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Microbial Pathogenesis

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The genus Aeromonas: A general approach

Rafael Bastos Gonçalves Pessoa, Weslley Felix de Oliveira, Diego Santa Clara Marques,Maria Tereza dos Santos Correia, Elba Verônica Matoso Maciel de Carvalho,Luana Cassandra Breitenbach Barroso Coelho∗

Departamento de Bioquímica, Centro de Biociências, Universidade Federal de Pernambuco (UFPE), Av. Prof. Moraes Rego, s/n, Cidade Universitária, CEP: 50670-420,Recife, Pernambuco, Brazil

A R T I C L E I N F O

Keywords:AeromonasVirulence factorsInfectionDiagnosis

A B S T R A C T

The genus Aeromonas comprises more than thirty Gram-negative bacterial species which mostly act as oppor-tunistic microorganisms. These bacteria are distributed naturally in diverse aquatic ecosystems, where they areeasily isolated from animals such as fish and crustaceans. A capacity for adaptation also makes Aeromonas able tocolonize terrestrial environments and their inhabitants, so these microorganisms can be identified from differentsources, such as soils, plants, fruits, vegetables, birds, reptiles, amphibians, among others. Infectious processesusually develop in immunocompromised humans; in fish and other marine animals this process occurs underconditions of stress. Such events are most often associated with incorrect practices in aquaculture. Aeromonas haselement diverse ranges, denominated virulence factors, which promote adhesion, colonization and invasion intohost cells. These virulence factors, such as membrane components, enzymes and toxins, for example, are dif-ferentially expressed among species, making some strains more virulent than others. Due to their diversity, nosingle virulence factor was considered determinant in the infectious process generated by these microorganisms.Unlike other genera, Aeromonas species are erroneously differentiated by conventional biochemical tests.Therefore, molecular assays are necessary for this purpose. Nevertheless, new means of identification have beenconsidered in order to generate methods that, like molecular tests, can correctly identify these microorganisms.The main objectives of this review are to explain environmental and structural characteristics of the Aeromonasgenus and to discuss virulence mechanisms that these bacteria use to infect aquatic organisms and humans,which are important aspects for aquaculture and public health, respectively. In addition, this review aims toclarify new tests for the precise identification of the species of Aeromonas, contributing to the exact and specificdiagnosis of infections by these microorganisms and consequently the treatment.

1. Introduction

It has been a journey of more than one hundred years structuringthe genus Aeromonas within the microbiological universe. It is believedthat the first isolates were reported in 1890 [1] and, throughout history,these bacteria have been classified and reclassified among the mostdiverse genera, such as Aerobacter, Pseudomonas, Escherichia, and Pro-teus, among others [2]. Although there is a plethora of classifications,these microorganisms share common characteristics, which include gasproduction from glucose and their aquatic distribution [1,2]. The termAeromonas (from the Greek words “Aer” meaning air or gas and “Mona”meaning units) was firstly proposed in 1936 by Kluyver and van Neil, toembrace gas-producing bacteria [3]. However, it was Stanier who in1943 officially used “Aeromonas” to denominate the genus where thesebacterial species were added [4].

Composing part of the class Gammaproteobacteria, orderAeromonadales and sharing the family Aeromonadaceae with two othergenera: Tolumonas and Oceanonimonas [3,5], the genus Aeromonas,currently with 32 recognized species [6], is constituted by facultativeanaerobic, Gram-negative, rod-shaped and non-spore-forming bacteriaof approximately 1–3 μm [2,7] in length. Moreover, they are oxidase-positive [8], capable of fermenting glucose [9] and characterized bytolerating increasing concentrations of NaCl varying from 0.3 to 5%[7]. Aeromonas are emerging pathogens capable of colonizing and in-fecting several hosts [10]. They are inhabitants of marine environ-ments, so fish and other seafood are the most common sources forisolating these microorganisms. Therefore, they are widely known inaquaculture as potentially infectious organisms [11] and can causediseases such as septicemia and furunculosis [12]. In addition, Aero-monas can also be isolated from foods, such as vegetables, dairy

https://doi.org/10.1016/j.micpath.2019.02.036Received 10 July 2018; Received in revised form 27 February 2019; Accepted 28 February 2019

∗ Corresponding author.E-mail address: luanacassandra@terra.com.br (L.C.B.B. Coelho).

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products, beef and pork [13–15]. In humans, these microorganisms areable to cause, most often, gastrointestinal system infections; however,such processes if not treated properly can evolve and reach systemiclevels, generating septicemia [7]. On the other hand, species of Aero-monas are capable of infecting soft tissues, the hepatobiliary system,causing ocular, respiratory and joint diseases and even bone infections,which is generally associated with a previous case of septicemia[16–19].

Aeromonas species produce a diverse and heterogeneous range ofvirulence factors. Expression of membrane components, toxins, en-zymes and several molecules contribute to bacterial pathogenicity [20]and act in different ways, such as tissue adhesion, immune responseevasion, and involvement of host cells [10]. In order to disseminatevirulence factors, Aeromonas have four types of secretion systems, re-sponsible for release of these cell products into the extracellular en-vironment or even directly into the host cell [21].

Identification of Aeromonas strains at the species level is still a greatchallenge. Due to the genetic heterogeneity existing in this genus, thereis no effective biochemical evidence for this task [10]. Although newmethods have been developed using molecular biology techniques,amplification of constitutive genes through polymerase chain reactionis still the best option to effectively identify species belonging to thisgenus [22].

2. Isolation and identification

Correct laboratory identification of the genus Aeromonas and itscomponents is still a great challenge. Several studies have been un-dertaken with the aim of making detection practical and reproducible,thus increasing the reliability of results.

The genus Aeromonas is divided, in relation to growth conditionsand biochemical characteristics, into two main groups: psychrophilic,composed of non-motile bacteria with good growth between 22 and25 °C; and mesophilic, which grow well at 35–37 °C and are motile [3,7]owing to a single polar flagellum, for the most part [2]. When grown ina laboratory, some differences related to the appearance of the coloniescan be noted. Aeromonas belonging to the psychrophilic group re-presented mainly by A. salmonicida, are characterized as the main fish[21] and reptile pathogens [7]. Bacterial colonies appear as pin-pointswithin the first 24 h of incubation at 20–22 °C, but after approximately4 days of incubation, they become circular and convex, with a diameterof 1–2mm [23]. Although bacteria belonging to the mesophilic group,such as A. hydrophila, A. caviae and A. veronii [2], are also reported to beharmful to marine animals [24], they are more frequently reportedcausing infections and other diseases in humans [3,7], such as gastro-intestinal diseases and septicemia [25]. Bacterial colonies of the me-sophilic group are 1–3mm in diameter and circular, convex andtranslucent, showing a buttery consistency after 24–48 h of incubationat 35 °C [5].

