Prevalence and Antimicrobial Susceptibility of Pathogenic Bacteria in Nile Tilapia, Oreochromis niloticus L

Nile tilapia, Oreochromis niloticus, is one of the most popular aquaculture fi sh species in the world. However, among several challenges, the presence of pathogenic bacteria causes high economic losses. Thus, the main objective of this study was to isolate and identify the potent bacterial pathogens from Nile tilapia reared at Hawassa Fish Research and Multiplication Station, Ethiopia. For this, infected fi sh samples were collected from the research station and subjected to microbiological and biochemical tests. The results of the study revealed that 75% of fi sh with the length group ranged from 14-17.9cm; 52% with the length group ranged from 18-21.9 cm and 33% with the length group ranged from 22-26.9cm were infected by different bacteria belonging to the genera Vibrio, Escherichia, Aeromonas, Pseudomonas, Salmonella and Streptococcus. Except for Streptococcus, all isolates belonged to gram negative bacteria. The bacterial population observed in fi sh organs was signifi cantly high in intestine (12.43±0.55 Log10 CFU -1g) than in liver (6.48±1.06 Log10 CFU -1g). An antibiogram test showed that isolated bacteria were sensitive to gentamycin, tetracycline and amoxicillin. In conclusion, the present results clearly indicate that cultivable fi shes are prone to infection by infectious and non-infectious and that it may affect fi sh and their product quality which leads to economic loss and livelihood of farmers who depend on small scale aquaculture. Research Article Prevalence and Antimicrobial Susceptibility of Pathogenic Bacteria in Nile Tilapia, Oreochromis niloticus L Begonesh Bekele, Kassaye Balkew Workagegn* and P Natarajan Department of Biology, Hawassa University (HU), PO Box 05, Hawassa, Ethiopia Received: 27 September, 2019 Accepted: 02 November, 2019 Published: 04 November, 2019 *Corresponding author: Kassaye Balkew Workagegn, Department of Biology, Hawassa University (HU), PO Box 05, Hawassa, Ethiopia, E-mail:


Introduction
The world's population is growing rapidly, being expected to reach 9.8 billion by 2050 [1]. To feed this population, food production must increase by 60% worldwide [2]. However, food production from the agricultural sector will fail to meet such high demand due to climatic effects. Thus, climate-smart aquaculture is vital to increase food production. It is one of the primary sources of cheap animal protein for the rapidly growing human population. According to FAO [3], aquaculture production has increased from 61.8 million tons in 2011 to 80 million tons in 2016. The bulk of this production came mainly from Asia and Latin America. Asia accounted for about 88.9% of the world aquaculture production. Although the contribution of African aquaculture to the world fi sh production is relatively low, it increased from 0.49% in 1995 to 2.3% in 2014 [4]. Aquaculture in Ethiopia remains more potential than the actual practice, even though the country's environmental conditions support its development. Extensive and semi-intensive aquaculture, in the form of stocking and enhancing artifi cial lakes, reservoirs and small water bodies, has been practiced since 1975 through the Sebeta National Fishery and Other Aquatic Life Research Center (SNFARC).
Although the country has rich fi sh biodiversity, the most dominant cultured fi sh species are Nile tilapia (Oreochromis niloticus), catfi sh (Clarias gariepinus) and common carp (Cyprinus carpio). Among these, Nile tilapia contributes about 50% of the overall fi sh production in the country [5,6]. This is because Nile tilapia has suitable cultivable characteristics such as effi cient use of natural and artifi cial feeds, resistance to diseases, tolerance to a wide range of environmental conditions, relatively fast growth rate, and excellent meat quality. In semi-intensive and intensive aquaculture production systems, however, disease is a primary constraint that affects the growth of many cultivable fi sh species, and is responsible for hampering production and expansion of the sector and thereby reducing socioeconomic development of many developing countries of the word. For instance Asia has been faced with mass mortalities of many cultured fi shes due to the occurrence of different bacterial diseases such as Aeromonas, Vibrio, and Pseudomonas [7]. In most cases, they cause infl ammation, ulcer and hemorrhages that lead to reduce the quality of fi sh and fi sh products. Noninfectious diseases due to pollution, algal toxins feed contamination and water quality also common in cultured fi shes which can have devastating effects on fi sh growth occasionally leading to crop loss [8]. Therefore, the main objective of this study was to isolate and identify the potential pathogenic bacterial in Nile tilapia, and to test their drugs sensitivity.  to 38 0 34' 0'' longtudes east and is situated 1,686m above mean sea level (Figure 1). recorded as CFU g -1 in each organ [8]. To determine the colony counts in CFU ml -1 of sample, the following formula was used:

Number of Colonies Dilution Factor Estimate of Microbial Load
Volume of Inoculum used  

Preparation of pure culture
Pure cultures of the isolates were identifi ed by using the standard procedures proposed by Barrow & Feltham [10]. In this regard, suspected colonies were picked up and re-streaked on new plates of selective medium (Mac Conkey Agar and TCBS agar). Identifi cation of the pure culture was made by examining the colony morphology, staining characteristics, motility, oxidase activity, and oxidation-fermentation properties [9].

Morphological and biochemical characterization of isolates
The isolates were identifi ed using morphological and biochemical characterization following the criteria proposed in the Bergey's Manual of Determinative Bacteriology [11]. In this regard colony morphological characterization was performed. Followed this, Gram's staining was performed using standard procedures and observed under oil immersion objectives to determine the shape and arrangement of bacteria. Later, different biochemical characterization such as catalase, sulfi de, indole tests and motility test were performed by following standard procedures as shown in Figure 2. Immediately, the sample was placed in 70% ethanol. From these, bacteria were isolated from the body surface, gills, liver, intestine and kidney of fi sh aseptically.

