Aerobic Metabolism see Metabolic Pathways: Release of Energy (Aerobic)

AEROMONAS

Contents
Introduction
Detection by Cultural and Modern Techniques

Introduction
MJ Figueras and R Beaz-Hidalgo, Universitat Rovira i Virgili, IISPV, Reus, Spain
Ó 2014 Elsevier Ltd. All rights reserved.
This article is a revision of the previous edition article by I.S. Blair, M.A.S. McMahon, D.A. McDowell, volume 1, pp. 25–30, Ó 1999, Elsevier Ltd.

Characteristics of the Genus Aeromonas gene, which is involved in the biosynthesis of folic and
aromatic amino acids (Table 2).
The genus Aeromonas includes Gram-negative oxidase positive At present, the genus comprises 25 species, some of which
bacilli considered autochthonous of the aquatic environments have been isolated from human clinical samples, drinking
that are commonly isolated from healthy or diseased fish, water, and food (Table 3). Three subspecies have been defined
a broad range of food products, animal and human feces, and from A. hydrophila, that is, subsp. hydrophila, ranae, and dha-
other clinical and environmental samples. It is included in the kensis. However, A. hydrophila subsp. dhakensis is considered
class Gammaproteobacteria, order Aeromonadales, and despite a synonym of Aeromonas aquariorum, a species first identified
being originally placed in the family Vibrionaceae, it was later from aquarium water and ornamental fish, then later from
recognized to be different enough to merit its own independent a wide range of human extraintestinal infections, and very
family Aeromonadaceae. The first descriptions of Aeromonas recently from chironomid egg masses, from where they can
species date back to 1891 and 1894 when the bacteria Bacillus contaminate drinking water systems. The species A. salmonicida,
hydrophillus fuscus (now Aeromonas hydrophila) and Bacillus de mainly implicated in fish disease, has been divided into five
Forellenseuche (now Aeromonas salmonicida) were linked to subspecies (salmonicida, masoucida, smithia, achromogenes, and
diseased frogs and trout, respectively. The formal description of pectinolytica), which are very difficult to differentiate.
the genus, by Stainer, was not made until 1943. The majority of On the basis of their phenotype, the species Aeromonas veronii
Aeromonas are mesophilic (they grow at 35–37
C), motile and has been divided into two biovars (bv. sobria and bv. veronii) that
nonpigmented, although one species, that is, A. salmonicida, cannot so far be distinguished genetically. Some species have
also includes nonmotile pigmented psychrophilic strains been synonymized with previously recognized species, such as
(optimum growth at 22–25
C). Aeromonas can be differenti-
ated from other closely related genera by several characteristics Table 1 Differential characteristics of Aeromonas
(Table 1).
In order to prevent confusion with other genera, a molec- Test Aeromonas Plesiomonas Vibrio
ular genus probe was developed in our laboratory that targets
the lipase glycerophospholipid-cholesterol acyltransferase gene Growth in: 0% NaCl þ þ –a
6% NaCl – – þ
(gcat). This lipase has been considered an important virulence
Resistance to 0/129b þ – –c
factor in Aeromonas strains, causing diseases in fish (Table 2). Fermentation of inositol – þ –
However, we now know that all the Aeromonas harbor the gcat
gene independent of their origin. Presumptive colonies can be þ, >90% positive; –, 0–10% positive.
a
Except Vibrio mimicus, Vibrio cholerae and Vibrio fluvialis.
confirmed as belonging to the genus by a gcat probe, by b
Vibriostatic agent (2,4-diamino-6,7-diisopropypteridine; 150 mg/disk).
amplifying this gene by PCR or by the amplification of the aroA c
Except V. cholerae serogroups 01 and 0139.

24 Encyclopedia of Food Microbiology, Volume 1 http://dx.doi.org/10.1016/B978-0-12-384730-0.00004-5

 AEROMONAS j Introduction 25

Table 2 Primers and PCR conditions for confirming strains as belonging to the genus Aeromonas

Gene (bp) Primer Conditions Reference

gcat (237) GCAT-F 50 - CTCCTGGAATCCCAAGTATCAG-30 95
C 3 min Chacón et al. (2002),a

GCAT-R 50 - GGCAGGTTGAACAGCAGTATCT-30 94
C 1 min
65
C 1 min  35
72
C 1 min
72
C 5 min
aroA (1236) PF1 50 -TTTGGAACCCATTTCTCGTGTGGC-30 94
C 2 min Naharro et al. (2010)
PR 50 -TCGAAGTAGTCCGGGAAGGTCTTGG-30 92
C 1 min
50
C 1 min  40
72
C 1 min
72
C 10 min
a
Previously described conditions are adapted for PCR reaction with an anealing temperature of 56
C.

