Occurrence and Characterization of Enterococcus Faecalis from Infected Farmed African Catfish in Ogun State, Nigeria

Background

In the past century, there has been a significant rise in demand for fisheries products, which has led to the rapid growth of the aquaculture industry [1]. Clarias gariepinus, the African catfish, is perfect for closed land-based aquaculture systems. The rearing of fish started in the early 1970s in central and western African countries. The species became widely accepted after it was found to be ideal for aquaculture and of high economic value. Since then, it has become the most widely raised fish in Nigeria and Africa [2]. The increased farming of African catfish is encouraged by capability to withstand high stocking density; ability to adapt to tropical environments; suitability for monoculture and polyculture with other freshwater fish species; ability to withstand handling stress; high fecundity; disease resistance; high weight gain; nutritional quality and palatability; and low production cost [3, 4].

Aquaculture encounters significant production losses for several reasons. The disease is the most significant obstacle among these reasons, causing harm to farmers’ livelihoods, reducing their income, and resulting in unemployment and food insecurity [5]. Diseases in stressed warm-water aquaculture are often caused by bacterial agents [6]. In recent years, severe outbreaks in aquaculture facilities have been linked to opportunistic bacterial fish pathogens. Enterococcus sp. has emerged as one of the most critical fish pathogens affecting aquaculture practices globally [7]. Enterococcus sp. may result in streptococcosis, meanwhile, streptococcosis outbreaks are usually triggered by stress factors, such as high temperatures, harvesting, improper handling, transportation, and poor water quality [8].

Fish diseases caused by Enterococcus sp were first reported in yellow-tail fish (Seriola quinqueradiata) in Japan [9] and then in Turbot (Scophthalmus maximus) [10] and tilapia [11]. Also, Enterococcus faecalis has been isolated from African catfish in Egypt [8]. However, E. faecalis has not been reported in infected African catfish in Nigeria. Hence, the present study aimed to determine the occurrence, isolate and characterize Enterococcus faecalis in infected farmed African catfish from Ogun State, Nigeria.

Study Area

The study area was Ogun state, which is a state in South Western Nigeria. It is situated in the tropical rainforest area, with latitudes 6.2°N to 7.8°N and longitudes 3.0° E to 5.0°E. The investigation area is 2200 square kilometers and the north side is confined by Oyo State, the west side by Benin Republic, the south by Lagos State, and the west by Ondo State [12]. It has a population of 3,751,140 and contains twenty local governments. Ogun state is a major catfish- producing state in the southwestern zone, with average yearly temperature and rainfall of 28°C and 1,270 mm .

Samples Collection

Infected African catfish (Clarias gariepinus) (n=128) with clinical signs of atrophied or reduced barbels, desquamation, or abrasion of the skin were purposively sampled during disease outbreaks from sixteen commercial farms in Ogun State. The samples were packaged, labeled, and transported on a sterile icepack at 40C to Aquatic Animal Medicine and Wildlife Laboratory (AAWL), Department of Veterinary Medicine, University of Ibadan for sampling. The samples were collected between October 2021 and June 2022. A total of 128 diseased catfish samples were collected from one Local Government Area in the three Senatorial Districts of the State as described in Table 1. The samples were collected from infected C. gariepinus fry to adults in various farms using earthen, concrete, or plastic ponds with stocking densities of 1,000 – 10,000 respectively. The farms were not on administration of antibiotics before and during sample collections.

Bacterial Isolation and Phenotypic Characterization

The skin, fin, liver, kidney lesions were swabbed with a sterile cotton swab, submerged in buffered peptone water;

and the stick swab was sealed in the tube. The swab was marked, labeled, and incubated for 24 hours at 300C, then the sample incubated in buffered peptone water was cultured on Tryptic Soy and MacConkey agar (HiMEDIA®, USA) at 30oC for 24 hrs. The dominant colonies were subcultured on Tryptic Soy agar (TSA) to obtain a pure culture. Suspected colonies were confirmed to be Enterococcus faecalis with the biochemical tests. The isolates were identified based on morphological and biochemical tests such as oxidase, catalase, motility, Gram staining, Simon citrate, Indole, Glucose (gas), Hydrogen sulfide production, Lysine, Ornithine and Urease, Methyl red, Vogues Proskauer, Triple sugar Iron agar slant culture as well as sugar fermentation test [13].

Genotypic Characterization

24-hour pure culture of Enterococcus faecalis was pelleted at 6000rpm for 10 mins and 1ml of DNA Extraction Buffer (DEB) containing proteinase K (0.05mg/ml) was added. The mixture was macerated in a sterile mortal and the extract was transferred into a 1.5ml Eppendorf tube.50µl of 20% Sodium Dodecyl Sulphate (SDS) was added and incubated in a water bath at 65°C for 30 minutes.100µl of 7.5M Potassium Acetate was added to each isolate and mixed briefly. The mixture was centrifuged at 13000rpm for 10 minutes and the supernatant was transferred into new fresh autoclaved tubes, 2/3 volumes of cold Isopropanol were added to each tube, inverted gently, incubated at -20°C for 1 hour, and centrifuged at 13, 000 rpm for 10 minutes. The supernatant was decanted and subsequently washed in 300μl of 70% ethanol. They were centrifuged again at 13, 000 rpm for 5 minutes and then incubated for 15 minutes at 37°C. The ethanol was decanted off and nucleic acids were air-dried at room temperature. The pellets were suspended in 30μl of sterile distilled water and stored at 4°C as stock solution. Forward primer 27F (5′ GGATTAGATACCTGGTAGTCC-3′ ′) and reverse primer 1495R (5′-TCGTTGCGGGACTTAACCCAAC -3′) were used to amplify 16S rRNA gene. The PCR reaction mixture (iTRON, Korea) was as follows: 2.5-unit Taq DNA polymerase, 2.5 mMmm dNTP, 1x reaction Buffer (10x), 1x Gel loading buffer, 5µl of template DNA, 1µL of primer (27F:10pmol/µ), 1µl of primer (1495R10 pmol/µl) and 15µl of distilled water mixed in a final volume of 25µl. The PCR conditions were; 94ºC for 5 minutes, 37 cycles of 94ºC for 1 minute, 58ºC for 1 minute, 72ºC for 1 minute, and final extension at 72ºC for 10 minutes. Amplification was done using a thermocycler system (BIO-RAD, USA). The PCR products were assessed by gel electrophoresis. One percent agarose gel with 0.5% ethidium bromide was mixed and 5μl of PCR product and ladder (iTRON, Korea) were transferred into separated wells in the gel. The electric current was allowed at 100 volts for 30 minutes, while UV Trans- illuminator (UVP, USA) was used for the observation of DNA bands [14]. The gene segments amplified from the isolates were sequenced in both directions from purified products after ethanol purification.

Bioinformatic Analysis

The 16S rRNA gene sequence was analyzed using BLAST [15] with a non-redundant NCBI GenBank database to find closely related bacterial 16S rRNA gene sequences. The 16S rRNA gene sequence of Enterococcus faecalis isolate was submitted to the NCBI database with accession number (OP595802.1). Ten high-quality 16S rRNA gene sequences were selected from the NCBI GenBank database and subjected to multiple sequence alignment using the Clustal W program [16] within BioEdit software [17]. The phylogenetic tree was constructed according to the maximum likelihood method using MEGA 11 software.

Statistical Analysis

The occurrence of Enterococcus faecalis isolates in infected Clarias gariepinus from selected local governments were calculated in percentage and the analysis of variance (ANOVA) was used to determine the level of statistical significance, a ‘less than 0.05 (p < 0.05) was considered as significant.