Aeromonas

TAXONOMY Back to top

The genus Aeromonas resides within the family Aeromonadaceae (16) and the newly

proposed orderAeromonadales, ord. nov., along with the

genera Oceanimonas and Tolumonas (45). Aeromonas is the only one of these three genera

that is pathogenic for humans. The use of frequent reclassifications and constant amended or

extended descriptions within Aeromonas taxonomy can often be initially puzzling to

microbiologists not working with these organisms on a daily basis. However, information in

this chapter should clarify the identification and significance of those species most often

associated with human disease (Table 1). DNA hybridization group numbers, which no longer

serve a meaningful purpose, and synonymous species designations for Aeromonas

veronii bv. sobria (A. ichthiosmia) and A. trota (A. enteropelogenes) (15) are not included,

for simplicity. Aeromonas group 501, which is made up of A. schubertii-like organisms,

and Aeromonassp. DNA hybridization group 11 (47), which is made up of A. eucrenophila/A.

encheleia-like organisms, are also not addressed in the table. These groups contain few

strains, their taxonomic status has yet to be resolved and is still highly debated, and most

importantly, neither group has been shown to be significant in human or animal disease.

Newly proposed Aeromonas species and subspecies since the publication of the previous

edition of this Manual include A. bivalvium sp. nov., isolated from bivalve mollusks (51); A.

tecta sp. nov., isolated from both clinical and environmental sources (19); A. piscicola sp.

nov., isolated from diseased fish (8); and A. aquariorum sp. nov., isolated from aquaria of

ornamental fish (49). However, there is controversy surrounding the proposal of A.

aquariorum, since this new species appears to be both phenotypically and genetically

identical to A. hydrophila subsp. dhakensis, proposed in 2002, and isolated from cases of

children with diarrhea in Bangladesh (46). Comparative studies between the two laboratories

are under way to try to resolve this taxonomic dilemma.

Because of its clinical significance, clinical strains formerly referred to as A. sobria are, in

fact, A. veronii bv. sobria (esculin hydrolysis and ornithine decarboxylase negative and

arginine dihydrolase positive) and should be reported as such. Nearly all rapid identification

databases, excepting API 20E strips (bioMerieux, Inc., Durham, NC), have converted their A.

sobria identifications to A. veronii bv. sobria. This is especially important because of A.

veronii bv. sobria ’s association with more severe, extraintestinal infections, such as

septicemia, meningitis following leech therapy, and disseminated intravascular gas

production (56, 67). It usually is not necessary to definitively separate members of the A.

hydrophila complex (A. hydrophila, A. bestiarum, and A. salmonicida) or the A.

caviae complex (A. caviae, A. media, and A. eucrenophila), especially when they are isolated

from feces (see “Evaluation, Interpretation, and Reporting of Results” below).

The type strain Aeromonas hydrophila subsp. hydrophila ATCC 7966 was the first aeromonad

to be completely sequenced, annotated, published, and deposited in GenBank (as CP000462)

(66). This was followed just recently by the publication of the complete genome sequence

of Aeromonas salmonicida subsp. salmonicidaA449, an agent of furunculosis (a bacterial

septicemia of salmonid fish), which was deposited in GenBank as NC 00938. Comparing this

aeromonad genome with the A. hydrophila ATCC 7966T genome, which has one

chromosome, showed that the A449 A. salmonicida genome harbored one chromosome and

two large plasmids, carried multiple inversions in the chromosome, and additionally had an

approximately 9% difference in gene content compared with the A.

hydrophila subsp. hydrophila ATCC 7966 type strain (60).

DESCRIPTION OF THE GENUS Back to top

Members of the genus Aeromonas are gram-negative facultative anaerobes that are straight,

coccobacillary to bacillary cells with rounded ends, 0.3 to 1.0 μm in diameter and 1.0 to 3.5

μm in length. They can occur singly, in pairs, or, rarely, in short chains. Most species are

motile by a single, polar flagellum with a 1.7-μm wavelength, but peritrichous flagella may

be formed on solid media in young cultures and lateral flagella occur in some species.

Aeromonads are usually oxidase positive and catalase positive and are generally resistant to

150 μg of the vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129). They are

chemoorganotrophic, displaying oxidative and fermentative metabolism of glucose. Acid, and

often acid with gas, is produced from many carbohydrates, especially glucose, and nitrate is

reduced to nitrite. A variety of exoenzymes such as arylamidases, amylase, DNase,

esterases, peptidases, proteases, chitinase, chondroitinase, and hemolysins are produced.

