Prevalence and characterization of virulence-associated genes and antimicrobial resistance in Aeromonas hydrophila from freshwater finfish farms in Andhra Pradesh, India

Aeromonas hydrophila, a natural inhabitant of aquatic environments is known to become pathogenic in fish under favourable conditions. It has also been reported to cause infections in humans and animals. The prevalence, virulence-associated genes, and antimicrobial resistance of 57 A. hydrophila isolates from freshwater fish farms in Andhra Pradesh, India, were studied. The isolates showed resistance to trimethoprim (35.9%), co-trimoxazole (21.1%), oxytetracycline (19.3%), doxycycline (17.5%), and enrofloxacin (8.93%), but were completely sensitive to ciprofloxacin. The majority of the A. hydrophila isolates were strong (33%), moderate (50%), and weak (17%) biofilm formers, playing roles in virulence and colonization in hosts. The virulence gene distribution in representative A. hydrophila isolates was very high for enolase (enol, 96.4%), followed by flagellin (fla, 94.7%), cytotonic enterotoxins (alt, 91.2%), cytotoxic enterotoxins (act, 84.2%), lipase (lip, 77.1%), serine protease (ahp, 54.3%), aerolysin (aerA, 50.8%), and elastase (ahyB, 43.8%), respectively. 80% of the isolates carried a maximum of four virulence genes, whereas none carried the DNAase (exu) or haemolysin (hly) genes. Hemolytic assays on blood agar demonstrated that 70% of the isolates were β-haemolytic. The findings of the study will help to create awareness among aquaculturists of the potential risk of the spread of isolates and the virulence-associated genes across other sectors and the need to adopt best management practices in aquaculture.


Introduction
Freshwater fish farming contributes significantly to the aquaculture industry, notably in Asian countries, providing food, employment, and economic benefits (Buentello et al. 2007).A large diversity of fish species has been cultivated in various geographical regions, contributing to worldwide aquaculture fish production (Cai et al. 2019).Labeo rohita, Catla catla, Cirrhinus mrigala, Cyprinus carpio, Pangasianodon hypophthalmus, and Channa spp.are the most cultivable fishes in Andhra Pradesh, India, due to their fast growth, consumer preference, and good market prices.This has facilitated the rapid expansion of the culture area and made them candidate species for aquaculture (Katiha et al. 2005;Mishra et al. 2017a, b).In response to rising demand for fish protein, culture systems have shifted from traditional to intensive culture methods (Mishra et al. 2017a;Mzula et al. 2020;Patil et al. 2021;Van Boeckel et al. 2015).Modern farming systems are distinguished by high stocking densities and high organic matter, which create stress conditions that eventually, result in disease outbreaks (Mitra et al. 2019;Nhinh et al. 2021).Disease outbreaks are seen as significant constraints to aquaculture production and sustainability, contributing about 10-15% of total production costs (Tavares-Dias and Martins 2017).Disease-causing Aeromonas spp. in finfish include A. caviae, A. veronii, A. hydrophila, A. salmonicida, A. sobria, and A. bestiarum (Chandrarathna et al. 2018;Esteve et al. 2012;Hoai et al. 2019;Kumar et al. 2022;Liu et al. 2016;Long et al. 2016;Muduli et al. 2021;Thomas et al. 2013;Yao et al. 2018).A. hydrophila, which comes under the family Aeromonadaceae, is an opportunistic bacterial pathogen that causes Motile Aeromonas Septicemia (MAS) in freshwater finfish, displaying signs of ulceration, abdominal distension, exophthalmia, haemorrhage, and fin erosion, which lead to cumulative mortalities (Awan et al. 2018;Janda and Abbott 2010).More critically, due to its zoonotic nature, the fish infected with A. hydrophila have been proposed as a potential threat to human and animal health via foodborne contamination (Palu et al. 2006).
The pathogenicity and toxicity of bacterial pathogens are determined by virulence factors (Gao et al. 2018).The virulence factors in A. hydrophila include aerolysins (aer), cytotoxic enterotoxins (act, ast), cytotonic enterotoxins (alt), polar flagella (fla), serine protease (ser, ahp), elastase (ahyB), enolase (enol), lipase (lip), hemolysisn (hlyA), DNases (exu), glycerophospholipid cholesterol acyltransferase (gcaT), and type III secretion system (ascV), which play a prominent role in bacterial pathogenesis (Nawaz et al. 2010).In addition, they are also capable of producing biofilms, which create bacterial resistance against antimicrobial agents, making pathogen difficult to treat (Cai et al. 2019;Lynch and Wiener-Kronish 2008).Aeromonas spp.has evolved numerous regulatory mechanisms for biofilm formation that are intimately coupled to virulence factors (Rasmussen-Ivey et al. 2016b).Antibiotics and other chemical compounds are commonly used in aquaculture to prevent and control bacterial diseases, either through medicated feed or direct addition into the culture water (Mishra et al. 2017b).High levels of antibiotic use in animal food production jeopardize the aquatic environment by allowing antimicrobial resistance (AMR) and antibiotic resistance genes (ARGs) to enter the environment (Kummerer 2001;Liu et al. 2017;Lulijwa et al. 2020;Sawyer et al. 2015).Antibiotic-resistant bacteria accumulate in water, sediment, farmed animals, wild stock, and in and around farms, limiting effective treatment options and threatening aquatic food supply and animal welfare (Mishra et al. 2017b).
The state of Andhra Pradesh is leading in fish production in India.The sampling areas are the main centres for the culture of Indian major carps, exotic carps, and other commercially important freshwater fishes.Due to the high density culture, diseases often become a major constraint in freshwater aquaculture in this region.A. hydrophila has been identified as one of the most troublesome pathogens causing mortality outbreaks in a diversity of cultured fish species and has been responsible for huge economic losses.Hence, to address the challenges that the freshwater aquaculture sector would confront, a study was designed to investigate the prevalence, distribution of the virulence genes, and antibiotic resistance in A. hydrophila isolated from freshwater fish farms in Andhra Pradesh, India.

