Distribution, Extracellular Virulence Factors and Drug Resistance of Motile Aeromonads in Fresh Water Ornamental Fishes and Associated Carriage Water

During last decades there has been a continuous growth of aquaculture industries all over the world and taking into consideration the spurt in freshwater ornamental fish aquaculture and trade in Kerala, the present study was aimed to assess the prevalence of various motile Aeromonas spp. in fresh water ornamental fishes and associated carriage water. The extracellular virulence factors and the antibiogram of the isolates were also elucidated. Various species of motile aeromonads such as Aeromonas caviae, A. hydrophila, A. jandaei, A. schubertii, A. sobria, A. trota and A. veronii were detected. Aeromonas sobria predominated both fish and water samples. Extracellular enzymes and toxins produced by motile aeromonds are important elements of bacterial virulence. The production of extracellular virulence factors proteases, lipase, DNase and haemolysin by the isolates were studied. All the isolates from both fish and water samples produced gelatinase and nuclease but the ability to produce lipase, caseinase and haemolysins was found to vary among isolates from different sources. Among the 15 antibiotics to which the isolates were tested, all the isolates were found to be sensitive to chloramphenicol, ciprofloxacin and gentamicin and resistant to amoxycillin. Local aquarists maintain the fish in crowded stressful conditions, which could trigger infections by the obligate/ opportunistic pathogenic members among motile aeromonads.


Introduction
Keeping colourful and fancy fishes known as ornamental fishes, aquarium fishes, or live jewels is one of the oldest and most popular hobbies in the world. The growing interest in aquarium fishes has resulted in a steady increase in aquarium fish trade globally. The global scope and scale of the ornamental fish trade and growing popularity of pet fish are strong indicators of the myriad economic and social benefits the pet industry provides. Culture of ornamental fish in the backyards of households requires very little space, skill and time, and has the potential to improve the economic condition of the household. 'Earning a regular income, unlike seasonal work in agriculture, provides further motivation (Shaleesha and Stanley, 2000). Aquaculture is an emerging industrial sector which requires continued research with scientific and technical developments, and innovation. Over one billion ornamental fish comprising more than 4000 freshwater and 1400 marine species are traded internationally each year, making it one of the most important components of the global fish trade. Freshwater species make up 90% of this trade as they are the most popular and widely kept aquarium pets worldwide (Krishnakumar et al., 2009). The trade in ornamental (pet) fish is greater than 1 billion animals per year globally. More than 45 million fish per year are imported into the United Kingdom (UK) alone from a wide range of countries, in particular those in South East Asia (Wittington and Chong, 2007).
Fish diseases are a scourge of ornamental fish industry bringing huge economic loss. Bacterial organisms may be the primary cause of disease, or they may be secondary invaders. The majority of bacterial fish pathogens are natural inhabitants of the aquatic environment. Infections caused by motile members of the genus Aeromonas, are amongst the most common and troublesome diseases diagnosed in cultured warm water fishes and have been referred to by various names, including motile aeromonad septicemia (MAS), motile aeromonad infection (MAI), hemorrhagic septicemia, red pest, and red sore. Aeromonas bacteria causing these infections are called aeromonads (Camus et al., 1998). Aeromonas infections are a serious threat to fresh water fish production, bringing enormous economic loss to ornamental fish industry.
The detection of virulence factors in Aeromonas is a key component in the determination of potential pathogenicity, because more than two virulence factors act multifunctionally and multifactorially it seems necessary to continue surveying the distribution of known virulence determinants in currently circulating Aeromonas strains.
The disease problems are treated with antibiotics, the indiscriminate use of which can result in the rapid spread of multi-drug resistant pathogens across the system. It is also important for the ornamental fish industry to recognize the extent to which the bacteria associated with ornamental fish have developed antimicrobial resistance. This fact along with the financial crisis caused by the mortality of ornamental fishes makes the study of different geographical isolates of aeromonads important.
Aquaculture is in a phase of rapid growth and development. Fish diseases are among the most important problems and challenges confronting fish culturing. Among the etiological agents of bacterial fish disease, the motile Aeromonas group is considered important. Aeromonas spp. is ubiquitous in natural waters and comprises mesophilic motile and psychrophilic non motile gram-negative bacteria. Worldwide studies have demonstrated that Aeromonas spp. are universally distributed and widely isolated from clinical, environmental and animal sources, food samples and aquatic environment (Janda and Abbott, 2010). In aquatic environments, they are found in ground water, surface water, estuarine environments, sewage effluents, lakes and rivers (Galindo and Chopra, 2007) and in public drinking water and tap water (Pablos et al., 2009;Kivanc et al., 2011).
The widespread distribution of these bacteria in the aquatic environment and the stress induced by intensive culture practices predisposes fish to infections. A number of putative virulence factors that may play an important role in the development of disease, have been described in several species of the genus Aeromonas, which includes haemolysins, cytotoxins, enterotoxins, proteases, lipases, DNases and adhesins (Sen and Rodgers, 2004). The pathogenesis of Aeromonas infections is multifactorial, and no single virulence factor can be unequivocally pinpointed as responsible for particular symptoms or disease stages. Pathological conditions attributed to members of the motile aeromonad complex may include dermal ulceration, tail or fin rot, ocular ulceration, hemorrhagic septicemia and scale protrusion disease. Outbreaks of motile aeromonad septicaemia can reach epidemic proportions in farmed aquatic animals, with high rates of mortality (Liles et al., 2011). The disease problems are treated with antibiotics, but the emergence of antibiotic-resistant bacteria imposes a substantial burden on aquaculture. Antibiotic resistance is particularly relevant in pathogenic Aeromonas species in which, besides the classical resistance to β-lactam antibiotics, multiple-resistance has been frequently identified (Goñi-Urriza et al., 2000;Vila et al., 2002;2003). These bacteria can receive and transfer antibiotic resistance genes to other Gram negative bacteria (Marchandin et al., 2003).
Diseases in intensive freshwater aquaculture have brought great economic loss to India in recent years. Infections due to Aeromonas are common and pose a threat to the development of the aquaculture enterprise. Therefore the present study was carried out to assess the prevalence of various motile Aeromonas spp. in fresh water ornamental fishes and associated carriage water. The extracellular virulence factors and the antibiogram of the isolates were also studied.