Table 3 Species included in the genus Aeromonas.a–d Isolation from is from feces of patients with diarrhea, then from wounds, and
clinical and environmental samples finally from blood. The enteropathogenicity of Aeromonas has
been demonstrated by the production of an intestinal secretory
Species Year and origin of the type strain
immunoglobulin A in the intestinal mucosa in response to the
A. hydrophila a–d 1943/Milk exoproteins produced by strains of these bacteria. Three species
A. salmonicida a–d 1953/Salmon clearly predominate in clinical samples, which in our experience
A. sobria a,b,d 1976/Fish account for 90% of all clinical isolates when genetic identifica-
A. media a–d 1983/Water tion methods have been applied, that is, Aeromonas caviae (49%),
A. veronii a–d 1987/Sputum A. veronii (34%), and A. hydrophila (7%). A. veronii (bv. sobria) is
A. caviae a–d 1988/Guinea pig clearly the species of clinical importance rather than Aeromonas
A. eucrenophila a–d 1988/Freshwater fish
sobria that is frequently mentioned in the literature. In fact,
A. schubertii a,d 1988/Skin abscess
A. sobria sensu stricto has seldom been isolated from clinical
A. jandaei a,b,d 1991/Human feces
A. trota a 1991/Human feces samples but is a typical environmental species frequently
A. allosaccharophila a,c,d 1992/Eel recovered from diseased fish (especially trout).
A. encheleia a,d 1995/Eel The recent discovery of new clinical species like
A. bestiarum a–d 1996/Diseased fish A. aquariorum (misidentified as A. caviae or A. hydrophila using
A. popoffii a,b 1997/Drinking water biochemical methods) has changed the mentioned prevalence
A. simiae c 2004/Monkey feces of the species. For instance, in a study carried out in Australia
A. molluscorum d 2004/Bivalve mollusks that sequenced the rpoD and gyrB genes, A. aquariorum was the
A. bivalvium d 2007/Bivalve mollusks most frequently isolated species in clinical and water samples.
A. tecta a,d 2008/Child feces
Furthermore, we sequenced the rpoD in our laboratory from
A. aquariorum a–d 2008/Ornamental fish
138 biochemically identified Aeromonas recovered from human
A. piscicola d 2009/Diseased salmon
A. fluvialis b 2010/River water extraintestinal infections in Taiwan, and 73 (52.9%) corre-
A. taiwanensis a 2010/Wound infection sponded to A. caviae, 22 (15.9%) to A. aquariorum, 17 (12.3%)
A. sanarellii a 2010/Wound infection to A. hydrophila, 16 (11.6%) to A. veronii, 5 (3.6%) to Aeromonas
A. diversa a 2010/Leg wound media, 3 (2.1%) to A. trota, 1 (0.7%) to Aeromonas sanarellii, and
A. rivuli b 2011/Water rivulet 1 (0.7%) to Aeromonas taiwanensis.
a Other less prevalent clinical species are A. veronii (bv. ver-
Clinical samples (the most prevalent species in bold).
b
Water. onii), Aeromonas jandaei, Aeromonas schubertii, Aeromonas bestia-
c
Meat. rum, A. salmonicida, Aeromonas eucrenophila, Aeromonas encheleia,
d
Fish and seafood.
A. allosaccharophila, Aeromonas popoffii, and A. sobria, but the real
prevalence of these species is not known. Again, there is a lot of
Aeromonas ichthiosmia and Aeromonas culicicola with A. veronii and inaccurate information in the clinical literature due to the
Aeromonas enteropelogenes with Aeromonas trota, and the species erroneous biochemical identification of the species. Therefore,
A. sharmana has been recognized as not belonging to the genus speculation about the predominance of phenotypically iden-
Aeromonas. However, some of these names are still being reported tified A. sobria or A. hydrophila or about their virulence, antibi-
in the literature because these are the names of 16S rRNA gene otic resistance, and clinical characteristics has to be regarded as
sequences deposited at the GenBank. unreliable.

