of the primer
combination AHCF1-AHCR1 for the clinical, environmental,
and food isolates was justified by the fact that almost all of the
isolates received in our laboratory for this investigation were in
HG1, HG2, HG3, HG4, HG5A, HG5B, or HG8. The refer-
ence strains in these HGs always reacted positively or nega-
tively with both primers AHCF1 and AHCR1 and the cocktail
master mixture. Of the 55 isolates which were not character-
ized to the genotype (HG) level because they could not grow
on the culture medium required for identification by the
FAME method or which were only serotyped (fish isolates), 31
(56%) were positive for one of the virulence factors.
Specificity of primers. The specificity of the primer combi-
nation AHCF1-AHCR1 and the cocktail master mixture for
the amplification of the 232-bp amplicon in Aeromonas spp.
was demonstrated by the negative PCR results obtained with
all of the non-Aeromonas reference strains (Table 2).
PCR-RFLP characterization. Results of the characterization
of the reference strains and isolates by PCR-RFLP are pre-
sented in Tables 2 and 3, respectively. The characterization of
the 232-bp PCR product from reference strains by restriction
enzyme digestion (PCR-RFLP) using endonuclease HpaII re-
vealed three types of amplicons, as predicted by the Clone
Manager software (Fig. 1 and 2). PCR-RFLP type 1 (PCR-
RFLP 1), exhibiting two restriction fragments of 66 and 166 bp,
was found in HG6, HG7, HG8, HG10, and HG11 (e.g., strain
A926; Table 2). PCR-RFLP 2, displaying three restriction frag-
ments of 18, 66, and 148 bp, was found in HG1, HG2, HG3,
and HG11 (e.g., strain A1653; Table 2). PCR-RFLP 3, with
four fragments of 7, 20, 66, and 139 bp, was found only in
HG13. PCR-RFLP characterization of amplicons of Aeromo-
nas isolates from clinical, environmental, and food samples by
either FAME, MEE, or ribotyping generally produced the
same values as the reference strains. All 18 amplicons of the A.
veronii complex isolates tested were found to be of PCR-RFLP
1. Of the 57 amplicons of HG1, HG2, HG3, and the HG4/
5A/5B complex tested, only 2 (1 of HG1, isolated from water in
Belgium, and 1 from a clinical isolate of HG4 from Bang-
ladesh) were of PCR-RFLP 1 instead of PCR-RFLP 2. Diver-
gent results were found when the HGs of the isolates were not
identified or when they were serotyped by the Danish National
Veterinary Laboratory serotyping system, except for A. salmo-
nicida, for which all six of the amplicons tested were of PCR-
RFLP 2.
PCR-ASA characterization. The results of PCR-ASA for the
reference strains demonstrated the existence of three PCR-
ASA groups in Aeromonas (Table 2). Similarly to PCR-RFLP
1, PCR-ASA group 1 (PCR-ASA 1) includes strains of the A.
veronii complex in HG6, HG7, HG8, HG10, and HG11. All six
of the positive strains of this complex tested for this analysis
showed sequence similarities ranging from 76 to 78% com-
pared to the reference amplicon of the AHCYTOEN gene.
PCR-ASA 2, with 82% sequence similarity, was found exclu-
sively in HG13. PCR-ASA 3 contained HG1, HG2, HG3, and
HG11, with sequence similarities to the reference amplicon of
the AHCYTOEN gene varying from 91 to 99%. The results of
PCR-ASA of amplicons from clinical, environmental, and food
isolates characterized by either FAME, MEE, or ribotyping as
for PCR-RFLP characterization were similar to those obtained
with the reference strains. Again, as for the PCR-RFLP test,
the isolates which were not characterized with respect to HG
5296 BIN KINGOMBE ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 2. Results of PCR and characteristics of the 232-bp amplicons of reference strains
No. of
% Similarity
Organism Reference no. Source Country of origin PCRa PCR-RFLPb Gene similarity
by PCR-ASAc
fragments

A. hydrophila (HG1)
A17d LMG 13439 Unknown Germany ⫹ 3 96 Hemolysin-enterotoxin
A19 LMG 13440 Unknown Germany ⫺
A22 Not available Feces Switzerland ⫺
A34 LMG 13441 Feces Switzerland ⫺
A52 LMG 13442 Feces Switzerland ⫺
A82 LMG 13656 Feces Switzerland ⫺

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A145 LMG 13657 Feces Switzerland ⫹ 3 99 Hemolysin-enterotoxin
A162 LMG 13658 Feces Switzerland ⫺
A167 LMG 13659 Feces Switzerland ⫹ 3 98 Hemolysin-enterotoxin
A306 ATCC 7966Te Milk United States ⫹ 3 97 Hemolysin-enterotoxin

A. bestiarum (HG2)
A2 LMG 13445 Human Germany ⫹ 3 92 Hemolysin-enterotoxin
A13 LMG 13446 Environmental Germany ⫺
A39 LMG 13447 Water Switzerland ⫹ 3 91 Hemolysin-enterotoxin
A169 LMG 13448 Feces Switzerland ⫹ 3 94 Hemolysin-enterotoxin
A307 CDC 9533-76T Fish France ⫹ 3 94 Hemolysin-enterotoxin
A908 ATCC 14715 Fish France ⫹ 3 94 Hemolysin-enterotoxin
A936 LMG 13664 Unknown United States ⫹ 3 94 Hemolysin-enterotoxin
A937 LMG 13665 Unknown United States ⫹ 3 94 Hemolysin-enterotoxin
A976 LMG 13666 Water United States ⫹ 3 94 Hemolysin-enterotoxin
A1613 LMG 13667 Water United States ⫹ 3 95 Hemolysin-enterotoxin