Distribution of Aeromonas Species
Motile Aeromonas spp. was isolated from ornamental fish samples and associated carriage water samples. One hundred and seventy five isolates from the fish samples and one hundred and eighty two isolates from the water samples were characterized to species level, Aeromonas sobria predominated in both cases -40.57% in fish samples and 34.80% in water samples.
A. caviae was the second dominant spp. in both samples but its percentage of occurrence was much higher in fish samples, (31.43%), when compared to water samples (16.57%). A. hydrophila, A. jandaei, A. schubertii and A. veronii predominated in water samples when compared to fish samples as shown in Figure 1. Prevalence of motile aeromonads at various parts of the body of fresh water ornamental fishes is given in Figure 2. A. hydrophila, A. veronii and A. jandaei were frequently encountered on gill surface when compared to body surface and intestine.

Extracellular Virulence Factors of Aeromonas Species
All the isolates of Aeromonas spp. obtained exhibited gelatinase and DNase activity. All the isolates of Aeromonas sobria exhibited caseinase and β haemolysin production. Similarly all the isolates of A.
hydrophila and A. jandaei were capable of producing β haemolysin. β haemolysin production was infrequent in A. caviae. Lipase was produced by all the isolates of A. hydrophila, A. jandaei and A. veronii but their production among the isolates of other spp. varied. The production of extracellular virulence factors by the Aeromonas isolates obtained from fish and water samples are given Table 1 and Table 2 respectively.