The main cellular fatty acids produced are hexadecanoic acid (16:0), hexadecenoic acid

(16:1), and octadecenoic acid (18:1). Human (mesophilic) strains grow between 10 and

42°C, but occasional isolates may be more active in some biochemical assays at 22 to 25°C.

Psychrophilic strains from fish and the environment (A. popoffii and A. salmonicida) seldom

grow above 37°C and preferentially grow at 22 to 25°C. In brain heart infusion broth at

28°C, growth occurs between pH 4.5 and 9.0 and at salt concentrations between 0 and 4%.

The mol% G+C of the DNA is 57 to 63%.

EPIDEMIOLOGY AND TRANSMISSION Back to top

Aeromonads are inhabitants of aquatic ecosystems worldwide such as groundwater,

reservoirs, and clean or polluted lakes and rivers. Aeromonas may also be found in marine

environments but only in brackish water or water with a low saline content.

Most Aeromonas species, particularly those associated with human infections, are found in a

wide variety of fresh produce, meat (beef, poultry, and pork), and dairy products (raw milk

and ice cream) (32). A.veronii bv. sobria is a symbiont in the gut of medicinal leeches, where

it may grow as a pure culture (26). Infections in frogs, pigs, cattle, birds, and marine

animals have also been reported (32).

Most clinical infections with aeromonads are related to an exposure to some type of aquatic

source, whether the clinical specimen is feces or extraintestinal, and, to a lesser extent, to

the ingestion of foods. The majority of studies have found a seasonal relationship between

the recovery of aeromonads from specimens and the warmer months of the year (37). This

is not surprising since the optimal temperature for the growth of mesophilic aeromonads

would be that occurring in the warmer months. This would therefore increase the likelihood

of recreational human exposure to these bacteria, thereby resulting in an increased risk of

colonization and/or infections with these indigenous aquatic microorganisms.

Since Aeromonas is not a reportable condition in the United States or in most other

countries, the true incidence of Aeromonas infections worldwide is not known. Estimates

from England/Wales and the United States for septicemia with aeromonads in 2004 revealed

an incidence of 1.5 per million population (34). However, any estimates of incidence would

most likely be an underestimation, particularly as relates to exposure through drinking

water.

CLINICAL SIGNIFICANCE Back to top

Aeromonas gastroenteritis ranges from an acute watery diarrhea (most common form) to

dysenteric illness to chronic illness. Stools from acute watery diarrhea are loose (take the

shape of their container), and erythrocytes and fecal leukocytes are absent. Accompanying

symptoms include abdominal pain (60 to 70%), fever and vomiting (20 to 40%), and nausea

(40%) (35). Infections are usually self-limiting, but children may require hospitalization due

to dehydration. A. caviae is the most common species associated with these infections,

and A. caviae infection can even mimic inflammatory bowel disease in children (74). A.

veronii bv. sobria strains may be associated with rare cholera-like disease characterized by

abdominal pain (60%) and fever and nausea (20%) (32). In dysenteric diarrhea resembling

shigellosis, patients suffer from severe abdominal pain and have bloody stools containing

mucus and polymorphonuclear leukocytes. About 10 to 15% of patients with either choleralike

or dysenteric diarrhea are coinfected with another enteric pathogen(s).

A comprehensive Bangladesh study found that the presence of loose stools or more severe

watery diarrhea was associated with Aeromonas strains possessing an alt gene (for a heatlabile

cytotonic enterotoxin) alone or both alt and ast (for a heat-stable cytotonic

enterotoxin), respectively (6). A large traveler’s diarrhea study in Spain found the

predominant species to be A. veronii bv. veronii and A. caviae (75). A third large study in

India found seven different species among hospitalized patients with diarrhea, with A.

caviae predominating, followed by A. hydrophila and A. veronii bv. sobria, along with the

presence of the alt and ast genes as well as the act gene, which encodes a well-established

cytotoxic enterotoxin often present in clinical aeromonad isolates (68).

Finally, in a large acute diarrheal outbreak in Brazil that involved 2,170

cases, Aeromonas was the species that was recovered in 19.5% of those cases (28).

Although most diarrheal cases are generally self-limited, a combination of supportive therapy

and antimicrobials are often indicated in the pediatric, geriatric, and immunocompromised

populations (35). A 2007 article gives a nice summary of the latest data and theories related

to the association of Aeromonas with diarrhea (21).

Complications from Aeromonas diarrheal disease include hemolytic-uremic syndrome (9, 20)

and kidney disease requiring kidney transplantation (23). These more severe infections are

usually associated with A. hydrophila or A. veronii bv. sobria. Also, nonresolvable,

intermittent diarrhea can occur months after the initial infection and may persist for months

or several years.