Cultivation and isolation of Aeromonas in a laboratory can be per-formed in a variety of culture media (Fig. 1). Tryptic soy agar (TSA) andtryptic soy broth (TSB) have been routinely applied for the maintenanceof samples isolated from contaminated fish and water tanks [26].Starch-Ampicillin agar was used for isolation of A. hydrophila in com-mercially obtained foods. After growth of the bacterial colonies, Lugoliodine solution was added and those that were amylase-positive werepresumed to be A. hydrophila [27]. Media such as cefsulodin-irgasan-novobiocin agar (CIN), MacConkey agar and blood agar enriched withampicillin were used when aiming to do a presumptive identification ofAeromonas colonies from fecal samples [28]. In addition, for the sametype of sample, taurocholate-tellurite-gelatin agar (TTGA) was used,which was also tested for sow rectal swabs. In this case, oxidase-positivecolonies showing a gray aspect without a black center but a zone ofopacity were tested for Aeromonas species [29]. Ampicillin Dextrin Agar(ADA) has also been used for isolation of this genus and the color of thecolonies displayed in the medium is what differentiates Aeromonas from

other bacteria. However, there is the possibility that the colors ex-hibited by bacterial colonies in ADA may vary, depending on themanufacturer of the medium used, since some authors report thatpresumptive Aeromonas colonies were yellow [30] while others report awhite color [31] when sown in ADA.

Use of selective media for Gram-negative bacteria is also commonwith regard to the isolation of this bacterial genus. In MacConkey agar,which is widely used in clinical laboratories, the mesophilic group ofAeromonas generally grows as a non-lactose fermenter, with A. caviaebeing an exception, as it is most often a fermenting species [5]. Anotherselective medium used is Hektoen Enteric Agar, due to its ability toinhibit Gram-positive bacteria. Combination of this medium withCHROMagar Salmonella Plus and evaluation by MALDI-TOF has beenreported as a reliable and practical alternative for detecting Aeromonas[32].

The use of media that mainly differentiate the genus Aeromonasfrom similar genera is of extreme importance, since some may exhibitsimilarity in phenotypic and even metabolic characteristics, which canlead to an erroneous isolation and consequently an incorrect classifi-cation. Pseudomonas, for example, share similar physical aspects andoxidase tests [7]; apparently, ampicillin-enriched culture media cancontribute to differentiation [33]. Isolates were seeded in GlutamateStarch Phenol Red (GSP) agar base for selection of Aeromonas colonies,which showed yellow in color, while Pseudomonas colonies were pink[34].

Another genus to which these bacteria are sometimes erroneouslyclassified is Vibrio. In order to avoid misunderstanding, two media wereused: Aerosmart AH medium, which produces a yellow color withAeromonas colonies, and Thiosulfate Citrate Bile Salts Sucrose agar(TCBS), where Vibrio species possess a yellowish aspect [35]. A re-sistance test to vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine(O/129) is also recommended to promote differentiation between thesetwo genera, since bacteria belonging to the Vibrionaceae family arecharacterized as sensitive [7]. However, this methodology does notprovide a totally reliable result, since there are reports of Vibrio speciesresistant to the substance O/129 [36].

Conventional biochemical tests, as well as automated systems, are oflimited utility in the identification of some Aeromonas species [37]. Inaddition, their accuracy is affected by constant reclassification amongcomponents of this genus [22]. Some biochemical profiles are reportedas standards for taxonomic distinction. According to Bergey's Manual ofSystematics of Archaea and Bacteria, the genus Aeromonas comprises D-glucose fermentative facultative anaerobic bacteria that are D-glucosefermenters with or without gas production. In addition, they are cata-lase and oxidase positive, reduce nitrate to nitrite, produce severalenzymes and are capable of using other carbohydrates besides glucose[5]. However, there are some methodologies that present more variedresults than those previously established for Aeromonas identification.Strains were classified as Aeromonas sober with a positive result forarginine dihydrolase (ADH), Voges-Proskauer and gelatin hydrolysis,contrary to what had previously been proposed in the literature [38].Another study reported the same result, in what the first test was pre-valent of all 25 A. sobria strains [39]. A divergence was identified forelastase production between A. hydrophila and A. sobria, which con-tributed to the differentiation of these two species [38]. Similar positiveresults have been described for salicylin, rhamnose and elastase be-tween A. salmonicida and A. bestiarum [40]. Moreover, most of themwere negative for lactase production, which is not specific amongAeromonas, although they mainly grow on MacConkey agar [41]. Dif-ferences have also been identified that support the distinction among A.veronii, A. sobria and A. encheleia, as well as distinct profiles that dis-tinguish the last two. A. sobria and A. veronii biovar. sobria strains weredifferentiated by starch hydrolysis and arabinose fermentation [42]; thelast test was predominantly negative in A. veronii biovar. sobria [43].The diversity of results reflects the difficulty in promoting conclusiveidentification based on biochemical methods.

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Due to the challenges described, molecular techniques are still thebest option for identification and taxonomic classification of the genusAeromonas. A specific molecular marker for this genus that has beenused to identify and distinguish Aeromonas from other genera is thegene-encoding glycerophospholipid cholesterol acyltransferase (GCAT).Its amplification by conventional molecular methods, such as poly-merase chain reaction (PCR), has demonstrated qualitative results inthe identification of Aeromonas in several samples [44,45]. However,the reliability of this method is better assured when associated withother techniques that aim at the taxonomic classification of evaluatedstrains. The most common technique involves the 16S rRNA gene, themain component of the 30S ribosomal subunit [46]. Its sequencing isuniversally known in relation to the establishment of phylogenetic re-lationships among bacteria [47], however, accuracy of this method islimited when analyzing strains whose sequences are very similar [48].The amplification of 16S rRNA for sequencing is generally done byconventional polymerase chain reaction (PCR), using extracted bac-terial DNA. However, it has been reported that sequencing of the 16SrRNA gene amplified by direct PCR colony, a method that excludesDNA extraction and purification of the PCR product, is not only cheaperbut also more efficient in the taxonomic classification of Aeromonasspecies (whose sequences are not similar) than the amplification of the16S rRNA gene using bacterial DNA previously extracted and purified[49].

As the low accuracy of 16S rRNA sequencing is due to the highsimilarity among the sequences [22], the amplification of so-calledhousekeeping genes [50,51] is presented as the best way to do taxonomicclassification, since they have a greater discriminatory capacity [25].

Specifically, gyrB, encoding the B subunit of the enzyme DNA gyrase,and rpoD, which encodes one of the factors involved in RNA polymeraseactivity, are the most widely used housekeeping genes in taxonomicstudies and allow greater reliability in the phylogenetic classification inAeromonas [52]. Although the 16S rRNA has only limited distinguishingpower for this genus, it can be used in conjunction with gyrB; the latterbeing confirmatory [53], since its ability to identify species and strainsis proven [54].

The genes gyrB and rpoD offer advantages to phylogenetic studies;however, a concatenated analysis of not only two but seven house-keeping genes guarantees a more advanced and concrete way of ob-taining a taxonomic classification from this class of genes. This tech-nique is called Multilocus Phylogenetic Analysis (MLPA) [55]. Itconsists of the individual and joint evaluation of sequences of the genes:gyrB, rpoD, recA, dnaJ, gyrA, dnaX, and atpD, which are classified ashousekeeping genes. This method is capable of providing consistentphylogenetic data, which aid in the taxonomic classification of Aero-monas as well as in the discovery of new species [55].