Sample collection
The present study was carried out in selected fish farms in Krishna (16°36'21.22"N,80°42'56.39"E)and West Godavari (16°53'55.65"N,81°18'9.30"E)districts in Andhra Pradesh, India, from June 2021 to October 2022.Indian major carps (IMC) Catla catla, Labeo rohita, and Cirrhinus mrigala are being cultured together as a composite fish culture, while for Pangasianodon hypophthalmus monoculture is practiced in this area.Fish samples from 100 finfish farms were collected from IMC (n = 73) and Pangasius fish farms (n = 27), including healthy (n = 210) and diseased (n = 43) fishes.From each farm, an average of 2-3 fish was collected for bacterial isolation and identification.The fish samples were then transported to the laboratory within 2-3 h in sterile, sealed bags under iced conditions for clinical examination.Clinical signs and gross features of all fish were observed and recorded as per the standard protocols (Austin et al. 2007).

Isolation and identification of A. hydrophila
Gill, liver, and kidney samples were collected and transferred into trypticase soya broth supplemented with ampicillin (30 µg/ml) for enrichment (Nawaz et al. 2010).After the enrichment, the culture was streaked onto Aeromonas selective agar (Himedia, India) followed by incubation at 32 °C for 18-24 h.The presumptive colonies of round, reddish orange-colored and clear to slightly opalescent bacteria were selected and re-isolated three times for their purity, followed by the determination of colony and bacterial morphology (M1890, Himedia).Further, the isolates were tested for Gram's reaction, motility, oxidase, catalase, Voges-Proskauer (VP), sensitivity to 0/129 (150 µg), indole production, citrate utilization, glucose fermentation, H 2 S production, urease production, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, arabinose fermentation, inositol fermentation, mannitol fermentation, and gelatin hydrolysis according to Bergey's Manual of Determinative Bacteriology (Holt et al. 1994).Based on phenotypic and biochemical characterization, the suspected Aeromonas isolates were confirmed and further characterized using an automated bacterial identification system, BD Phoenix™ M50 (BD Diagnostics, USA), with a confidence interval of 90-98%.A. hydrophila strain ATCC 7966 was used as positive control.