Clinical Relevance
Nowadays, Aeromonas are considered to be the etiological agents
of several infections that can occur in both immunocompetent
and immunocompromised people. The most common isolation
Molecular versus Phenotypic Identification
Numerous biochemical schemes have been proposed for the
characterization of Aeromonas species, and even though some
26 AEROMONAS j Introduction
may include a large number of biochemical tests they are not
accurate and produce a lot of errors. Comparison of the results
obtained using genetic identification methods with those
using commercial identification systems (i.e., API20E, Vitek,
BBL Crystal, and MicroScan W/A) and/or conventional
biochemical methods has revealed that the latter two can
identify erroneously up to 70% of the strains as belonging to
A. hydrophila. As a result, there is a clear bias toward an over-
estimation of the clinical and environmental importance
attributed to A. hydrophila, with a lot of studies concentrating
only on this species. For instance, the U.S. Food and Drug
Administration considers only A. hydrophila as an emerging
food borne pathogen of concern when, in fact, many other
species are more relevant. This overestimation has given rise to
the development of specific molecular methods that target
only this species in different types of food products, and to
many studies that evaluate its behavior and survival charac-
teristics under different conditions. Biochemical identification
is therefore not recommended for an accurate identification of
Aeromonas species.
The phylogeny of the genus Aeromonas was originally
studied in the early 1990s using the 16S rRNA gene, and at that
time this gene was already recognized as highly conserved.
Despite the extensive use of this gene for identification of
bacteria, it is not useful for the genus Aeromonas because species
share a high percentage of similarity (99.8–100%). For
instance, strains from the species A. salmonicida, A. bestiarum,
and Aeromonas piscicola can share identical 16S rRNA gene
sequences or have at most two nucleotide differences in the
complete gene (1503 bp). Short fragments of this gene
(200–500 bp), routinely used for identification of many
bacteria or microbial communities, are totally unreliable for
identification of Aeromonas species (i.e., strains identified as
A. sobria were shown to belong to a new species Aeromonas rivuli
or to A. encheleia). Other housekeeping genes that codify
essential proteins for the survival of the bacteria, such as gyrB
(b-subunit DNA gyrase) or rpoD (s70 factor), have a higher
resolution than the 16S rRNA gene and are therefore more
useful for separating Aeromonas species. The phylogenetic
analysis of the genus using the concatenated sequences of seven
genes (gyrB, rpoD, recA, dnaJ, gyrA, dnaX, and atpD) was pub-
lished very recently as well as the first open-access multilocus
sequence typing scheme (http://pubmlst.org/aeromonas).
Many molecular methods (different from sequencing) have
been proposed for identifying Aeromonas spp., but they target
only a few species and have rarely been validated by comparing
the results with those of other bonafide molecular methods. The
restriction fragment length polymorphism (RFLP) of the aroA
gene has been used for identifying food strains (Table 2). It
uses the HaeII nuclease and generates species-specific patterns
for 11 Aeromonas spp. An RFLP method of the 16S rRNA gene
described by our group recognizes the 14 species described in
the genus up to the year 2000. The 16S rRNA-RFLP method
involved an initial digestion of 1503 bp of the 16S rRNA
amplified gene with two restriction enzymes (AluI þ MboI) and
a comparison of the obtained banding patterns with those
defined as specific for 10 species or as a common pattern for
A. salmonicida, A. bestiarum, A. encheleia, and A. popoffii. The
differentiation of these species required further digestions with
other enzymes. However, the addition of 11 new species in the
genus since 2000 limits the usefulness of this method because
the same patterns are obtained for some species (i.e.,
A. aquariorum and A. caviae), even though new species-specific
RFLP patterns have been obtained for the recently described
species Aeromonas tecta, Aeromonas molluscorum, Aeromonas
simiae, and A. rivuli. This RFLP method has been applied in
several studies, enabling the authors to recognize several new
species and mutations in the 16S rRNA gene. For instance, it
was noticed that the prevailing Aeromonas species to which 82
isolates from frozen freshwater fish (Tilapia) sold in markets in
Mexico D.F. belonged were A. salmonicida (n ¼ 52), A. bestiarum
(n ¼ 16), A. veronii (n ¼ 4), A. encheleia (n ¼ 3), and
A. hydrophila (n ¼ 2). It was also observed that five strains (6%)
did not belong to the genus, despite being considered so when
they were identified with biochemical methods.
There can be a lot of confusion when using commercial
identification systems. The Vitek-GNI system identified 81
strains isolated from the intestines of catfish (Ictalurus puncta-
tus) collected from different geographical regions of the United
States as A. hydrophila (n ¼ 23), A. trota (n ¼ 7), A. veronii
(n ¼ 42), A. caviae (n ¼ 6), and A. jandaei (n ¼ 3). However,
when they were evaluated with the mentioned 16S rDNA-RFLP,
all the 81 strains were identified as A. veronii. In that instance,
the errors in phenotypic identification were masking the
importance that the species A. veronii may have in catfish
pathology and the possible implications this may have for
human health, considering that this is a very common species
associated with human disease.
In a more recent study, the results of the 16S rDNA-RFLP
identification method for 90 strains recovered from fish and
shellfish were verified by sequencing the rpoD gene. The only
strains which could not be differentiated by the 16S rRNA gene
were those belonging to A. bestiarum, A. salmonicida, and the
new species discovered in this study, A. piscicola as they shared
99.8–100% similarity. Furthermore, it was recognized that
under the phenotypically identified strains of the species
A. hydrophila eight other species were uncovered (i.e., A. sobria,
A. media, A. bestiarum, A. piscicola, A. salmonicida, A. eucrenophila,
A. caviae, and A. tecta).
One of the most complete studies whose methodological
approach can be used as a model is one that used gyrB gene
sequences for the identification of Aeromonas species recovered
from pork and from abattoir environments in Portugal. The
species that they found, in order of prevalence, were A. media
(23.1%), A. hydrophila (19.8%), A. salmonicida (19.8%),
A. allosaccharophila (14%), A. veronii (9.9%), A. caviae (8.3%),
A. bestiarum (3.3%), A. aquariorum (0.8%), and A. simiae
(0.8%). In fact, this is the first report of the species A. simiae
since its description in 2004. Among the species isolated in
these slaughterhouses were the clinically relevant species
A. media, A. hydrophila, A. veronii, A. caviae, and A. aquariorum.
Applying genotyping techniques, they were able to trace the
origin of some of the Aeromonas strains within the abattoir,
which will be discussed later.
We recommend laboratories that are unable to properly
identify the strains by molecular methods to refer their isolates
as Aeromonas spp. If not, they should send their strains to
reference laboratories before publication to ensure reliable
identification by sequencing the rpoD or gyrB genes. In our
view, this is the only way to clarify the true relevance of the
 AEROMONAS j Introduction 27