Other molecular-level methods can be applied for effective phylo-genetic studies in Aeromonas. Restriction Fragment LengthPolymorphism (RFLP) analysis allows the study of small variant re-gions, or polymorphisms, in DNA using restriction enzymes [56]. Ap-plication of this technique in 16S rRNA analyses has been reported as afast and effective way to identify some Aeromonas species [57,58].Additionally, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method is considered apowerful tool in the identification of microorganisms [59]. This tech-nique consists of the ionization of samples and generation of gas-phase

Fig. 1. Aeromonas hydrophila in distinct media. MacConkey Agar (a); Aeromonas Medium (b); Tryptic Soy Agar (c) and Nutrient Agar (d).

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ions which, after particle acceleration in an electric field, are detectedaccording to their speed (depending on mass and ionic charge) uponarriving at the detector [60]. MALDI-TOF MS allows for the analysis ofthe protein content of a microorganism, constituted largely of ribo-somal proteins, but also cytosolic proteins, such as heat-shock proteins.Particular differences in the protein composition among microorgan-isms are what effectively guarantees the success of the technique intaxonomic distinction, not only at the level of genus, but also of species[59,60]. The application of MALDI-TOF in phylogenetic study andprotein characterization in Aeromonas has already been reported[9,61]. In addition, there is a molecular tool capable of accurately anduniversally providing bacterial characterization through the analysis ofthe nucleotide sequence of multiple chromosome sites, the MultilocusSequence Typing (MLST) [62]. Such a technique detects changes inDNA quickly and reproducibly without the need for reagent exposureand does not require living bacterial suspensions or even high-qualitygenomic material, sparing the difficulties associated with the transportand handling of bacterial samples [63].

Genomic sequence of a microorganism represents the extreme levelof information that can be used in phylogenetic studies [64]. The pro-portion of guanines (G) and cytosines (C) in microbial DNA is theparameter most frequently evaluated in genome-based phylogeneticclassification. The DNA-DNA hybridization technique (DDH) consists ofdetermining the genetic distance between two microorganisms basedon their G + C content [65]. Such evaluation establishes a limit insimilarity percentage as a criterion to classify two organisms as be-longing or not to the same species. In the case of DDH, when genomicsequences present below 70% similarity, the evaluated microorganismsare considered distinct species, whereas when they have similaritygreater than 70%, they are classified as belonging to the same species[66]. For a long time, this form of evaluation was considered the goldstandard with respect to the taxonomic classification of Archaea andBacteria domains. However, since there are several limiting factors: thecomplexity of the technique, a high probability of errors, and the needfor several other tests for statistical evidence of results, analyses of si-milarity among 16S rRNA gene sequences was used instead, as dis-cussed above [65]. Nevertheless, in order to make the results obtainedby the DDH useful, other techniques have been developed. The in silicoDDH (isDDH) method is able to produce values close to those obtainedfrom DDH, establishing limit values by standardized parameters. Thus,bioinformatic assessments can be facilitated and the variations in DDHovercome [67]. Due to the scientific advances in and availability ofcomplete genomic sequences, new comparison indices have been cre-ated to calculate similarities without the need for functional steps, in anobjective and reproduceable way [68]. For example, Genome BlastDistance Phylogeny (GBDP), Maximal Unique Matches Index (MUMI)and, most commonly, Average Nucleotide Identity (ANI), consideredthe possible gold standard of its generation for species distinction. ANIis the mean of identity values and similarity between two genomes[64]. In general, it is calculated in two steps, consisting of genomefragmentation into sequences of approximately 1020 bp and, with theaid of Blast program, comparing each fragment generated over the se-quence of another genome; therefore, ANI is calculated according tovalues of identical nucleotides [68].

3. Virulence factors and correlated genes

Aeromonas virulence is complex, since several factors contributesignificantly to the development of an infectious process [69] as theefficacy of the host immune system decreases [70]. Structural compo-nents, toxins and extracellular products [21], acting jointly or in-dividually [71], enable these microorganisms to colonize and infecthosts [25]. The Aeromonas virulence complex, due to this diversity, hasnot yet been established [72].

3.1. Structural components

The structural composition of a bacterial cell has a great influenceon the infectious process. Several factors such as flagella, pili, proteinsand membrane antigens, among others, have been studied and relatedto roles in bacterial pathogenicity, for example, locomotion, adhesionto host tissue, protection against bactericidal agents and immunesystem cells [73].

Flagella are structures whose main function is to promote bacterialocomotion; they may be present at different sites [74]. They arecomposed of an external filament and an inner portion, denominatedthe basal body, attached to the membrane [75]. Two flagella types havebeen identified in most Aeromonas species belonging to the mesophyllgroup, polar and lateral, which confer motility in liquid environmentsand solid surfaces, respectively [74]. In addition, they perform func-tions relating to cell adhesion and persistence in the infectious process[3].

Two major types of motility are observed according to the sort offlagellum produced. Swimming motility is associated with liquid en-vironments [75] and promoted mainly by the polar flagellum, while thelateral type promotes swarming movement on solid surfaces [3]. Mo-tility types have been verified in Aeromonas species isolated from retailsushi, whose analysis has been made by sowing strains in media such asnutrient agar and LB, measuring the colonies growth diameter from thecenter to periphery [25]. Several genes are involved in the productionof flagella [21]. Virulence genes have been investigated by multiplexpolymerase chain reaction (PCR) in Aeromonas strains isolated fromclinical and water samples, identifying the fla gene, which encodes thepolar flagellum, in most isolates from both sources [76]. The same genewas also detected in Aeromonas species isolated from aquaculture en-vironments and slaughterhouses [71]. However, the gene coding thelateral flagellum, laf, was only found in slaughterhouse samples. Oc-currence of mutations in these genes can affect both adhesion capacityand biofilm formation [73], leading to virulence reduction. A. hydro-phila mutant strains without the flgC and flgE genes, responsible forproduction of the flagellum inner portion and hook filament, respec-tively, had partial and total reduction in motility, respectively, as wellas defects related to adhesion capacity and chemotaxis [77].

