Bacteria, one of the smallest and first life forms to appear on earth are present in almost every environment, including aquatic systems where they are abundant. Fish infected by bacteria within the water environment calls for the need to differentiate between environmental and fish pathogenic bacteria.
Liver, kidney, and spleen collected aseptically from apparently sick African catfish farmed in Delta and Ogun states, were transported on ice to the University of Ibadan for bacterial culture, isolation, and identification. DNA extraction, 16S rRNA PCR and Sanger sequencing were conducted at the International Institute of Tropical Agriculture (IITA). Enriched tissue samples in peptone water were later cultured in Nutrient Agar and Salmonella-Shigella Agar. Isolates suspected as Edwardsiella tarda and Klebsiella spp. by biochemical tests, had their DNA extracted, 16S rRNA gene amplified by PCR, and Sanger sequenced for molecular identification and classification. Nucleotide sequences were blasted on National Centre for Biotechnology Information (NCBI). Phylogenetic trees were then used to determine the genetic relatedness of E. tarda and Klebsiella spp. with other references deposited on the database.
A BlastN results from suspected Edwardsiella spp were Providencia strains, Moellerella spp., Proteus spp., Xenorhabdus spp., Enterobacillus spp, Edwardsiella spp., e.t.c, all with 90 to 99% identity while genetic relatedness of 1_907R (Ogun state) and 2_907R (Delta state) returned 100% identity to E. tarda isolates from Singapore (AB050828.1; AB050829.1), and the USA (AF015259.1). E. tarda from Ogun state had 70% similarity with TS6-1 strain from China (DQ884466.1), while Edwardsiella spp from Delta state had 72% similarity with AB050832.1 (Singapore) and AB050833.1 (Singapore) and 68% with FJ405292.1 (Korea). BlastN results from suspected Klebsiella aerogenes (3_907R and 4_907R) was Providencia, Pseudochrobactrum spp., Stenotrophomonas maltophilia, Brucellaceae, Ochrobactrum spp., while the specific blast search was returned as Klebsiella pneumonia and Klebsiella aerogene. The Klebsiella spp isolate from Ogun state (3 907R) was 58% identical to K. pneumonia from Oyo state (KOy2), and Lagos state (KL1 and KL2) and 92% similar to MK024386.1 (Saudi Arabia), MK024386.1 (India), FJ971886 (Spain). The Klebsiella spp isolate from Delta state (4_907R) had 64% similarity with K. pneumonia strains from Bangladesh (MN658387 and MN625862). The two Klebsiella spp isolates (3_907R and 4_907R) also had 64% intraspecific similarity.
The result showed the validity of culture identification and biochemical test, especially for developing countries, which still depends largely on this technique for clinical diagnoses of fish pathogens. It should be considered that disease-causing bacterial organisms of fish share the same environment with other resident and opportunistic pathogens with the possible sharing and transfer of genetic materials. Therefore, the use of NCBI blast with all the accompanying filter tools need to be engaged for accurate diagnoses of fish pathogens.
Keywords: Edwardsiella spp, Klebsiella spp., Genotypic Differentiation, Fish Diseases, Environmental pathogens