Aeromonas can also be isolated from a variety of extraintestinal sites, although blood and

wounds are the most common sources. Aeromonas septicemia occurs rarely in

immunocompetent hosts; most cases are in patients with liver disease and hematological

malignancies and can be accompanied by necrotizing fasciitis (40, 41). The species more

commonly isolated from septicemia are A. hydrophila, A. veronii bv. sobria, and A.

jandaei. Wound infections are usually preceded by traumatic injury that occurs in contact

with water, where the predominant species is A. hydrophila. These infections range from

uncomplicated cases of cellulitis to myonecrotic infections with a poor prognosis (4, 52). Two

such scenarios are the reported outbreaks of wound infections with A. hydrophila associated

with mud football (73) and wound infections among both the 2004 Asian tsunami survivors

(44) and the 2005 Hurricane Katrina survivors in New Orleans, LA (59). Surveys indicate that

only 17 to 52% of Aeromonas wound infections are monomicrobic (35). Use of medicinal

leeches postoperatively to enhance blood flow to surgical sites has resulted in wound

infection rates of 20%, primarily with A. veronii bv. sobria (26, 65).

Other extraintestinal infections include ocular, respiratory, surgical, and urinary tract

infections; meningitis; osteomyelitis; cholecystitis; pneumonia; endocarditis; peritonitis;

portal pyemia; and pancreatic abscess (12,17, 18, 33, 42, 50, 71, 72). A few such examples

were the isolation of A. caviae from keratitis associated with contact lens wear (58) and

isolation of A. caviae and A. popoffii from separate cases of urinary tract infection (5,29).

The newest disease association with Aeromonas hydrophila is spa bath folliculitis, but this is

in keeping with the ubiquitous nature of this aquatic microorganism (54).

COLLECTION, TRANSPORT, AND STORAGE OF

SPECIMENS Back to top

Aeromonads survive well in specimens, and any of the widely used transport media are

acceptable for transport (Amies, Cary-Blair, modified Stuart’s, and buffered glycerol in

saline), with Cary-Blair generally considered to be the best (see chapter 16). Feces are

always preferable to rectal swabs for isolation of enteric pathogens, and stools should be

collected in the acute phase of disease. Most strains grow equally well at room temperature

(20 to 25°C) and incubator temperature (35 to 37°C). Because isolates being kept for longterm

storage do not survive well at room or refrigerator temperature in the laboratory for

long periods (>1 month), placing aeromonads in media, such as Trypticase soy broth with

30% glycerol, and deep freezing at -80°C is recommended for their long-term storage.

DIRECT EXAMINATION Back to top

The direct microscopic examination of wound or skin/superficial specimens or positive blood

culture specimens would be somewhat unremarkable, in that the presence of aeromonads

would be denoted as straight, gram-negative bacilli with or without the presence of white

cells, not unlike the presentation of a similar infection with either enterics or pseudomonads.

It is possible to rarely see somewhat elongated bacilli in blood or urine specimens with

aeromonads if the patient is undergoing antimicrobial therapy.

Although there have been several DNA probe and real-time PCR methods described for the

possible identification of aeromonads from either water, food, or veterinary sources, there

are no widely recognized antigen detection and/or nucleic acid detection methods available

for detection within clinical specimens.

ISOLATION PROCEDURES Back to top

Aeromonads generally grow well on a variety of enteric differential and selective agars,

although sucrose- and/or lactose-fermenting strains usually resemble nonpathogens on these

media. Blood agar with 20 μg of ampicillin per ml had previously been considered useful for

isolating all Aeromonas species; however, a substantial percentage (15 to 57%) of A.

caviae isolates are resistant to ampicillin, and certain species, like A. trota, are intrinsically

susceptible to ampicillin (10, 38). In fact, a recent environmental sampling study to detect

aeromonads showed that when ampicillin is used as a selective agent, a significant portion

(17.3%) of the aeromonad population, in at least some environments, could not be isolated

using such media (30). Therefore, laboratories should use caution when medium with

ampicillin is used in the setup of stool specimens for detecting the presence of all clinically

relevant aeromonad species as bacterial enteropathogens.

Modified cefsulodin-Irgasan-novobiocin (CIN) (4 μg of cefsulodin per ml, versus 15 μg/ml in

unmodified CIN) is also an excellent isolation medium for aeromonads. On this

medium, Aeromonas colonies have a pink center with an uneven, clear apron and are

indistinguishable from Yersinia enterocolitica morphologically. One can incubate CIN at 25°C

to enhance the recovery of Yersinia and still be able to recover Aeromonas within 24 h at this

temperature.