Antimicrobial Resistance
All the aeromonad isolates tested were resistant to amoxycillin and sensitive to ciprofloxacin, chloramphenicol and gentamicin regardless of their source. While all the Aeromonas isolates from water were sensitive to nitrofurantoin, 1.14% of isolates from fish samples was resistant to nitrofurantoin. Resistance to ceftazidime was found in 13.33% of A.
veronii and 5.63% of A. sobria isolates from fish samples and 8% of A. veronii isolates from water samples. All the other isolates were sensitive to this antibiotic. All the isolates except 3.63% of A. caviae from fish samples and 20% of A. hydrophila, 12.69% to other antibiotics by the isolates from fish and water samples are given in Table 3 and Table 4 respectively.

Discussion
The prevalence and distribution of bacteria belonging to the genus Aeromonas in aquatic environments is of great public health concern since Aeromonas spp. can cause infections and epizootics in a variety of animals.
Motile aeromonads have been recognized as occasional pathogens of cultured fishes and the most common bacteria in freshwater habitats throughout the world. It is the etiological agent for motile aeromonad septicaemia (MAS) in fish. Aeromonas has also been frequently isolated from the lesions of epizootic ulcerative syndrome (EUS) fishes (Torres et al., 1990;Subasinghe et al., 1990;Roberts et al., 1990). This disease is a serious threat to the freshwater fish production of Southeast Asian countries. It causes mass mortalities in both cultured and wild fish species every year. The prevalence of different species of Aeromonas is likely to vary with geographical locations. In the present study Aeromonas sobria was the predominant species isolated from fish samples  (Rahman et al., 2002). A.
sobria, has been identified as a causative agent of disease in farmed perch Perca fluviatilis L. in Switzerland (Wahli et al., 2005). Aeromonas veronii has been isolated from the ascitic fluid of Oscar Astronotus ocellatus showing signs of infectious dropsy in India (Sreedharan et al., 2011). Hatha et al., (2005) reported A. hydrophila to be the predominant species in the intestine of farm-raised fresh water fish followed by A. caviae and A. sobria. In the present study A. sobria was frequently encountered in the intestinal samples of the ornamental fishes.
Motile Aeromonas species are ubiquitous bacteria in aquatic environments. These bacteria can be found in both polluted and unpolluted fresh water, in sewage, in drinking water, private wells, in unchlorinated as well as chlorinated water. In recent years, the presence of Aeromonas spp. in municipal drinking water supplies has become an emerging public health problem (Haunninen, 1994). In the present study, in terms of prevalence and abundance in water samples, the most predominant species was found to be The pathogenesis of Aeromonas infections is multifactorial, as aeromonads produce a wide variety of virulence factors. Several virulence factors have been studied in Aeromonas including aerolysin/hemolysin, enterotoxins, proteases, lipases and deoxyribonucleases (Chopra et al., 2000;Janda, 2001;Chacón et al., 2003). Nevertheless, it is apparent that some exo-enzymes are important pathogenicity factors. The haemolytic and proteolytic activities of motile and mesophilic aeromonads were reported in most studies as virulence-associated factors (Esteve et al., 1995;Gonzalez-Serrano et al., 2002;Rahman et al., 2002). The high rate of hemolytic activity detected in Aeromonas spp. is remarkable. The haemolytic activity is strongly associated with enterotoxin production in members of the genus Aeromonas (Burke et al., 1983) A. hydrophila strains producing cytotoxins, proteases and aerolysin were commonly isolated from both healthy and moribund fish (Cahill, 1990).
Potentially pathogenic Aeromonas species are present in diseased as well as healthy fish.
Widespread haemolytic, caseinolytic and gelatinolytic activity was encountered among the motile aeromonads isolated in the present study. These factors were considered as pathogenicity markers by (Kozińska, 2007). The isolates obtained from healthy fishes in this study are also potentially pathogenic as revealed by the production of extracellular virulence factors by the isolates. Though extracellular virulence factors cannot be considered as definite marker for pathogenicity of the isolates, the poor water quality conditions of the aquaria maintained by local farmers in this region might trigger disease outbreak by opportunists. Shome et al. (1999) report that the production of enzymes or toxins is not reflective of biological virulence and they do not satisfy a strain to be virulent or avirulent even though these appear to enhance the disease process in-vivo. The whole process of pathogenesis is a complex interaction among the host, agent and environmental determinants. Yucel et al., (2003)  jandaei isolates are lipase producers. Possibility of caseinolytic (Mateos et al., 1993) and gelatinolytic activity (Shome et al., 1999) with virulence is substantiated by the above research groups who observed that all the A. hydrophila isolates from diseased fishes with dropsy and EUS had caseinolytic and gelatinolytic activity.