different Aeromonas species in different types of foods (infor- (up to 80% of cultivated trout) so-called carrier fish, meaning
mation that is largely missing at present). This will help to that fish do not show external lesions or clinical signs of disease
prevent the continual bias toward A. hydrophila that is obvious but are indeed able to shed Aeromonas at high concentrations
at present in the literature. (105–106 CFU per fish per hour). These carriers increase the
likelihood of transmission of the microbe to other susceptible
Aeromonas in Water and Their Interaction with Food fish or to humans. In fact, 20% of ready-to-eat salted herring
samples have contained Aeromonas, as frequently has marinated
Aeromonas are considered inhabitants of mainly freshwater
raw fish. Despite the common belief that the Aeromonas spp. that
environments (lakes, rivers, and reservoirs) although they have
cause diseases in fish (i.e., A. salmonicida) do not infect humans,
been isolated from chlorinated and unchlorinated drinking
the latter species have in fact been isolated from human samples
water systems, bottled water, and swimming pools as well as
and recently linked to an episode of peritonitis in a 68-year-old
reused water, brackish waters, and seawater. The presence of
diabetic woman, who had to be treated by continuous ambu-
Aeromonas in drinking water systems is linked to poor main-
latory peritoneal dialysis after she ate fish. In fact, many cases of
tenance, characterized by low levels of disinfectant (chlorine,
Aeromonas diarrhea or bacteremia have been linked to having
etc.), high concentrations of organic matter, and presence of
eaten raw fish or shellfish on the preceding days.
biofilms. Relatively high amounts of biodegradable organic
Furthermore, several species linked to human clinical
carbon, together with warm temperatures and low chlorine
samples have been recovered from both diseased and healthy
residual concentrations, can allow Aeromonas and other
fish. Strains of A. veronii have been recovered from appar-
microorganisms to multiply during drinking water storage and
ently healthy freshwater fish (tilapia) that had been sold
distribution. Some countries like the Netherlands have estab-
frozen in markets in Mexico D.F. or together with catfish in
lished quality standards of 20 colony-forming units (CFUs)
the United States, as have other species such as A. encheleia,
100 ml1 of Aeromonas for finished water and 200 CFU
A. allosaccharophila, A. jandaei, A. media, A. eucrenophila,
100 ml1 for the water in the distribution system. Drinking
A. aquariorum, and A. tecta.
water systems are affected by seasonal variations, with higher
numbers of Aeromonas being present in the warmer months.
This is coincidentally the time when cases of gastroenteritis and
septicemia attributed to this microbe are generally higher.
Epidemiology
The Aeromonas species prevalent in drinking water have
As commented before, ingestion of contaminated water or food
commonly been considered different from those found in
(meat, fish, and bivalves such as oysters and mussels) are
clinical cases, but recent studies have shown the most prevail-
considered the principal routes of Aeromonas transmission. An
ing species identified molecularly to be A. veronii, followed by
infectious level of Aeromonas in humans, based on the limited
A. salmonicida, A. hydrophila, A. media, and A. jandaei. Free
oral exposure studies, ranges from 103 to 109 CFU g1, but
chlorine residuals in these samples were between <0.05 and
some strains might also have a lower infective level in immu-
1.5 ppm. Also, recently the same strains have been found in
nocompromised people or children. In fact, it has been found
both drinking water and in cases of diarrhea.
that meat and fish suspected of being responsible for a gastro-
Aeromonas can easily enter the food chain through
enteritis outbreak had concentrations of 106–107 bacterial cells
contaminated water (through irrigation in the case of vegeta-
per gram of food product.
bles or washing) or during many of the ‘farm-to-table’
It has recently been reported that the exact global incidence
manipulation processes. Many products sold in supermarkets
of Aeromonas infections is unknown because it is not manda-
and stores, such as meat, milk, cheese, ready-to-eat foods,
tory to report these infections. Despite that, based on data
salads, vegetables, fish, and shellfish, have been found to
collected from 219 patients over a 12-month period in
contain aeromonads, and in most cases water was considered
California, the overall incidence of Aeromonas infections was
the source of contamination. Flies and mosquitoes can also be
estimated to be 10.6 cases per million of the population.
mechanical and biological vectors, and the feces of pets like
However, this seems relatively low considering that many
dogs, cats, and horses can be additional reservoirs of Aeromonas,
studies worldwide on the incidence of Aeromonas in diarrhea
as can soil, chironomid egg masses, and plankton because this
cases estimate it at around 2% (the most coincidental value).
bacterium has been recovered from all these origins. Vegetables
This incidence is also similar to that found in cases of Aero-
that are eaten raw have been reported to contain Aeromonas in
monas traveler’s diarrhea.
26–40% of samples, meat such as lamb, veal, pork, poultry,
Suspected food borne Aeromonas disease outbreaks have
and ground beef have been found to be positive in 3–70% of
mainly been linked to raw fish and seafood such as prawns,
the cases, and those from shellfish (31%) and fish (72%)
oysters, and shrimp, and may affect just a few or many people
reportedly contain the most positive samples. However, in
and have incubation periods as short as 24 h. It is in the
most of these studies the majority of Aeromonas were recovered
investigation of such outbreaks that molecular fingerprinting of
after an enrichment step, which indicates that concentrations
Aeromonas isolates is required to determine strain relatedness.
must have been relatively low.
Random amplified polymorphic DNA (RAPD), enterobacterial
repetitive intergenic consensus (ERIC) sequences, pulsed-field
Aeromonas and Fish
gel electrophoresis (PFGE), or amplified fragment length
Members of the genus Aeromonas are considered important polymorphism (AFLP), among others, have all been tested in
pathogens of fish, and therefore fish can be an important reser- Aeromonas as well as MLST approaches. A comparative study
voir of these microbes. It has been estimated that there are many using several of these typing methods has revealed that ERIC
28 AEROMONAS j Introduction