A. hydrophila (HG3)
A7 LMG 13452 Environmental Germany ⫹ 3 91 Hemolysin-enterotoxin
A8 LMG 13453 Environmental Germany ⫹ 3 91 Hemolysin-enterotoxin
A14 LMG 13674 Environmental Germany ⫹ 3 91 Hemolysin-enterotoxin
A15 LMG 13675 Environmental Germany ⫹ 3 91 Hemolysin-enterotoxin
A63 LMG 13450 Feces Switzerland ⫹ 3 91 Hemolysin-enterotoxin
A99 LMG 13449 Feces Switzerland ⫹ 3 91 Hemolysin-enterotoxin
A220 LMG 13661 Feces Switzerland ⫹ 3 91 Hemolysin-enterotoxin
A308 CDC 043-84 Water France ⫹ 3 91 Hemolysin-enterotoxin
A938 Not available Unknown United States ⫹ 3 91 Hemolysin-enterotoxin
A1422 Not available Unknown United States ⫹ 3 91 Hemolysin-enterotoxin

A. salmonicida (HG3)
Not available ATCC 33658T Fish United Kingdom ⫹ 3 91 Hemolysin-enterotoxin
Not available ATCC 33659 Fish United Kingdom ⫺

A. caviae (HG4)
A1 LMG 13454 Human Germany ⫺
A3 Not available Unknown Germany ⫺
A309 ATCC 15468T Guinea pig France ⫺

A. caviae (HG5A)
A6 LMG 13460 Environmental Germany ⫺
A75 LMG 13461 Feces Switzerland ⫺
A310 ATCC 51107 Fish France ⫺

A. caviae (HG5B)
A117 LMG 13465 Feces Switzerland ⫺
A137 Not available Feces Switzerland ⫺
A213 LMG 13466 Feces Switzerland ⫺

A. eucrenophila (HG6)
A311 ATCC 23309 Fish Unknown ⫹ 2 76 Hemolysin
A914 CDC 9179 Unknown Unknown ⫹ 2 76 Hemolysin

A. sobria (HG7)
A312 ATCC 43979T Fish France ⫹ 2 76 Hemolysin
A915 LMG 13469 Fish United States ⫺

A. veronii biovar sobria (HG8)

A10 LMG 13067 Environmental Germany ⫹ 2 76 Hemolysin
A11 LMG 13693 Environmental Germany ⫹ 2 78 Hemolysin
A27 LMG 13694 Unknown Switzerland ⫹ 2 78 Hemolysin
A132 Not available Feces Switzerland ⫹ 2 76 Hemolysin
Continued on following page
VOL. 65, 1999 PCR DETECTION OF AEROMONAS VIRULENCE GENES 5297

TABLE 2—Continued
No. of
% Similarity
Organism Reference no. Source Country of origin PCRa PCR-RFLPb Gene similarity
by PCR-ASAc
fragments

A. veronii biovar veronii (HG10)

A901 ATCC 35624T Human United States ⫹ 2 77 Hemolysin

A. jandaei (HG9)
A179 LMG 13064 Feces Switzerland ⫺
A919 LMG 13065 Feces United States ⫺
A1642 ATCC 49568T Feces United States ⫺

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A. encheleia (HG11)
A902 ATCC 35941 Human New Zealand ⫺
A926 LMG 13076 Water United States ⫹ 2 76 Hemolysin
A1653 LMG 13061 Water Germany ⫹ 3 99 Hemolysin-enterotoxin

A. schubertii (HG12)
A903 ATCC 43700T Human United States ⫺
A922 LMG 13473 Human United States ⫺

A. trota (HG14)
A1645 ATCC 49659 Human United States ⫹ 4 82 Hemolysin
A1646 ATCC 49657T Feces India ⫹ 4 82 Hemolysin
A1647 ATCC 49660 Feces Thailand ⫹ 4 82 Hemolysin

Non-Aeromonas spp.
Bacillus cereus ATCC 11778 Unknown United States ⫺
Bacteroides coagulans ATCC 7050 Milk United Kingdom ⫺
Clostridium perfringens ATCC 13124 Cow United Kingdom ⫺
C. perfringens CIP 60-19 Sheep France ⫺
C. perfringens CIP 60-60 Dog France ⫺
Candida albicans ATCC 10231 Unknown United States ⫺
Escherichia coli ATCC 25922 Clinical United States ⫺
Listeria monocytogenes ATCC 7644 Human Unknown ⫺
Micrococcus luteus ATCC 9341 Soil United States ⫺
Proteus vulgaris ATCC 13315 Unknown United Kingdom ⫺
Pseudomonas aeruginosa ATCC 27853 Blood United States ⫺
Salmonella typhimurium ATCC 14028 Bovine United States ⫺
Staphylococcus aureus ATCC 25923 Clinical United States ⫺
S. aureus ATCC 33862 Unknown Unknown ⫺
S. epidermidis ATCC 12228 Unknown United States ⫺
Streptococcus agalactiae ATCC 12386 Unknown Unknown ⫺
Vibrio parahemolyticus ATCC 17802 Food Japan ⫺
V. vulnificus CIP 75.4 Blood United States ⫺
V. vulnificus CIP 81.90 Blood France ⫺
Yersinia enterocolitica ATCC 23715 Blood Unknown ⫺
a
PCR positivity.
b
PCR-RFLP fragments obtained when using HpaII endonuclease. PCR-RFLP 1 has two fragments, PCR-RFLP 2 has three fragments, and PCR-RFLP has four
fragments.
c
PCR-ASA indicating similarity to the cytolytic enterotoxin gene (AHCYTOEN, GenBank accession no. M84709). PCR-ASA 1 has 76 to 78% similarity, PCR-RFLP
2 has 82% similarity, and PCR-ASA 3 has 91 to 99% similarity to the AHCYTOEN gene.
d
Altwegg reference number.
e
T, type strain in HG.