Antimicrobial susceptibility testing (AST)
Antimicrobial susceptibility testing (AST) of 57 A. hydrophila isolates was carried out following the disc diffusion method (Bauer 1966) and zones of inhibition were interpreted in accordance with the Clinical and Laboratory Standard Institute (CLSI 2022) standards.All the antibiotic discs were procured from Himedia, India, and maintained at -20 o C until use.Overnight-grown bacterial cultures were adjusted to 0.5 McFarland standard, and 100 µL of bacterial culture was transferred onto Muller-Hinton agar plates and incubated at 28 °C for 18-24 h.The antibiotic panel used for AST included oxytetracycline (30 µg), doxycycline (30 µg), ciprofloxacin (5 µg), enrofloxacin (10 µg), trimethoprim (30 µg), and co-trimoxazole (25 µg).The zones of inhibition were recorded and categorized as susceptible, intermediate, and resistant according to the zone diameter interpretation standard described in the CLSI M100, 2022.

Biofilm formation assay
The biofilm formation assay was performed using the tissue culture plate (TCP) method described by Mathur et al. (2006).Briefly, individual colonies of isolates were inoculated into brain heart infusion (BHI) broth supplemented with 2% sucrose (Himedia, India).200 µL of the bacterial suspensions were carefully added into a 96-well microtitre plate.A negative control with 200 µL of sterile BHI broth was also maintained.Plates were incubated at 37 °C for 24 h.The wells were washed three times with sterile deionized water to remove non-adherent bacteria.After 45 min of air drying, each well was filled with 200 µL of 0.2% (v/v) crystal violet solution (HiMedia, India) and further incubated for 45 min at room temperature, after which the wells were washed four times with sterile, deionized water.The dye was solubilized in each well with 200 µL of 33% glacial acetic acid (HiMedia, India), and the optical density (OD) was measured at 650 nm using the iMark™ Microplate Reader (Biorad, USA).Isolates with mean OD values greater than 0.108 were classified as strong biofilm formers, 0.108 − 0.083 as moderate biofilm formers, and 0.083 as weak biofilm formers (Hassan et al. 2011).

Hemolytic activity
A. hydrophila strains were examined for hemolytic activity on blood agar plates (Himedia, India) supplemented with 5% sheep blood.Aliquots of the overnight cultures were streaked onto the plates and incubated at 37 °C for 18 h, as described by Rahim et al. (2004).

Screening of virulence genes
For molecular identification of virulence genes, crude DNA was extracted according to the method of Kpoda et al. (2018).Briefly, exponential bacterial culture were suspended in 200µL of TE buffer and incubated at 95 °C for 10 min in a dry bath (IKA®, Dry block heater, India).The cell culture suspension was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected and used as a template for the polymerase chain reaction (PCR).A total of 57 isolates were screened for 10 potential virulence genes, namely aerA coding for aerolysins, act coding for cytotoxic enterotoxins, alt coding for cytotonic enterotoxins, ahp coding for serine protease, ahyB coding for elastase, enol coding for enolase, fla coding for polar flagella, exu coding for DNases, lip coding for lipase, and hlyA coding for hemolysin using PCR.The PCR reaction mixture of 25µL consisted of 12.5µL of EmeraldAmp GT PCR Master Mix (Takara, Japan), 1 µL of specific primers (25µM of forward and reverse primer), 2 µL of DNA template, and 8.5 µL of nuclease-free water.The primers used for target gene amplification, amplicon size, and annealing temperatures are shown in Table 1.The PCR amplification of virulence genes were carried out in Applied Biosystems™ Proflex™ (Thermo Fisher Scientific, USA), which consisted of an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 40s, annealing for 40s (Table 1), extension at 72 °C for 1 min, and a final extension at 72 °C for 7 min.The PCR products run on 1.5% agarose (80 volts for 40 min) in an electrophoresis unit were captured digitally using a gel image system (Biorad, USA).