Pili, or fimbriae, are slightly smaller structures than the flagella [21]and are mainly responsible for ensuring bacterial cell adhesion to hosttissue [74] and other solid surfaces [78]. Four types of pili are found inGram-negative bacteria, with types I and IV described in Aeromonasspecies [21]. Type I pili are structurally characterized as short-rigid [3]and are often reported in environmental samples; generally, thesestructures are unrelated to the pathogenicity of the bacterium [74] andmost of the time are present in A. hydrophila [3]. A mutant strain wasdeveloped of A. salmonicida which lacks the gene cluster related to theproduction of type I pili and tested in vivo in Atlantic salmon (Salmosalar) species. Adhesion efficacy to host tissue was detected, while in-vasive capacity remained intact [79]. Type IV pili are characterized asflexible-wavy [3] or long-wavy [74] and are more related to bacterialpathogenicity [21]. In addition, they are generally detected in isolatesfrom clinical samples [74] and usually found in A. hydrophila, A. veronii,A. caviae and A. trota [3]. There are three main genes related to theexpression of type IV pili. The first is the tap gene, associated with thetapABCD cluster, which exerts different functions [73]. TapA is re-sponsible for the production of class A pili, the type IV pili-formingsubunit. An A. salmonicida strain containing a mutation in the tapA genewas less pathogenic to rainbow trout (O. mykiss) [80]. Moreover, pre-viously challenged specimens, which were exposed to wild strains(containing the tapA gene), exhibited a higher resistance to infectionthan those challenged only with the mutant strains, indicating im-munogenic potential to products expressed by this gene. The tapB andtapC genes are responsible for securing subunits within pili while thetapD gene contributes to protein processing and maturation [80]. Thesecond formative gene of type IV pili is flp, coding biosynthetic proteins

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of peritrichous pili. Challenging Atlantic salmon (S. salar) specimenswith mutant strains of A. salmonicida subsp. salmonicida revealed thatthe absence of the tap gene contributed more to strain virulence thanthe flp gene, whose contribution was minimal and even nonexistent[78]. The Msh gene encodes grouped filaments of type IV pili and theseare considered the biggest explanation for bacterial colonization ability[74]. Thus, it is also known as the bfp (bundle-forming pili) gene [21].The importance of this gene was revealed when it was verified thatmutant strains from A. hydrophila, which did not contain the mshQgene, presented deficient adhesion and biofilm production; the piliexpressed by this gene are crucial in the adherence of this micro-organism to solid surfaces [81].

The protein composition of the outer membrane plays a relevantrole in Aeromonas virulence. Outer membrane proteins (OMP) havefunctions related to osmoregulation and nutrient absorption, amongothers [21] and have been researched for the development of vaccines.The products of the ompA1, tdr and tbpA genes, encoding the outermembrane protein A (OMPA), TonB-dependent receptor and transferrinbinding protein A, respectively, have been evaluated as candidates forthe development of vaccines against A. hydrophila [82]. The first twogenes cited were able to provide strong immunological protection tocatfish (I. punctatus) specimens. The cell wall of Aeromonas species alsocontains substances involved in the pathogenic process [83]. Tetra-gonally arranged proteins present on the superficial layer contribute tothe adhesion to membrane components of the host cell [3,74] and playan important role in protection from immune system responses andphagocytic cell actions [84]. Surface proteins (A-proteins) denominatedA-layers or S-layers (superficial layers), are encoded by the vapA gene,also referred to as ashA by some authors [74,84]. Tests with culturemedium containing bright coomassie blue can indicate the presence ofthese proteins [21]. These structures were predominant in membraneprotein extracts from A. salmonicida strains, composing 60% of proteinconcentration after separation by two-dimensional electrophoresis[83]. The amino acid sequence of A-proteins from five strains of A.salmonicida subsp. salmonicida revealed that four sequences wereidentical, and one presented a difference in only one amino acid [84].The effects of an A. hydrophila mutant strain, lacking the vapA gene, onpathogenicity to rats, rainbow trout (O. mykiss) and larval zebrafish(Danio rerio) specimens have been evaluated [85]. After intraperitonealinjection of the strain, there was no change in virulence, but a con-tribution to the inflammatory process due to induction of interleukinproduction.

The outer membrane of Aeromonas spp. is composed of moleculesthat, besides assisting in the adhesion process, has immunostimulatoryactivity [21]. The so-called membrane lipopolysaccharides (LPS) areamphiphilic complexes composed mainly of 3 domains [86,87]. Themolecule's lipid portion is characterized of a highly conserved bioactivesubunit, named lipid A, covalently bound to the polysaccharide struc-ture and to the outer membrane [3,21]. On the other hand, the LPSpolysaccharide complex is formed by a central region and an oligo-saccharide sequence, containing from one to six carbohydrate mole-cules, which confer hydrophilic and antigenic characteristics to mem-brane lipopolysaccharides, denominated the O antigen [3,87]. Oantigens, also known as specific O polysaccharides, confer intraspecificdivisions within most bacterial species called serovars or serogroups[86], as well as contribute to the development of the immune response,being inducers and targets of specific antibodies [21]. Genes encodingthe LPS central region belong to a cluster, named cluster wa, and areidentified in three regions [88]. The wa 1 region has genes related toproduction of the upper portion of the molecule, which is bound to theO antigen, whereas wa 2 and wa 3 are responsible for the synthesis ofthe lower portion, which binds to lipid A [86,89]. These genes arespatially close to the coding clusters of other structural components,such as the already mentioned flagella and pili [21]. Although theimmunostimulatory capacity of LPS is known, the inflammatory processinduced is generic, that is, despite the O antigen inducing the

production of specific antibodies, as explained, the content of in-flammatory response modulators is not substantially altered [90].

In addition to all this content, the bacterial cell outer membrane isenveloped by a hydrated structure composed mainly of monosaccharideunits, linked together by glycosidic bonds, denominated capsule [3,75].However, capsules of some bacteria can also have polypeptides in theirconstitution [21,74]. They serve the function of conferring resistance toimmunological reactions, such as phagocytosis and complementarysystem actions [3]. In a cluster responsible for capsule expression in anA. hydrophila strain contained 13 genes divided into three regions, thefirst and third had transport-related genes, while the second was re-sponsible for structure synthesis [91]. The two genes orf1 and wcaJ,belonging to the cluster of capsule formation, have been reported [92].The relevance was justified since strains of A. hydrophila mutants didnot develop capsules even in propitious medium.

Aeromonas are also well known for being biofilm producers [93].Biofilms are an extracellular polymer matrix of three-dimensional ar-chitecture composed mainly of proteins, but also of polysaccharides andDNA molecules [94]. Moreover, microorganisms, which interact witheach other, account for part of the total volume of the biofilm; takingadvantage of the nutrients extracted from the environment where thismicrobial community is formed [95]. Biofilm formation is considered avirulence factor by confering resistance to bactericidal agents [73] andpromoting adhesion to abiotic solid surfaces as well as to host cells,which characterize the first stage of the infection process [93]. Biofilmformation of Aeromonas spp. strains was identified by culturing themicroorganisms in 96-well polystyrene plates [71]. Following succes-sive washes, an optical density reading was performed after addition ofcrystal violet, which stained the bacterial cells adhered to plate. Biofilmformation is not limited to the presence of nutrients, but also to theirconcentrations, and depending on the nutrient, they can act in an in-hibitory way. Effects of glucose concentration variation on biofilmproduction have been evaluated by A. hydrophila strains isolated fromdifferent sources [96]. Bacteria cultured in media of up to 0.05% glu-cose did not present significant alterations; concentrations between0.25% and 2.5% were shown to be inhibitory. Expression of lateralflagella is also related to biofilm production. Molecular methods iden-tified the presence of fla and laf genes coding polar and lateral flagella,respectively, and their relationship to biofilm formation capacity of A.caviae samples from different sources [97]. Production was confirmedonly in fla- and laf-positive samples. Sets of samples that only had thepolar flagellum gene or those that did not have either of the two genestested did not form biofilms. Antigenic capacity of this virulence factorhas also been investigated. Channa striatus specimens fed with biofilm-containing rations have been identified with high antibody titers thatpromoted a high survival rate when challenged by harmful doses of A.hydrophila [98].