and serotype gave divergent results (Table 3), except for iso-
lates identified as A. salmonicida by serotyping. All six of these
isolates of A. salmonicida had the PCR-ASA 3 profile.
DISCUSSION
The results of alignment analysis have shown that high se-
quence homologies exists between the AHCYTOEN gene and
other aerolysin genes, ranging from 73.1 to 96.7%, thus con-
firming clearly the high level of DNA relatedness of Aeromo-
nas sp. virulence factors. This enabled us to retrieve specific
primers from their common regions (Table 1). We found that
the ORF2 region within the AHCYTOEN gene was the most
common region of the aligned genes. The systematic search for
specific primers from ORF2 within the AHCYTOEN gene led
to the selection of primers AHCF1 (forward) and AHCR1
(reverse). These primers were selected for their uniqueness to
this gene, but when these primers were compared to sequences
in the GenBank database, high sequence similarities to eight
other described virulence genes of the Aeromonas complex
were revealed by gene alignment analysis (Table 1). From the
primer comparison results, six more primers were generated,
including AHCF2, AHCF3, AHCR2, AHCR3, AHCR4, and
AHCR5, each with the ability to amplify the same amplicon
from its specific gene. The master mixture using all eight prim-
ers (cocktail master mixture) also produced the 232 bp in
reference strains.
Among the described virulence factors of Aeromonas spp.,
two major groups are frequently associated with its pathoge-
nicity, namely, the aerolysins and the enterotoxins (11). The
5298 BIN KINGOMBE ET AL.
FIG. 1. PCR-RFLP maps of the 232-bp amplicon showing the three types of
HpaII endonuclease restriction fragment patterns. The upper map represents
PCR-RFLP 1 with two fragments of 66 and 166 bp. The second map represents
PCR-RFLP 2 with three fragments of 18, 66, and 148 bp. The third map repre-
sent PCR-RFLP 3 with fragments of 7, 20, 66, and 139 bp. The lower map
represents the scaled 232-bp amplicon. The arrows flanking the PCR-RFLP 1
map represent primers which may be AHCF1, -2, or -3 and AHCR1, -2, -3, -4, or
-5.
specific and rapid detection of the virulence genes (Table 1) by
means of molecular techniques has been the subject of many
studies during the last decade (8, 14, 35, 41, 47). However,
many of these investigations were limited to the detection of
one specific virulence gene. Theoretically, primers AHCF1 and
AHCR1 used in this investigation were designed to target both
the AHCYTOEN gene and the extracellular hemolysin gene
with EMBL database accession no. X65045 (22) of A. hy-
drophila on the basis of 100% sequence homology. In practice,
however, these primers produced amplicons in almost all of the
HGs of the reference strains except HG9, HG10, HG11,
HG12, and HG13. Also, we observed that these particular HGs
were practically absent in the wild-type strains we received
from different laboratories for this investigation. This observa-
tion demonstrates the multigene detection characteristics of
the two-primer set AHCF1-AHCR1 used in this work for wild-
type isolates and its greater usefulness than previously de-
scribed primers (14, 46, 49).
The results of PCR-RFLP analysis of Aeromonas reference
strains with endonuclease HpaII have revealed that three main
types or clusters of virulence genes coexist in the 15 HGs of
Aeromonas spp. (Fig. 1 and 2).
As for the PCR-RFLP, the PCR-ASA of the 232-bp ampli-
cons showed that representatives of all known Aeromonas HGs
can also be divided into three types (Tables 2 and 3). In
Aeromonas sp. taxa, amplicons from A. hydrophila HG1 and A.
encheleia HG11 (e.g., strain A1653; this strain was recently
moved from HG6 [A. eucrenophila] to HG11 [A. encheleia]) ref-
erence strains were most closely related to the AHCYTOEN
gene sequence, with homologies between 97 and 99%. The
strains of A. bestiarum HG2 were more versatile in their ho-
mology to the AHCYTOEN gene, with 91 to 95% sequence
homology, whereas all of the reference strains of HG3, includ-
ing A. hydrophila and A. salmonicida, exhibited 91% of homol-
ogy to the AHCYTOEN gene. These data clearly illustrated
the stability of this gene in these HGs (Table 2).
The reference strains of A. caviae (HG4/5A/5B) could not be
classified in any of the defined PCR-RFLP or PCR-ASA types
because all representatives were negative for the 232-bp am-
plicon with the primer set AHCF1-AHCR1 and the cocktail
APPL. ENVIRON. MICROBIOL.
master mixtures. The lack of these genes in A. caviae (HG4/
5A/5B) is in agreement with previous reports (3, 21, 31, 32, 53)
which suggested that this group of aeromonads are less virulent
and less cytotoxigenic than other Aeromonas taxa. Neverthe-
less, recent studies have demonstrated the production of cyto-
toxin by A. caviae strains under specific culture conditions and
its implications for the generation of diarrhea in very young
children and old people (40, 45). Negative 232-bp amplicon
detection results were also obtained with the A. jandaei (HG9)
and A. schubertii (HG12) reference strains. Since only a limited
number of strains were included, it is not possible to report on
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the distribution of hemolytic or enterotoxin genes in these
Aeromonas HGs.
The PCR-RFLP and PCR-ASA assays performed on Aero-
monas reference strains have proven to be helpful tools for the
detection and characterization of potential enterotoxigenic
and hemolytic aeromonads. This characterization can be per-
formed in any molecular laboratory, depending on the avail-
ability of equipment, principally a thermocycler and the restric-
tion endonuclease HpaII. The application of these procedures
to clinical, environmental, and food isolates will permit not
only the detection of the virulence genes but also their char-
acterization and the distribution of these genes in wild isolates
from different sources or in their HGs.
Of the total of 350 clinical, environmental, and food isolates
of Aeromonas spp. included in this study, 65% harbored viru-
lence genes. Fifty percent of the human clinical isolates origi-
nating from Bangladesh, Ivory Coast, and Switzerland were
positive by PCR for virulence factors and belonged mainly to
the A. caviae complex (HG4/5A/5B), A. hydrophila (HG1), and
A. veronii (HG8/10) (Table 3). Although it is very likely that
clinical isolates possess at least one virulence gene, it should be
kept in mind that Aeromonas spp. are well recognized as op-
portunistic organisms that may be present in diarrheal stool as
commensals rather than as primary pathogens (3). In addition,
the primers used for PCR identification in this study may not
FIG. 2. PCR amplicon of 232 bp and PCR-RFLP types of 232-bp amplicons
of different HGs of reference strains of Aeromonas spp. PCR amplicon and
PCR-RFLP types of reference and wild-type strains of Aeromonas spp. on 4%
Metaphor agarose gel (Bioconcept, Allschwil, Switzerland) stained with ethidium
bromide at 0.5 ␮g/ml (Bio-Rad). Lanes: 1 and 15, DNA size marker VIII (Roche
Diagnostics AG; catalog no. 1336 045); 2, a PCR-RFLP 2 of A17 (ATCC 7966),
a reference strain of HG1 isolated from canned milk from the United States; 3,
PCR-RFLP 2 of A307 (ATCC 51108), a reference strain of HG2 isolated from
a fish in France; 4, PCR-RFLP 2 of A14 (LMG 13674), a reference strain of HG3
that is an environmental isolate from Germany; 5, PCR-RFLP type 1 of A914
(CDC 9179), a reference strain of HG6 (source unknown); 6, PCR-RFLP 1 of
A312 (ATCC 43979), a reference strain of HG7 that is a fish isolate from France;
7, PCR-RFLP 1 of A10 (CCUG 30356), a reference strain of HG8 that is an
environmental isolate from Germany; 8, PCR-RFLP 1 of A901 (ATCC 35624),
a reference strain of HG10 that is a clinical isolate (sputum) from the United
States; 9, PCR-RFLP 1 of A926 (CDC 3136), a reference strain of HG11 that is
a water isolate from the United States; 10, PCR-RFLP 3 of A1645 (ATCC
49659), a reference strain of HG13 that is a clinical isolate from the United
States; 11, uncut 232-bp amplicon of strain A1645; 12, PCR-RFLP 2 of a wild-
type strain (BP 3) of HG1 that is a clinical isolate of HG1 from Bangladesh; 13,
PCR-RFLP 2 of a wild-type strain (CBK 45) of HG3 that is a pork meat isolate
from Switzerland; 14, PCR-RFLP 1 of a wild-type strain (BE 6) of HG8 that is
an environmental isolate from Bangladesh.
VOL. 65, 1999 PCR DETECTION OF AEROMONAS VIRULENCE GENES 5299