Aeromonas agar, available from Lab-M (http://www.lab-m.com), is a relatively new

alternative medium to CIN agar that uses D-xylose (which aeromonads do not ferment) as a

differential characteristic (7).

Since most clinically relevant species are beta-hemolytic, including an increasing number

of A. caviae strains, beta-hemolytic colonies on blood agar should be screened with oxidase

and a spot indole test. Any colonies positive by both tests should be characterized further,

although occasional indole-negative A. caviae and nearly all known A. schubertii isolates

(which are generally associated with severe aquatic wounds) are indole negative (2).

Thiosulfate-citrate-bile salts-sucrose medium is usually inhibitory to aeromonads. Enrichment

in alkaline peptone water enhances recovery of Aeromonas from populations that generally

would be expected to shed low numbers of organisms (carriers, convalescent-phase patients,

and those with subclinical infections). For patients with acute diarrhea, enrichment is

probably unnecessary (61).

IDENTIFICATION Back to top

Aeromonas spp. are most easily confused in the laboratory with other oxidase-positive

fermenters, i.e., Vibrioand Plesiomonas spp. Plesiomonas is easily differentiated

from Aeromonas by positive reactions in Moeller’s lysine, ornithine, and arginine tests and by

fermentation of m-inositol. Vibrios may be more difficult to distinguish from aeromonads (1),

which is particularly true for Vibrio fluvialis and A. caviae, and in laboratories where the sole

means of identification is a rapid miniaturized system (31, 69). Resistance to O/129

vibriostatic agent (150 μg) and the inability to grow in salt concentrations of ≥6% usually

indicate the genus Aeromonas. Vibrio cholerae O139, a cholera toxin-positive, non-saltrequiring,

O/129 vibriostatic agent-resistant vibrio, is a major exception to this rule.

However, the decarboxylase pattern (positive for lysine and ornithine) and negative reactions

for arginine dihydrolase, production of gas from glucose, and fermentation of salicin separate

this organism from most aeromonads. Unfortunately, strains of ornithine decarboxylasepositive

A. veronii bv. veronii will often yield an excellent to very good identification for V.

cholerae with the rapid identification API-20-E strip (bioMerieux, Inc.), and serotyping and/or

additional testing is required to resolve the issue. A. veronii bv. veronii would be string test

negative, O/129 resistant, and able to produce gas from glucose fermentation; would not

require additional salt for growth; and would be inhibited on thiosulfate-citrate-bile saltssucrose

agar. V. cholerae strains would have the opposite reactions. Once it has been

determined that you have a glucose-fermenting, oxidase-positive, motile gram-negative rod

that is resistant to O/129, a small number of biochemical tests can be used for

separating Aeromonas species into the three major species complexes (Table 2). If

warranted, even more discriminatory results for separating members of each complex can be

found in bolded text in Table 3 (2), which should replace earlier published tests for species

identification (3).

Other Identification Methods

The sequencing of a single housekeeping gene 16S rRNA (48), followed by the development

of an extended method using 16S ribosomal DNA (restricted fragment length polymorphism)

analysis (22), were both initially promising as methods to identify aeromonads to the species

level. However, data on the intragenomic heterogeneity within the 16S rRNA gene

in Aeromonas strains suggest caution in using this gene for anything beyond genus level

identification (53). Therefore, the use of other housekeeping genes as multiple molecular

markers, such as gyraseB and rpoD (70) and dnaJ (55), or an even broader approach using

multilocus sequence typing with several different genes, seems to be the future avenue for

accurate species identification. Extensive studies by Chopra et al. have delineated several

DNA probes for the detection of a number of possible virulence-related factors. This was the

result of the public release of the Aeromonas hydrophila ATCC 7966T genome sequence and

comparative work with the diarrheal Aeromonas hydrophila SSU strain (13). These include,

but are not limited to, the discovery of a new hemolysin, the presence of a functional type VI

secretion system, a cold shock exoribonuclease R (VacB), and a surface-associated enolase.

SEROLOGIC TESTS Back to top

Most serologic assays that have been used to detect antibodies to Aeromonas (tube

agglutination, immunoblotting, and enzyme-linked immunoassay) have low sensitivity and

specificity and are not considered reliable.

