Gene Sequencing and Haemolysis of Bacteria in Clarias gariepinus From Ajilete Location on Yewa River

Gene sequencing and haemolysis of bacteria in Clarias gariepinus post juveniles sampled from Ajilete location on Yewa River, Ogun state, Nigeria was carried out. Bacteria were isolated from the gut, gills and skin of the fish. Thirty bacteria isolates were selected and the DNAs were extracted using hexadecyltrimethylammonium bromide (CTAB) method and Polymerase Chain Reaction (PCR) amplification of the isolates was carried out using universal primer for bacteria. The purification of PCR product was done using ethanol precipitation and thereafter the DNAs were sequenced using automated DNA sequencer. The sequence data were analyzed on the GenBank database (NCBI) using Basic Local Alignment Search Tool (BLAST) to find the nearest related sequence, and to construct the phylogenetic relationship. The haemolysis of the selected isolates were also carried out. Data collected were subjected to descriptive statistics. Nine genera of bacteria isolated were Escherichia, Staphylococcus, Enterobacter, Proteus, Pseudomonas, Klebsiella, Citrobacter, Streptococcus and Bacillus . The 16S rRNA gene sequencing method identified the isolates as strains of Brevibacillus agri, Bacillus cereus, Ignatzschineria indica, Comamonas kerstersii, Proteus vulgaris, Lysinibacillus sphaericus, Proteus hauseri, Clostridium bifermentans, Proteus mirabilis, Pseudomonas putida, Escherichia coli, Comamonas jiangduensis, Enterobacter cloacae, Serratia marcescens, Alcaligenes faecalis, Lysinibacillus fusiformis, Myroides odoratimimus . Haemolysis showed that M. odoratimimus , B. cereus , C. kerstersii , P. vulgaris , C. bifermentans , E. coli and E. cloacae all had β -haemolysis. A. faecalis displayed α haemolysis while, B. agri , I. indica , L. sphaericus , P. hauseri , L. fusiformis , S. marcescens were non-haemolytic. This study confirms the reliability of the 16S rRNA gene sequencing method. This study also concludes that there are different pathogenic bacteria species that are associated with C. gariepinus .


INTRODUCTION
Fish is a major source of nutrients for humans, providing a significant portion of the protein intake in the diets of a large proportion of people in developing countries where it represents one of the cheapest sources of animal protein. However, freshly harvested fish is highly perishable; and depending on harvesting techniques and handling, may deteriorate and spoil within six hours of landing [1]. Apart from the high perishability of fish, consumer safety is an issue to be considered because fish is a good medium for rapid bacteria multiplication particularly when exposed to unsanitary conditions. [2] observed that bacteria associated with freshly caught fish are principally a function of the environment where it is caught. Thus there is a need to investigate the bacteria associated with fish if only to safeguard public health. Moreover, contamination of water body by various activities of man has been frequently associated with transmission of diseases causing bacteria such as Vibrio, Salmonella, Bacillus, Staphylococcus, E. coli [3]. Diverse group of microorganisms exist in the aquatic environment where they live as saprophytic, pathogenic or heterotrophic organisms. Microorganisms share common environment with fish in their natural habitat and thus become resident fish flora, but they can cause epizootics under stressful conditions. These stressful conditions may be brought about by chemical, physical and or biotic factors [4]. [5] showed that there is an established connection between public health and fish consumption in terms of communicable diseases, malnutrition and non-communicable diseases. However, the presence of potential human pathogens suggests that fish improperly handled, consumed raw or undercooked may lead to disease to susceptible host. Therefore, the objective of this study is to identify bacteria flora from gills, guts and skin of C. gariepinus post juveniles using 16S rRNA gene sequence so as to know if there are different organism associated with these organs and to construct a cluster analysis to find evolutionary ties among the bacteria and also to evaluate the bacteria haemolysis.

Samples Collection
The study was carried out at Ajilete station on Yewa River which has six landing site. It is situated at 6º 22' North and 2º 55' East. Bacteria samples were taken from the gill, skin and gut of C. gariepinus with the use of swab stick according to [6]. All swab sticks were streaked on both Nutrient agar and Mac Conkey agar by BioMark Laboratory, the samples were later incubated for 18 hours at 37ºC.

Water Test and Morphometric of Fish Sample
The standard length, total length, the head length in centimeter (cm) were measured using a measuring ruler and recorded after weighing the fish sample in grams (g) using sensitive weighing balance. The water quality was tested on Temperature, Dissolved Oxygen and pH from difference point on the landing site.

Isolation of Bacteria and Morphological Characterization
Isolation of bacteria from the gills, guts and skin of the fish samples was carried by standard method. Each of the swab stick taken from the gills, guts and skin was inoculated on Nutrient agar and MacConkey agar (Oxoid, UK) and incubated at 37ºC for 24 hours. Bacteria colonies obtained were purified on Nutrient agar and incubated at 37ºC for 24 hours for growth pure discrete colonies.
Colonial and cellular morphology was performed by phenotypic examination and Gram staining respectively. Each bacteria isolates were biochemically characterized according to [7].

