Identification and characterization of virulent Aeromonas hydrophila Ah17 from infected Channa striata in river Cauvery and in vitro evaluation of shrimp chitosan

Abstract Aeromonas hydrophila, an inhabitant in the aquatic ecosystem is considered as an important foodborne bacterial zoonotic pathogen in aquaculture. The present study aimed to identify virulent A. hydrophila from naturally infected Channa striata in river Cauvery and in vitro evaluation of shrimp chitosan. Rimler Shotts (RS) and blood agar medium identified the presence of pathogenic Aeromonas sp. from the infected C. striata. A. hydrophila Ah17 was identified using 16S rRNA nucleotide sequence. Extracellular enzymes such as amylase, lipase, and protease were screened in A. hydrophila Ah17. Antibiotic susceptibility tests showed A. hydrophila Ah17 was highly resistant against β‐lactam, glycopeptide, macrolides, phosphonic, fucidin, and oxazolidinone classes of antibiotics. Virulent genes such as hemolysin (aer and hly), heat‐labile enterotoxin (act), cytotonic heat‐stable enterotoxin (ast), elastase (ahyB), and lipase (lip) were identified. Growth and the viable cell population of virulent A. hydrophila Ah17 were significantly reduced in a dose‐dependent manner against shrimp chitosan (CHS) from Penaeus indicus (P. indicus). Thus, the present study isolated virulent A. hydrophila Ah17 from the environmental source and characterized in vitro with shrimp chitosan.


| Collection of naturally infected fish
Channa striata displayed with the clinical signs of disease were collected from the river Cauvery, Pallipalayam, Erode District, Tamil Nadu, India (lat: 11 o 21'39.1N and long: 77 o 44'35.2E).

| Isolation of virulent Aeromonas hydrophila
Ulcerated regions (skin-lesions/muscle) of infected C. striata were wiped with a sterile cotton swab and suspended in physiological saline (0.85% NaCl) under aseptic conditions. The suspension was serial diluted and plated on RS agar medium (supplemented with novobiocin (5mg/L)) and incubated at 37ºC. After overnight incubation, isolates were patched on tryptic soy agar (TSA) medium for further analysis.

| PCR amplification of 16S rRNA region of β hemolysin-positive isolates
16S rRNA region was amplified for all the β hemolysin-positive isolates using the primers as described by Dorsch, Ashbolt, Cox, and Goodman (1994), and A. hydrophila ATCC 7966 strain was used as the positive control. Briefly, genomic DNA was extracted from β hemolysin-positive isolates using Bacterial Genomic DNA Purification Kit (HiMedia, Mumbai, India). Quality and quantity of genomic DNA were measured using Nanodrop™ (Thermo Fisher Scientific) and resolved using 0.7% agarose gel electrophoresis. Details of the primers and their product size are provided in Table 1. 16S rRNA gene was amplified using SureCycler 8,800 Thermal Cycler (Agilent Technologies), and the PCR product was eluted using PureLink™ Quick Gel Extraction Kit (Thermo Fisher Scientific). The eluted PCR product was cloned into TA cloning vector pXcmKn12 (Thermo Fisher Scientific) and transformed into Escherichia coli DH5-α.
Transformants were selected on Luria Bertani (LB) agar ampicillin (50 µg/ml) plate by Blue-white selection method and confirmed by colony PCR. All the clones were sequenced in automated DNA sequencer (Xcelris Labs Limited, Ahmedabad, India).

| Molecular evolutionary relationship of A. hydrophila Ah17
Similarity search was carried out for 16S rRNA nucleotide sequences of the selected isolates in nucleotide BLAST search engine tool on NCBI database (https ://blast.ncbi.nlm.nih.gov/). The molecular phylogenetic tree was constructed using the 16S rRNA sequence of A.
The following 16S rRNA nucleotide sequences of A. hydroph-

| Screening of extracellular enzymes
Production of extracellular enzymes such as amylase, lipase, and protease was screened in A. hydrophila Ah17. Briefly, for amylase activity, the isolate was patched on starch agar medium (HiMedia) and incubated at 37ºC. After incubation, the surface of the culture was flooded with Gram's iodine (HiMedia), and appearance of the zone of clearance around the colonies was indicated as amylase-positive isolate (Yang & Fang, 2003).
For lipase activity, the isolate was patched on tributyrin agar base (HiMedia) containing 10 ml of tributyrin and incubated at 37°C.
The appearance of the zone of clearance around the colonies was indicated as lipase-positive isolate (Collee, Duguid, Fraser, Marmion, & Simmons, 1996).
For proteolytic activity, the isolate was patched on skim milk agar (HiMedia) and incubated at 37ºC. The appearance of the zone of clearance around the colonies was indicated as protease-positive isolate (Yang & Fang, 2003). A. hydrophila ATCC 7966 was used as the positive control for the study.