TABLE 3. PCR results and characteristics of virulence gene amplicons in Aeromonas sp. isolates
No. of
No. of No. (%) % Similarity
Sample Origin Organism HG HpaIIa PCR-RFLP
isolates PCR positive by PCR-ASAb
fragmentsc

Food
Beef Switzerland A. bestiarum 2 2 1 1 93 3
A. caviae 5A 1 0
A. hydrophila 3 12 10 9 90, 91, 92 3
A. veronii 8/10 2 1 1 77 2
Aeromonas sp. NDd 4 3 ND ND

Total 21 15 (71) 11

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Minced meat Switzerland A. bestiarum 2 1 1 ND ND
A. caviae 5A 2 0 ND ND
A. hydrophila 3 9 9 ND ND
Aeromonas sp. ND 2 2 ND ND

Total 14 12 (86)

Pork Switzerland A. hydrophila 3 6 6 6 90, 91, 92 3

Aeromonas sp. ND 4 3 ND ND

Total 10 9 (90) 6

Poultrye Switzerland A. hydrophila 3 6 6 6 91, 92 3

European Union A. veronii 8/10 1 1 1 77 2
Aeromonas sp. ND 10 3 ND

Total 17 10 (59) 7

Seafoodf European Union A. caviae 5A 1 0 ND ND

A. hydrophila 3 5 5 1 78 2
Aeromonas sp. ND 6 4 ND ND

Total 12 9 (75) 1

Perch European Union A. bestiarum 2 1 1 ND ND

A. encheleia 6B 1 0 ND ND
A. hydrophila 3 4 4 1 91 3
Aeromonas sp. ND 5 4 ND ND

Total 11 9 (82) 1

Salmon European Union A. bestiarum 2 1 1 1 94 3

A. hydrophila 1 1 1 1 95 3
A. hydrophila 3 15 15 5 91, 93 3
Aeromonas sp. ND 5 3 ND ND

Total 22 20 (91) 7

Vegetables Belgium A. bestiarum 2 2 0 ND ND

A. caviae 4/5A/5B 15 2 ND ND
A. caviae Complex 6 4 ND ND
A. hydrophila 3 1 1 ND ND
Aeromonas sp. ND 6 1 ND ND
Switzerland Aeromonas sp. ND 4 1 ND ND

Total 34 9 (27)

Environmental
Water Bangladesh A. veronii 8/10 5 5 5 77, 78 2
Belgium A. bestiarum 2 4 4 4 94, 95 3
Continued on following page
5300 BIN KINGOMBE ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 3—Continued
No. of
No. of No. (%) % Similarity
Sample Origin Organism HG HpaIIa PCR-RFLP
isolates PCR positive by PCR-ASAb
fragmentsc