Identification of the Bacteria Isolates by 16S rRNA Gene Amplification
Bacteria isolate was grown overnight in Nutrient Broth and spun at 14,000 rpm for 2 mins, the DNA was extracted using CTAB method [8]. The DNA was later resuspended in 100 µl of sterile distilled water. DNA concentration of the samples was measured and the genomic purity was determined. PCR analysis was done using MJ Research Thermal Cycler (PTC-200 model). The primer used for PCR amplification was 16S universal primer for bacteria, the sequence for the forward primer was 5'AGAGTTTGATCCTGGCTCAG3' and reverse primer was 5'ACGGCTACCTTGTTACGACTT3' The PCR mix comprises of 1 µl of 10X buffer, 0.4 µl of 50 mM MgCl 2, 0.5 µl of 2.5 mM dNTPs, 0.5 µl 5 mM Forward primer, 0.5 µl of 5 mM Reverse primer, 0.05 µl of 5 units/µl Taq with 2 µl of template DNA and 5.05 µl of distilled water. The PCR profile used has initial denaturation temperature of 94ºC for 3 mins, followed by 30 cycles of 94ºC for 60 seconds, 56ºC for 60 seconds 72ºC for 120 seconds and the final extension temperature of 72ºC for 5 minutes and the 10ºC hold forever.
The PCR product was further purified before the sequencing using 2M Sodium Acetate washing techniques. The pellet was resuspended in 5 µl sterile distilled water. The PCR mix used includes 0.5 µl of BigDye Terminator Mix, 1µl of 5X sequencing buffer, 1 µl of 16S Forward primer with 6.5 µl Distilled water and 1 µl of the PCR product making a total of 10 µl. The PCR profile for Sequencing is a Rapid profile, the initial Rapid thermal ramp to 96ºC for 1 min followed by 25 cycles of Rapid thermal ramp to 96ºC for 10 seconds Rapid thermal ramp to 50ºC for 5 seconds and Rapid thermal ramp to 60ºC for 4 minutes, then followed by Rapid thermal ramp to 4ºC and hold forever. The PCR sequence product was purified before the sequencing using 2M Sodium Acetate washing techniques. The pellet was re-suspended in 5 µl sterile distilled water [9]. The combination of 9µl of Hi di Formamide with 1 µl of Purified sequence making a total of 10 µl was prepared and loaded on Applied Biosystem (AB1 3130xl model).

Haemolysis Determination
Antibiotic susceptibilities of the isolated bacteria were analysed by using the Kirby-Bauer disc diffusion method. An overnight culture was standardized to a turbidity equivalent to 0.5 McFarland standards (1.5×10 8 cfu/ml) with sterile 1% peptone water. The standardized overnight broth culture was spread on Mueller-Hinton agar plates using sterile swabs. Antibioticimpregnated discs (Abtek, U.K) were placed on seeded plates and were incubated at 37ºC for 24 hours. Zones of inhibition were measured after 24 hours of incubation [10]. The strains were classified as 'resistant (R)', 'intermediate sensitive (I)' or 'sensitive (S)' using standard recommendations of Clinical and Laboratory Standards Institute.

Nucleotide Sequence and Statistical Analysis
Corrected sequences were aligned to 16S rRNA gene sequences in the Genbank DNA database and the homology of the sequences were analyzed using Basic Local Alignment Search Tool (BLAST) program of the National Centre for Biotechnology Information (NCBI) in order to determine the bacterial identities and the statistical analysis to determine the phylogenetic tree was done using Darwin software while descriptive statistics was used to analyze data on morphometric and water parameter.

Water Parameters and Morphometric Characteristics of the Fish
There is a range of biological, physical and chemical components that affect the quality of water. These variables provide general indication of water pollution, whereas others enable a direct tracking of the sources of pollution [11]. The Physico-chemical parameters recorded at the study site are temperature, pH and Dissolved oxygen. The temperature value range between 28.7-29.4ºC, this value recorded was similar to [12] who recorded temperature value range of 26.8º-27ºC, this result is within WHO standard. The pH recorded was 6.6 which was within the WHO standard, The Dissolved Oxygen value was 3.15 mg/l, this value is low and it is below the WHO standard limit of 5 mg/l for sustenance of aquatic life, any value below the standard will affect aquatic biological life adversely, a concentration below 2 mg/l may eventually lead to death of most fishes [12].

Morphology of Bacteria
The morphology of the isolated bacteria is presented in Table 3. Enterobacter spp is a gram negative, round-shaped, motile organism, Pseudomonas fluorescens is a gram negative, round-shaped, motile organism while Streptococcus spp is a gram positive, roundshaped, non-motile organism. The total bacterial count in the guts, gills and skin of advanced C. gariepinus juveniles showed that gut has the highest bacteria count (4.72×10 4 ), followed by the skin with 3.5×10 4 while the gill recorded the least count of 2.53×10 4 .

Molecular Characterization by Gene Sequencing
The size of the amplified band with 16S universal primer was 1600bp for the 26 samples, this is similar to the work of [9]. The DNA target for amplification using PCR technique was 16S ribosomal RNA, this is because 16S rRNA has its presence in all bacteria [13], often existing as a multi gene family, or operons and the function of the 16S rRNA gene over time has not changed, suggesting that random sequence changes are a more accurate measure of time (evolution); and the 16S rRNA gene (1,500bp) is large enough for informatics purposes.     domains are bacterial toxins that tend to function by assembling identical subunits in a membranespanning pore [14]. Out of the 17 bacteria strains identified, 10 (58.82%) exhibited β-haemolysis; only Alcaligenes faecalis strain displayed partial haemolysis while 6 (35.29%) were nonhaemolytic in blood agar. In addition, the concentration of the blood in the agar influence the observed haemolysis; high concentrations may cause β-haemolytic organisms appear as non-haemolytic organisms while low concentrations may make αor β-haemolysis difficult to determine.

CONCLUSION
This study showed that the isolated bacteria from the gills, gut and skin of Clarias gariepinus post juveniles from Ajilete location on Yewa River included genera strains of