Preparation of serial dilution and incubation
One gram samples from the above mentioned organs of each fi sh were taken and mixed with 100ml of 0.1% sterile peptone water in sterile bottles. Similarly, mixed with 9ml of 0.1% sterile peptone water in sterile test tubes. The bottles were then shaken thoroughly, and a 10-fold serial dilution was carried out. In this regard, 1ml of the original mixed sample was transferred to the fi rst test tube and mixed thoroughly.
From this solution, 1 ml was taken from the fi rst test tube and added to the second test tube and mixed thoroughly. This procedure continued until the tenth serial dilution. Later, from each serially diluted sample, 0.1ml was transferred to nutrient agar using a pipette and dispensed and cultured by the glass spread method [9]. All the incubated nutrient agar plates were incubated in an inverted position at a temperature of 37 0 C for 24 hours. Later, the bacterial colonies found on all plates were counted using a colony counter. Plates containing 30-300 colonies were used to calculate the bacterial population and

Antibiotic sensitivity test
The antibiotic sensitivity test was done by using the disc diffusion method. A total of seven types of antibiotic discs including Ampicillin (10μg), Streptomycin (10μg), Tetracycline

Prevalence and load of isolates
The prevalence of bacteria in the naturally infected Nile tilapia reared at the research station is shown in Tables 1,2. The results showed that 75% within the 14-17.9cm length group, 52% within the 18-21.9cm length group and 33% with the 22-26.9cm length group were infected with bacteria with a prevalence of 55%. The isolates observed in fi sh organs was highest in the intestine (12.43±0.55 Log 10 CFU -1 g) followed by gill (12.10±0.42 Log 10 CFU -1 g) and skin (10.30±1.29 Log 10 CFU -1 g). and least in the liver (6.48±1.06 Log 10 CFU -1 g). External observation of infected fi sh showed clinical signs of disease ( Figure 3). The results of the study showed that infection was found to be higher in gills and intestine and lower in liver and kidney.

Morphological and biochemical characteristics of bacteria
The bacteria isolated from fi sh tissues are presented in Table 3. The results of this study showed that the isolated bacteria belonged to the genera Vibrio, Escherichia, Aeromonas, Pseudomonas, Salmonella and Streptococcus. All the genera, except the Streptococcus, were gram negative, motile, oxidase, catalase, indole and H 2 S positive (Table 3 and Figures 4,5).

Antibiotic sensitivity
The results of the antibiotic tests of the isolates are presented in Figure 6. The results showed that gentamycin was the best antibiotic followed by Tetracycline while Ampicillin and Erythromycin were least effective.

Discussion
Intensive aquaculture production systems are carried out with high stocking densities, intensive feeding and improved management practices. In such situations, the cultured organisms are subjected to several ecological stressors, which in turn cause diseases in fi sh. The major diseases associated with fi sh are due to parasites, bacteria, viruses and fungi that reduce fi sh production by affecting the normal physiology of fi sh leading to mass mortalities. Among these, bacterial infection constitutes one of the major constraints for aquaculture that results in large-scale economic loss [12].
The occurrence of more number of pathogenic bacterial isolates found in the semi-intensive farming systems in the present study may be due to poor production management, including poor/overfeeding [13]. With different length groups,     the results revealed that smaller fi sh were more sensitive to bacterial infection than the larger fi sh. This implies that as size of fi sh increases the prevalence of infection decreases, and this might be related to the ability of fi sh to withstand infections at later age. In semi-intensive and intensive aquaculture high temperature, overcrowding, occasional water restoration rate and failure to remove injured and dead fi shes lead to a rise in bacterial ailments [14].
Most of the morphological and biochemical characteristics of bacterial isolates observed in the present study agreed with the reports of earlier workers [15][16][17][18]. The high bacterial count found in the intestine and gills could be associated with poor water quality and feeding practices as reported by Beveridge et al., [19], who stated that voracious feeding behavior of Nile tilapia, presence of organic matter and poor water quality of the system were responsible for the higher incidence of bacterial population in the intestine and gills of fi sh. The higher load of bacterial isolates in gills was also due to the fact that gills play a vital role in fi ltering microscopic organisms [20]. Several studies also suggested that smaller particles are entrapped by the gill fi laments in a mucous leading to higher level of bacterial population [19, 21,22]. Due to their broad body surface of the fi sh and their frequent contact with the sediment and water, skin also showed higher bacterial count. In addition, the scales could also trap detritus particles which serve as substratum for the growth of different types bacteria. On the other hand, liver showed the lowest bacterial load, which could be due to its detoxifi cation function [22].
The present results of the antibiotic test showed that bacterial isolates were highly susceptible to gentamycin, tetracycline and amoxicillin. Isolates were moderately sensitive to Kanamycin and Streptomycin which confi rm the reports of Shaw et al., [23], Sudh et al., [24] and Yu et al., [25]. The isolates found in the present study were resistant to ampicillin and erythromycin which is in agreement with the fi ndings of others [26,27]. In conclusion, the present results clearly indicate that cultivable fi shes are prone to infection by infectious and non-infectious and that it may affect fi sh and their product quality which leads to economic loss and livelihood of farmers who depend on small scale aquaculture. It is also a fact that commercial culture will also seriously suffer due to fi sh diseases as the type of culture practice followed is intensive. Hence, there is an imperative and urgent need for an integrated approach to fi sh health, especially general husbandry and management strategies.