Table 4 Major virulence factors in the genus Aeromonas

Virulence factor (genes)a Function

Filamentous adhesins
Polar flagella (flaA,B,G,H,i, maf-1, fliA) Swimming and swarming motility, enhances adherence, biofilm formation, host attachment,
Lateral flagella (lafA-U, maf-5, flhAL, fliU) and cellular invasion.
Pili or fimbriae types I–IV (tapA-D, flp)
Nonfilamentous adhesins
Capsule (kpsC,D,E,M,S,T) Enhances resistance to nonspecific immune defenses.
A or S-layerb (vapA) Protects the bacteria against the host defense.
Lipopolysaccharide (wahA-F, waaA,C,E,F) Responsible for the inflammatory activity.
Extracellular products
Cytotoxic enterotoxin (act) Inhibits the host’s phagocytic activity, produces hemolysis and increases levels of necrosis
tumoral factor-a, interleukine 1b, etc.
Cytotonic enterotoxins (alt, ast) Increases the level of cAMPc and prostaglandins in the intestinal mucosa.
Hemolysins (aerA, hylA) Creates pores in host cell membranes and lysis.
Proteases (tagA) and lipases (gcat, pla) Facilitates invasion and tissue damage.
Shiga-like toxins (stx1, stx2) Inactivates ribosomes (stops protein synthesis) of the vascular endothelium cells, leading to
cell death.
Secretion systems
T2SS (exeA, exeB, exeD) Secretion of amylases, proteases, aerolysine, etc.
T3SS (ascF-G, ascV) and T6SS (vasH, vasK) Injection of effector proteins into the host cells.
T4SS (traA-K, VirB1-11, VirD) Delivery of proteins and DNA into the host cells acting as a bacterial conjugation system.
a
Only some genes are listed.
b
It is an outer membrane protein.
c
cAMP (Cyclic adenosine monophosphate).

