Isolation, genetic characterization, and virulence profiling of different Aeromonas species recovered from moribund hybrid catfish (Clarias spp.)

Background and Aim: The high diversity of Aeromonas spp. results in various pathogenicity levels. This group of bacteria causes a serious disease named motile Aeromonas septicemia (MAS) in catfish (Clarias spp.). This study aimed to characterize the species and virulence gene diversity of Aeromonas spp. isolated from diseased catfish. Materials and Methods: Nine Aeromonas spp. were isolated from infected catfish cultivated in Java, Indonesia, and they were identified at the phenotypic and molecular levels (16S rDNA). The virulence genes assessed included aer/haem, alt, ast, flaA, lafA, and fstA. Results: Phylogenetic analysis identified nine isolates of Aeromonas spp.: Aeromonas hydrophila (11.11%), Aeromonas caviae (11.11%), Aeromonas veronii bv. veronii (44.44%), and Aeromonas dhakensis (33.33%). Virulence genes, such as aer/haem, alt, ast, flaA, lafA, and fstA, were detected in all isolates at frequencies of approximately 100%, 66.67%, 88.89%, 100%, 55.56%, and 66.67%, respectively. This study is the first report on A. dhakensis recovered from an Indonesian catfish culture. Furthermore, our study revealed the presence of A. veronii bv veronii, a biovar that has not been reported before in Indonesia. Conclusion: This finding confirms that MAS was caused by multiple species of Aeromonas, notably A. dhakensis and A. veronii bv veronii, within Indonesian fish culture. The presence of these Aeromonas species with multiple virulence genes poses a significant threat to the freshwater aquaculture industry.

Most Aeromonas spp.cause aeromoniasis or motile Aeromonas septicemia (MAS), which features specific clinical signs of erosion and hemorrhage in the mouth and on the body surface, erosion, hyperemia, congestion, abscess, necrosis, dropsy, abdominal dropsy, abdominal ascites, and exophthalmia [12][13][14][15][16][17][18].Infection by Aeromonas spp.also causes histological changes in the organs of the affected fish.Such conditions result in decreased levels of hemocyte aggregation in the digestive system of infected fish, massive aggregation of hemocytes and pyknotic nuclei in the hepatopancreas, and hemocyte aggregation and necrotic cells in gills [19].In humans, Aeromonas spp.infect skin, soft tissues, and gastrointestinal tract and causes hepatobiliary disease, diarrhea, and bacteremia [11,20,21].
Twenty-four species of the Aeromonas complex had been identified as of 2010 [22].By 2017, this number had grown to 32 [23].The Aeromonas complex includes 36 species as reported in 2020 [24], which indicates the possibility of further expansion of this genus.
Based on biochemical characterization and 16S rDNA sequences, the main etiologic agents for aeromoniasis in eels in Europe, Japan, and Korea include Aeromonas hydrophila, Aeromonas veronii, Aeromonas sobria, Aeromonas caviae, Aeromonas jandaei, Aeromonas aquariorum, Aeromonas media, and Aeromonas trota [25][26][27].These species may also infect other aquaculture species, including catfish.In addition to its high species diversity, most members of the genus Aeromonas include are pathogenic.Their pathogenicity is related to their virulence factors.The complex virulence of Aeromonas features is caused by some factors contributing to the development of the infection process [28].The virulence is strictly related to the expression of genes recognized in Aeromonas spp.Extracellular products, such as hemolysin, protease and lipase, toxin, and structural components (flagella, fimbriae, lipopolysaccharide, and outer-membrane protein), whether acting together or individually, enable the microorganisms to colonize and infect the host [29].Several virulence genes have been identified to play a role in the pathogenicity of Aeromonas spp.[30].
The total catfish production in Indonesia reached 1.06 million tons in 2021 and 1.03 million tons in 2020, which indicate a production increment of 2.8% [31].Disease outbreaks have resulted in a low increment in total fish production in Indonesia in 2020-2021.The previous reports by Mulia et al. [32], Marnis and Iswanto [33], Sarjito et al. [34], Rejeki et al. [35], and Yuliantoro et al. [36] have described Aeromonas spp. as a causative disease agent in catfish.The Aeromonas species identified from fish in Indonesia include A. hydrophila, Aeromonas salmonicida, Aeromonas sobria, A. caviae, A. media, Aeromonas taiwanensis, and A. jandaei [37][38][39], while those from pond water comprise A. hydrophila and A. veronii [40].Records of the diverse types of pathogenic Aeromonas are not yet available at the global level or in Indonesia.Therefore, a study on the etiological agents of catfish aeromoniasis is required.In addition, the identification of Aeromonas spp.based on phenotype characteristics along is very difficult and inaccurate.Thus, genotypic identification supported by phenotypic characters and virulence data is required.
This study aimed to characterize the species and virulence gene diversity of Aeromonas spp.isolated from diseased catfish.