Identification of Aeromonas spp.
All of the presumptive bacterial isolates grown on Aeromonas selective agar, exhibiting round, reddish orangecoloured, clear to slightly opalescent colonies of Gramnegative, rod-shaped, and motile bacilli, oxidase-positive, catalase-positive, and O/129-resistant bacteria, were tentatively identified as Aeromonas spp.Out of 79 Aeromonas spp., 57 isolates were confirmed to be A. hydrophila, 16 to be A. veronii, and 6 to be A. caviae using an automated bacterial identification system, BD Phoenix™ M50 (BD Diagnostics, USA).All 57 confirmed A. hydrophila isolates were included in this study.

Distribution of A. hydrophila
A total of 253 fish were collected from 100 freshwater fish farms in Andhra Pradesh, India, across the Krishna and West Godavari districts.The total prevalence of A. hydrophila from the IMC (C.catla and L. rohita) and Pangasius fish farms (P.hypophthalmus) included in the study was found to be 27%.The incidence of A. hydrophila was higher in IMC (C.catla and L. rohita) than in P. hypophthalmus.Regardless of farm-wise distribution, a total of 57 strains were collected from 253 fish (100 fish farms) and included L. rohita (n = 24), C. catla (n = 18), and P. hypophthalmus (n = 15).Out of 57 isolates, 20 and 37 were from healthy and diseased fish, respectively.Clinical examination of infected fish revealed pale gills, dark skin, abdominal distension, exophthalmia, haemorrhages on the body surface and internal organs, and an enlarged liver.

Antimicrobial susceptibility
The antimicrobial resistance profiles of Aeromonas spp.
(n = 57) isolates against six different antibiotics was assessed.The findings demonstrated that all Aeromonas isolates recovered from L. rohita, C. catla, and P. hypophthalmus exhibited varying degrees of susceptibility, intermediate and total resistance to all the antibiotics tested.The highest of resistance was found against trimethoprim (35.9%), followed by co-trimoxazole (21.1%), oxytetracycline (19.3%), doxycycline (17.5%), and enrofloxacin (8.9%), respectively.None of the isolates were found resistant to ciprofloxacin.The isolates from P. hypophthalmus showed high resistance in comparison to the isolates from IMC (C.catla and L. rohita).

Biofilm formation capabilities in A. hydrophila
A. hydrophila isolates were categorized as strong biofilm formers (SBF), moderate biofilm formers (MBF), and weak biofilm formers (WBF).33%, 50% and 17% of the isolates were categorized as strong, moderate and weak biofilm formers, respectively.In the present study, all strong and moderate biofilm formers harbored the fla gene, which is responsible for virulence and biofilm formation in many Aeromonas spp.

β-Hemolysis
A. hydrophila strain interestingly demonstrated a very clear β-hemolysis in the form of clearance zone along the streak on blood agar plate within 24 h of incubation at room temperature.70% of the strains were β-hemolytic in nature and remaining had no haemolytic activity.All the haemolytic isolates harboured either aerA and act or both (Table 2).

