Prevalence of Bacterial Microbiota in Tilapia Guineensis Harvested from Buguma Brackish Water Environment River State, Nigeria

Brackish water fishes usually harbour human pathogenic bacteria particularly the enterics and coliform groups in their mucosal surfaces. Thus, this study was undertaken to investigate the prevalence of skin and gills microbiota of Tilapia guineensis as to understand their microbial assemblages and as well provide useful insights into the broad dynamics of the fish host-microbial relationship and health status. Thirty samples of Tilapia guineensis were collected from Buguma brackish water environment, River State, Nigeria. The skin and the gills of the tilapia were analyzed microbiologically. Specimen were collected by swabbing aseptically over the skin and gills segment of the fish and then processed for total bacterial count, followed by isolation and identification of isolated bacteria. Microbial load analysis revealed that the total viable count of bacteria ranged from 2.4 to 7.6 x 10 5 cfu/ml for skin and 2.1 to 5.7 x 10 6 cfu/ml for gills, and it was observed to fall within the recommended microbiological standard safety limit. A total of thirty-seven (37) bacteria, belonging to fifteen different genera in the family of Vibrionaceae, enterobacteriaceae and Morganellaceae were isolated from the fish. The prevalent rate of the bacterial shows that Proteus vulgaris recorded the highest frequency of occurrence of 16.22%, followed by Vibrio parahaemolyticus with 10.81% occurrence and Provindencia rettgeri, Serratia mercescens and Salmonella sp. trailed them with 8.11%. Kluyyera ascorbate, Citrobacter amalonaticus, Proteus mirabilis, Enterobacter aerogenes, plasiomonas shigelloides and Pseudomonas sp. have the least frequency of occurrence with 2.7% respectively for each. Though the bacterial count of Tilapia guineensis harvested from the water and fish sample did not exceed the standard recommended safety limit. There were several potential pathogenic and spoilage bacteria isolated as part of the fish flora. The presence of these bacteria could poses or constitute a public health risk.


Introduction
Tilapia Guineensis is a widespread fish species with great aquaculture potentials in all the tropical and sub-tropical regions of the world [1]. Tilapia Guineensis have been classified prominent in the ecology of tropical waters as well as in the resources of their aquatic systems [2]. Tilapia Guineensis is very common in the brackish water zone of Nigeria and most especially in the Niger Delta region [3]. There is an increasing interest in the Tilapia guineensis production due to their several nutritional advantage of being firm in texture, possession of low cholesterol and relatively easy to cook [4]. Regardless of these numerous advantages, Tilapia guineensis and other fish species in general are susceptible to contamination with pathogenic bacterial organisms because of their flesh texture, their living habits as well as their habitant which is loaded with pathogenic bacteria. Among these pathogenic bacteria are the Vibrio sp., Aeromonas sp., Edwardsiella sp., and Salmonella sp., which belong to water and food borne group of pathogens. These bacteria are widely distributed in brackish water environment and they have been implicated as opportunistic pathogens causing gastroenteritis and other severe health conditions to man. Their occurrence has been reported in some African, Asian, European countries and virtually across the globe. Several researchers have reported that the release of pathogenic bacteria in faeces dispersed into aquatic environments can contaminate fish and shellfish harvested from these waters [5]. Once these bacteria are in the aquatic environment, plasmid exchange between the bacteria will be readily facilitated and can result in a higher frequency of multiple antibiotic resistant strains which perhaps could exert selective pressures to influence the antibiotic resistance [6].
Bacteria, a large group of single-celled, prokaryotic and ubiquitous microscopic organisms whose single cells have neither a membrane bounded nucleus nor other membrane bounded organelles like mitochondria and chloroplasts [7]; and are noted for most fish diseases, fish spoilage and threats to public health [8].
Evaluation of the prevalence of bacterial microbiota associated with the skin and gills of Tilapia guineensis harvested from brackish water environment usually give information on the relationship between the fish and the environment. Moreover, Water with high organic load have been reported to predisposes fish to disease condition [9]. Therefore, this study was embarked on to investigate the prevalence of bacterial microbiota associated with the skin and gills of Tilapia guineensis caught from Buguma brackish water environment.

Materials and Methods
Thirty (30) fish samples of Tilapia guineensis were collected from Buguma brackish water environment, River State, Nigeria, and transported in an oxygenated bag to Nigerian Institute for Oceanography and Marine Research, Lagos, where the skin and the gills of the fish sample were aseptically swabbed with a sterile swab stick for bacteriological studies.

Total Bacteria Count Analysis
Swabs were aseptically taking from the gills and skin of the fish with a clean sterile swab stick. The swabs were immersed into a 100 ml conical flask containing buffered peptone water (0.1%) (Merck, Germany). After overnight incubation for 18 hours at 37°C, 1 ml was transferred for further analysis from the peptone water to Tryptone soy agar. Ten-fold serial dilutions (10 -5 ) were carried out, and viable bacterial counts of the samples were obtained.