Ethical approval
The management, conditions, and procedures of the experiment in this study were approved by the Ethical Clearance Commission of Universitas Gadjah Mada (approval # certificate: 00137/04/LPPTI/|/201).

Study period and location
The study was conducted from May 2017 to January 2019.The samples were collected from aquaculture ponds in four provinces in the Special Region of Yogyakarta, Central Java, West Java, and East Java, Indonesia.

Isolation and bacterial culture
Sixty-seven fish with a size of 3.5-30 cm was obtained.Bacteria were isolated from the kidneys of diseased catfish The bacteria were cultured in glutamate starch phenyl (GSP) medium (Merck, Darmstadt, Germany), a selective medium for Aeromonas and Pseudomonas, at 30°C for 24 h.Aeromonas and Pseudomonas grew in yellow and red colonies, respectively.Furthermore, yellow single colonies were recovered in the tryptic soy broth (TSB) medium (Merck).The isolates were stored in TSB medium (Merck) with 20% glycerol at −80°C for further assay.

Phenotypic identification of Aeromonas spp.
Phenotypic identification was performed by observing the colonies, cell morphology, and biochemical properties of the bacteria.The bacteria were grown on tryptic soy agar (TSA) medium (Merck) at 30°C for 24 h.Biochemical characterizations included the oxidative/fermentative (F), Voges-Proskauer (VP) test, citrate utilization, lysine utilization, ornithine utilization, urease production, phenylalanine deamination, nitrate reduction, H 2 S production, glucose utilization and gas production, adonitol, lactose, arabinose, sorbitol utilization, and aesculin hydrolysis which were conducted using the biochemical identification kit (HiMedia, India).

Bacterial genomic DNA extraction
A total of 1 mL of bacterial culture in TSB medium was incubated at 30°C for 24 h and centrifuged at 13,000× g for 2 min.The bacterial genomic DNA was extracted using a bacterial DNA kit (Promega, Madison, USA) in accordance with the manufacturer's protocol.The extracted bacterial DNA was stored at −20°C for further assay.

Clinical signs of diseased catfish
Figure-1 shows the clinical signs of diseased catfish.The clinical signs observed included skin depigmentation, necrosis, hemorrhagic, hyperemia, ulcer, abdominal dropsy, abdominal ascites, abscess, and paleness in the kidney.

Molecular identification of Aeromonas spp. based on 16S rDNA
The results of 16S rDNA amplification for nine isolates revealed base lengths of approximately 1500 bp (Figure -2).The 16S rDNA sequences of these isolates were compared with those of 28 isolates from the GenBank database (NCBI, USA).The homology search based on the 16S rDNA sequence (1410-1479 bp) showed that the nine isolates demonstrated similarities to A. hydrophila, A. caviae, A. veronii bv veronii, and Aeromonas dhakensis.Queries reached up to 96%-100% with a similarity range of 97%-99.86%(Table -3).

