Microbial Pathogenesis 130 (2019) 81–94

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Microbial Pathogenesis

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The genus Aeromonas: A general approach T 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

ARTICLE INFO ABSTRACT

Keywords: The genus Aeromonas comprises more than thirty Gram-negative bacterial species which mostly act as oppor- Aeromonas tunistic microorganisms. These bacteria are distributed naturally in diverse aquatic ecosystems, where they are Virulence factors easily isolated from animals such as fish and crustaceans. A capacity for adaptation also makes Aeromonas able to Infection colonize terrestrial environments and their inhabitants, so these microorganisms can be identified from different Diagnosis sources, such as soils, plants, fruits, vegetables, birds, reptiles, amphibians, among others. Infectious processes usually develop in immunocompromised humans; in fish and other marine animals this process occurs under conditions of stress. Such events are most often associated with incorrect practices in aquaculture. Aeromonas has element diverse ranges, denominated virulence factors, which promote adhesion, colonization and invasion into host 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, no single 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 been considered 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 Aeromonas genus 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 to clarify new tests for the precise identification of the species of Aeromonas, contributing to the exact and specific diagnosis of infections by these microorganisms and consequently the treatment.

1. Introduction Composing part of the class Gammaproteobacteria, order Aeromonadales and sharing the family Aeromonadaceae with two other It has been a journey of more than one hundred years structuring genera: Tolumonas and Oceanonimonas [3,5], the genus Aeromonas, the genus Aeromonas within the microbiological universe. It is believed currently with 32 recognized species [6], is constituted by facultative that the first isolates were reported in 1890 [1] and, throughout history, anaerobic, Gram-negative, rod-shaped and non-spore-forming bacteria these bacteria have been classified and reclassified among the most of approximately 1–3 μm[2,7] in length. Moreover, they are oxidase- diverse genera, such as Aerobacter, Pseudomonas, Escherichia, and Pro- positive [8], capable of fermenting glucose [9] and characterized by teus, among others [2]. Although there is a plethora of classifications, tolerating increasing concentrations of NaCl varying from 0.3 to 5% these microorganisms share common characteristics, which include gas [7]. Aeromonas are emerging pathogens capable of colonizing and in- production from glucose and their aquatic distribution [1,2]. The term fecting several hosts [10]. They are inhabitants of marine environ- Aeromonas (from the Greek words “Aer” meaning air or gas and “Mona” ments, so fish and other seafood are the most common sources for meaning units) was firstly proposed in 1936 by Kluyver and van Neil, to isolating these microorganisms. Therefore, they are widely known in embrace gas-producing bacteria [3]. However, it was Stanier who in aquaculture as potentially infectious organisms [11] and can cause 1943 officially used “Aeromonas” to denominate the genus where these diseases such as septicemia and furunculosis [12]. In addition, Aero- bacterial species were added [4]. monas can also be isolated from foods, such as vegetables, dairy

∗ Corresponding author. E-mail address: [email protected] (L.C.B.B. Coelho). https://doi.org/10.1016/j.micpath.2019.02.036 Received 10 July 2018; Received in revised form 27 February 2019; Accepted 28 February 2019 Available online 05 March 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved. R.B. Gonçalves Pessoa, et al. Microbial Pathogenesis 130 (2019) 81–94 products, beef and pork [13–15]. In humans, these microorganisms are other bacteria. However, there is the possibility that the colors ex- able to cause, most often, gastrointestinal system infections; however, hibited by bacterial colonies in ADA may vary, depending on the such processes if not treated properly can evolve and reach systemic manufacturer of the medium used, since some authors report that levels, generating septicemia [7]. On the other hand, species of Aero- presumptive Aeromonas colonies were yellow [30] while others report a monas are capable of infecting soft tissues, the hepatobiliary system, white color [31] when sown in ADA. causing ocular, respiratory and joint diseases and even bone infections, Use of selective media for Gram-negative bacteria is also common which is generally associated with a previous case of septicemia with regard to the isolation of this bacterial genus. In MacConkey agar, [16–19]. which is widely used in clinical laboratories, the mesophilic group of Aeromonas species produce a diverse and heterogeneous range of Aeromonas generally grows as a non-lactose fermenter, with A. caviae virulence factors. Expression of membrane components, toxins, en- being an exception, as it is most often a fermenting species [5]. Another zymes and several molecules contribute to bacterial pathogenicity [20] selective medium used is Hektoen Enteric Agar, due to its ability to and act in different ways, such as tissue adhesion, immune response inhibit Gram-positive bacteria. Combination of this medium with evasion, and involvement of host cells [10]. In order to disseminate CHROMagar Salmonella Plus and evaluation by MALDI-TOF has been virulence factors, Aeromonas have four types of secretion systems, re- reported as a reliable and practical alternative for detecting Aeromonas sponsible for release of these cell products into the extracellular en- [32]. vironment or even directly into the host cell [21]. The use of media that mainly differentiate the genus Aeromonas Identification of Aeromonas strains at the species level is still a great from similar genera is of extreme importance, since some may exhibit challenge. Due to the genetic heterogeneity existing in this genus, there similarity in phenotypic and even metabolic characteristics, which can is no effective biochemical evidence for this task [10]. Although new lead to an erroneous isolation and consequently an incorrect classifi- methods have been developed using molecular biology techniques, cation. Pseudomonas, for example, share similar physical aspects and amplification of constitutive genes through polymerase chain reaction oxidase tests [7]; apparently, ampicillin-enriched culture media can is still the best option to effectively identify species belonging to this contribute to differentiation [33]. Isolates were seeded in Glutamate genus [22]. Starch Phenol Red (GSP) agar base for selection of Aeromonas colonies, which showed yellow in color, while Pseudomonas colonies were pink 2. Isolation and identification [34]. Another genus to which these bacteria are sometimes erroneously Correct laboratory identification of the genus Aeromonas and its classified is Vibrio. In order to avoid misunderstanding, two media were components is still a great challenge. Several studies have been un- used: Aerosmart AH medium, which produces a yellow color with dertaken with the aim of making detection practical and reproducible, Aeromonas colonies, and Thiosulfate Citrate Bile Salts Sucrose agar thus increasing the reliability of results. (TCBS), where Vibrio species possess a yellowish aspect [35]. A re- The genus Aeromonas is divided, in relation to growth conditions sistance test to vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine and biochemical characteristics, into two main groups: psychrophilic, (O/129) is also recommended to promote differentiation between these composed of non-motile bacteria with good growth between 22 and two genera, since bacteria belonging to the Vibrionaceae family are 25 °C; and mesophilic, which grow well at 35–37 °C and are motile [3,7] characterized as sensitive [7]. However, this methodology does not owing to a single polar flagellum, for the most part [2]. When grown in provide a totally reliable result, since there are reports of Vibrio species a laboratory, some differences related to the appearance of the colonies resistant to the substance O/129 [36]. can be noted. Aeromonas belonging to the psychrophilic group re- Conventional biochemical tests, as well as automated systems, are of presented mainly by A. salmonicida, are characterized as the main fish limited utility in the identification of some Aeromonas species [37]. In [21] and reptile pathogens [7]. Bacterial colonies appear as pin-points addition, their accuracy is affected by constant reclassification among within the first 24 h of incubation at 20–22 °C, but after approximately components of this genus [22]. Some biochemical profiles are reported 4 days of incubation, they become circular and convex, with a diameter as standards for taxonomic distinction. According to Bergey's Manual of of 1–2mm[23]. Although bacteria belonging to the mesophilic group, Systematics of Archaea and Bacteria, the genus Aeromonas comprises D- such as A. hydrophila, A. caviae and A. veronii [2], are also reported to be glucose fermentative facultative anaerobic bacteria that are D-glucose harmful to marine animals [24], they are more frequently reported fermenters with or without gas production. In addition, they are cata- causing infections and other diseases in humans [3,7], such as gastro- lase and oxidase positive, reduce nitrate to nitrite, produce several intestinal diseases and septicemia [25]. Bacterial colonies of the me- enzymes and are capable of using other carbohydrates besides glucose sophilic group are 1–3 mm in diameter and circular, convex and [5]. However, there are some methodologies that present more varied translucent, showing a buttery consistency after 24–48 h of incubation results than those previously established for Aeromonas identification. at 35 °C [5]. Strains were classified as Aeromonas sober with a positive result for Cultivation and isolation of Aeromonas in a laboratory can be per- arginine dihydrolase (ADH), Voges-Proskauer and gelatin hydrolysis, formed in a variety of culture media (Fig. 1). Tryptic soy agar (TSA) and contrary to what had previously been proposed in the literature [38]. tryptic soy broth (TSB) have been routinely applied for the maintenance Another study reported the same result, in what the first test was pre- of samples isolated from contaminated fish and water tanks [26]. valent of all 25 A. sobria strains [39]. A divergence was identified for Starch-Ampicillin agar was used for isolation of A. hydrophila in com- elastase production between A. hydrophila and A. sobria, which con- mercially obtained foods. After growth of the bacterial colonies, Lugol tributed to the differentiation of these two species [38]. Similar positive iodine solution was added and those that were amylase-positive were results have been described for salicylin, rhamnose and elastase be- presumed to be A. hydrophila [27]. Media such as cefsulodin-irgasan- tween A. salmonicida and A. bestiarum [40]. Moreover, most of them novobiocin agar (CIN), MacConkey agar and blood agar enriched with were negative for lactase production, which is not specific among ampicillin were used when aiming to do a presumptive identification of Aeromonas, although they mainly grow on MacConkey agar [41]. Dif- Aeromonas colonies from fecal samples [28]. In addition, for the same ferences have also been identified that support the distinction among A. type of sample, taurocholate-tellurite-gelatin agar (TTGA) was used, veronii, A. sobria and A. encheleia, as well as distinct profiles that dis- which was also tested for sow rectal swabs. In this case, oxidase-positive tinguish the last two. A. sobria and A. veronii biovar. sobria strains were colonies showing a gray aspect without a black center but a zone of differentiated by starch hydrolysis and arabinose fermentation [42]; the opacity were tested for Aeromonas species [29]. Ampicillin Dextrin Agar last test was predominantly negative in A. veronii biovar. sobria [43]. (ADA) has also been used for isolation of this genus and the color of the The diversity of results reflects the difficulty in promoting conclusive colonies displayed in the medium is what differentiates Aeromonas from identification based on biochemical methods.