All the isolates from the water samples in the present study are producers of gelatinase and nuclease. β haemolysin was produced by 100% of A. hydrophila and A. sobria (Tables 1 and 2). β haemolytic activity among all the isolates of A. hydrophila and A. sobria and α haemolytic activity among all the isolates of A.
caviae from water sample are reported earlier (Gibotti et al., 2000). The extracellular virulence factors among Aeromonas spp. isolated from Bhavani river, South India was studied by Bagyalakshmi et al. (2009). Several of these virulence factors have been identified in strains isolated from water (Sechi et al., 2003). In the present study also, all these virulence factors have been identified in strains isolated from water.
There have been conflicting reports on the susceptibility of motile Aeromonads to commonly used antibiotic agents. High level of resistance against amoxycillin, and carbenecillin and 100% sensitivity to chloramphenicol, ciprofloxacin and gentamicin was noticed in the isolates from both fish and water samples. Motile aeromonads isolated from fish and water samples, in India exhibiting 100% sensitivity to, ciprofloxacin and gentamicin and high level of susceptibility to chloramphenicol is reported by Rathore et al. (2005). Complete sensitivity to chloramphenicol, ciprofloxacin and gentamicin by the isolates is reported by Penders and Stobberingh (2008). All the isolates from water samples in the present study were susceptible to nitrofurantoin and only a 3.63% of A. caviae isolates from fish samples exhibited resistance to the antibiotic. High level of susceptibility to nitrofurantoin by the isolates is also reported by Castro-Escarpulli et al. (2003) but in contrast to our finding he has reported 100% sensitivity to nalidixic acid and more than 50% resistance to gentamicin and varying degrees of resistance to chloramphenicol and ciprofloxacin.
Among the motile aeromonads isolated from fish samples in India, relatively low level (<10%) of resistance to nalidixic acid, gentamicin and ciprofloxacin is reported by Hatha et al., (2005). However, resistance to nalidixic acid was considerably high among the motile aeromonads encountered in the present study (49.71%), while all the isolates were sensitive to gentamicin and ciprofloxacin. Varying levels of resistance to nalidixic acid, gentamicin and ciprofloxacin has been reported among motile aeromonads from water samples (Koksal et al., 2007;Evangelista-Barreto et al., 2010). Similar to the findings of these researchers, we also observed complete sensitivity of our isolates to nitrofurantoin, chloramphenicol and ciprofloxacin. The development of antibiotic resistant strains is a great extent attributed to the misuse of antibiotics in culture systems for treatment and for better feed conversion, these levels are bound to vary according to local practises.
Resistance to ceftazidime among the isolates from water and fish are found to vary from 8 to 15% among The present study reveals the prevalence of motile aeromonads in ornamental fishes and associated carriage water. Most of the motile aeromonads were capable of producing extracellular virulence factors and had acquired multidrug resistance. Poor water management practices could trigger infections caused by these potential pathogens/opportunists which might spell doom for this emerging industry in Kerala. Prudent use of antibiotics and proper water management practices may be promoted for sustainable development of ornamental fish industry, which can sustain the livelihoods of large number of rural people, who are currently engaged in this industry.

Collection of Samples
Live, healthy ornamental fish samples (Poecilia sphenops and Poecilia reticulate) and associated carriage water samples were collected from three different aquarium vendors in and around Cochin. The fish samples were transported to the laboratory in sterile polythene bags and water samples in sterile bottles. The samples were then analyzed for aeromonads within 4 hours of collection.