Phylogenetic tree
Phylogenetic analyses showed that Ah-01 and Ac-01 isolates were included in the clade of A. hydrophila ATCC 7966 and A. caviae ATCC

Detection of virulence genes
Six virulence genes (aer/haem, alt, ast, flaA, lafA, and fstA) of the Aeromonas spp.were detected (Figure -3 and Table-4).The aer/haem gene was found in all isolates, and the alt gene was present in +, >90% of strains positive; -, >90% of strains negative; v, 11%-89% of strains positive; F=Fermentative, TSA=Tryptic soy agar, GSP=Glutamate starch phenyl, O/F=Oxidative/Fermentative six isolates.The virulence gene ast was discovered in eight isolates, and flaA was amplified in all isolates.The virulent gene lafA was found in five isolates, and fstA was found in six isolates.The four isolates of A. dhakensis harbored aer/haem, alt, flaA, and fstA but not lafA.However, three isolates harbored ast (Table-4 and Figure -3).The aer/haem, ast, flaA, and lafA were detected in all isolates of A. veronii bv veronii.However, alt and fstA were detected in two isolates, whereas only A. caviae isolate had aer/haem, ast, flaA, lafA, and fstA, but not alt.The genes aer/haem, alt, ast, and flaA were detected in A. hydrophila but lafA and fstA were missing.
The genes aer/haem, alt, ast, and flaA were detected in A. hydrophila, but lafA and fstA were absent.A. caviae isolate contained the aer/haem, ast, flaA, lafA, and fstA, but not alt.The aer/haem, ast, flaA, and lafA were detected in all isolates of A. veronii bv veronii.However, alt and fstA were detected in two isolates.The three isolates of A. dhakensis harbored aer/haem, alt, flaA, and fstA but did not harbor the lafA gene.However, two isolates harbored ast (Table-4 and Figure -3).