82 R.B. Gonçalves Pessoa, et al. Microbial Pathogenesis 130 (2019) 81–94

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

Due to the challenges described, molecular techniques are still the Specifically, gyrB, encoding the B subunit of the enzyme DNA gyrase, best option for identification and taxonomic classification of the genus and rpoD, which encodes one of the factors involved in RNA polymerase Aeromonas. A specific molecular marker for this genus that has been activity, are the most widely used housekeeping genes in taxonomic used to identify and distinguish Aeromonas from other genera is the studies and allow greater reliability in the phylogenetic classification in gene-encoding glycerophospholipid cholesterol acyltransferase (GCAT). Aeromonas [52]. Although the 16S rRNA has only limited distinguishing Its amplification by conventional molecular methods, such as poly- power for this genus, it can be used in conjunction with gyrB; the latter merase chain reaction (PCR), has demonstrated qualitative results in being confirmatory [53], since its ability to identify species and strains the identification of Aeromonas in several samples [44,45]. However, is proven [54]. the reliability of this method is better assured when associated with The genes gyrB and rpoD offer advantages to phylogenetic studies; other techniques that aim at the taxonomic classification of evaluated however, a concatenated analysis of not only two but seven house- strains. The most common technique involves the 16S rRNA gene, the keeping genes guarantees a more advanced and concrete way of ob- main component of the 30S ribosomal subunit [46]. Its sequencing is taining a taxonomic classification from this class of genes. This tech- universally known in relation to the establishment of phylogenetic re- nique is called Multilocus Phylogenetic Analysis (MLPA) [55]. It lationships among bacteria [47], however, accuracy of this method is consists of the individual and joint evaluation of sequences of the genes: limited when analyzing strains whose sequences are very similar [48]. gyrB, rpoD, recA, dnaJ, gyrA, dnaX, and atpD, which are classified as The amplification of 16S rRNA for sequencing is generally done by housekeeping genes. This method is capable of providing consistent conventional polymerase chain reaction (PCR), using extracted bac- phylogenetic data, which aid in the taxonomic classification of Aero- terial DNA. However, it has been reported that sequencing of the 16S monas as well as in the discovery of new species [55]. rRNA gene amplified by direct PCR colony, a method that excludes Other molecular-level methods can be applied for effective phylo- DNA extraction and purification of the PCR product, is not only cheaper genetic studies in Aeromonas. Restriction Fragment Length but also more efficient in the taxonomic classification of Aeromonas Polymorphism (RFLP) analysis allows the study of small variant re- species (whose sequences are not similar) than the amplification of the gions, or polymorphisms, in DNA using restriction enzymes [56]. Ap- 16S rRNA gene using bacterial DNA previously extracted and purified plication of this technique in 16S rRNA analyses has been reported as a [49]. fast and effective way to identify some Aeromonas species [57,58]. As the low accuracy of 16S rRNA sequencing is due to the high Additionally, the matrix-assisted laser desorption/ionization time-of- similarity among the sequences [22], the amplification of so-called flight mass spectrometry (MALDI-TOF MS) method is considered a housekeeping genes [50,51] is presented as the best way to do taxonomic powerful tool in the identification of microorganisms [59]. This tech- classification, since they have a greater discriminatory capacity [25]. nique consists of the ionization of samples and generation of gas-phase