has a better discriminatory power than PFGE and AFLP when
those methods were applied to a study of A. popoffii strains. In
fact, earlier studies based on ribotyping found that some
oysters were responsible for an outbreak of Aeromonas. The
same PFGE pattern was revealed recently from Aeromonas
strains recovered from drinking water and from the feces of
patients with diarrhea.
Genotyping methods have been used to compare the
isolates recovered from pigs at slaughterhouses in the north of
Portugal and one strain of A. caviae isolated from pig feces was
recognized to share an identical ERIC pattern of a strain
obtained from the floor at the same slaughterhouse. Also,
strains of A. hydrophila, showing the same genetic patterns, were
isolated from diaphragm muscle and pig feces collected on the
same day. This suggested a direct role of feces in the contami-
nation of the meat. Applying these genotyping methods can
therefore be very useful for tracing such food-chain contami-
nation problems and may be relevant for the Hazard Analysis
of Critical Control Point assessment.
Virulence Factors
Like many other bacteria, aeromonads possess many virulence
factors that participate in infecting the host tissues that can be
classified into three groups: (1) structural components, such as
filamentous adhesins (i.e., flagella) and fimbriae and nonfila-
mentous adhesins (i.e., lipopolysaccharide, capsule, and outer
membrane proteins), (2) extracellular proteins (exoenzymes or
exotoxins), such as cytotoxic and cytotonic enterotoxins,
hemolysins, lipases, proteases that may pose a health risk due
to their capacity to produce tissue damage, infection, spoilage
of food, or intoxication, and finally (3) some of the six secre-
tion systems (T1SS–T6SS) that have been molecularly
characterized in Gram-negative bacteria, that is, types II (T2SS),
III (T3SS), IV (T4SS), and VI (T6SS) specialized in delivering
specific toxins into the host cells. The principal genes encoding
these factors as well as their virulence-associated properties are
summarized in Table 4.
The virulence properties associated with the mentioned
secretion systems and with the other factors listed in Table 4 have
been demonstrated, generating mutant strains for the implicated
genes and comparing their behavior with the wild-type strains.
For instance, mutant deficient strains for a structural gene (ascV)
of the T3SS have been shown to be less toxic or virulent in
different cell lines. Mutated strains for two genes (vasH, vasK)
encoding components of the T6SS were also less toxic to murine
macrophages and human epithelial cells than the nonmutated
strains. There has been a dramatic decrease in the adherence,
biofilm formation, and invasive ability of mutated strains for
some of the genes encoding the polar (flaA, flab, flaH, maf-1, fliA,
among others) and lateral (lafA1, lafA1, lafK, maf-5, flhAL, fliU,
among others) flagella. In addition, a mutant for the Act cyto-
toxic enterotoxin encoded by the act gene did not cause any
damage to the small intestinal epithelium of mice, whereas the
wild-type strain caused complete destruction of the microvilli.
A typical food borne disease linked to the consumption of
contaminated meat products is the hemolytic uremic syndrome
(HUS), which causes hemolytic anemia, thrombocytopenia,
and acute renal failure due to the production of Shiga toxins
(Stx1 and/or Stx2). HUS normally occurs after a gastrointes-
tinal infection produced by a Shiga-toxin-producing Escherichia
coli (O157:H7). However, cases attributed to Aeromonas have
also been described, and the genes involved (stx1 and stx2)
have been detected in clinical and environmental strains of
A. hydrophila, A. caviae, and A. veronii and were sequenced for
the first time. The stx genes found in Aeromonas were highly
homologous to those of the most virulent variants of E. coli, but