ANTIMICROBIAL SUSCEPTIBILITIES Back to top

Two of the earliest articles on Aeromonas antimicrobial susceptibilities (36, 57) included only

strains well characterized to the species level and expanded previously known susceptibility

information on aeromonads isolated less frequently from clinical specimens. A general

antimicrobial susceptibility profile for Aeromonasderived from both of these investigations as

well as other studies (32, 39, 76) is given in Table 4. There are CLSI (Clinical and Laboratory

Standards Institute) testing guidelines for the major clinical Aeromonas species as related to

antimicrobial dilution and disk susceptibility testing in document M45-A for infrequently

isolated or fastidious bacteria (14).

Ciprofloxacin, commonly used to treat gram-negative infections, was initially reported as

active against all species of Aeromonas, with little or no resistance reported in studies in the

United States and most of Europe (36, 57). However, 2 to 3% of A. caviae, A.

hydrophila, and A. veronii bv. sobria strains in Asia have been reported to be ciprofloxacin

resistant, as early as 1996 (39). Aeromonas species can express three chromosomal β-

lactam-induced β-lactamases, including a group 1 molecular class C cephalosporinase, a

group 2d molecular class D penicillinase, and a group 3 molecular class B metallo-β-

lactamase (carbapenemase) (63). The presence of these β-lactamases in Aeromonas, in

particular the carbapenemase, may not be detected by conventional susceptibility methods

(63). CphA, one of several enzymes responsible for resistance to carbapenems, hydrolyzes

nitrocefin poorly or not at all, indicating that the nitrocefin test is not reliable for detecting

carbapenemases (27, 63). A case of sepsis due to an extended-spectrum β-lactamase

(ESBL)-producing A. hydrophila strain in a pediatric patient with diarrhea and pneumonia

(62) and a case of A. hydrophila necrotizing fasciitis with probable in vivo transfer of a TEM-

24 plasmid-borne ESBL gene fromEnterobacter aerogenes have been reported (24).

A 2009 report on the development of imipenem resistance in an Aeromonas veronii bv.

sobria clinical isolate recovered from a patient with cholangitis warrants concern among

physicians as to the possible emergence of multidrug resistance with this species (64). Much

more disturbing are two reports of plasmid-mediated single-resistance and multiresistance

determinants among environmental aeromonad isolates (11, 25).

Antimicrobial susceptibility testing of local isolates is necessary for the detection of speciesrelated

patterns, because susceptibilities may differ from one geographic area to another.

This was very apparent in a study on the in vitro activities of tigecycline, a novel

glycylcycline antimicrobial agent, against clinical isolates ofAeromonas in Taiwan. It was

found that 200 of 201 Aeromonas isolates were susceptible to tigecycline, with 1A.

caviae isolate having an MIC of 4 μg/ml, and the species-related patterns that varied with

geographic areas were confirmed (43).

EVALUATION, INTERPRETATION, AND REPORTING OF

RESULTS Back to top

Regardless of the site of isolation (intestinal or extraintestinal), aeromonads should be

identified either as belonging to the A. hydrophila or A. caviae complex or as A.

veronii complex and not “A. sobria,” which is now A. veronii bv. sobria. For routine isolates

recovered from uncomplicated cases of gastroenteritis, this level of identification may be

sufficient. Although there is strong evidence that some aeromonads are gastrointestinal

pathogens, there is no convincing evidence, at present, that all fecal isolates

of Aeromonas are involved in diarrheal disease. Thus, the significance of the recovery of

aeromonads from stool specimens should be interpreted cautiously and must rely on both

laboratory information and clinical interpretation. Because of this, the relative quantity

of Aeromonas organisms recovered on enteric media (few colonies, moderate growth, or

predominant organism) should be reported in conjunction with the Aeromonas complex or

species identification. For complicated cases of diarrhea, e.g., prolonged bloody diarrhea in

pediatric patients or chronic gastroenteritis of >1-month duration or in cancer patients with

positive fecal cultures (in whomAeromonas tends to disseminate), a definitive species

identification is warranted.

For extraintestinal isolates (from blood or wounds), the same general rules should apply to

species identification of aeromonads. Although it is clear that both the in vitro and in vivo

pathogenic potentials ofAeromonas species and strains vary considerably, for the present

time, there are no universal markers or indicators available that dictate when isolates should

be definitively identified to the species level. Thus, for extraintestinal isolates, identification

of aeromonads beyond complexes should be reserved for strains isolated from sterile body

sites (blood and cerebrospinal fluid) and serious wound infections (cellulitis and necrotizing

fasciitis); for strains exhibiting unusual resistance patterns, associated with nosocomial

outbreaks; and for publications describing traditional species associated with new disease

processes or newly described species isolated from new anatomic sites.