83 R.B. Gonçalves Pessoa, et al. Microbial Pathogenesis 130 (2019) 81–94 ions which, after particle acceleration in an electric field, are detected 3.1. Structural components according to their speed (depending on mass and ionic charge) upon arriving at the detector [60]. MALDI-TOF MS allows for the analysis of The structural composition of a bacterial cell has a great influence the protein content of a microorganism, constituted largely of ribo- on the infectious process. Several factors such as flagella, pili, proteins somal proteins, but also cytosolic proteins, such as heat-shock proteins. and membrane antigens, among others, have been studied and related Particular differences in the protein composition among microorgan- to roles in bacterial pathogenicity, for example, locomotion, adhesion isms are what effectively guarantees the success of the technique in to host tissue, protection against bactericidal agents and immune taxonomic distinction, not only at the level of genus, but also of species system cells [73]. [59,60]. The application of MALDI-TOF in phylogenetic study and Flagella are structures whose main function is to promote bacteria protein characterization in Aeromonas has already been reported locomotion; they may be present at different sites [74]. They are [9,61]. In addition, there is a molecular tool capable of accurately and composed of an external filament and an inner portion, denominated universally providing bacterial characterization through the analysis of the basal body, attached to the membrane [75]. Two flagella types have the nucleotide sequence of multiple chromosome sites, the Multilocus been identified in most Aeromonas species belonging to the mesophyll Sequence Typing (MLST) [62]. Such a technique detects changes in group, polar and lateral, which confer motility in liquid environments DNA quickly and reproducibly without the need for reagent exposure and solid surfaces, respectively [74]. In addition, they perform func- and does not require living bacterial suspensions or even high-quality tions relating to cell adhesion and persistence in the infectious process genomic material, sparing the difficulties associated with the transport [3]. and handling of bacterial samples [63]. Two major types of motility are observed according to the sort of Genomic sequence of a microorganism represents the extreme level flagellum produced. Swimming motility is associated with liquid en- of information that can be used in phylogenetic studies [64]. The pro- vironments [75] and promoted mainly by the polar flagellum, while the portion of guanines (G) and cytosines (C) in microbial DNA is the lateral type promotes swarming movement on solid surfaces [3]. Mo- parameter most frequently evaluated in genome-based phylogenetic tility types have been verified in Aeromonas species isolated from retail classification. The DNA-DNA hybridization technique (DDH) consists of sushi, whose analysis has been made by sowing strains in media such as determining the genetic distance between two microorganisms based nutrient agar and LB, measuring the colonies growth diameter from the on their G + C content [65]. Such evaluation establishes a limit in center to periphery [25]. Several genes are involved in the production similarity percentage as a criterion to classify two organisms as be- of flagella [21]. Virulence genes have been investigated by multiplex longing or not to the same species. In the case of DDH, when genomic polymerase chain reaction (PCR) in Aeromonas strains isolated from sequences present below 70% similarity, the evaluated microorganisms clinical and water samples, identifying the fla gene, which encodes the are considered distinct species, whereas when they have similarity polar flagellum, in most isolates from both sources [76]. The same gene greater than 70%, they are classified as belonging to the same species was also detected in Aeromonas species isolated from aquaculture en- [66]. For a long time, this form of evaluation was considered the gold vironments and slaughterhouses [71]. However, the gene coding the standard with respect to the taxonomic classification of Archaea and lateral flagellum, laf, was only found in slaughterhouse samples. Oc- Bacteria domains. However, since there are several limiting factors: the currence of mutations in these genes can affect both adhesion capacity complexity of the technique, a high probability of errors, and the need and biofilm formation [73], leading to virulence reduction. A. hydro- for several other tests for statistical evidence of results, analyses of si- phila mutant strains without the flgC and flgE genes, responsible for milarity among 16S rRNA gene sequences was used instead, as dis- production of the flagellum inner portion and hook filament, respec- cussed above [65]. Nevertheless, in order to make the results obtained tively, had partial and total reduction in motility, respectively, as well by the DDH useful, other techniques have been developed. The in silico as defects related to adhesion capacity and chemotaxis [77]. DDH (isDDH) method is able to produce values close to those obtained Pili, or fimbriae, are slightly smaller structures than the flagella [21] from DDH, establishing limit values by standardized parameters. Thus, and are mainly responsible for ensuring bacterial cell adhesion to host bioinformatic assessments can be facilitated and the variations in DDH tissue [74] and other solid surfaces [78]. Four types of pili are found in overcome [67]. Due to the scientific advances in and availability of Gram-negative bacteria, with types I and IV described in Aeromonas complete genomic sequences, new comparison indices have been cre- species [21]. Type I pili are structurally characterized as short-rigid [3] ated to calculate similarities without the need for functional steps, in an and are often reported in environmental samples; generally, these objective and reproduceable way [68]. For example, Genome Blast structures are unrelated to the pathogenicity of the bacterium [74] and Distance Phylogeny (GBDP), Maximal Unique Matches Index (MUMI) most of the time are present in A. hydrophila [3]. A mutant strain was and, most commonly, Average Nucleotide Identity (ANI), considered developed of A. salmonicida which lacks the gene cluster related to the the possible gold standard of its generation for species distinction. ANI production of type I pili and tested in vivo in Atlantic salmon (Salmo is the mean of identity values and similarity between two genomes salar) species. Adhesion efficacy to host tissue was detected, while in- [64]. In general, it is calculated in two steps, consisting of genome vasive capacity remained intact [79]. Type IV pili are characterized as fragmentation into sequences of approximately 1020 bp and, with the flexible-wavy [3] or long-wavy [74] and are more related to bacterial aid of Blast program, comparing each fragment generated over the se- pathogenicity [21]. In addition, they are generally detected in isolates quence of another genome; therefore, ANI is calculated according to from clinical samples [74] and usually found in A. hydrophila, A. veronii, values of identical nucleotides [68]. A. caviae and A. trota [3]. There are three main genes related to the expression of type IV pili. The first is the tap gene, associated with the tapABCD cluster, which exerts different functions [73]. TapA is re- 3. Virulence factors and correlated genes sponsible for the production of class A pili, the type IV pili-forming subunit. An A. salmonicida strain containing a mutation in the tapA gene Aeromonas virulence is complex, since several factors contribute was less pathogenic to rainbow trout (O. mykiss)[80]. Moreover, pre- significantly to the development of an infectious process [69] as the viously challenged specimens, which were exposed to wild strains efficacy of the host immune system decreases [70]. Structural compo- (containing the tapA gene), exhibited a higher resistance to infection nents, toxins and extracellular products [21], acting jointly or in- than those challenged only with the mutant strains, indicating im- dividually [71], enable these microorganisms to colonize and infect munogenic potential to products expressed by this gene. The tapB and hosts [25]. The Aeromonas virulence complex, due to this diversity, has tapC genes are responsible for securing subunits within pili while the not yet been established [72]. tapD gene contributes to protein processing and maturation [80]. The second formative gene of type IV pili is flp, coding biosynthetic proteins