A. caviae 4/5A 5 0
A. hydrophila 1 2 2 2 77, 94 2, 3
A. hydrophila 3 9 8 5 83, 91, 92 3
Switzerland A. bestiarum 2 6 6 6 90, 93, 94 3
A. caviae 4/5A/5B 9 0
A. eucrenophila 6 2 0
A. hydrophila 1 1 1 1 98 3

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A. hydrophila 3 4 3 2 91, 94 3
A. veronii 8/10 12 5 3 76, 78 2

Total 59 34 (58) 28

Clinical
Human Bangladesh A. caviae 4 10 4 4 78, 93, 96, 100 2, 3
A. hydrophila 1 13 2 ND ND
Ivory Coast A. hydrophila 1 5 2 2 100 3
A. veronii 8 6 6 6 76, 78 2
Switzerland A. bestiarum 2 1 1 1 94 3
A. caviae 4/5A/5B 11 0 ND ND
A. eucrenophila 6 1 1 ND ND
A. hydrophila 1 7 3 3 97, 98 3
A. veronii 8 17 17 6 76, 77, 78 2

Total 71 36 (50) 22

Fish Switzerland A. bestiarum 2 1 1 1 94 3

A. caviae 4 3 0 ND ND
A. eucrenophila 6 2 0 ND ND
A. hydrophila ND 5 3 2 77, 94 2, 3
A. hydrophila Ser.g 7 6 5 77, 78, 91, 94 2, 3
A. veronii 8/10 1 1 1 77 2
A. veronii ND 4 4 4 77, 78, 91 2, 3
A. veronii Ser. 3 3 3 76, 78 2
A. salmonicida Ser. 52 46 6 82, 91 3
A. sobria 7 1 0 ND ND

Total 79 64 (81) 23

Gross total 350 227 (65) 106

a
Number of amplicons used for restriction enzyme analysis with endonuclease HpaII.
b
PCR-ASA indicating similarity to cytolytic enterotoxin gene AHCYTOEN (GenBank accession no. M84709). PCR-ASA 1 has 76 to 78% similarity, PCR-ASA 2
has 82% similarity, and PCR-ASA 3 has 91 to 99% similarity to the AHCYTOEN gene.
c
Number of PCR-RFLP fragments obtained when using HpaII endonuclease. PCR-RFLP 1 has two fragments, PCR-RFLP has three fragments, and PCR-RFLP
3 has four fragments.
d
ND, not done.
e
Poultry including chicken, turkey, and duck.
f
Seafood including mussels, oysters, and scallops.
g
Ser., serotyped.

