A total of 15 different Gram-negative bacteria species A. sobria, C. koseri, C. freundii, E. tarda, E. aerogenes, E. cloacae, E. coli, K. pneumoniae, P. shigelloides, P. aeruginosa, S. paratyphi, S. typhi, S. dysenteriae, S. flexneri and V. parahemolyticus were isolated from 82 (79.6%) live fish (intestine, kidney and liver), 12 (11.6%) water and 9 (8.7%) processed fish samples. The majority of the isolates isolated from the live fish were originated from the intestines. This result is in line with the findings of Sedlacek et al. (2016) and Kassa and Mitiku, (2021) which shows higher bacterial load at intestine than other organs. In this study the differences in the frequency of each bacteria isolates between groups of the different variables (tissue samples) were more pronounced due to the variation of the frequency of bacteria isolates in the kidney and liver of fish, which were sampled during the study period. The most frequently isolated bacteria from live fish tissue samples were E. coli (2.4%) in the intestine and (3.6%) in the liver. And E. aerogenes is the most frequently isolated isolate from the kidney (2.1%). The least frequently isolated bacteria from live fish tissue samples were P. shigelloides (2.9%). The isolates E. coli, P. aeruginosa, A. sobria, Citrobacter spp, Shigella spp, P. shigelloides and V. parahemolyticus were found to be having statistically significant differences (P<0.05) between tissue samples. Characteristics of the microenvironment at various locations through the alimentary tract of each fish species also influence the taxonomic composition as well as the numerical abundance of bacteria present (Gufe et al., 2019).
Among the water samples examined bacteriologically during the study period, bacteria species A. sobria, Citrobacter spp, E. tarda, E. coli, K. pneumoniae, P. aeruginosa, S. paratyphi, S. dysenteriae and S. flexneri were isolated. (Gisain et al., 2013 and Hiko et al., 2018), studied fresh water (rivers and lakes) to investigate their microbial diversity leading to the identification of different bacterial species such as Shigella spp., E. coli, Aeromonas spp., Vibrio spp., Salmonella spp and Pseudomonas spp. This is similar to the present study findings where these and other different bacterial species were isolated. The presence of these bacteria both in the aquatic environment and in the fish has been often observed elsewhere (Egerton et al., 2018; Wang et al., 2017 and Marinho-Neto et al., 2019). The least frequently isolated bacteria from both live fish and water samples were A. sobria C. koseri and C .freundii with similar prevalence.
Different Gram-negative bacteria species were isolated from processed fish samples. Salmonella spp and fecal coliforms (E. coli, K. pneumoniae and E. aerogenes) bacteria were detected in 42% of water samples and 64% of processed fish samples. This result is in agreement with several studies which reported the isolation of E. coli, Enterobacter spp., and Citrobacter spp., and Klebsiella spp., P. aeruginosa, and Salmonella from fish products (Shafi et al., 2020 and Khater et al., 2021).This was indicative of post contamination of fish product in the processing system, fecal pollution and poor quality of processed fish which is linked with the practice of inadequate hygienic measure, mishandling, improper storage and use of dirty water during processing and all unhygienic condition of the processes.
Presence of E. coli in processed fish samples is attributed to contamination of the fish samples by man through handling and processing (Gufe et al., 2019). (Shafi et al., 2020), studied coliforms such as E. coli are usually present in live and fresh fish organs which indicate the faecal contamination from warm blooded animals. This is similar to the present study findings were different coliforms including E. coli was isolated. Among the isolated bacteria, E. coli was found in all the samples and majority of it was present in live fish followed by processed fish and water, and this might be due to contamination during handling in the processing center. This result is in agreement with the study of Rani et al. (2016). Even though Salmonella spp in fish products mainly originates from the environment rather than from poor standards of hygiene and sanitation, sometimes incidence of this bacterium in fish or similar foods of aquatic habitats may be happened due to external contamination. Shigella spp. and Salmonella spp. are pathogenic bacteria found in animal or human reservoir and contamination of fish products by these bacteria is almost always due to poor hygiene (poor personal hygiene, poor processing hygiene or poor water quality) (Gufe et al., 2019). This is similar to the findings of (Mzula et al., 2019) who studied the presence of spoilage bacteria (Salmonella spp) which make the control of spoilage microorganisms critical for fish product safety. Citrobacter, Enterobacter and Klebsiella are indigenous to general environment and frequently present in fish but most of the isolates are non-pathogenic environmental strains. The bacterial species isolated from processed fish (E. coli, K. pneumoniae and S. paratyphi) were also recovered from the water samples. However, the microbial status of processed fish was distantly related to the microbial conditions of the water.