84 R.B. Gonçalves Pessoa, et al. Microbial Pathogenesis 130 (2019) 81–94 of peritrichous pili. Challenging Atlantic salmon (S. salar) specimens production of specific antibodies, as explained, the content of in- with mutant strains of A. salmonicida subsp. salmonicida revealed that flammatory response modulators is not substantially altered [90]. the absence of the tap gene contributed more to strain virulence than In addition to all this content, the bacterial cell outer membrane is the flp gene, whose contribution was minimal and even nonexistent enveloped by a hydrated structure composed mainly of monosaccharide [78]. The Msh gene encodes grouped filaments of type IV pili and these units, linked together by glycosidic bonds, denominated capsule [3,75]. are considered the biggest explanation for bacterial colonization ability However, capsules of some bacteria can also have polypeptides in their [74]. Thus, it is also known as the bfp (bundle-forming pili) gene [21]. constitution [21,74]. They serve the function of conferring resistance to The importance of this gene was revealed when it was verified that immunological reactions, such as phagocytosis and complementary mutant strains from A. hydrophila, which did not contain the mshQ system actions [3]. In a cluster responsible for capsule expression in an gene, presented deficient adhesion and biofilm production; the pili A. hydrophila strain contained 13 genes divided into three regions, the expressed by this gene are crucial in the adherence of this micro- first and third had transport-related genes, while the second was re- organism to solid surfaces [81]. sponsible for structure synthesis [91]. The two genes orf1 and wcaJ, The protein composition of the outer membrane plays a relevant belonging to the cluster of capsule formation, have been reported [92]. role in Aeromonas virulence. Outer membrane proteins (OMP) have The relevance was justified since strains of A. hydrophila mutants did functions related to osmoregulation and nutrient absorption, among not develop capsules even in propitious medium. others [21] and have been researched for the development of vaccines. Aeromonas are also well known for being biofilm producers [93]. The products of the ompA1, tdr and tbpA genes, encoding the outer Biofilms are an extracellular polymer matrix of three-dimensional ar- membrane protein A (OMPA), TonB-dependent receptor and transferrin chitecture composed mainly of proteins, but also of polysaccharides and binding protein A, respectively, have been evaluated as candidates for DNA molecules [94]. Moreover, microorganisms, which interact with the development of vaccines against A. hydrophila [82]. The first two each other, account for part of the total volume of the biofilm; taking genes cited were able to provide strong immunological protection to advantage of the nutrients extracted from the environment where this catfish (I. punctatus) specimens. The cell wall of Aeromonas species also microbial community is formed [95]. Biofilm formation is considered a contains substances involved in the pathogenic process [83]. Tetra- virulence factor by confering resistance to bactericidal agents [73] and gonally arranged proteins present on the superficial layer contribute to promoting adhesion to abiotic solid surfaces as well as to host cells, the adhesion to membrane components of the host cell [3,74] and play which characterize the first stage of the infection process [93]. Biofilm an important role in protection from immune system responses and formation of Aeromonas spp. strains was identified by culturing the phagocytic cell actions [84]. Surface proteins (A-proteins) denominated microorganisms in 96-well polystyrene plates [71]. Following succes- A-layers or S-layers (superficial layers), are encoded by the vapA gene, sive washes, an optical density reading was performed after addition of also referred to as ashA by some authors [74 ,84]. Tests with culture crystal violet, which stained the bacterial cells adhered to plate. Biofilm medium containing bright coomassie blue can indicate the presence of formation is not limited to the presence of nutrients, but also to their these proteins [21]. These structures were predominant in membrane concentrations, and depending on the nutrient, they can act in an in- protein extracts from A. salmonicida strains, composing 60% of protein hibitory way. Effects of glucose concentration variation on biofilm concentration after separation by two-dimensional electrophoresis production have been evaluated by A. hydrophila strains isolated from [83]. The amino acid sequence of A-proteins from five strains of A. different sources [96]. Bacteria cultured in media of up to 0.05% glu- salmonicida subsp. salmonicida revealed that four sequences were cose did not present significant alterations; concentrations between identical, and one presented a difference in only one amino acid [84]. 0.25% and 2.5% were shown to be inhibitory. Expression of lateral The effects of an A. hydrophila mutant strain, lacking the vapA gene, on flagella is also related to biofilm production. Molecular methods iden- pathogenicity to rats, rainbow trout (O. mykiss) and larval zebrafish tified the presence of fla and laf genes coding polar and lateral flagella, (Danio rerio) specimens have been evaluated [85]. After intraperitoneal respectively, and their relationship to biofilm formation capacity of A. injection of the strain, there was no change in virulence, but a con- caviae samples from different sources [97]. Production was confirmed tribution to the inflammatory process due to induction of interleukin only in fla- and laf-positive samples. Sets of samples that only had the production. polar flagellum gene or those that did not have either of the two genes The outer membrane of Aeromonas spp. is composed of molecules tested did not form biofilms. Antigenic capacity of this virulence factor that, besides assisting in the adhesion process, has immunostimulatory has also been investigated. Channa striatus specimens fed with biofilm- activity [21]. The so-called membrane lipopolysaccharides (LPS) are containing rations have been identified with high antibody titers that amphiphilic complexes composed mainly of 3 domains [86,87]. The promoted a high survival rate when challenged by harmful doses of A. molecule's lipid portion is characterized of a highly conserved bioactive hydrophila [98]. subunit, named lipid A, covalently bound to the polysaccharide struc- ture and to the outer membrane [3,21]. On the other hand, the LPS 3.2. Extracellular products polysaccharide complex is formed by a central region and an oligo- saccharide sequence, containing from one to six carbohydrate mole- A remarkable variety of extracellular products is generated among cules, which confer hydrophilic and antigenic characteristics to mem- Aeromonas species [99]. Some enzymatic classes, such as hemolysins, brane lipopolysaccharides, denominated the O antigen [3,87]. O lipases and proteases besides toxins have been studied over time due to antigens, also known as specific O polysaccharides, confer intraspecific their role in the infectious process and host impairment [21]. divisions within most bacterial species called serovars or serogroups Hemolytic enzyme production is an alert factor related to several [86], as well as contribute to the development of the immune response, bacterial genera. Three major types of hemolysins have been identified being inducers and targets of specific antibodies [21]. Genes encoding among the products secreted by Aeromonas [100]. Aerolysins are pro- the LPS central region belong to a cluster, named cluster wa, and are teins capable of altering the permeability of blood cells, as well as other identified in three regions [88]. The wa 1 region has genes related to eukaryotic cells (and consequently promoting osmotic lysis) due to production of the upper portion of the molecule, which is bound to the polymerization of the structure induced by binding to a membrane- O antigen, whereas wa 2 and wa 3 are responsible for the synthesis of speci fic glycoprotein site [101]. The gene responsible for expression of the lower portion, which binds to lipid A [86,89]. These genes are this molecule, and studied by molecular methods, is named aer [102], spatially close to the coding clusters of other structural components, which is divided into three types: aerA, aerB and aerC, of which the first such as the already mentioned flagella and pili [21]. Although the is responsible for aerolysin production itself; the latter two are re- immunostimulatory capacity of LPS is known, the inflammatory process portedly involved in expression modulation and enzymatic activity induced is generic, that is, despite the O antigen inducing the [102,103]. Hemolysis types can be identified by sowing the bacterial

