Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1): 7-12 Research article DOI: https://doi.org/10.54796/njb.v10i1.225 ©NJB, BSN 7 Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley Shristi Prasai, Puja Shrestha, Sriniwas Pandey, Ishika Adhikari, Srijana Gurung, Kamil Prajapati St. Xavier’s College, Maitighar, Kathmandu, Nepal Received: 14 May 2022; Revised: 8 Jul 2022; Accepted: 15 Jul 2022; Published online: 30 Jul 2022 Abstract Microbial quality of Labeo rohita, Cyprinus carpio and Clarias batrachus collected from the markets of Kathmandu valley was evaluated. 9 freshwater fish (skin, gills, intestine) were sampled and were analyzed for Total Plate Count (TPC), Total Coliform Count (TCC) and Total Fecal Coliform Count (TFCC). The average TPC ranged from 4.1 x 107 to 1.02 x 108 cfu/gm, with the highest count in C. batrachus and the lowest in C. carpio, whereas the organ wise load was the highest in intestine with 1.3 x 108 cfu/gm and the lowest in skin with 1.02 x 107 cfu/gm. The highest TCC and TFCC was found in C. carpio and C. batrachus respectively, whereas organ wise distribution showed the highest count in intestine for both TCC and TFCC. The pathogens isolated from the samples were Escherichia coli, Staphylococcus aureus, Coagulase negative Staphylococcus (CoNS), Vibrio cholerae, Salmonella Typhi and S. Paratyphi. E. coli was isolated from 67% of L. rohita, 44.44% of C. carpio and 66.67% of C. batrachus. S. aureus was isolated from 44.44% of both L. rohita and C. batrachus whereas 55.55% of C. carpio. CoNS were isolated from 33.33% of L. rohita, 22.22% of C. carpio and 33.33% of C. batrachus. S. Typhi was isolated from 11.11% of C. carpio and 22.22% of C. batrachus. S. Paratyphi was isolated from 11.11% of both L. rohita and C. batrachus, V. cholerae was isolated from 11.11% of L. rohita, 33.33% of C. carpio and 22.22% of C. batrachus. The observation of this study showed higher bacterial load in all of the fishes above the acceptance level and presence of Total Coliform, Fecal Coliform and potential human pathogens suggests that the microbial quality of the fish available in the market is not satisfactory. Hence, the fishes possess a threat to public health safety and there is an urgent need to improve the Quality Control and Quality Assurance Systems for fish markets of Kathmandu valley. Keywords: Raw fish, microbial quality, E. coli, S. aureus, Vibrio cholerae. Corresponding author, email: kalkam2013@gmail.com Introduction Fish is one of the chief sources of protein and has remained an important part of consumption for many centuries[1]. The poikilothermic nature of fresh fish allows a wide variety of bacteria such as Pseudomonas, Moraxella, Acinetobacter, Shewanella, Flavobacterium, and Vibrio among Gram negative and Gram-positive bacteria such as Bacillus, Micrococcus, Clostridium, Lactobacillus, and Corynebacterium [2]. The non-indigenous ones that contaminate the fish or the habitat include Escherichia coli, Clostridium botulinum, Aeromonas, Shigella dysenteriae, Staphylococcus aureus, Listeria monocytogens and Salmonella spp. The indigenous bacterial pathogens that are found naturally in the fish habitat are Vibrio spp. and Aeromonas spp. [3]. Microbiological quality of raw fish results from microbiological load of aquatic habitat, methods of capture, transportation, chilling and storage conditions imposing a threat of food-borne infections as the pathogen can be conveyed to consumers at retail level through raw fish. Coliform, especially Escherichia coli are often used as criteria to assess the quality and safety of foods [1]. Extraneous bacteria, Escherichia coli is the fecal indicator capable of surviving in fish and found to be surviving and even multiplying in the digestive tract of rainbow trout (Oncorhynchus mykiss) [4]. Water being the habitat, fish is continually bathed in aqueous suspension of various microorganisms and their exterior surface, hence is in constant contact with these organisms. Some of the microorganisms may colonize the external parts of fish becoming the resident microflora. The presence of the microflora adds to the defense system of fish, thereby inhibiting the accession and consequent colonization by other potential pathogens. Obviously, the bacterial flora of fish depends on the fish’s recent intake