in Aeromonas these toxins are situated in plasmids, which tend
to be lost after regrowth of the strains in the laboratory, making
it very difficult to detect them.
The expression of many virulence factors (like the T3SS or
the Act toxin) is believed to be regulated by signal molecules
(the N-acylhomoserine lactones (AHLs)) produced by the
bacteria in response to their density through a phenomenon
defined as quorum sensing (QS). When the concentration of
the signal molecules reaches a limit (a minimum population
size), it induces the expression of certain genes like the earlier
mentioned virulence genes. It has been indicated that
compounds present in the food matrix and/or its storage
environment can influence the production of AHLs involved in
the QS phenomena. Several studies have used simulated food
culture agar media to find out if Aeromonas strains can produce
AHL molecules in similar conditions to those found in the real
food product. Regarding that, AHL production was observed in
simulated shrimp and fish agar media but practically none in
simulated vegetable media. Plant products (such as plant
exudates from the pea plant, Pisum sativum) that mimic bacte-
rial AHL-like activities and that can alter the dependent
behavior of cell density have been considered responsible for
false positive results obtained with the vegetable media. Further
insights into the ability of Aeromonas strains to produce AHLs in
food products or their conditions for preservation might lead to
a better understanding of the role of bacteria in food.
Virulence Genes and Complete Genomes
Complete genomes are now available for five Aeromonas species
– two strains of A. salmonicida (A449 and 01-B526) isolated
from diseased rainbow trout and infected brook trout, the type
strain of A. hydrophila (ATCC 7966T) isolated from milk, a strain
of A. caviae (Ae398) isolated from stool samples of a child,
a strain of A. veronii (B565) isolated from aquaculture pond
sediment, and a strain of A. aquariorum (AAK1) isolated from
blood of a cirrhosis patient. Comparative analyses enabled the
presence and functionality of known Aeromonas virulence genes
(like the T3SS, etc.) to be verified and new potential virulence
genes (like the T6SS) to be discovered. In comparison with the
others, the genome of A. salmonicida shows many insertions and
pseudogenes that are suggested to be the result of its evolution
and adaptation to a very specific host (fish). On the other hand,
the genome of A. hydrophila (ATCC 7966T) shows genes
encoding a greater diversity of metabolic pathways, reflecting
the versatility of this bacteria for living in a variety of different
environments, including aquatic ecosystems, foods, and a wider
range of hosts (i.e., humans, animals).
Detection of Virulence in Food Isolates
Many studies in recent years have investigated the presence of
some of the virulence genes mentioned in Table 4 in strains of
Aeromonas recovered from several food products using PCR
assays, while fewer studies have phenotypically evaluated
proteolytic, hemolytic, and cytotoxic behavior. The most
commonly sought genes by PCR have been aerA (aerolysin), hylA
(hemolysin), ast, alt (cytotonic enterotoxins), act (cytotoxic
AEROMONAS j Introduction 29
enterotoxin), and to a lesser extent those encoding elastase
(ahyB), GCAT, DNase, serine protease, or the T3SS. Many
conclusions about the virulence of the studied strains have been
derived from results on the presence or absence of the investi-
gated genes. However, these studies have serious limitations.
PCR assays can produce false negative reactions due to inhibi-
tion or to the presence of variability in the targeted DNA
sequence, but these possibilities are rarely taken into consider-
ation in the interpretation of the results. Furthermore, when
a positive reaction is obtained, the presence of the genes alone
does not guarantee its expression in vivo. In addition, the pres-
ence and expression of the virulence genes can be strain
dependent or influenced by the host and/or temperature
conditions (such as when evaluating the b-hemolysis). Several
reports indicate that clinical isolates express virulence traits more
frequently at 37
C, while isolates from food do this at refrig-
eration temperatures (2–10
C). Other studies report no differ-
ences between clinical and food Aeromonas strains in the
expression or presence of genes. In general, these types of studies
simply confirm the relative frequency of the studied genes, but
there is no evidence of their real implication in the development
of the infection. Despite that, it has been found that Aeromonas
strains recovered from food products can harbor and/or express
a high number of virulence genes, this reinforcing the potential
of this bacteria as a human pathogen. It is evident that some
aeromonad strains within certain species have true entero-
pathogenic potential in humans and that a high concentration
and prevalence of these pathogenic bacteria in ready-to-eat food
products can be a threat to public health. It would therefore be
advisable to control the density of these microbes both in food
production and in drinking water supplies.
Preservation and Control
Aeromonas are active spoilers of minimally processed food
products such as vegetables, fish, and meat, and the strains
recovered can express virulence factors even at low refriger-
ation temperatures. In fact, the majority of the members of
the genus have an optimal growth temperature similar to
mesophilic microbes (28–30
C), but most are capable of
acting as psychrophilic bacteria surviving and multiplying at
the lower refrigeration temperatures (2–10
C), highlighting
the importance of monitoring the presence of Aeromonas in
the cold chain. The number of Aeromonas in food products
can range from 102 to 105 CFU g1, but they can survive and
grow to higher numbers (increasing 10–1000 fold) during
7–10 day storage at 5
C. It has also been shown that they
can grow slowly at 0
C or even at temperatures as low
as 3
C.
Members of the genus Aeromonas are also able to survive
under other preservation measures such as vacuum package,
packaging under modified atmospheres, and high salt
concentrations. The recent use of vacuum and modified pack-
aging extends the storage time of many food products and
assures their safety in most cases. For instance, packing of the