85 R.B. Gonçalves Pessoa, et al. Microbial Pathogenesis 130 (2019) 81–94 strains in blood agar [25]. In addition to aerolysin, which induces β- prostaglandins in intestinal cells [75,119]. hemolysis, complete lysis of erythrocytes, β-hemolysins, another type of The act, asp and alp genes have been researched enough by mole- enzyme with hemolytic activity, can also be evaluated by molecular cular methods to evaluate Aeromonas virulence. Seven different methods [104]. Despite partial hemolysis promoted by α-hemolysin Aeromonas species from raw meats and clinical samples of diarrhea having a reversible effect, research into it is not as widespread as the have been isolated; most had at least one enterotoxin-encoding gene other two types [100]. [116]. All A. hydrophila strains contained the act and alt genes, while a The capacity of promoting damage to the intestinal epithelium of few had the ast gene. The absence of the alt gene in Aeromonas strains fish is conferred by lipolytic activity [21]. Lipases are enzymes re- isolated from clinical and environmental sources was reported; the act sponsible for the hydrolysis of triglycerides and are of important com- and ast genes were identified in a few isolates [41]. The act gene was mercial value [105]; different bacterial genera produce and secrete more prevalent than alt and ast among 176 strains isolated [118]. Two such molecules to perform distinct functions [106]. Genes such as lip, isolated strains that contained both act and ast genes in their genome, a lipH3 and pla are alternatives for lipase production [107]. Another type combination rarely found, have been reported [72]. of enzyme has been characterized, phospholipase C also known as Due to the fact that they are considered emerging pathogens related lectinase, which has been considered a virulence factor and presents to cases of diarrhea in humans [120], another toxin type reported cytotoxicity, with low or non-existent hemolytic activity [108]. The apl- mainly in the genus Shigella has been investigated among the Aeromonas 1 gene encodes this molecule [107]. In addition to the cited examples, species. The shiga-like toxins are exotoxins mainly causing hemolytic another type of lipase, glycerophospholipid:cholesterol acyltransferase uremic syndrome and can be harmful to the nervous system. Shiga (GCAT) was characterized as one of the most lethal virulence factors in toxins 1 and 2 are encoded by the stx1 and stx2 genes, respectively A. salmonicida [109]. Coded by the gene gcat, this enzyme can complex [121]. The production capacity of these molecules by Aeromonas is with LPS, becoming more toxic than its free form [21]. variable, like other virulence factors. Shiga-like toxins have been Extracellular proteases contribute to the process of bacterial mul- identified in animal isolates [34]; however, the presence of these genes tiplication, as well as act on host nutrient sequestration and the immune was not detected in bacteria isolated from retail sushi [25]. Both stx1 response [70]. Two major components have been identified in this class and stx2 were absent in a study from environmental isolates [122]. of enzymes [100]. Two different fractions of proteases have been identified in A. hydrophila, one with strong caseinolytic activity, called 3.3. Secretion systems serine protease, and another showing action against elastin and casein, known as metalloprotease [110]. This class of enzymes, beside usual Transfer of the virulence factors produced by bacteria to the ex- functions, also plays the role of activating other virulence factors. The tracellular medium and/or to host cells is extremely relevant to the participation of serine protease in GCAT activation of A. salmonicida has contamination and infection processes. Six different types of secretory been reported [111]. Molecular research aiming at amplifying genes systems have been detected in Gram-negative microorganisms [ 3]; four encoding extracellular enzymes is used to verify virulence potential of them reported in the genus Aeromonas, being types II, III, IV and VI among different Aeromonas strains. The presence of these enzymatic [21]. Aeromonas secretion systems, compositions and functions are re- virulence factors is common in isolates from frozen fish [112]. The presented in Fig. 2. genes lip, gcat and ser (coding serine protease) have been identified in Type II is known as a secretion pathway for substances, such as most of the isolated strains of pond-raised catfish (Ictalurus punctatus); toxins and mainly enzymes, to the extracellular environment through a however, the aerA gene was the most prevalent [113]. Similar results process that, in Gram-negative organisms, involves inner and outer have been reported in isolates from a trout farm [114]. cellular membranes, as well as the space between them, the so-called Great attention has been given to the effects generated in the gas- periplasmic space [123]. To have secretion of a given substrate, usually trointestinal system in cases of infections caused by the members of the polypeptide, by the type II pathway, its primitive form must be pro- genus Aeromonas; they are considered emerging pathogens to humans duced in the cytoplasm and then transported through the inner mem- [31]. The most common pathological condition is gastroenteritis [47], brane into the periplasmic space, where it reaches its final conforma- which may evolve with more aggravation into clinical manifestations tion. Two types of transport perform this function according to the [115]. Major contributors to development of gastrointestinal tract dis- molecular characteristics of the substrate. The general secretion (Sec) eases are different toxin types that bacteria of the genus Aeromonas are pathway carries protein filaments that do not need to be folded before capable of producing; among which cytotoxic enterotoxin and two cy- they are introduced into periplasmic space. However, the formation totonic enterotoxin types are well known [116]. process of some molecules requires a first folding of the structure in the Cytotoxic enterotoxin promotes degeneration of villi and mucus- cytoplasm; in this case, transport is carried out by the Twin Arginine producing cells [76], generally related to cases of bloody diarrhea in Translocation (Tat) pathway. The type II secretion system is composed humans [117]. In addition, it has hemolytic activity, which many au- of four components that communicate with one another: a protein base thors treat as being an aerolysin, a member of the hemolysin class [71]. located in the inner membrane; a cytoplasmic ATPase, which exhibits However, even though there is strong similarity, others consider them properties similar to type IV pili; the type II pseudo-pili; and finally a distinct molecules [69]. This enterotoxin, encoded by the act gene, protein referred to as D protein or secretin. Polymerization of secretin generates an inflammatory response in host cells, a factor that leads to leads to the formation of a channel in the outer membrane, the site of systemic involvement [118]. Morphological changes generated by cy- passage for substrates from the periplasmic space to the extracellular totoxic effects in hamster ovary (CHO) cells, human laryngeal epi- medium. In addition, it is also important for the formation of type II dermoid carcinoma (HEp-2) and African monkey kidney (VERO) cells pseudo-pili. In general, contact of proteins to be secreted with the se- have been detected. Cell rounding and shrinkage were identified, fol- cretin periplasmic portion stimulates retraction of pseudo-pili, which, lowed by cytoplasmic and cell membrane disorders [115]. by means of the energy supplied by cytoplasmic ATPase, pushes protein Unlike cytotoxic enterotoxin, cytotonic enterotoxins do not cause substrates through the secretin channel, to be released into the extra- degenerative damage to the intestinal epithelium [3] and are related to cellular space where they can exercise their respective biological cases of non-bloody diarrhea [117]. They are divided into two groups: functions [124,125]. heat-labile cytotonic enterotoxins, which are encoded by the alt gene Studies have been performed with the aim of uncovering the com- and can be degraded at 56 °C for 10 min. While heat-stable cytotonic ponents, as well as their respective functions, of the type II secretion enterotoxins, with degradation at 100 °C for 30 min, are expressed by system in A. hydrophila and A. salmonicida strains. The participation of the ast gene [3,69]. These toxins promote elongation in CHO and se- two inner membrane proteins, ExeA and ExeB, has been described as cretion of liquids in rat ileum, but also induce increased cyclic AMP and the key to the secretion process [126]. ExeA has two main domains, one