Isolation and identification of Aeromonas
Different parts of the body (body surface, gill and intestine) of fish samples were analyzed for motile aeromonads. The body surface and the gill of the fishes were repeatedly swabbed using sterile cotton swabs. Using a pair of scissors, an incision was made near the vent of the fish facilitating the swabbing of intestine. The swabs were then transferred to alkaline peptone water (APW pH 8.4) which was used as the enrichment medium. After incubation at 37℃ for 18 h, a loopful of the APW culture was streaked on Starch Ampicillin Agar (Ampicillin 10 mg/L) which was used as the selective isolation medium and incubated at 37 o C for 18-24 h (Palumbo et al., 1985).
The water samples were serially diluted and bacterial isolation was done by spread plate method using Starch Ampicillin Agar plates. The plates were incubated at 37 ℃ for 18-24 h and then flooded with approximately 5 ml of Lugol's iodine solution and amylase positive yellow to honey colored colonies were isolated. The isolated cultures were then purified by repeated streaking on nutrient agar plates. Those strains that were gram negative bacilli, motile, oxidase and catalase positive, glucose fermenting, nitrate reducing, urease negative and which do not grow in 6% NaCl were further tested for arginine dihydrolase, lysine decarboxylase, and ornithine decarboxylase activity. Additional tests include acid production from mannitol, sucrose, arabinose, Voges-Proskauer reaction, hydrolysis of esculin and indole production. The isolates were identified to the species level according to Aerokey II (Carnahan et al., 1991).

Study of Extracellular Virulence Factors 3.2.1 Production of gelatinase
Pure cultures of the isolates were spot inoculated on gelatin agar plates (2%w/v gelatin), and the plates were incubated at 28 ℃ for 24-48 hrs. Zone of clearance around the colonies, after the plates were flooded with saturated solution of ammonium sulphate indicated that gelatin has been hydrolyzed.

Production of caseinase
The test organisms were heavily spot inoculated on skim milk agar plates and the plates incubated at 28℃ for 24 hrs -48 hrs. Caseinase production was detected by the presence of clear zones around the test colonies.

Production of lipase
Tributyrin or glyceryl tributyrate is commonly used for studying lipolytic activities. The test organisms were heavily spot inoculated on tributyrin agar plates and the plates incubated at 28℃ for 24 hrs -48 hrs. A positive result was indicated by a zone of clearance around the colonies of lipolytic organisms, where the tributyrin has been hydrolyzed (Rhodes, 1959).

Production of DNase
A plate test for the demonstration of bacterial decomposition of nucleic acid was performed. The test organisms were heavily spot inoculated on DNA agar plates and the plates incubated at 28℃ for 24 hrs -48 hrs. After incubation the plates were flooded with 1M HCl. The appearance of clear zone around the colonies indicated that the bacteria has elaborated DNase and hydrolyzed the DNA. The rest of the plate with the intact DNA turned opaque white, on addition of 1M HCl.

β-Haemolytic assay
Haemolytic activity was determined using blood agar International Journal of Aquaculture, 2013, Vol.3, No.17, 92-100 http://ija.sophiapublisher.com 99 medium containing 5% human blood. Pure cultures of bacterial isolates were spot inoculated onto blood agar plates and β haemolytic activity was recorded as clear zone around the colonies after incubation at 37℃ for 24 h.
Pure cultures of Aeromonas were inoculated into nutrient broth and incubated at 37℃ for 6 h-8 h. The cultures were then seeded onto Mueller Hinton agar plates. Antibiotic discs were placed on the surface of the agar with sterile forceps and pressed down gently to ensure even contact. Zone of inhibition around the antibiotic discs was measured after 16 h-18 h incubation at 37 ℃ and susceptibility/resistance interpretation was performed according to the manufacturer's interpretative table supplied by the Hi-media laboratories, Bombay as a matching criteria.