Discussion
The transition from extensive to intensive farming has aided the rapid development of animal food production, which has historically been accompanied by a rising trend of diseases in farmed animals (Schar et al. 2020;Van Boeckel et al. 2015;Boeckel et al. 2017).The genus Aeromonas contains a number of opportunistic pathogens causing diseases in human beings and aquatic animals (Schar et al. 2021), of which A. hydrophila has been described as a significant pathogen for both humans and aquatic animals (Cui et al. 2007).There is evidence that Aeromonas infections exist and that the correlation between virulence-associated factors and virulence increases in fish and other animals (Li et al. 2011;Wang et al. 2021), under conditions of stress.The present study found that 27% of A. hydrophila isolates were prevalent in freshwater fish culture farms in Andhra Pradesh, India, revealing an opportunistic distribution of bacterial pathogens.A similar study was done by Nhinh et al. (2021), who reported that A. hydrophila prevalence was found to be 46.4% in different freshwater fishes in Vietnam, posing high potential pathogenicity to fish.The present study confirmed the presence of A. hydrophila in freshwater fish species with obvious clinical signs and noticeable gross lesions, which may lead to mortalities.The presence of A. hydrophila isolates in healthy fish suggests that stress-related infections can be brought on by opportunistic bacterial pathogens in the aquatic environment.Antimicrobial compounds are unrestrictedly used in intensive fish farming to prevent or control infectious diseases.As a result, reservoirs of antimicrobial-resistant bacteria have developed in fish and other aquatic species as well as in the aquatic environment (Akinbowale et al. 2006;Heuer et al. 2009).According to earlier reports, A. hydrophila isolates from freshwater fish showed varying degrees of resistance to a wide range of antibiotics used to treat bacterial infections (Aiyahya et al. 2018;Chang et al. 2007;Dubey et al. 2022;Jacobs and Chenia 2007;Muduli et al. 2021;Nawaz et al. 2010;Stratev et al. 2015;Zhang et al. 2019).Plasmids, transposons, and integrases, which are important in the acquisition and transmission of AMR and virulence genes among other bacteria, have been implicated in the development of antibiotic resistance in Aeromonas spp.(Dubey et al. 2022;Palu et al. 2006;Sorum et al. 2003).In this study, A. hydrophila isolates from IMC (C.catla and L. rohita) and P. hypophthalmus displayed varying degrees of antimicrobial resistance to trimethoprim, cotrimoxazole, oxytetracycline, doxycycline, and enrofloxacin.The higher levels of antibiotic resistance in A. hydrophila from P. hypophthalmus compared to IMC could indicate that antimicrobial drugs are used more frequently in Pangasius fish farming.It was observed that high stocking densities (n = 8000-10,000/acre) without proper farm management are major potential problems resulting in excessive use of chemicals in order to maintain the fish's health.The dissimilarity in resistance patterns of A. hydrophila isolates among fish species may indicate historical variations in the frequency, quantity, method of application, and drug species used in fish production systems (Yuan et al. 2019).The pathogenicity of A. hydrophila is linked to the expression of virulence factors (Kumar et al. 2022).There is evidence that greater pathogenicity in fish and other hosts is strongly correlated with the presence of virulence-associated components (Oliveira et al. 2012).In the current study, 80% of A. hydrophila isolates had a minimum of four virulence genes.Aerolysins, cytotoxic enterotoxins, cytotonic enterotoxins, serine proteases, elastases, enolases, polar flagella, DNases, lipases, and hemolysins were found in A. hydrophila, indicating that they may be extremely pathogenic (Jiravanichpaisal et al. 2009;Nawaz et al. 2010).These virulence genes have been extensively utilized to investigate the potential pathogenicity of Aeromonas spp.