86 R.B. Gonçalves Pessoa, et al. Microbial Pathogenesis 130 (2019) 81–94

Fig. 2. Aeromonas secretion systems, compositions and functions. cytoplasmic, linked to ATPase, and another periplasmic, which has a tested had all the genes studied; sequence analysis revealed less genetic peptidoglycan-binding site. In the presence of the ligand, ATPase gen- variability in AexT, indicating that its function is conserved for this erates energy for the formation of the ExeAB complex, which is crucial species [127]. The ascV mutation attenuated the virulence of A. sal- for multimerization of ExeD monomers in the inner membrane, which monicida subsp. salmonicide, since mutant strains were phagocytized by are then translocated to the outer membrane for secretin formation. In leukocytes, contrary to what happens in wild isolates [129]. When the the absence of the ExeAB complex, ExeD is maintained in the inner aopB gene, responsible for the formation of the translocon in A. hy- membrane, interfering in the secretory process. In addition, the im- drophila strains, was deleted, a reduction of the toxicity in cultured cells portance of ExeB in this process was investigated and it was concluded followed [128]. In addition, the mutant isolates for both aopB and act that such a protein acts to support the structure formed by the three (coding for cytotoxic enterotoxin) were avirulent in the in vivo assay molecules in association with the binding peptidoglycan [126]. The using rats. The presence of the type III secretion system in Aeromonas influence of this ligand on the formation of the ExeAB complex from spp. was detected through the amplification of the ascV gene. Among 64 ExeA was also emphasized [123]. strains tested, 29 were positive for the search performed and these were Current knowledge suggests that the type III secretion is perhaps the quite harmful to HEp-2 cells [115]. Amplification of the ascV and aopB most widespread system in relation to Aeromonas virulence. It consists genes with a similar purpose was accomplished and the presence of of a thorn-shaped or syringe structure responsible for injecting proteins these genes was verified as being more prevalent in Aeromonas strains harmful to cellular metabolism directly into the host-cell cytoplasm isolated from diseased fish, which caused the highest mortality rates [127]. Unlike type II, the base of the type III secretory complex is a when injected intraperitoneally into Nile tilapia (O. niloticus) specimens structure composed of approximately 15 proteins, which interconnects [130]. the inner and outer membranes, known as a basal body [125]. At the Participation of the type III pathway in the development of the base of the system a protein arrangement known as “injectisome” is immune response has also been studied. The presence of antigens of this exposed to the extracellular medium [128]. This structure has an inner secretory system hampers immunological activity in rainbow trout (O. channel and a complex of proteins at the tip with sensory activity, mykiss), since the vaccination of fish with mutant strains of the ascV which detects contact with the membrane of host cells. Thus, promoting gene induced a significant survival rate in relation to those vaccinated the structuring of the third component of the type III system, the with antigens from wild strains [131]. Infections generated by Aero- translocon, which acts together with sensory proteins to form a pore in monas containing this system, functional or inhibited, have been iden- the eukaryotic cell [125]. Thus, products to be secreted have free tified; they developed suppression of the immune system in rainbow passage between the two cytoplasmic environments without ever being trout (O. mykiss)[132]. However, those classified as lethal were only exposed to the extracellular medium. developed by strains that had the structure of the fully functional type Research involving the detection and deletion of genes related to the III secretion pathway; those devoid of such a system did not cause expression of components of the type III secretion pathway, as well as deficits in the immune response and were not lethal to the fish tested. its effector molecules, contributes to the understanding of the im- Exchange of genetic material among bacteria is a process that guaran- portance of this system in bacterial virulence. The prevalence of genes tees survival of these microorganisms under established environmental encoding the type III secretion system, asc-V and ascF-ascG, as well as conditions. The most important clinical consequence of this event is the two effector proteins of the complex, AexU and AexT, characterized as spread of antibiotic-resistant genes among bacterial species, generating ADP-ribosylating toxins has been analyzed. Twenty A. veronii strains what are known as multidrug-resistant microorganisms [133].