3.2. Extracellular products

A remarkable variety of extracellular products is generated amongAeromonas species [99]. Some enzymatic classes, such as hemolysins,lipases and proteases besides toxins have been studied over time due totheir role in the infectious process and host impairment [21].

Hemolytic enzyme production is an alert factor related to severalbacterial genera. Three major types of hemolysins have been identifiedamong the products secreted by Aeromonas [100]. Aerolysins are pro-teins capable of altering the permeability of blood cells, as well as othereukaryotic cells (and consequently promoting osmotic lysis) due topolymerization of the structure induced by binding to a membrane-specific glycoprotein site [101]. The gene responsible for expression ofthis molecule, and studied by molecular methods, is named aer [102],which is divided into three types: aerA, aerB and aerC, of which the firstis responsible for aerolysin production itself; the latter two are re-portedly involved in expression modulation and enzymatic activity[102,103]. Hemolysis types can be identified by sowing the bacterial

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strains in blood agar [25]. In addition to aerolysin, which induces β-hemolysis, complete lysis of erythrocytes, β-hemolysins, another type ofenzyme with hemolytic activity, can also be evaluated by molecularmethods [104]. Despite partial hemolysis promoted by α-hemolysinhaving a reversible effect, research into it is not as widespread as theother two types [100].

The capacity of promoting damage to the intestinal epithelium offish is conferred by lipolytic activity [21]. Lipases are enzymes re-sponsible for the hydrolysis of triglycerides and are of important com-mercial value [105]; different bacterial genera produce and secretesuch molecules to perform distinct functions [106]. Genes such as lip,lipH3 and pla are alternatives for lipase production [107]. Another typeof enzyme has been characterized, phospholipase C also known aslectinase, which has been considered a virulence factor and presentscytotoxicity, with low or non-existent hemolytic activity [108]. The apl-1 gene encodes this molecule [107]. In addition to the cited examples,another type of lipase, glycerophospholipid:cholesterol acyltransferase(GCAT) was characterized as one of the most lethal virulence factors inA. salmonicida [109]. Coded by the gene gcat, this enzyme can complexwith LPS, becoming more toxic than its free form [21].

Extracellular proteases contribute to the process of bacterial mul-tiplication, as well as act on host nutrient sequestration and the immuneresponse [70]. Two major components have been identified in this classof enzymes [100]. Two different fractions of proteases have beenidentified in A. hydrophila, one with strong caseinolytic activity, calledserine protease, and another showing action against elastin and casein,known as metalloprotease [110]. This class of enzymes, beside usualfunctions, also plays the role of activating other virulence factors. Theparticipation of serine protease in GCAT activation of A. salmonicida hasbeen reported [111]. Molecular research aiming at amplifying genesencoding extracellular enzymes is used to verify virulence potentialamong different Aeromonas strains. The presence of these enzymaticvirulence factors is common in isolates from frozen fish [112]. Thegenes lip, gcat and ser (coding serine protease) have been identified inmost of the isolated strains of pond-raised catfish (Ictalurus punctatus);however, the aerA gene was the most prevalent [113]. Similar resultshave been reported in isolates from a trout farm [114].

Great attention has been given to the effects generated in the gas-trointestinal system in cases of infections caused by the members of thegenus Aeromonas; they are considered emerging pathogens to humans[31]. The most common pathological condition is gastroenteritis [47],which may evolve with more aggravation into clinical manifestations[115]. Major contributors to development of gastrointestinal tract dis-eases are different toxin types that bacteria of the genus Aeromonas arecapable of producing; among which cytotoxic enterotoxin and two cy-totonic enterotoxin types are well known [116].

Cytotoxic enterotoxin promotes degeneration of villi and mucus-producing cells [76], generally related to cases of bloody diarrhea inhumans [117]. In addition, it has hemolytic activity, which many au-thors treat as being an aerolysin, a member of the hemolysin class [71].However, even though there is strong similarity, others consider themdistinct molecules [69]. This enterotoxin, encoded by the act gene,generates an inflammatory response in host cells, a factor that leads tosystemic involvement [118]. Morphological changes generated by cy-totoxic effects in hamster ovary (CHO) cells, human laryngeal epi-dermoid carcinoma (HEp-2) and African monkey kidney (VERO) cellshave been detected. Cell rounding and shrinkage were identified, fol-lowed by cytoplasmic and cell membrane disorders [115].

Unlike cytotoxic enterotoxin, cytotonic enterotoxins do not causedegenerative damage to the intestinal epithelium [3] and are related tocases of non-bloody diarrhea [117]. They are divided into two groups:heat-labile cytotonic enterotoxins, which are encoded by the alt geneand can be degraded at 56 °C for 10min. While heat-stable cytotonicenterotoxins, with degradation at 100 °C for 30min, are expressed bythe ast gene [3,69]. These toxins promote elongation in CHO and se-cretion of liquids in rat ileum, but also induce increased cyclic AMP and

prostaglandins in intestinal cells [75,119].The act, asp and alp genes have been researched enough by mole-

cular methods to evaluate Aeromonas virulence. Seven differentAeromonas species from raw meats and clinical samples of diarrheahave been isolated; most had at least one enterotoxin-encoding gene[116]. All A. hydrophila strains contained the act and alt genes, while afew had the ast gene. The absence of the alt gene in Aeromonas strainsisolated from clinical and environmental sources was reported; the actand ast genes were identified in a few isolates [41]. The act gene wasmore prevalent than alt and ast among 176 strains isolated [118]. Twoisolated strains that contained both act and ast genes in their genome, acombination rarely found, have been reported [72].

Due to the fact that they are considered emerging pathogens relatedto cases of diarrhea in humans [120], another toxin type reportedmainly in the genus Shigella has been investigated among the Aeromonasspecies. The shiga-like toxins are exotoxins mainly causing hemolyticuremic syndrome and can be harmful to the nervous system. Shigatoxins 1 and 2 are encoded by the stx1 and stx2 genes, respectively[121]. The production capacity of these molecules by Aeromonas isvariable, like other virulence factors. Shiga-like toxins have beenidentified in animal isolates [34]; however, the presence of these geneswas not detected in bacteria isolated from retail sushi [25]. Both stx1and stx2 were absent in a study from environmental isolates [122].

3.3. Secretion systems

Transfer of the virulence factors produced by bacteria to the ex-tracellular medium and/or to host cells is extremely relevant to thecontamination and infection processes. Six different types of secretorysystems have been detected in Gram-negative microorganisms [3]; fourof them reported in the genus Aeromonas, being types II, III, IV and VI[21]. Aeromonas secretion systems, compositions and functions are re-presented in Fig. 2.