Bacterial Enumeration and Identification
Enumeration of bacteria was carried out using spread plate method. The plates containing Tryptone Soy Agar (Himedia, India) were inoculated and incubated at 37°C for 24 hours after which the discrete colonies were observed and subcultured to get pure colonies of the isolates. Pure colonies of the isolates were further sub-cultured on Eosin methylene blue (EMB) agar, Salmonella-Shigella agar, Thiosulfate Citrate bile salts sucrose (TCBS) agar and Mannitol salt agar. The bacteria isolates were identified using some parameters such as Gram staining reaction, cultural and morphological characteristics, and series of biochemical tests to confirm the presence of the suspected microorganism by their reactions to the tests according to Bergey's manual of determinative bacteriology, 8th Edition [10].

Results
Total bacteria count result of water sample, skin and gills of Tilapia guineensis from Buguma brackish water, River State are shown in Table 1. The bacterial load from the skin are significantly lower (P<0.05) than the bacteria count from the gills and the water sample. The highest microbial load was observed in the water sample, followed by the gills. All the bacterial isolated from the water sample, skin and gills of Tilapia guineensis from Buguma are shown in Table 2.  The cultural and morphological characteristics used in the identification processes of the bacterial isolates from Buguma are represented in Table 3. Table 4 shows the Gram stain reaction and biochemical tests for presumptive confirmation of the bacterial isolates obtained from the gills and skin of the fish samples. Table 5, on the other hand represent the percentage frequency of occurrence of the bacterial isolates from Tilapia guineensis.

Discussion
Total bacteria count in brackish water sample, skin and gills of the Tilapia guineensis varies significantly. The bacterial load in each of the sample analyzed was high but does not exceed the standard microbiological recommended limit. The high bacterial load in each of the sample segment could be as a result of high temperature where the fish samples were collected which is close to optimum temperature for several mesophilic bacterial [10]. Total bacteria load in the gills ranged from 2.1 to 5.7 x 10 6 , 2.4 to 7.6 x 10 5 for the skin and 3.3 to 8.5 x 10 6 for the water sample. High bacteria load observed from this study is in agreement with the study of [11] who reported the presence of high bacterial load in tilapia gills, but in contrast with the work of Chessbrought [12], who reported that bacteria associated with the gills are actively maintained at low level to enable the fish keep the bacterial number low, and therefore afford the fish some degree of protection against bacterial invasion by the gills micro flora.
The bacterial flora from the water sample, skin and gills of Tilapia guineensis were majorly gram negative bacteria belonging to the family of enterobacteriaceae, Vibrionaceae and Morganellaceae, and it is a reflection of the bacteria composition from their environment. This finding is in agreement with an earlier report by Al-Harbi and Uddin [13] that the microflora of caught fish and other aquatic specimens is largely a reflection of the microbial quality of the water where they were harvested.  [14] reported different bacterial species in their study of Characterization of bacterial flora of tilapia harvested from four lakes in the north of Cameroon. Proteus sp., Serratia mercescens, Vibrio species, Salmonella sp., Morganella morganii, Provindencia rettgeri dominated other bacterial groups in the water sample, skin and gills of the Tilapia guineensis as shown in Table 2. However, the commensal bacterial flora from this fish is a facultative opportunistic pathogen which under stress could give rise to fish disease and could as well be zoonotic in nature. Salmonella sp., E. coli, Shigella sp., and Vibrio sp. have been implicated in so many fish borne infection of human.
A total of thirty-seven [37] bacteria, belonging to fifteen different genera in the family of Vibrionaceae, enterobacteriaceae and Morganellaceae were isolated from the fish sample [15].
The prevalence rate shows that Proteus vulgaris recorded the highest frequency of occurrence of 16.22%, followed by Vibrio parahaemolyticus with 10.81% occurrence and Provindencia rettgeri, Serratia mercescens and Salmonella sp. trailed them with 8.11%. Kluyyera ascorbate, Citrobacter amalonaticus, Proteus mirabilis, Enterobacter aerogenes, plesiomonas shigelloides and Pseudomonas sp. have the least frequency of occurrence with 2.7% respectively for each. In conclusion, Though the bacterial count of Tilapia guineensis harvested from the water and fish sample did not exceed the standard recommended limit. There were several potential pathogenic and spoilage bacteria isolated as part of the fish flora. The presence of these bacteria could poses or constitute a public health risk. This study showed that the Tilapia guineensis and the water sample from Buguma brackish water environment host a ridiculous and diverse bacterial flora. However, Microbiota of fishes and brackish water from Buguma, River State, Nigeria is mainly composed of Gramnegative bacilli.