87 R.B. Gonçalves Pessoa, et al. Microbial Pathogenesis 130 (2019) 81–94

Moreover, the aquatic environment, since it receives discharges from ions. This path is known as siderophore-independent [144]. urban and industrial effluents, favors this process by conferring greater To support various environmental conditions in which they are in- contact among wild strains and drug substances [134]. This, in theory, serted, bacteria proliferate in populations, which may follow various suggests that Aeromonas, since they naturally inhabit such environ- mechanisms to develop a favorable medium for the maintenance and ments, are potentially capable of acquiring antimicrobial multi- protection of bacterial cells, such as biofilms [95]. Intercellular com- resistance. munication in a bacterial population is performed through QS [21], Unlike protein transport, the translocation of DNA molecules is which is a chemical signaling pathway that controls members' behavior more complex [135]. In Gram-negative bacteria, this function is con- of a given bacterial population through gene expression [145]. Signal ferred by the type IV secretion system [124]. This pathway is divided molecules secreted in this communication pathway vary depending on into three subgroups with different functions, the most prevalent one the bacterial classification. In Gram-positive bacteria they are small being for bacterial conjugation [136]. In addition to the genetic ma- peptides, while in Gram-negative, as is the case of Aeromonas, they are terial, several other types of molecules can be secreted and destined to acylated homoserine lactones (AHLS)[21,146]. In general, each bac- recipient cells, which may be other bacteria (not necessarily of the same terial cell in a population is able to secrete and respond to these che- species) or eukaryotes [125]. mical stimuli by different ways, either to increase nutrient uptake, to Neither the components nor structure of the type IV system in respond to competition with other organisms or to promote defense Aeromonas have yet been well elucidated. However, some products against phagocytic cells, among other actions [21,147]. Signaling mo- secreted by this route have been identified. Plasmid genes related to lecules produced by a LuxI synthase enzyme, are recognized by a spe- tetrodotoxin (TTX) biosynthesis as well as their regulation have been cific receptor, LuxR, a transcriptional regulator of several genes. The detected [137]. Moreover, the strains tested also contained genes as- ligand-receptor complex binds to DNA and initiates gene expression, sociated with expression of the type IV system, which suggests a strong including those expressing LuxI, which makes this system self-inducing relationship between the product and its secretory complex. [145,148]. The activated gene regulator decays slowly, reducing pro- Little is known about the mechanisms related to the type VI secre- duction, with accumulation of signal molecules [146]. tion system; it is generally responsible for contact-dependent translo- cation of proteins from one bacterial cell to another, as well as to eu- 4. Ecology of Aeromonas karyotes [125]. It is possible that the secretory structure of this system derived from phagocytic mechanisms [138]. Like type III, it also se- As mentioned before, Aeromonas can be isolated from several nat- cretes effector molecules for deregulation of the recipient cell. The ural sources [23], particularly aquatic environments [31]. Contact with existence of a gene cluster, VasH, was reportedly responsible for pro- fish and other aquatic animals develops in a continuous and almost duction of the hemolysin-coregulated protein (hcp), an effector enzyme inevitable way [149]. Thus explaining the variety of species from which that has a role in the modulation of the immune response, preventing these microorganisms have already been isolated, such as Nile tilapia phagocytosis [138,139]. Another effector, a component of the VgrG (Oreochromis niloticus)[53], rainbow trout (Oncorhynchus mykiss)[24], family of proteins, that is VgrG1, was identified which required prior channel catfish (Ictalurus punctatus)[150], Japanese eel (Anguilla ja- contact between a secretory bacterial cell and a host cell to be trans- ponica)[151], crayfish (Pacifastacus leniusculus)[152], tambaqui fish, ferred by the type VI system [140]. Genes homologous to hcp and VgrG Colossoma macropomum [153], oysters [154], and ornamental fish located outside the cluster of the type VI secretion system were iden- [9,117]. Aeromonas are considered opportunistic pathogens [21]; they tified; the deletion of these genes did not affect secretion of effector also can act as secondary agents in other previously installed patholo- proteins, suggesting redundancy in maintaining the structure of this gical dysfunctions [9]. Most of the time, the main trigger for develop- secretion pathway [138]. ment of infections is stressful conditions generated in aquatic animals by environmental changes [152], such as alterations in water char- 3.4. Other virulence factors acteristics and constituents [21]. Despite expectations, captive-bred species are more susceptible to stress than wild-bred species [70]. Aeromonas possess a complex system of virulence factors, which Fish and other seafood are sources of numerous nutrients. have been uncovered over time, clarifying their ability to infect several Consumption of these foods can considerably reduce risks for the de- hosts and colonize the most varied environments. However, bacterial velopment of inflammatory diseases, such as atherosclerosis, acute cell survival depends not only on how harmful it may be but also on myocardial infarction, stroke, among others [155], which explains how well it is able to maintain itself in situations unfavorable to its these products being universally consumed in diets [156]. With the growth. Thus, the means of acquiring essential nutrients and of bac- expected world population growth, there is an increasing demand in terial intercellular communication are also considered virulence fac- food production. However, natural stocks of fish and other seafood are tors, which, in Aeromonas, refers to the mechanisms of iron acquisition not able to keep up with this reality [157]. Thus, the practice of and a mechanism named Quorum Sensing (QS), respectively. aquaculture emerged to reduce extractive activities and to ensure uni- Iron is an essential nutrient for the bacterial cell since it assists in versal access to products of aquatic origin [157,158], thereby reducing the maintenance of several metabolic functions [21] that guarantee environmental degradation and promoting species preservation [153]. their survival in the host, thus promoting the infection process [141]. This activity has grown over time and is recognized as an important Due to iron's low availability in free form, bacteria have developed contributor to the production of fish and other species derived from throughout the evolutionary process some effective molecular systems aquatic environments [159]. The main obstacles of aquaculture are to obtain iron in its most diverse states, the siderophores [142]. The low diseases and infectious processes that lead to the death of specimens, molecular weight chelators of ferric ion (Fe3+) are structures re- which consequently affects their production and blocks sales of these sponsible for extracting the iron bound to host proteins and in- products in the market [117]. Adoption of practices such as poor water corporating it into the bacterial cell. In the case of Aeromonas, the main treatment, excessive handling, proximity among species, transport and siderophores produced are enterobactin and amonabactin [21]. How- storage are responsible for generating a stressful environment for the ever, not all iron uptake by bacteria is mediated by siderophores, since cultivated animals, making them susceptible to infections although they have high ion-binding affinity, they cannot extract it [21,70,153,160]. Twenty-six strains of Aeromonas were isolated from from more complex protein molecules such as hemoglobin, lactoferrin water samples of aquaculture [71]. When submitting specimens of and transferrin [143]. Thus, bacteria have specific receptors, such as tambaqui (C. macropomum) to physiological stress by confinement, the heme receptor on the outer membrane, capable of binding to the species of Aeromonas spp. were also obtained [153]. These results complex molecule and then promoting dissociation of existing ferric corroborate with the information mentioned above. A. veronii samples