be specific for other virulence genes of Aeromonas spp. (Table
1). Interestingly, all of the Swiss human and environmental
isolates of A. caviae were found to be negative for the targeted
genes whereas 25% of the A. caviae isolates originating from
Bangladesh harbored virulence genes. This may confirm the
previously published observations that geographical variation
in virulence factors for this Aeromonas species may exist (3).
A total of 22 amplicons from human clinical isolates belong-
ing to A. bestiarum (n ⫽ 1), A. caviae (n ⫽ 4), A. hydrophila
(n ⫽ 5), and A. veronii (n ⫽ 12) were included for PCR-RFLP
and PCR-ASA assays. All positive A. caviae isolates from Ban-
gladesh were classified as members of HG4 and belonged to
PCR-RFLP 2 and PCR-ASA 3 with amplicon sequence ho-
mologies ranging from 93 to 100%. Clearly, these amplicons
show high relatedness to the reference sequence of the multi-
virulence AHCYTOEN gene found mostly in A. hydrophila
HG1. In this study, amplicons of reference strains and clinical
and environmental isolates of HG1 exhibited 97 to 100% ho-
mology with the AHCYTOEN gene (Table 3). In addition, it
was found that the PCR-RFLP and PCR-ASA profiles of HG1
isolates were similar to those of HG1 reference strains, sug-
gesting that these isolates possess a multiple-virulence gene
comparable to the AHCYTOEN gene. Previous clinical stud-
ies (37, 43) have suggested that the expression of multiple
biological activities, as in the case of the AHCYTOEN gene, is
necessary for the expression of microbial pathogenicity.
All of the 23 clinical isolates of A. veronii included in this
investigation contained the 232-bp amplicon. Of the 12 ampli-
VOL. 65, 1999
cons characterized by PCR-RFLP and PCR-ASA assays, all
were of PCR-ASA 1 and PCR-RFLP 1, regardless their origin.
From the literature, it is known that A. veronii represents one
of the more virulent species in the genus Aeromonas, as was
proven by its pronounced invasiveness and lower 50% lethal
doses (17). Similar to A. hydrophila HG1, A. veronii isolates
originated mainly from clinical and environmental samples,
suggesting that the aquatic environment acts as a reservoir of
potentially virulent Aeromonas spp.
Fresh water is known to be a source and reservoir of Aero-
monas spp. Among the 60 water isolates, 58% were found to be
potentially pathogenic. Virulence genes were found in a high
proportion in all species but in none of 15 A. caviae (HG4/5A/
5B) isolates. All A. bestiarum and A. hydrophila isolates were
found to be of PCR-ASA 3 and PCR-RFLP 2. A strain of A.
hydrophila of HG1 and one of A. caviae of HG4 were found to
be of PCR-ASA 1 and PCR-RFLP 1; these were the only cases
of FAME-characterized isolates to be found so among all of
the reference strains and wild isolates.
The Aeromonas sp. fish isolates under study were dominated
by A. salmonicida (n ⫽ 52), followed by A. hydrophila (n ⫽ 12)
and A. veronii (n ⫽ 8), as determined by serotyping. In contrast
to the reference strains, the distribution of virulence genes
determined by the PCR-RFLP and PCR-ASA tests among the
PCR-RFLP 1 or 2 and PCR-ASA 1 or 2 amplicons in isolates
classified as A. veronii or A. hydrophila demonstrated the lack
of specificity of the serotyping technique in the classification of
Aeromonas spp. Furthermore, it was interesting to note such a
proportion (85%) of PCR-positive A. salmonicida isolates,
among which almost all of the amplicons belonged to PCR-
RFLP 2 and PCR-ASA 3. The fact that all of the fish isolates
were isolated from moribund fish may confirm the high patho-
genicity of A. salmonicida for fish.
A total of 67% of the food isolates originating from beef,
minced meat, pork, poultry, seafoods, perch, salmon, and veg-
etables possessed one of the targeted virulence genes. The
majority of these food isolates were identified as A. bestiarum
HG2 and A. hydrophila HG3. The virulence genes of these
isolates, as shown by molecular analysis, belonged to PCR-
RFLP 2 and PCR-ASA 2. Representatives of the A. caviae
complex were rarely isolated from foods of animal origin, and
none of them was positive for the virulence genes. These
strains mostly originated from vegetables sampled in Belgium,
and 29% were found to be positive for the targeted virulence
genes. Also, A. veronii HG8/10 was not frequently found
among the food isolates; the few positive amplicons all be-
longed to PCR-RFLP 1 and PCR-ASA 1.
In conclusion, we found that the alignment and comparison
analysis of the described aerolysins, hemolysins, and AHCYT
OEN genes of Aeromonas spp. confirm the high homologies
reported in the literature. The primers designed from the 10
Aeromonas sp. virulence factor genes permit the detection of
any of the above virulence markers according to a specific
need. The results of the application of the PCR-RFLP and
PCR-ASA assays to Aeromonas reference strains and clinical,
environmental, and food isolates demonstrated that the detec-
tion and identification of potentially pathogenic Aeromonas
spp. have become more specific, faster, and easier to perform
than conventional phenotyping methods. The PCR procedure
did not detect the targeted virulence genes in reference strains
of A. caviae; however, there were 10 (15%) of 68 well FAME-
characterized positive A. caviae isolates, including 4 clinical
isolates of HG4 from Bangladesh and 6 vegetable isolates from
Belgium, of which 4 were identified as belonging to the A.
caviae complex. In this context, the geographic variation in
virulence and/or the failure of actual characterization systems
PCR DETECTION OF AEROMONAS VIRULENCE GENES 5301
for A. caviae isolates should be considered. Virulence genes
were detected and characterized in well-typed reference
strains. They were also found and characterized in 65% of wild
isolates of Aeromonas spp. from around the world which orig-
inated from different samples, proving the universality of the