pearlspot fish (Etroplus suratensis), a popular brackish water fish
species from India, in a modified atmosphere containing 60%
CO2/40% O2 has inhibited the growth of Aeromonas and other
bacteria and has extended the life of the product. On the other
30 AEROMONAS j Introduction
hand, low levels of these bacteria have been isolated from
vacuum-packaged fresh pork.
Sodium chloride is a common food preservative for raw
meat and fish products. Even though Aeromonas are generally
considered to be unable to grow at 6% NaCl (though they do
grow from 0 to 4% NaCl), some strains have been shown to
be tolerant to this concentration, reaching levels of 105 viable
bacterial cells per ml when measured by flow cytometry.
Using this latter technique, from an initial inocula of 107 cells
per ml in nutrient broth containing 6% NaCl, it has been
observed that the number of viable cells remains stable and
relatively high (104–105 cells per ml) after storage for 188
days at 4
C and at 24
C. However, after that time only
103 CFU ml1 of the cells that proved to be viable by flow
cytometry at 4
C and 10 CFU ml1 at 24
C were recovered in
culture. These results indicate that at 6% NaCl concentration,
the temperature affects the ability of the cells to grow on solid
media but does not affect their viability. The physiological
adaptation of bacterial cells to high NaCl concentrations has
been linked to modification of the transport of Naþ ions
across the bacterial cell membrane. It seems that higher
temperatures hamper this physiological adaptation and
therefore enhances the permeability of the membrane to Naþ,
thus inducing cytotoxicity of the bacteria and inhibiting their
growth. Several studies have reported that these two impor-
tant factors limiting bacterial growth, that is, low temperature
and high salt concentration, do not always reduce the viability
of Aeromonas and cannot always assure safety, especially in the
case of high levels of this bacteria contaminating food
products.
Another way to control bacterial pathogens in food is to
lower the pH, such as use of lime juice to prepare raw fish
dishes (such as ceviche). However, it has been demonstrated
that a pH of 5 (obtained with lime juice) is not sufficient to kill
or even to reduce Aeromonas counts.
Radiation has been shown to be another effective method
for eliminating food borne pathogens. Gamma radiation has
a high penetration power and can inactivate pathogens that
may have entered the tissues of vegetables, meat, or fish flesh. It
has been proven that radiation treatment with a 1.5 kGy dose
has completely eliminated 105 CFU g1 of Aeromonas spp. from
mixed sprouts, chicken, and fish samples.
Aeromonas are also able to colonize and/or form biofilms on
food surfaces and drinking water distribution systems. In the
latter, it has been demonstrated that single strains seem to
dominate these bacterial populations. Eliminating and
controlling aeromonad numbers in a distribution system that
contains biofilms can take some time and needs concentrations
of chlorine of 0.2 mg l1.
See also: Aeromonas: Detection by Cultural and Modern
Techniques; Classification of the Bacteria: Traditional;
Biochemical and Modern Identification Techniques: Food-
Poisoning Microorganisms; Chilled Storage of Foods: Use of
Modified-atmosphere Packaging; Shellfish Contamination and
Spoilage; Water Quality Assessment: Routine Techniques for
Monitoring Bacterial and Viral Contaminants; Water Quality
Assessment: Modern Microbiological Techniques.
Further Reading
Beaz-Hidalgo, R., Figueras, M.J., 2012. Molecular detection and characterization of
furunculosis and other Aeromonas fish infections. In: Carvalho (Ed.), Health and
Environment in Aquaculture. InTech, Brazil, pp. 97–132. (http://www.intecho-
pen.com/articles/show/title/updated-information-of-aeromonas-infections-and-
furunculosis-derived-from-molecular-methods-).
Chacón, M.R., Castro-Escarpulli, G., Soler, L., Guarro, J., Figueras, M.J., 2002. A DNA
probe specific for Aeromonas colonies. Diagnostic Microbiology and Infectious
Disease 44, 221–225.
Chopra, A.K., Graf, J., Horneman, A.J., Johnson, J.A., 2009. Virulence factor-activity
relationships (VFAR) with specific emphasis on Aeromonas species. Journal of
Water and Health (7 Suppl. 1), S29–S54.
Edberg, S.C., Browne, F.A., Allen, M.J., 2007. Issues for microbial regulation: Aero-
monas as a model. Critical Reviews in Microbiology 33, 89–100.
Figueras, M.J., 2005. Clinical relevance of Aeromonas. Reviews in Medical Microbi-
ology 16, 145–153.
Figueras, M.J., Soler, L., Chacón, M.R., Guarro, J., Martínez-Murcia, A.J., 2000. Use
of restriction fragment length polymorphism of the PCR-amplified 16S rRNA gene
for the identification of Aeromonas spp. Journal of Clinical Microbiology 38,
2023–2025.
Figueras, M.J., Beaz-Hidalgo, R., Collado, L., Martínez-Murcia, A.J., 2011. Point of
view on the recommendations for new bacterial species description and their
impact on the genus Aeromonas and Arcobacter. The Bulletin of Bergey’s Inter-
national Society for Microbial Systematics 2, 1–16.
Fontes, M.C., Saavedra, M.J., Martins, C., Martínez-Murcia, AJ., 2011. Phylogenetic
identification of Aeromonas from pigs slaughtered for consumption in slaughter-
houses at the north of Portugal. International Journal of Food Microbiology 146,
118–122.
Janda, J.M., Abbott, S.L., 2010. The genus Aeromonas, taxonomy, pathogenicity, and
infection. Clinical Microbiology Reviews 23, 35–73.
Martin-Carnahan, A., Joseph, S.W., 2005. Family I. Aeromonadaceae Colwell,
MacDonell and DeLey 1986. In: Brennan, D.J., Krieg, N.R., Staley, J.T.,
Garrity, G.N. (Eds.), Bergey’s Manual of Systematic Bacteriology, second ed., vol.
2. Springer-Verlag, New York, pp. 556–578.
Martínez-Murcia, A., Morena, A., Saavedra, M.J., et al., 2011. Multilocus phylogenetic
analysis of the genus Aeromonas. Systematic and Applied Microbiology 34,
189–199.
Naharro, G., Riaño, J., de Castro, L., Álvarez, S., Luengo, J.M., 2010. Aeromonas. In:
Dongyou, L. (Ed.), Molecular Detection of Foodborne Pathogens. CRC Press, Boca
Raton, FL, pp. 273–287.
Pablos, M., Huys, G., Cnockaert, M., et al., 2011. Identification and epidemiological
relationships of Aeromonas isolates from patients with diarrhea, drinking water and
foods. International Journal of Food Microbiology 147, 203–210.