| Biochemical characterization
Biochemical characterization of A. hydrophila Ah17 was performed by Bergey's manual of systematic bacteriology (Garrity, 2007), and A. hydrophila ATCC 7966 was used as the reference strain for the study.

| Antibiotic susceptibility profile
Antibiotic susceptibility profile for A. hydrophila Ah17 was determined by the Kirby-Bauer disk diffusion method (Bauer, Kirby, Sherris, & Turck, 1966). The following antibiotics were tested: Antibiotic discs were placed on the MHA medium and incubated at 37°C for 24-48 hr. The diameter of the zone of inhibition was measured, and susceptibility was categorized according to the manufacturer's protocol.

| Antimicrobial activity of shrimp chitosan against A. hydrophila Ah17
Antibacterial activity of CHS against A. hydrophila Ah17 was studied at pH-6.5. Briefly, A. hydrophila Ah17 culture was taken and cells were harvested by centrifugation at 10,000 rpm for 10 min.

| Bacterial cell viability assay
The relative cell viability of A. hydrophila Ah17 against CHS was

| Statistical analysis
One-way analysis of variance (ANOVA) and Dunnett's multiple comparison test were performed to scrutinize the data of bacterial cell viability assay. For all comparison, p < .05 was considered as statistically significant. All the statistical analysis was performed using the GraphPad Prism 7.0 software.

| Identification of A. hydrophila Ah17 strain in the river Cauvery
Satellite view of the collection site and representative image of naturally infected C. striata from the river Cauvery are represented in (Figure 1a,b). A total of 430 colonies were obtained from the infected C. striata after screening with the RS agar medium. Among 430 colonies, 20 isolates were positive for β-hemolytic activity on blood agar medium (Table 3). Out of twenty isolates (β hemolysin-positive isolates), five isolates were amplified (686 bp, data not shown) using Aeromonas sp.-specific 16S rRNA primers ( Figure 2).  (Figure 3).

| Screening of extracellular enzymes and biochemical characterization in A. hydrophila Ah17
The appearance of the zone of clearance on starch, tributyrin, and skim milk agar medium indicated the production of extracellular enzymes such as amylase, lipase, and protease in A. hydrophila Ah17 ( Figure 4). Biochemical properties such as ornithine decarboxylase were negative whereas oxidase, Voges Proskauer, motility, H 2 S production, glucose fermentation (D-glucose), lysine decarboxylase, and arginine dihydrolase were positive for A. hydrophila Ah17 (Table 4).

| Antimicrobial susceptibility profile of A. hydrophila Ah17
Antimicrobial susceptibility profile of A. hydrophila Ah17 is provided in Figure 5. A. hydrophila Ah17 showed resistance to β-lactam antibiotics such as amoxicillin, ampicillin, methicillin and penicillin G. The resistance was also observed with glycopeptide class of antibiotics

| Putative virulent factors in A. hydrophila Ah17
PCR-based identifications of putative virulent factors such as cytotoxins, hemolysins, lipases, and proteases were evaluated in A.