Type II is known as a secretion pathway for substances, such astoxins and mainly enzymes, to the extracellular environment through aprocess that, in Gram-negative organisms, involves inner and outercellular membranes, as well as the space between them, the so-calledperiplasmic space [123]. To have secretion of a given substrate, usuallypolypeptide, by the type II pathway, its primitive form must be pro-duced in the cytoplasm and then transported through the inner mem-brane into the periplasmic space, where it reaches its final conforma-tion. Two types of transport perform this function according to themolecular characteristics of the substrate. The general secretion (Sec)pathway carries protein filaments that do not need to be folded beforethey are introduced into periplasmic space. However, the formationprocess of some molecules requires a first folding of the structure in thecytoplasm; in this case, transport is carried out by the Twin ArginineTranslocation (Tat) pathway. The type II secretion system is composedof four components that communicate with one another: a protein baselocated in the inner membrane; a cytoplasmic ATPase, which exhibitsproperties similar to type IV pili; the type II pseudo-pili; and finally aprotein referred to as D protein or secretin. Polymerization of secretinleads to the formation of a channel in the outer membrane, the site ofpassage for substrates from the periplasmic space to the extracellularmedium. In addition, it is also important for the formation of type IIpseudo-pili. In general, contact of proteins to be secreted with the se-cretin periplasmic portion stimulates retraction of pseudo-pili, which,by means of the energy supplied by cytoplasmic ATPase, pushes proteinsubstrates through the secretin channel, to be released into the extra-cellular space where they can exercise their respective biologicalfunctions [124,125].

Studies have been performed with the aim of uncovering the com-ponents, as well as their respective functions, of the type II secretionsystem in A. hydrophila and A. salmonicida strains. The participation oftwo inner membrane proteins, ExeA and ExeB, has been described asthe key to the secretion process [126]. ExeA has two main domains, one

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cytoplasmic, linked to ATPase, and another periplasmic, which has apeptidoglycan-binding site. In the presence of the ligand, ATPase gen-erates energy for the formation of the ExeAB complex, which is crucialfor multimerization of ExeD monomers in the inner membrane, whichare then translocated to the outer membrane for secretin formation. Inthe absence of the ExeAB complex, ExeD is maintained in the innermembrane, interfering in the secretory process. In addition, the im-portance of ExeB in this process was investigated and it was concludedthat such a protein acts to support the structure formed by the threemolecules in association with the binding peptidoglycan [126]. Theinfluence of this ligand on the formation of the ExeAB complex fromExeA was also emphasized [123].

Current knowledge suggests that the type III secretion is perhaps themost widespread system in relation to Aeromonas virulence. It consistsof a thorn-shaped or syringe structure responsible for injecting proteinsharmful to cellular metabolism directly into the host-cell cytoplasm[127]. Unlike type II, the base of the type III secretory complex is astructure composed of approximately 15 proteins, which interconnectsthe inner and outer membranes, known as a basal body [125]. At thebase of the system a protein arrangement known as “injectisome” isexposed to the extracellular medium [128]. This structure has an innerchannel and a complex of proteins at the tip with sensory activity,which detects contact with the membrane of host cells. Thus, promotingthe structuring of the third component of the type III system, thetranslocon, which acts together with sensory proteins to form a pore inthe eukaryotic cell [125]. Thus, products to be secreted have freepassage between the two cytoplasmic environments without ever beingexposed to the extracellular medium.

Research involving the detection and deletion of genes related to theexpression of components of the type III secretion pathway, as well asits effector molecules, contributes to the understanding of the im-portance of this system in bacterial virulence. The prevalence of genesencoding the type III secretion system, asc-V and ascF-ascG, as well astwo effector proteins of the complex, AexU and AexT, characterized asADP-ribosylating toxins has been analyzed. Twenty A. veronii strains

tested had all the genes studied; sequence analysis revealed less geneticvariability in AexT, indicating that its function is conserved for thisspecies [127]. The ascV mutation attenuated the virulence of A. sal-monicida subsp. salmonicide, since mutant strains were phagocytized byleukocytes, contrary to what happens in wild isolates [129]. When theaopB gene, responsible for the formation of the translocon in A. hy-drophila strains, was deleted, a reduction of the toxicity in cultured cellsfollowed [128]. In addition, the mutant isolates for both aopB and act(coding for cytotoxic enterotoxin) were avirulent in the in vivo assayusing rats. The presence of the type III secretion system in Aeromonasspp. was detected through the amplification of the ascV gene. Among 64strains tested, 29 were positive for the search performed and these werequite harmful to HEp-2 cells [115]. Amplification of the ascV and aopBgenes with a similar purpose was accomplished and the presence ofthese genes was verified as being more prevalent in Aeromonas strainsisolated from diseased fish, which caused the highest mortality rateswhen injected intraperitoneally into Nile tilapia (O. niloticus) specimens[130].

Participation of the type III pathway in the development of theimmune response has also been studied. The presence of antigens of thissecretory system hampers immunological activity in rainbow trout (O.mykiss), since the vaccination of fish with mutant strains of the ascVgene induced a significant survival rate in relation to those vaccinatedwith antigens from wild strains [131]. Infections generated by Aero-monas containing this system, functional or inhibited, have been iden-tified; they developed suppression of the immune system in rainbowtrout (O. mykiss) [132]. However, those classified as lethal were onlydeveloped by strains that had the structure of the fully functional typeIII secretion pathway; those devoid of such a system did not causedeficits in the immune response and were not lethal to the fish tested.Exchange of genetic material among bacteria is a process that guaran-tees survival of these microorganisms under established environmentalconditions. The most important clinical consequence of this event is thespread of antibiotic-resistant genes among bacterial species, generatingwhat are known as multidrug-resistant microorganisms [133].

Fig. 2. Aeromonas secretion systems, compositions and functions.

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Moreover, the aquatic environment, since it receives discharges fromurban and industrial effluents, favors this process by conferring greatercontact among wild strains and drug substances [134]. This, in theory,suggests that Aeromonas, since they naturally inhabit such environ-ments, are potentially capable of acquiring antimicrobial multi-resistance.

Unlike protein transport, the translocation of DNA molecules ismore complex [135]. In Gram-negative bacteria, this function is con-ferred by the type IV secretion system [124]. This pathway is dividedinto three subgroups with different functions, the most prevalent onebeing for bacterial conjugation [136]. In addition to the genetic ma-terial, several other types of molecules can be secreted and destined torecipient cells, which may be other bacteria (not necessarily of the samespecies) or eukaryotes [125].

Neither the components nor structure of the type IV system inAeromonas have yet been well elucidated. However, some productssecreted by this route have been identified. Plasmid genes related totetrodotoxin (TTX) biosynthesis as well as their regulation have beendetected [137]. Moreover, the strains tested also contained genes as-sociated with expression of the type IV system, which suggests a strongrelationship between the product and its secretory complex.