(Nawaz et al. 2010;Nhinh et al. 2021).In the present study, virulence gene distribution in representative A. hydrophila isolates was very high for enolase (enol, 96.4%), followed by flagellin (fla, 94.7%), cytotonic enterotoxins (alt, 91.2%), cytotoxic enterotoxins (act, 84.2%), lipase (lip, 77.1%), serine protease (ahp, 54.3%), aerolysin (aerA, 50.8%), and elastase (ahyB, 43.8%), respectively.Enolases are secreted and surface-expressed glycolytic enzymes work as transcriptional regulators in A. hydrophila and A. dhakensis by binding to host structures and breaking down blood plasma, both of which are required for survival in the host environment (Fernandez-Bravo and Figueras 2020).Similarly, the fla gene plays a vital role in the development of biofilms by Aeromonas spp.and could be an important factor that contributes to the pathogenicity of the host (Ibrahim et al. 2020;Kumar et al. 2022).In this study, fla virulent genes were detected in 94.7% of the isolates examined, which is almost similar to the study reported by Hayati et al. (2018), where 96.3% of isolates recovered from farmed and wild tilapia, climbing perch, and catfish in Malaysia harboured the fla gene.This demonstrates a close relationship between the role of flagella in Aeromonas spp.biofilm formation, which is important for disease development and conferring resistance to bactericidal drugs (Cai and Arias 2017;Rasmussen-Ivey et al. 2016).It was discovered that all A. hydrophila strains can form strong (33%), moderate (50%), and weak (17%) biofilms, and that 100% of the strong and moderate biofilm formers carried the fla gene in this study.Cytotonic enterotoxins (alt), cytotoxic enterotoxins (act), and aerolysins (aerA) promote tissue damage and fluid buildup in fish intestinal epithelial cells, responsible for enterotoxic, hemolytic, and cytotoxic effects (Sha et al. 2002).The incidence of the alt, act, and aerA genes in A. hydrophila has also been linked to disease outbreaks in freshwater aquaculture (EI-Bahar et al. 2019, Nhinh et al. 2021).In the present study, 91.2%, 84.2%, and 50.8% of the A. hydrophila isolates harboured alt, act, and aerA genes, respectively.Hemolysis and cytotoxicity are caused by aerolysin (aerA) and hemolysins (hlyA), which are important virulence factors, in a variety of species and cell types (Elbahnaswy and Elshopakey 2020).However, in our study, the haemolysin (hlyA) gene was not found in the A. hydrophila isolates tested.Previous studies have found a relationship between the ability of the aerA, hly, and act genes to induce hemolytic activity and the pathogenic potential of Aeromonas spp.(Singh and Sanyal 1992).In the present study, 70% of the A. hydrophila strains possessed β-hemolytic activity, which may be responsible for haemolysis and immune suppression.These findings are in line with earlier findings made by Messi et al. (2003), who reported that A. hydrophila infections in fish are frequently associated with hemolytic and cytotoxic activities.Similarly, the extracellular lipases secreted by Aeromonas spp.(lip, lipH, pla, and plc) actively contribute to the alteration of the host plasma membrane, increasing the severity of infection.In the current investigation, the lip gene was present in 77.1% of the isolates.Extracellular serine protease (ahp) and elastase (ahyB) secretions promote the availability of nutrients and resistance to the host immune response, and associated with host tissue damage (Mzula et al. 2020).In the current study, serine protease (ahp) and elastase (ahyB) prevalence were 54.3% and 43.8%, respectively.Falco et al. (2012) reported that regulation and secretion of extracellular serine proteases (ahp) and lipases (lip) play a coherent and integrated function in the pathogenicity of furunculosis disease caused by A. salmonicida in cold water fishes.Additionally, it has been suggested that serine protease may change the virulence of Aeromonas spp.by activating aerolysin (aerA) and other extracellular enzymes (Cahill 1990).Aerolysins, cytotoxic enterotoxins, cytotonic enterotoxins, enolases, flagellins, elastases, serine proteases, DNases, lipases, and haemolysins are the key contributors to the pathogenicity of Aeromonas strains in humans and animals (Chen et al. 2013;Fernandez-Bravo and Figueras 2020;Figueras and Beaz-Hidalgo 2015;Hu et al. 2012;Muduli et al. 2021;Nawaz et al. 2010;Roges et al. 2020).Hence, the presence of these genes in the bacterial isolates has public health significance.

Conclusion
The prevalence of A. hydrophila has been determined at 27% across fish farms in the selected area of study in Andhra Pradesh.Although the AMR prevalence was found to be low, most of the isolates harboured a number of virulence genes that posed a high potential for pathogenicity in fish and even in humans.Therefore, these findings may serve as a caution to farmers and aquaculture entrepreneurs for reducing the incidence of pathogenic A. hydrophila in fish culture systems by implementing best management practices.

Table 1
List of primers, expected amplicon size and their corresponding annealing temperatures used in this study