| D ISCUSS I ON
In recent year's great attention have been made to the genus Aeromonas due to its pathogenic nature in aquatic organisms as well as in humans (Furmanek-Blaszk, 2014). Generally, it is difficult to screen pathogenic A. hydrophila directly from the environmental sources. Hemolytic activity is considered as one of the major characteristics property to distinguish virulent and avirulent strains in A. hydrophila (Wang et al., 2003). Production of the hemolytic toxin has been considered as the pathogenic potential trait in Aeromonads (Santos et al., 1999), and moreover, β-hemolysin has been reported as one of the major virulent factors in motile Aeromonads (Majeed & MacRae, 1993). In line with these arguments, the identified A. hydrophila Ah17 strain in the river Cauvery showed characteristic βhemolytic activity in blood agar medium.
Over the last decades, strategies have been employed for the rapid and direct identification of foodborne pathogenic A. hydrophila strains from the environmental sources. PCR-based microbial typing emerged as the most rapid and reliable ways to characterize and identify microbes from the environmental source (Van Belkum,
With the help of molecular markers, identification of specific microbial taxa and their phylogeny was explored over several decades (Bartual et al., 2005). Among these molecular markers, 16S rRNA gene sequencing is widely used for the assessment of phylogenetic relatedness of organisms due to its functional constancy, and thus, it is considered as an effective molecular chronometer for the mo- AL09-71 produce amylase and protease which contributes pathogenicity to the host organism (Sandkvist, 2001). In addition, extracellular lipase has been reported with virulence in many pathogens (Stehr, Kretschmar, Kröger, Hube, & Schäfer, 2003). In the present study, A.
hydrophila Ah17 secretes amylase, lipase, and protease and it may contribute pathogenicity to the host. Ulcerative lesions and depigmentation on the caudal fins were observed during the course of A.
Generally, Aeromonas sp. exhibited high resistance toward wide groups of antibiotics which are considered as the concerning factor for the treatment of Aeromonas infection. A. hydrophila Ah17 showed resistance toward most of the β-lactam antibiotics except third-generation antibiotics such as cephalosporins, cefixime which are displaying antagonistic property. It was observed that antibiotic resistance of A. hydrophila was mediated by chromosome-associated β-lactamse gene (Jacobs & Chenia, 2007).
Pathogenesis of A. hydrophila is multifactorial, associated with the number of virulent factors (Albert et al., 2000). PCR-based approach identified possible virulent genes which are associated with the pathogenicity of any A. hydrophila strains. Therefore, in the present study, PCR-based approaches have been carried out to detect one or more virulent genes which contribute to the pathogenicity of A. hydrophila Ah17, in agreement with the previous reports (Furmanek-Blaszk, 2014;Kingombe et al., 1999;Sechi, Deriu, Falchi, Fadda, & Zanetti, 2002;Sen & Rodgers, 2004;Wang, Tyler, Munro, & Johnson, 1996). Based on epidemiological studies, the presence of these virulent factors is being used as the genetic markers to discriminate between pathogenic and nonpathogenic strains of Aeromonas sp. (Kingombe et al., 1999;Sen & Rodgers, 2004;Wang et al., 2003).
Studies on cryo-electron microscopy showed that the bacterial PFTs are generally secreted as water soluble monomers and binds with target membranes and assemble into the circular oligomers, which undergoes the conformational changes that allow membrane insertion leading to pore formation and finally potential cell death (Iacovache et al., 2016). In the present study, the strain Ah17 harbors both aerolysin (aer) and hemolysin (hly) genes and the presence of these genes evidently supports the pathogenic nature of A. hydrophila Ah17.
Besides, cytotoxic enterotoxin act exhibits hemolytic activity and the gene encoding these activities different from aerolysin and hemolysin (Chopra & Houston, 1999). In the present study, act and ast were identified in A. hydrophila Ah17 and our results are in good agreement with the earlier studies (Kingombe et al., 1999;Sen & Rodgers, 2004).
In addition to that, the presence of elastase and lipase was evaluated in A. hydrophila Ah17 strain which is responsible for the invasion of intestinal mucosa and establishment of the infection into the host.
Mutation studies confirmed that the presence of temperature stable metalloprotease with elastolytic activity becomes more important for A. hydrophila virulence when tested against cold water fish Oncorhynchus mykiss (Cascon et al., 2000). Earlier reports confirmed that the presence of phospholipase contributes to the virulent nature of bacterial pathogens (Konig, Jaeger, Sage, Vasil, & König, 1996;Merino et al., 1999). Pathogenic strains with lipase and aerolysin genes together involved in altering the structure of the cytoplasmic membrane of the host and thereby, aggravate the pathogenic nature of A. hydrophila (Nawaz et al., 2010). Both ahyB and lip genes were identified in A. hydrophila Ah17.
Studies proved that chitosan acts as the antimicrobial agent against many foodborne pathogens such as Candida sp. (Rhoades & Roller, 2000), Staphylococcus aureus, E· coli (Chung, Kuo, & Chen, 2005), hydrophila Ah17 in a dose-dependent manner. Studies proved that the growth of pathogenic S. aureus inhibited when DD of chitosan is high (Takahashia, Imai, Suzuki, & Sawai, 2008). Further, CHS significantly reduced the viable cell population of A. hydrophila Ah17 when compared to the control group. Our study is in good agreement with the study conducted by Lin, Lin, and Chen (2009) in which, viable cell populations of A. hydrophila were reduced at higher concentrations. Thus, the present study confirmed that the CHS with DD value of 84% showed good antimicrobial response against virulent A. hydrophila Ah17.

| CON CLUS ION
In conclusion, A. hydrophila Ah17 was isolated from naturally infected freshwater fish harbouring six virulent factors (aer, hly, act, ast, ahyB, and lip). In vitro characterization demonstrated that shrimp chitosan can able to control the growth of virulent A. hydrophila Ah17 in a dose-dependent manner. In future, it is necessary to recognize and monitor the potential reservoirs of pathogenic bacteria and ensure their control measurements in an eco-friendly manner, which are essentially important in epidemiological and environmental studies to prevent possible health risks.

ACK N OWLED G M ENTS
The authors would like to acknowledge DBT-MKU IPLS programme for the financial support and Genomics Common Instrumentation facility at SBS, MKU.

CO N FLI C T S O F I NTE R E S T
We declare that we have no conflict of interest.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.