diet and the extent of contamination in the food [5]. A study performed on fish skin sampled from the lake Hawassa of Southern Ethiopia by [6], resulted in finding of the pathogenic strains of E. coli to be contaminating the fish with statistically significant results as defense to the fact that fish are contaminated enough to cause food- Nepal Journal of Biotechnology Publisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 Journal Homepage: https://nepjb.com/index.php/NJB ISSN (Print): 2091-1130 https://orcid.org/0000-0002-9739-4848 mailto:kalkam2013@gmail.com Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1): 7-12 Prasai et al. ©NJB, BSN 8 borne illness. According to Sichewo et al. (2014) [7], inspection of various organs of fish such as skin, intestine, gills and mouth collected from different fish ponds of Zimbabwe showed presence of Salmonella Typhi from Nhengo, Imbayago and Nyamakwe. According to Kumari et al. (2001) [8], gill and intestine samples of Labeo rohita when processed revealed to be contaminated with coagulase negative Staphylococcus spp. and Staphylococcus aureus. According to Xu et al. (2019) [9], V. cholerae was detected from the intestines of ten freshwater fish species collected, including Astatotilapia flaviijosephi, Barbus longiceps, C. idella, Cyprinus carpio, Mugil cephalus, Myripristis murdjan, Oreochromis aureus, Sarotherodon galilaeus, and Tilapia spp. and Tilapia zilli. Improper handling or consumption of undercooked or raw fish may contribute to the intake of pathogens which can potentially cause diseases. Diseases in human that can be caused by bacteria present in fish includes food poisoning and gastroenteritis, diarrhoea, superficial wound infections and ulcers, bacillary dysentery (shigellosis), clonorchiasis, dracunculiasis and paragonimiasis due to larvae and metacercariae ingested in fish and crustaceans, cholera, typhoid and paratyphoid, etc. [4]. Salmonella, Staphylococcus spp., Escherichia spp., Vibrio parahaemolyticus, Clostridium perfringens, Clostridium botulinum E and Enteroviruses are held responsible for majority of fish concerned with food borne diseases [2]. The safety concerns instigated the need for the study i.e. to investigate the presence of any human pathogenic bacteria from live freshwater fishes that are popularly consumed in Kathmandu valley. Materials and methods Collection of fish sample Simple random sampling method was used for selecting samples. The freshly slaughtered 9 fish samples were collected on different day from different retail markets of Kathmandu valley (Sundhara, Baneshwor, Thapagaun, Gairigaun, Ghatthaghar) and brought to the laboratory in an icebox. Fishes were killed by the retailers without causing any physical injury just before the sample collection. The three species of fish, L. rohita (rohu), C. carpio (common carp) and C. batrachus (mungri) were chosen based on popularity among the consumers and commercial availability. Sample preparation and processing Sample preparation was done according to Sichewo et al. (2014) [7]. Fish was cut ventrally to collect intestine and gills using sterile surgical blades and forceps in aseptic condition. One g of intestine and gills were taken and crushed into fine solution by adding 10ml of normal saline in sterile mortar, from where one ml of aliquot volume was taken and serially diluted up to 10-5. Skin samples were taken by rolling sterile cotton swabs all over the skin surfaces of all 9 fish and then inoculated into 10ml of normal saline. It was then serially diluted up to a dilution of 10-5. Bacteriological analysis of fish samples For total plate count (TPC), 0.1ml sample from 10-3 and 10-5 dilutions were taken and spread plating was done on PCA agar. The plates were incubated for 24 hours at 37°C. For total coliform count (TCC) and total fecal coliform count (TFCC), spread-plating was done from 0.1 ml of every sample of 10-3 dilutions in VRBA agar plates and incubated at 37°C and 44.5°C, respectively for 24 hours. For isolation of Salmonella spp. one ml of sample was inoculated in 9 ml of enrichment media, Selenite F Broth, from where a loopful of sample was taken and cultured on SS Agar. For Vibrio cholerae, one ml of sample was inoculated in 9ml of enrichment media, alkaline peptone water, incubated at 37°C for 24 hours. A