Little is known about the mechanisms related to the type VI secre-tion system; it is generally responsible for contact-dependent translo-cation of proteins from one bacterial cell to another, as well as to eu-karyotes [125]. It is possible that the secretory structure of this systemderived from phagocytic mechanisms [138]. Like type III, it also se-cretes effector molecules for deregulation of the recipient cell. Theexistence of a gene cluster, VasH, was reportedly responsible for pro-duction of the hemolysin-coregulated protein (hcp), an effector enzymethat has a role in the modulation of the immune response, preventingphagocytosis [138,139]. Another effector, a component of the VgrGfamily of proteins, that is VgrG1, was identified which required priorcontact between a secretory bacterial cell and a host cell to be trans-ferred by the type VI system [140]. Genes homologous to hcp and VgrGlocated outside the cluster of the type VI secretion system were iden-tified; the deletion of these genes did not affect secretion of effectorproteins, suggesting redundancy in maintaining the structure of thissecretion pathway [138].

3.4. Other virulence factors

Aeromonas possess a complex system of virulence factors, whichhave been uncovered over time, clarifying their ability to infect severalhosts and colonize the most varied environments. However, bacterialcell survival depends not only on how harmful it may be but also onhow well it is able to maintain itself in situations unfavorable to itsgrowth. Thus, the means of acquiring essential nutrients and of bac-terial intercellular communication are also considered virulence fac-tors, which, in Aeromonas, refers to the mechanisms of iron acquisitionand a mechanism named Quorum Sensing (QS), respectively.

Iron is an essential nutrient for the bacterial cell since it assists inthe maintenance of several metabolic functions [21] that guaranteetheir survival in the host, thus promoting the infection process [141].Due to iron's low availability in free form, bacteria have developedthroughout the evolutionary process some effective molecular systemsto obtain iron in its most diverse states, the siderophores [142]. The lowmolecular weight chelators of ferric ion (Fe3+) are structures re-sponsible for extracting the iron bound to host proteins and in-corporating it into the bacterial cell. In the case of Aeromonas, the mainsiderophores produced are enterobactin and amonabactin [21]. How-ever, not all iron uptake by bacteria is mediated by siderophores, sincealthough they have high ion-binding affinity, they cannot extract itfrom more complex protein molecules such as hemoglobin, lactoferrinand transferrin [143]. Thus, bacteria have specific receptors, such asthe heme receptor on the outer membrane, capable of binding to thecomplex molecule and then promoting dissociation of existing ferric

ions. This path is known as siderophore-independent [144].To support various environmental conditions in which they are in-

serted, bacteria proliferate in populations, which may follow variousmechanisms to develop a favorable medium for the maintenance andprotection of bacterial cells, such as biofilms [95]. Intercellular com-munication in a bacterial population is performed through QS [21],which is a chemical signaling pathway that controls members' behaviorof a given bacterial population through gene expression [145]. Signalmolecules secreted in this communication pathway vary depending onthe bacterial classification. In Gram-positive bacteria they are smallpeptides, while in Gram-negative, as is the case of Aeromonas, they areacylated homoserine lactones (AHLS) [21,146]. In general, each bac-terial cell in a population is able to secrete and respond to these che-mical stimuli by different ways, either to increase nutrient uptake, torespond to competition with other organisms or to promote defenseagainst phagocytic cells, among other actions [21,147]. Signaling mo-lecules produced by a LuxI synthase enzyme, are recognized by a spe-cific receptor, LuxR, a transcriptional regulator of several genes. Theligand-receptor complex binds to DNA and initiates gene expression,including those expressing LuxI, which makes this system self-inducing[145,148]. The activated gene regulator decays slowly, reducing pro-duction, with accumulation of signal molecules [146].

4. Ecology of Aeromonas

As mentioned before, Aeromonas can be isolated from several nat-ural sources [23], particularly aquatic environments [31]. Contact withfish and other aquatic animals develops in a continuous and almostinevitable way [149]. Thus explaining the variety of species from whichthese microorganisms have already been isolated, such as Nile tilapia(Oreochromis niloticus) [53], rainbow trout (Oncorhynchus mykiss) [24],channel catfish (Ictalurus punctatus) [150], Japanese eel (Anguilla ja-ponica) [151], crayfish (Pacifastacus leniusculus) [152], tambaqui fish,Colossoma macropomum [153], oysters [154], and ornamental fish[9,117]. Aeromonas are considered opportunistic pathogens [21]; theyalso can act as secondary agents in other previously installed patholo-gical dysfunctions [9]. Most of the time, the main trigger for develop-ment of infections is stressful conditions generated in aquatic animalsby environmental changes [152], such as alterations in water char-acteristics and constituents [21]. Despite expectations, captive-bredspecies are more susceptible to stress than wild-bred species [70].

Fish and other seafood are sources of numerous nutrients.Consumption of these foods can considerably reduce risks for the de-velopment of inflammatory diseases, such as atherosclerosis, acutemyocardial infarction, stroke, among others [155], which explainsthese products being universally consumed in diets [156]. With theexpected world population growth, there is an increasing demand infood production. However, natural stocks of fish and other seafood arenot able to keep up with this reality [157]. Thus, the practice ofaquaculture emerged to reduce extractive activities and to ensure uni-versal access to products of aquatic origin [157,158], thereby reducingenvironmental degradation and promoting species preservation [153].This activity has grown over time and is recognized as an importantcontributor to the production of fish and other species derived fromaquatic environments [159]. The main obstacles of aquaculture arediseases and infectious processes that lead to the death of specimens,which consequently affects their production and blocks sales of theseproducts in the market [117]. Adoption of practices such as poor watertreatment, excessive handling, proximity among species, transport andstorage are responsible for generating a stressful environment for thecultivated animals, making them susceptible to infections[21,70,153,160]. Twenty-six strains of Aeromonas were isolated fromwater samples of aquaculture [71]. When submitting specimens oftambaqui (C. macropomum) to physiological stress by confinement,species of Aeromonas spp. were also obtained [153]. These resultscorroborate with the information mentioned above. A. veronii samples

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were isolated from Nile tilapia at two private fish farm sites and thequality of the water in which the species were maintained was ana-lyzed. Significant variations in temperature, pH, dissolved oxygen,ammonia (NH3), nitrate (NO3) and nitrite (NO2) concentrations wereidentified in the period of approximately one year; such alterationsprobably contributed to the development of the infectious process [53].

Mesophile and psychrophile groups of the genus Aeromonas cancause infections in fish. A. salmonicida is known to be a causative agentof furunculosis [70], an ulcerative disease affecting mainly salmonids[74]. A. hydrophila, as well as A. veronii [53], are considered to causehemorrhagic septicemia [152]. Rainbow trout (O. mykiss) specimenswere exposed to strains of different Aeromonas species; the group in-fected with A. salmonicida, A. hydrophila, A. veronii and A. bestiarumpresented abnormal swimming, signs of anorexia and hyperpigmenta-tion [24]. Moreover, they identified that these bacteria promoted ahigher mortality rate in the specimens tested. Nile tilapia infected by A.veronii exhibited different clinical signs, such as insensitivity, lethargyin movements and absence of reflex; they also had ulcerations, palespots and hemorrhages along their body [53]. A. veronii, A hydrophila,A. salmonicida and A. media strains were isolated from fish, whichshowed signs such as hemorrhages in the mouth, eyes and other organs;some also exhibited swelling with the presence of ascetic fluid [50].Infected crayfish (P. leniusculus) specimens revealed small spots on thecuticle and/or showed lethargy and weakness [152]. Frequency ofdiseases and outbreaks can be related with geographic differences,though the genus Aeromonas is widely distributed in diverse environ-ments [149].