procedures described here. The characterization of the PCR
products by PCR-RFLP using endonuclease HpaII and PCR-
ASA revealed three major types or clusters of amplicons. It
may suggest the classification of pathogenic Aeromonas sp.
virulence genes into three main groups (aerolysins-hemolysins,
cytolytic enterotoxins, and cytotonic enterotoxins) with PCR-
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RFLP 1, 2, and 3 and PCR-ASA 1, 2, and 3. The PCR, PCR-
RFLP, and PCR-ASA systems may prove to be important tools
for the detection, identification, differentiation, and distribu-
tion of virulence markers in HGs. These tools will give micro-
biologists an alternative way to understand pathogenicity in
Aeromonas spp. and their distribution in isolates from different
sources and HGs.
ACKNOWLEDGMENTS
We thank M. Altwegg and J. Lüthy-Hottenstein, University of Zü-
rich, Zürich, Switzerland; T. Wahli and J. Graf, University of Bern,
Bern, Switzerland; M. Jermini of the Cantonal Health Laboratory of
Ticino, Lugano, Switzerland; M. Uyttendaele and K. Neyts of the
University of Ghent for providing us with Aeromonas sp. isolates; A.
Caminada of the Cantonal Institute of Bacteriology, Lugano, Switzer-
land; P. Meyer of Perkin-Elmer Switzerland; and E. Lüthi and D.
Howald of the Swiss Federal Veterinary Office for their exceptional
technical support.
REFERENCES
1. Abbott, S. L., W. K. W. Cheung, S. Kroske-Bystrom, T. Malekzadeh, and
J. M. Janda. 1992. Identification of Aeromonas strains to the genospecies
level in the clinical laboratory. J. Clin. Microbiol. 30:1262–1266.
2. Abeyta, C., Jr., C. A. Kaysner, M. M. Wekell, J. J. Sullivan, and G. N. Stelma.
1986. Recovery of Aeromonas hydrophila from oysters implicated in an out-
break of foodborne illness. J. Food Prot. 49:643–646.
3. Altwegg, M. 1985. Aeromonas caviae: an enteric pathogen? Infection 13:228–
230.
4. Altwegg, M., and H. K. Geiss. 1989. Aeromonas as human pathogen. Crit.
Rev. Microbiol. 16:253–286.
5. Altwegg, M., G. Martinetti Lucchini, J. Lüthy-Hottenstein, and M. Rohr-
bach. 1990. Aeromonas-associated gastroenteritis after consumption of con-
taminated shrimp. Eur. J. Clin. Microbiol. Infect. Dis. 10:44–45.
6. Altwegg, M., and J. Lüthi-Hottenstein. 1991. Methods for the identification
of DNA hybridization groups in the genus Aeromonas. Experientia 47:403–
406.
7. Anguita, J., L. B. R. Aparicio, and G. Naharro. 1993. Purification, gene
cloning, amino acid sequence analysis, and expression of an extracellular
lipase from an Aeromonas hydrophila human isolate. Appl. Environ. Micro-
biol. 59:2411–2417.
8. Baloda, S. B., K. Krovacek, L. Eriksson, T. Linne, and I. Mansson. 1995.
Detection of aerolysin gene Aeromonas strain isolates from drinking water,
fish, and foods by polymerase chain reaction. Comp. Immunol. Microbiol.
Infect. Dis. 18:17–26.
9. Balows, A., W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J.
Shadomy (ed.). 1991. Manual of clinical microbiology, 5th ed. American
Society for Microbiology, Washington, D.C.
10. Buchanan, R. L. 1984. The new pathogens. An update of selected examples.
Assoc. Food Drug Off. Q. Bull. 48:142–155.
11. Burke, V., J. Robinson, J. Beaman, M. Gracey, M. Lesmana, R. Rockhill, P.
Echeverria, and J. M. Janda. 1983. Correlation of endotoxicity with biotype
in Aeromonas spp. J. Clin. Microbiol. 18:1196–1200.
12. Cahill, M. M. 1990. Virulence factors in motile Aeromonas species: a review.
J. Appl. Bacteriol. 69:1–16.
13. Carnahan, A. M., and M. Altwegg. 1996. Taxonomy, p. 1–38. In B. Austin, M.
Altwegg, P. J. Gosling, and S. Joseph (ed.), The genus Aeromonas. John
Wiley & Sons, Chichester, United Kingdom.
14. Cascón, A., J. Anguita, C. Hernanz, M. Sánchez, M. Fernández, and G.
Naharro. 1996. Identification of Aeromonas hydrophila hybridization group I
by PCR assays. Appl. Environ. Microbiol. 62:1167–1170.
15. Chakraborty, T., B. Huhle, H. Bergbauer, and W. Goebel. 1986. Cloning,
expression, and mapping of the Aeromonas hydrophila aerolysin gene deter-
minant in Escherichia coli. J. Bacteriol. 167:368–374.
16. Chopra, A. K., C. W. Houston, J. W. Peterson, and G.-F. Jin. 1993. Cloning,
5302 BIN KINGOMBE ET AL.
expression, and sequence analysis of a cytolytic enterotoxin gene from Aero-
monas hydrophila. Can. J. Microbiol. 39:513–523.
17. Daily, O. P., S. W. Joseph, J. C. Coolbaulgh, R. I. Walker, B. R. Merrell,
D. M. Rollins, R. J. Seidler, R. R. Colwell, and C. R. Lissner. 1981. Asso-
ciation of Aeromonas sobria with human infection. J. Clin. Microbiol. 13:
769–777.
18. Foster, E. M. 1997. Historical overview of key issues in food safety. Emerg.
Infect. Dis. 3:481–482.
19. George, W. L., J. M. Jones, and M. M. Nakata. 1986. Phenotypic character-
istics of Aeromonas species isolated from adult humans. J. Clin. Microbiol.
23:1026–1029.
20. Gobat, P.-F., and T. Jemmi. 1994. Comparison of seven selective media for
the isolation of mesophilic Aeromonas species in fish and meat. Int. J. Food
Microbiol. 24:375–384.
21. Gracey, M., V. Burke, and J. Robinson. 1982. Aeromonas-associated gastro-
enteritis. Lancet ii:1304–1306.
22. Hirono, I., T. Aoki, T. Asao, and S. Kozaki. 1992. Nucleotide sequences and
characterization of hemolysin genes from Aeromonas hydrophila and Aero-
monas sobria. Microb. Pathog. 13:433–446.
23. Hirono, I., and T. Aoki. 1993. Cloning and characterization of hemolysin
genes from Aeromonas salmonicida. Microb. Pathog. 15:269–282.
24. Howard, S. P., and J. T. Buckley. 1986. Molecular cloning and expression in
Escherichia coli of the structural gene for the hemolytic toxin aerolysin from
Aeromonas hydrophila. Mol. Gen. Genet. 204:289–295.
25. Howard, S. P., W. J. Garland, M. J. Green, and J. T. Buckley. 1987. Nucle-
otide sequence of the gene for the hole-forming toxin aerolysin of Aeromo-
nas hydrophila. J. Bacteriol. 169:2869–2871.
25a.Hui, Y. H., et al. (ed.). 1994. Foodborne disease handbook, diseases caused