88 R.B. Gonçalves Pessoa, et al. Microbial Pathogenesis 130 (2019) 81–94 were isolated from Nile tilapia at two private fish farm sites and the A. hydrophila has been identified as the cause of acute gastroenteritis in quality of the water in which the species were maintained was ana- meat consumers in a Bhutanese village, in the Himalayas; among 55 lyzed. Significant variations in temperature, pH, dissolved oxygen, people who reported consumption, about 33 developed the disease in ammonia (NH3), nitrate (NO3) and nitrite (NO2) concentrations were question [177]. In an evaluation of Aeromonas distribution in meat from identified in the period of approximately one year; such alterations different animals, mutton was found to be the source of the greatest probably contributed to the development of the infectious process [53]. number of microorganisms [116]. Hygienic practices and correct food Mesophile and psychrophile groups of the genus Aeromonas can processing techniques are major contributors to avoiding contamina- cause infections in fish. A. salmonicida is known to be a causative agent tion by Aeromonas as well as other types of microorganisms. In relation of furunculosis [70], an ulcerative disease affecting mainly salmonids to the techniques of food decontamination, susceptibility of these bac- [74]. A. hydrophila, as well as A. veronii [53], are considered to cause teria to heat, acidity, disinfectants and irradiation itself have already hemorrhagic septicemia [152]. Rainbow trout (O. mykiss) specimens been reported [178]. were exposed to strains of different Aeromonas species; the group in- fected with A. salmonicida, A. hydrophila, A. veronii and A. bestiarum 5. Aeromonas as a public health problem presented abnormal swimming, signs of anorexia and hyperpigmenta- tion [24]. Moreover, they identified that these bacteria promoted a Aeromonas are known to cause several diseases in humans. The higher mortality rate in the specimens tested. Nile tilapia infected by A. genetic plasticity and diversity of virulence factors present in the genus veronii exhibited different clinical signs, such as insensitivity, lethargy make them very versatile microorganisms. They are considered emer- in movements and absence of reflex; they also had ulcerations, pale ging pathogens [19], since Aeromonas are related to clinical cases of spots and hemorrhages along their body [53]. A. veronii, A hydrophila, gastroenteritis and infections in various organs and tissues A. salmonicida and A. media strains were isolated from fish, which [7,179–181]. showed signs such as hemorrhages in the mouth, eyes and other organs; There are increasingly frequent reports of pathologies attributed to some also exhibited swelling with the presence of ascetic fluid [50]. the Aeromonas genus. Due to complications that occur with the correct Infected crayfish (P. leniusculus) specimens revealed small spots on the taxonomic identification of the genus, clinical data on frequency and cuticle and/or showed lethargy and weakness [152]. Frequency of etiology of disease outbreaks may have excluded the presence of diseases and outbreaks can be related with geographic differences, Aeromonas due to the lack of technical capacity to properly identify though the genus Aeromonas is widely distributed in diverse environ- these bacteria. ments [149]. Their high production of toxins, ability to adapt to the most diverse Although water is the main means for propagation of Aeromonas habitats, tolerance of environmental stress and recent reports of the species, they can also be isolated from animals other than inhabitants of emergence of resistance to antibiotics [11,73,182], highlight the con- aquatic environments. A. hydrophila is the primary cause of red-leg cern with the monitoring of these bacteria in hospital settings. For a syndrome in amphibians, a disease characterized by redness in the legs long time Aeromonas were believed to be pathogens of an essentially and abdomen of frogs and salamanders, promoting anemia, lethargy opportunistic nature; however, there is already evidence of virulent and other symptoms [161]. Analysis of swabs from the cloacal and strains causing severe septicemia in immunocompetent patients [179]. pharyngeal region of free-living birds identified the presence of three Moreover, in humans, this genus also causes gastrointestinal, wound species of Aeromonas: A. hydrophila, A. sobria and A. veronii [162]. and soft tissue infections [7,183–186]. Muscle infections, skin diseases, Bacteria belonging to this genus could be identified in healthy pigs' eye infections, pneumonia and septicemia are examples of secondary feces [163] as well as promoting bronchopneumonia with purulent pathologies triggered by virulent strains of Aeromonas [187]. exudate in a wild boar [164]. In relation to reptiles, A. hydrophila has The gastrointestinal tract is still the main target of infection by these been found as part of the bacterial diversity existing in the oral cavity of bacteria, with diarrhea being a common symptom [187]. Gastro- venomous snakes [165,166]. The same species was also identified as intestinal infections caused by Aeromonas can be confused with those of causing septicemia in two types of crocodiles. Tissue analysis found cholera [188]. Other symptoms associated with gastrointestinal infec- extensive respiratory and intestinal lesions [167]. A. hydrophila was tions caused by Aeromonas include fever, abdominal pain and dehy- also the only Aeromonas species isolated from domestic reptiles [168]. dration [187]. These bacteria can cause necrosis and septic shock as a The ability of these bacteria to colonize insects has been reported, since result of infection in soft tissues [189]. strains of A. hydrophila and A. veronii were identified in intestinal One of the characteristics that most favor these bacteria with respect samples of house flies (Musca domestica L.) [169]. to contamination is their ecological adaptability, since they have a di- Environmental adaptation strategies, especially tolerance to low versified metabolism, which allows Aeromonas to be present in almost temperatures, make Aeromonas important colonizers of food, whether any environment and to be transmitted by diverse routes and vectors of vegetable or animal origin [170,171]. Although some strains of [6,25,182,190]. Species of Aeromonas most frequently associated with Aeromonas have already been isolated directly from soil [172,173], diseases in humans are A. hydrophila (14.5%), A. caviae (37.6%), A. these microorganisms can be introduced into food by different ways, veronii bv. Sobria (27.2%) and A. dhakensis (16.5%), representing about such as animal feces, handling without previous hygiene and especially 96% of gastroenteritis cases [191,192]. However, as the taxonomy of the water itself used in irrigation systems in agricultural practice. About the genus is constantly changing, new species have arisen and many 15 types of plants have been tested for the presence of Aeromonas; the strains have been reclassified into different taxa. most prevalent species was A. caviae, followed by A. hydrophila. How- Aeromonas infects human hosts mainly via consumption of con- ever, in the same evaluation, no microorganism was detected in four of taminated food and water, besides direct contact with aquatic en- 15 vegetables tested, one of them being lettuce [174]. Moreover, this vironments colonized by the genus [187,193]. Risk of contamination is result does not exempt such vegetables from being contaminated by higher in aquatic environments during the summer, when water tem- Aeromonas, since there are reports of isolation of this genus from lettuce peratures are elevated and bacterial populations are larger [194]. samples showing the prevalence of A. caviae and A. hydrophila [175]. Aeromonas are present in dairy, pork or beef products, fish, seafood and Aeromonas can also be found in foods of animal origin, such as meats vegetables [13–15,25, 183], as well as being frequently isolated from and derivatives, milk, and cheese, among others [175,176]. Comparing diverse activities of animal husbandry such as aquaculture, ranching, the number of strains isolated, meat products had a greater number in crustacean breeding and aviculture [71,151,191,195–198]. relation to vegetables, being identified as A. caviae, A. hydrophila and A. Aeromonas represents the challenges faced in modern clinical mi- sobria [174]. Except for A. caviae, the same species were also found in crobiology: constant change in virulence caused by the acquisition of another analysis using samples of meat, milk and dairy products [170]. genetic determinants through lateral transference and emergence of

89 R.B. Gonçalves Pessoa, et al. Microbial Pathogenesis 130 (2019) 81–94 multiresistance [199], including resistance to antibiotics of last choice Aeromonas-associated diseases in humans, Pol. J. Microbiol. 67 (2) (2018) [200]. Therefore, study of the aspects that constitute the biology of 137–149, https://doi.org/10.21307/pjm-2018-020. ff [17] N.M. Clark, C.E. Chenoweth, Aeromonas infection of the hepatobiliary System : these bacteria is fundamental for the development of e ective strategies report of 15 cases and review of the literature, Clin. Infect. Dis. (2003) 60612. in the fight against these highly harmful microorganisms. [18] M.T.M. Roberts, D.A. Enoch, K.A. Harris, J.A. Karas, Case Report Aeromonas veronii biovar sobria bacteraemia with septic arthritis confirmed by 16S rDNA PCR in na immunocompetent adult, J. Med. Microbiol. (2006) 241–243, https://doi.org/10. 6. Conclusions 1099/jmm.0.46295-0. [19] I.H. Igbinosa, E.U. Igumbor, F. Aghdasi, M. Tom, A.I. 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