Although water is the main means for propagation of Aeromonasspecies, they can also be isolated from animals other than inhabitants ofaquatic environments. A. hydrophila is the primary cause of red-legsyndrome in amphibians, a disease characterized by redness in the legsand abdomen of frogs and salamanders, promoting anemia, lethargyand other symptoms [161]. Analysis of swabs from the cloacal andpharyngeal region of free-living birds identified the presence of threespecies of Aeromonas: A. hydrophila, A. sobria and A. veronii [162].Bacteria belonging to this genus could be identified in healthy pigs'feces [163] as well as promoting bronchopneumonia with purulentexudate in a wild boar [164]. In relation to reptiles, A. hydrophila hasbeen found as part of the bacterial diversity existing in the oral cavity ofvenomous snakes [165,166]. The same species was also identified ascausing septicemia in two types of crocodiles. Tissue analysis foundextensive respiratory and intestinal lesions [167]. A. hydrophila wasalso the only Aeromonas species isolated from domestic reptiles [168].The ability of these bacteria to colonize insects has been reported, sincestrains of A. hydrophila and A. veronii were identified in intestinalsamples of house flies (Musca domestica L.) [169].

Environmental adaptation strategies, especially tolerance to lowtemperatures, make Aeromonas important colonizers of food, whetherof vegetable or animal origin [170,171]. Although some strains ofAeromonas have already been isolated directly from soil [172,173],these microorganisms can be introduced into food by different ways,such as animal feces, handling without previous hygiene and especiallythe water itself used in irrigation systems in agricultural practice. About15 types of plants have been tested for the presence of Aeromonas; themost prevalent species was A. caviae, followed by A. hydrophila. How-ever, in the same evaluation, no microorganism was detected in four of15 vegetables tested, one of them being lettuce [174]. Moreover, thisresult does not exempt such vegetables from being contaminated byAeromonas, since there are reports of isolation of this genus from lettucesamples showing the prevalence of A. caviae and A. hydrophila [175].Aeromonas can also be found in foods of animal origin, such as meatsand derivatives, milk, and cheese, among others [175,176]. Comparingthe number of strains isolated, meat products had a greater number inrelation to vegetables, being identified as A. caviae, A. hydrophila and A.sobria [174]. Except for A. caviae, the same species were also found inanother analysis using samples of meat, milk and dairy products [170].

A. hydrophila has been identified as the cause of acute gastroenteritis inmeat consumers in a Bhutanese village, in the Himalayas; among 55people who reported consumption, about 33 developed the disease inquestion [177]. In an evaluation of Aeromonas distribution in meat fromdifferent animals, mutton was found to be the source of the greatestnumber of microorganisms [116]. Hygienic practices and correct foodprocessing techniques are major contributors to avoiding contamina-tion by Aeromonas as well as other types of microorganisms. In relationto the techniques of food decontamination, susceptibility of these bac-teria to heat, acidity, disinfectants and irradiation itself have alreadybeen reported [178].

5. Aeromonas as a public health problem

Aeromonas are known to cause several diseases in humans. Thegenetic plasticity and diversity of virulence factors present in the genusmake them very versatile microorganisms. They are considered emer-ging pathogens [19], since Aeromonas are related to clinical cases ofgastroenteritis and infections in various organs and tissues[7,179–181].

There are increasingly frequent reports of pathologies attributed tothe Aeromonas genus. Due to complications that occur with the correcttaxonomic identification of the genus, clinical data on frequency andetiology of disease outbreaks may have excluded the presence ofAeromonas due to the lack of technical capacity to properly identifythese bacteria.

Their high production of toxins, ability to adapt to the most diversehabitats, tolerance of environmental stress and recent reports of theemergence of resistance to antibiotics [11,73,182], highlight the con-cern with the monitoring of these bacteria in hospital settings. For along time Aeromonas were believed to be pathogens of an essentiallyopportunistic nature; however, there is already evidence of virulentstrains causing severe septicemia in immunocompetent patients [179].Moreover, in humans, this genus also causes gastrointestinal, woundand soft tissue infections [7,183–186]. Muscle infections, skin diseases,eye infections, pneumonia and septicemia are examples of secondarypathologies triggered by virulent strains of Aeromonas [187].

The gastrointestinal tract is still the main target of infection by thesebacteria, with diarrhea being a common symptom [187]. Gastro-intestinal infections caused by Aeromonas can be confused with those ofcholera [188]. Other symptoms associated with gastrointestinal infec-tions caused by Aeromonas include fever, abdominal pain and dehy-dration [187]. These bacteria can cause necrosis and septic shock as aresult of infection in soft tissues [189].

One of the characteristics that most favor these bacteria with respectto contamination is their ecological adaptability, since they have a di-versified metabolism, which allows Aeromonas to be present in almostany environment and to be transmitted by diverse routes and vectors[6,25,182,190]. Species of Aeromonas most frequently associated withdiseases in humans are A. hydrophila (14.5%), A. caviae (37.6%), A.veronii bv. Sobria (27.2%) and A. dhakensis (16.5%), representing about96% of gastroenteritis cases [191,192]. However, as the taxonomy ofthe genus is constantly changing, new species have arisen and manystrains have been reclassified into different taxa.

Aeromonas infects human hosts mainly via consumption of con-taminated food and water, besides direct contact with aquatic en-vironments colonized by the genus [187,193]. Risk of contamination ishigher in aquatic environments during the summer, when water tem-peratures are elevated and bacterial populations are larger [194].Aeromonas are present in dairy, pork or beef products, fish, seafood andvegetables [13–15,25,183], as well as being frequently isolated fromdiverse activities of animal husbandry such as aquaculture, ranching,crustacean breeding and aviculture [71,151,191,195–198].

Aeromonas represents the challenges faced in modern clinical mi-crobiology: constant change in virulence caused by the acquisition ofgenetic determinants through lateral transference and emergence of

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multiresistance [199], including resistance to antibiotics of last choice[200]. Therefore, study of the aspects that constitute the biology ofthese bacteria is fundamental for the development of effective strategiesin the fight against these highly harmful microorganisms.

6. Conclusions

Aeromonas is a genus still on the rise in microbiology althoughisolated bacteria have been reported for approximately two centuries.Since they are emerging pathogens, widespread knowledge of thesebacteria is lacking. The authors hope that the approaches of this reviewmay assist those seeking to know Aeromonas and its effects on animaland human health.

Acknowledgments

The authors are grateful to the Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq) for fellowships(MTSC and LCBBC) and research grants. In addition, the Coordenaçãode Aperfeiçoamento de Pessoal de Nível Superior (CAPES) andFundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco(FACEPE) are acknowledged.

Conflicts of interest

The authors declare that there are no conflicts of interest.

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