by bacteria, vol. 1, p. 1–27. Marcel Dekker Inc., New York, N.Y.
26. Husslein, V., B. Huhle, T. Jarchau, R. Lurz, W. Goebel, and T. Chakraborty.
1988. Nucleotide sequence and transcriptional analysis of the AerCaerA
region of Aeromonas sobria encoding aerolysin and its regulatory region.
Mol. Microbiol. 2:507–517.
27. Huys, G., M. Vancanneyt, R. Coopman, P. Janssen, E. Falsen, M. Altwegg,
and K. Kersters. 1994. Cellular fatty acid composition as a chemotaxonomic
marker for the differentiation of phenospecies and hybridization groups in
the genus Aeromonas. Int. J. Syst. Bacteriol. 44:651–658.
28. Huys, G., R. Coopman, P. Janssen, and K. Kersters. 1996. High-resolution
genotypic analysis of genus Aeromonas by AFLP fingerprinting. Int. J. Syst.
Bacteriol. 46:572–580.
29. Huys, G., I. Kesters, R. Coopman, P. Janssen, and K. Kersters. 1996. Ge-
notypic diversity among Aeromonas isolates recovered from drinking water
production plants as revealed by AFLP™ analysis. Syst. Appl. Microbiol.
19:428–435.
30. International Commission on Microbiological Specifications for Food. 1996.
Aeromonas, p. 5–19. In Microorganisms in foods. 5. Microbiological specifi-
cations of food pathogens. Blackie Academic & Professional, London, En-
gland.
31. Janda, J. M., M. Reitano, and E. J. Bottone. 1984. Biotyping of Aeromonas
isolates as correlate to delineating a species-associated disease spectrum.
J. Clin. Microbiol. 19:44–47.
32. Janda, J. M., R. B. Clark, and R. Brenden. 1985. Virulence of Aeromonas
species as assessed though mouse lethality studies. Curr. Microbiol. 12:163–
168.
33. Janda, J. M. 1991. Recent advances in study of the taxonomy, pathogenicity,
and infectious syndromes associated with the genus Aeromonas. Clin. Mi-
crobiol. Rev. 4:397–410.
34. Joseph, S. W., M. Janda, and A. Carnahan. 1988. Isolation, enumeration and
identification of Aeromonas spp. J. Food Safety 9:23–35.
35. Kaznowski, A. 1998. Identification of Aeromonas strains of different origin to
APPL. ENVIRON. MICROBIOL.
the genomic species level. J. Appl. Microbiol. 84:423–430.
36. Khan, A. A., E. Kim, and C. E. Cerniglia. 1998. Molecular cloning, nucleo-
tide sequence, and expression in Escherichia coli of a hemolytic toxin (aero-
lysin) gene from Aeromonas trota. Appl. Environ. Microbiol. 64:2473–2478.
37. Kirov, S. M., B. Rees, R. C. Wellock, J. M. Goldsmid, and A. D. Van Galen.
1986. Virulence characteristics of Aeromonas spp. in relation to source and
biotype. J. Clin. Microbiol. 24:827–834.
38. Kühn, I., G. Allestam, G. Huys, P. Janssen, K. Kersters, K. Krovacek, and
T.-A. Stenström. 1997. Diversity, persistence, and virulence of Aeromonas
strains isolated from drinking water distribution systems in Sweden. Appl.
Environ. Microbiol. 63:2708–2715.
39. Kühn, I., M. J. Albert, M. Ansaruzzaman, N. A. Bhuiyan, S. A. Alabi, M.
Sirajul Islam, P. K. B. Neogi, G. Huys, P. Janssen, K. Kersters, and R.
Möllby. 1997. Characterization of Aeromonas spp. isolated from humans
Downloaded from http://aem.asm.org/ on May 19, 2017 by UNIVERSITAS AIRLANGGA
with diarrhea, from healthy controls, and from surface water in Bangladesh.
J. Clin. Microbiol. 35:369–373.
40. Kuijper, E. J., H. C. Zanen, and M. F. Peeters. 1987. Aeromonas-associated
diarrhea in The Netherlands. Ann. Intern. Med. 106:640–641.
41. Lior, H., and W. M. Johnson. 1991. Application of polymerase chain reaction
(PCR) to detection of the aerolysin gene in whole cell cultures of B-hemo-
lysin Aeromonas hydrophila. Experientia 47:421–4249.
42. Molenda, J. R., and J. M. Cottingham. 1998. Emerging infectious diseases of
particular relevance to environmental health professionals. Dairy Food En-
viron. Sanit. 18:427–435.
43. Morgan, D. R., P. C. Johnson, H. L. Dupont, T. K. Satterwhite, and L. V.
Wood. 1985. Lack of correlation between known virulence properties of
Aeromonas hydrophila and enteropathogenicity for humans. Infect. Immun.
50:62–65.
44. Nakano, H., T. Kameyama, K. Venkateswaran, H. Kawamaki, and H. Hashi-
moto. 1990. Distribution and characterization of hemolytic and enteropatho-
genic motile Aeromonas in aquatic environment. Microbiol. Immunol. 34:
447–458.
45. Namdari, H., and E. J. Bottone. 1990. Microbiological and clinical evidence
supporting the role of Aeromonas caviae as a pediatric enteric pathogen.
J. Clin. Microbiol. 28:837–840.
46. Pin, C., Y. Benito, M. L. Carcia, D. Selgas, J. Tormos, and C. Casas. 1996.
Influence of temperature, pH, sodium chloride, and sodium nitrite on the
growth of clinical and food motile Aeromonas sp. strains. Arch. Lebensmit-
telhyg. 47:35–56.
47. Pollard, D. R., W. M. Johnson, H. Lior, S. D. Tyler, and K. R. Rozee. 1999.
Detection of the aerolysin gene in Aeromonas hydrophila by the polymerase
chain reaction. J. Clin. Microbiol. 28:2477–2481.
48. Schweizer, R., M. Kaderli, and U. Spahr. 1995. Aeromonas hydrophila in
Rohmilch in der Schweiz. Schweiz. Milchwirtsch. Forsch. 24:9–11.
49. Shibata, M., K. Morita, N. Wanatabe, H. Wada, T. Okitsu, S. Yamai, K. Itoh,
T. Shimada, H. Wanatabe, and M. Kanamori. 1996. Rapid detection of the
hemolysin genes in Aeromonas sobria by polymerase chain reaction. Kansen-
shogaku Zasshi 70:1266–1270.
50. Tonolla, M., A. Demarta, and R. Peduzzi. 1991. Multilocus genetic relation-
ships between clinical and environmental Aeromonas strains. FEMS Micro-
biol. Lett. 81:193–200.
51. Umadatt, S. 1997. Isolation and identification of Aeromonas spp. from
ground meats in eastern Canada. J. Food Prot. 60:125–130.
52. Wang, G., K. D. Tyler, C. K. Munro, and W. M. Johnson. 1996. Character-
ization of cytotoxic, hemolytic Aeromonas caviae clinical isolates and their
identification by determining presence of a unique hemolysin gene. J. Clin.
Microbiol. 34:3203–3205.
53. Watson, I. M., J. O. Robinson, V. Burke, and M. Gracey. 1985. Invasiveness
of Aeromonas spp. in relation to biotype virulence factors and clinical fea-
tures. J. Clin. Microbiol. 22:48–51.