Discussion
This study investigated the diversity of Aeromonas spp.causing MAS in Indonesia and infected catfish.Specifically, this study aimed to determine the biodiversity of Aeromonas spp. in  diseased catfish cultivated in four provinces on Java Island, Indonesia.
The clinical signs of diseased catfish included depigmentation of the skin, necrosis, hemorrhagic, hyperemia, ulcer, abdominal dropsy, abdominal ascites, abscess, and paleness of the kidney (Figure -1).Similar findings have been reported by Austin and Austin [46] and Dias et al. [47].Aeromonas spp.can break the blood vessels, which results in ulcerative lesions with a hemorrhagic appearance throughout the tegument [48].In this study, clinical signs of diseased catfish indicated typical signs of aeromoniasis.This finding suggests that the isolates of Aeromonas spp.considered in this study were pathogenic and infectious with remarkable clinical signs.
Based on phenotypic characteristics, this study identified nine isolates as presumptive Aeromonas species.The isolates were characterized based on their growth on GSP selective agar medium: yellow colonies, rod shape, Gram-negative, motile, and production of acid from glucose (Table-2).These characteristics have also been reported previously by El-Sharaby et al. [5].In addition, Aeromonas can ferment glucose.A common characteristic of Aeromonas is their ability to produce gases from glucose.However, this study found only one isolate with such a trait, which suggests the inconsistency of this ability among Aeromonas [49].In the present study, all Aeromonas isolates reduced nitrate, as previously reported by Vega-Sanchez et al. [50] for A. caviae, A. veronii bv veronii, and A. hydrophila.However, Aeromonas cannot produce acetyl methyl carbinol (VP), urease, and H 2 S. The previous study reported by Khor et al. [51] have also reported that all Aeromonas isolates are unable to produce urease and H 2 S. The ability of Aeromonas to produce acetyl methyl carbinol (VP) varies.Aeromonas hydrophila and A. veronii bv veronii manufacture this substance [52], but A. caviae does not [53].The previous study by Khor et al. [51] have also revealed that Aeromonas varies in their ability to use citrate.This research found characteristics that are different from those of previous study by Borty et al. [52], revealing that A. hydrophila cannot utilize lysine; in addition, some species cannot utilize ornithine [51], but A. veronii bv veronii cannot specifically use arabinose [50,54,55].
In contrast to the findings of previous studies, A. hydrophila and A. veronii bv veronii cannot utilize sorbitol [50,52], and hydrolyze aesculin [53], but can hydrolyze aesculin [50,52,54,55]; meanwhile, 50% of A. caviae can hydrolyze aesculin [52].These results indicate variations in the phenotypic characteristics of Aeromonas.This condition has also been reported previously by Liu [49] and Martin-Carnahan and Joseph [56].Phenotypic variation is thought to have clinical significance for determining the relationship between biotype and enterotoxin production [53].
The identification of members of Aeromonas spp. at the species level remains a huge challenge given their genetic heterogeneity.Biochemical characteristics are ineffective for the identification of Aeromonas at the species level [57]; thus, species identification should be supported by genotypic analysis.Molecular identification using 16S rDNA is very helpful for accurate species identification [58,59].Furthermore, we proved the biodiversity of Aeromonas spp.by 16S rDNA sequencing.We used 16S rDNA sequence rather than gyrB sequence for molecular identification in this study.We performed preliminary screening for Aeromonas spp.using GSP medium based on its morphological characteristics, further confirmed using a series of biochemical tests that highly aligned with Aeromonas characteristics, and finally, 16S rDNA phylogenetical analysis with the inclusion of Aeromonas type strains.Results from our phylogenetic tree also revealed that our isolates formed a tight clade with their corresponding type strains.In addition, the numbers of 16S rRNA gene database in NCBI are much higher available than that of gyrB gene sequence.Based on the availability of the database, the initial molecular identification is likely more appropriate using the 16S rDNA sequence to avoid the mismatch results of similarity analysis.
The previous study has also identified Aeromonas spp.from catfish (Clarias gariepinus) in Egypt using genotypic analysis, these bacteria consisted of A. hydrophila (56%) and A. veronii (44%) [62], A. hydrophila (50.4%), and A. caviae (5.6%) according to phenotypic analysis [63].Other research on catfish in Uganda discovered A. hydrophila (33%) and A. sobria (33%) through genotypic analysis [13].In the present study, 16S rDNA molecular identification in catfish revealed the high biodiversity of Aeromonas spp., that is, four species, compared with a previous study, which detected one or two Aeromonas species.The high biodiversity of Aeromonas spp. is presumably due to the large sampled locations, which covered three provinces in Java Island: DIY Yogyakarta, Central Java, and West Java.The former surveys only involved West Java of Indonesia [33], center Java of Indonesia [34], and Qena in Egypt [62], as well as the central region and northern region of Uganda [13].In addition, the differences in type and number of Aeromonas species identified from catfish may be due to the differences in individual fish, cultivation systems, climates, and environmental influences.
In line with this study, A. hydrophila, A. veronii, and A. caviae have frequently been isolated from diseased catfish.Unexpectedly, in this study, the prevalence of A. dhakensis was observed.Although A. dhakensis is rarely found in fish, this species is virulent to Nile tilapia (Oreochromis mossambicus) in Mexico [64] and other fish in Australia [65].In addition, A. dhakensis was first reported to cause acute hemorrhagic septicemia in experimentally infected pacu fish (Piaractus mesopotamicus) in Brazil [66].Aeromonas dhakensis was the predominant Aeromonas species isolated from freshwater fish in nine fish farms in Malaysia [67].Aeromonas dhakensis is a major emerging pathogen of striped catfish in Vietnam [68].Aeromonas dhakensis is possibly an important pathogen in catfish and other freshwater fishes, either in Indonesia or around the world.In addition, A. veronii bv veronii is the first A. veronii biovar, which has been previously reported as A. veronii, reported in Indonesia [34,39].
Numerous virulence genes associated with the pathogenicity of Aeromonas spp.act synergistically with each other to exhibit their pathogenicity.In this study, the biodiversity of the Aeromonas complex was due to variations in virulence genes.The pathogenicity of the Aeromonas complex is multifactorial and assumed to involve the products of some genes, such as aerolysin (aer) and hemolysin (haem) (extracellular products), two cytotoxic, heat-labile (alt) and heat-stable (ast) genes, polar flagellum (fla) (structural components), lateral flagellum (laf), and ferric siderophore receptor (fst) [22,69].To spread their virulence factors, Aeromonas employs four secretion systems that discharge cellular products into the extracellular environment or directly to the host cell [70].
To recognize the potential virulence of Aeromonas spp., scientists regard the detection of virulence genes as a practical method for screening a large number of Aeromonas isolates with a suitable virulence potential.The previous studies by Mulia et al. [16] and Pessoa et al. [29] have also reported the detection and characterization of virulence factors in Aeromonas spp.In the present study, six virulence genes, that is, aer/haem, alt, ast, flaA, lafA, and fstA, were detected .This study also showed that Aeromonas have at least four virulence genes.Similar results have been reported for aer/haem and flaA [30,62].Aerolysin is a hemolytic extracellular product coded by the aerolysin gene and plays a significant role in the pathogenicity of Aeromonas spp.It is the most important toxin among potential virulence factors in Aeromonas [71].Aeromonas has two types of flagella, that is, polar and lateral, which are coded by flaA and lafA genes, respectively [70].Alt and ast have been detected in some Aeromonas spp.[65].The present study detected fstA in some isolates of Aeromonas spp.species but it not in A. hydrophila.In contrast, other research on a variety of sources (seawater, water, diseased fish, bivalves, cake, and wound exudate) of Aeromonas detected fstA in A. salmonicida, but not in A. hydrophila, A. caviae, and A. veronii [70].
All findings of this study provide important information regarding the characteristics and species diversity of Aeromonas, virulence genes of Aeromonas spp., and the presence of A. dhakensis in catfish.Aeromonas dhakensis showed virulence potential due to some virulence genes it possesses, such as aer/haem, alt, flaA, and fstA genes (100%), as well as the ast gene (66.67%).Such information may be useful for determining strategies for the control of MAS disease in catfish and other freshwater fishes.