Except A.sobria all the others bacterial isolate were isolated from the tilapia (Oreochromis niloticus). Similarly all the others bacterial isolate were isolated from the catfish (Clarias gariepinus) with the exception of P. shigelloides. However, C. freundii, C, koseri and V. parahemolyticus were not isolated from carps (Cyprinus carpio). Even though differences in frequency and percentage of each particular bacteria species were found among different fish species, stastically significant differences (P<0.05) were shown in E. coli, A. sobria, Citrobacter spp, P. shigelloides, S. flexneri and V. parahemolyticus. This result is in agreement with the study of Wamala et al. (2020) and Feliatra et al. (2020) which indicate the isolation of different pathogenic bacteria; Aeromonas sobria, Edwardsiella tarda, Plesiomonas shigelloides, Pseudomonas aeruginosa, Citrobacter spp and Klebsiella spp) from tilapia (Oreochromis niloticus) and catfish (Clarias gariepinus), whereby they could be considered a reservoir or vehicle for food-borne infections and, therefore, a threat to public health.
Characterization of the bacterial isolates from diseased fish of this study revealed that majority of them are A. sobria, E. tarda, P. shigelloides, P. aeruginosa, S. paratyphi, S. typhi, S. dysenteriae, S. flexneri and V. parahemolyticus which were considered as a potential pathogenic bacteria. This result is in agreement with several studies which reported the isolation of pathogenic Gram-negative bacteria from tissue samples of different diseased fish showing clinically sign of disease and healthy (Tesfaye et al., 2018 and Wamala et al., 2020). Similarly Naim et al. (2019) also reported the presence of Gram-negative bacteria particularly E. coli, K. pneumoniae, E. aerogenes, E. cloacae, C. freundii, C. koseri, P. vulgaris, P. mirabilis, Salmonella spp., Shigella spp., Serratia spp., Pseudomonas spp., Yersinia spp., Aeromonas spp. and Vibrio spp. in raw fish sampled from Karachi, Pakistan. The presence of the microorganisms in internal fish organs could indicate the breakdown of immunological defense mechanisms (Chandrarathna et al., 2019; Hossain et al., 2019 and Mzula et al., 2019).
All the fifteen (15) bacterial isolates were isolated from lake Hawassa fish samples. Except C. freundii the others bacterial isolate were also isolated from lake Ziway fish samples. But only 66.7% of the bacterial isolates were isolated from lake Langanoo fish samples. Even though differences in frequency and percentage were recorded at any particular bacterial isolates among the three sampling lakes; statistically significant difference was only recorded in E. coli, A. sobria, Citrobacter spp, E. cloacae, P. aeruginosa and V. parahemolyticus(P<0.05). It is accepted that fish possess a specific intestinal microbiota, but nutritional status or feeding habits, species (attribute to complexity of the fish digestive system) and the environmental conditions (salinity of the habitats and the bacterial load in the water) are the most influential factor which change the fish intestinal microbiota composition (Egerton et al., 2018).
Among the isolated bacteria from lake Hawassa majority of the isolates were obtained from Amora Gedel (S1) (22.9%) and Referal hospital area (S4) sites (18.6%). In lake Langanoo most isolates were isolated from O’etu (S2) site (10%). Similarly, most of the bacteria were isolated from Cafeteria (S3) (18.8%) and Abosa (S1) (13.8%) sites of lake Ziway. During the study period differences were observed in the bacterial prevalence and frequency across the sampling sites of each lake. This may be generally attributed to the relative distance and degree of exposure to the nearby point source pollution around the study area. Most of E. coli (2.4%) was isolated during this study period from the intestine of fish. The occurrence of faecal coliforms in fish intestine reflects the warm-blooded animal pollution level of the water and also indicates that the organism can probably survive and multiply when fish and water temperatures are 280C or higher (Gufe et al., 2019).
The physicochemical parameters of water samples indicate that mean values of pH, temperature, dissolved oxygen and nitrate in all sampling sites of the three lakes found to be within the normal range at which most freshwater fish species become non-stressed. The concentrations of nitrate in each lake were within the limit of WHO a standard showing that the selected sites of each lake was less polluted by nitrogenous waste materials. However, highest concentration of phosphate (4.48 mg/L) was recorded at lake Langanoo O’etu site (S2) which were higher than the limit of WHO standards (WHO, 2008). These could be due to pollution from different domestic sewages, surface runoff from phosphate containing fertilizers and certain industrial wastes that led to eutrophication and can lead to low productivity of the lake. Unlike pH and temperature of lake Ziway, temperature and EC of lake Hawassa and lake Langanoo indicates to much differences between water samples during the study period. The mean dissolved oxygen were high indicating that there was a good aeration at each lake during the study period, which may be attributed to lower temperature and good flow rate (Garg et al., 2010). Studied temperature oscillated between 19 and 28.7°C among sampling sites of each lake. The effect of stress on fish depends on the severity of the stress, its duration and the physiological state of the fish.