Conclusion
Four species of Aeromonas spp., namely, A. hydrophila, A. caviae, A. veronii bv veronii, and A. dhakensis were identified in the current study.Virulence genes, including aer/haem, alt, ast, flaA, lafA, and fstA were detected at prevalence rates of 100%, 66.67%, 88.89%, 100%, 55.56%, and 66.67%, respectively.This study is the first report on the isolation of A. dhakensis from aquaculture species in Indonesia.Two isolates of A. dhakensis harbored five virulence genes, and one isolate possessed four virulence genes.In addition, the A. veronii bv veronii isolated in this study is the first A. veronii biovar isolated from aquaculture species in Indonesia.The isolates of A. veronii bv veronii possess six, five, or four virulence genes.This study has successfully revealed the diversity of Aeromonas spp.and their virulence genes.The current report contributes considerably to the basic consideration for the development of an Aeromonas vaccine.

Figure- 3 :
Figure-3: Phylogenetic tree constructed from the 16S rDNA sequences of Aeromonas sp. and other Aeromonas species (class of Gammaproteobacteria) and detected virulence genes.Pseudomonas aeruginosa was used as an outgroup.The topology was obtained by maximum likelihood with bootstraps of 1000 replications.The scale bar signifies 0.02 substitutions per nucleotide position (Knuc).Colored circles indicate the presence of the virulence genes for each isolate,  aer/haem: aerolysin/hemolysin;  alt: heat-labile cytotonic enterotoxin;  ast: heat-stable cytotonic enterotoxin;  flaA: polar flagellum;  lafA: lateral flagellum;  fstA: ferric siderophore receptor.

Table - 1
: Primer sets used in this study.

Table - 4
: Virulence genes in the Aeromonas spp.isolated from catfish.