loopful of sample was taken from enrichment broth and cultured on TCBS Agar. A loopful of original sample was streaked on MacConkey Agar (for E. coli) and on Mannitol Salt Agar plate (for Staphylococcus aureus) and the plates were incubated at 37°C for 24 hours. The colonies obtained from TCBS, SS, MA and MSA were further sub-cultured on NA [10] and identified by gram staining and biochemical tests (IMViC, TSIA, Urease, Catalase, Oxidase, Oxidative/fermentative test). For Vibrio cholerae string test was also performed while Staphylococcus aureus was confirmed by coagulase test. Results Table 1. Average TPC of fish samples Sample Organs (cfu/gm in average) Skin Gills Intestine Average LR 6.5 x 106 8.2 x 107 8.05 x 107 5.6 x 107 CC 1.23 x 107 2.96 x 107 8.03 x 107 4.1 x 107 CB 1.2 x 107 6.6 x 107 2.3 x 108 1.02 x 108 Average 1.02 x 107 5.9 x 107 1.3 x 108 Note: LR = Labeo rohita, CC = Cyprinus carpio and CB = Clarias batrachus In this study, 9 fish sample of 3 different varieties, L. rohita (LR), C. carpio (CC) and C. batrachus (CB) were analyzed for its microbial quality and antimicrobial susceptibility testing was done for the isolated strain. The average TPC of LR, CC and CB ranged from 4.1 x 107 to 1.02 x 108 cfu/gm (Table 1). The highest bacterial load was found in C. batrachus whereas the lowest in C. carpio. Among the Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1): 7-12 Prasai et al. ©NJB, BSN 9 body parts, intestine was found to be highly loaded with the bacteria in all the fishes. Total coliforms were present in all parts of the three species of fishes. TCC ranged from 2.47 x 105 to 8.7 x 105 cfu/gm while TFCC was found to be 1.6 x 105 to 3.07 x 105 cfu/gm (Table 2). Fecal coliforms were present in all parts of all the analyzed fishes except for skin of L. rohita, and skin and gills of C. carpio. All parts of C. batrachus showed presence of fecal coliforms. Six different organisms were isolated from gills, skin and intestine of the three fish samples (Table 3). In case of L. rohita, gills were found to harbor the diverse species of bacterial pathogens compared to skin and intestines. Gills were found to harbor S. aureus (66.67%), V. cholerae (33.33%), E. coli (100%) and CoNS (33.33%). E. coli, S. aureus and CoNS each were present in 66.67% of skin sample. E. coli was found in 100% and S. Paratyphi in 33.33% of intestine. S. Typhi was not isolated from any of the three body parts. As for C. carpio, S. aureus was present in 100% of gills, 66.67% of skin. V. cholerae was found in 66.67% of gills and 33.33% of intestine. E. coli was found in 66.67% of gills and 33.33% of both skin and intestine. CoNS were present in 33.33% of both skin and gills. S. Typhi was present in only 33.33% of intestine whereas S. Paratyphi was not isolated from any of the three body parts. In case of C. batrachus, gills showed the prevalence of most of the type of bacteria. E. coli was present in 100% of gills, 66.67% of intestines and 33.33% of skin. S. aurues was in 100% of skin and 33.33% of gills. CoNS was present in 66.67% of skin and 33.33% of gills. S. Typhi was present in 33.33% of gills and intestines each. Discussions This study was carried out in the quest to determine the microbial quality of raw and freshly killed freshwater fishes available in retail markets of Kathmandu valley. The skin, gills, and intestine of the samples were analyzed for TPC, TCC and TFCC. The average total plate count of L. rohita, C. carpio and C. batrachus was found to be 5.6 x 106, 4.1 x 107, 1.02 x 108, respectively indicating that latter had higher microbial load than the other two (Table 1). As for organ-wise distribution, skin of L. rohita contained least microbial load compared to gills and intestine where as in C. batrachus intestine had the highest microbial load. However, in C. carpio, all three organs had similar Table 2. Average TCC and TFCC of fish samples Sample Total Coliform Count (cfu/gm) Total Fecal Coliform Count (cfu/gm) Skin Gills Intestine Average Skin Gills Intestine Average LR 1.5 x 105 3.5 x 105 2.4 x 105 2.47 x 105 No growth 9.4 x 106 2.6 x 105 1.77 x 105 CC 1.2 x 106 2.1 x 105 1.2 x 106 8.7 x 105 No growth No growth 1.6 x 105 1.6 x 105 CB 7.6 x 105 2 x 105 TMTC 4.8 x 105 1.7 x 105 2.9 x 105 4.6 x 105 3.07 x 105 Average 7.03 x 105 2.53 x 105 7.2 x 105 1.7 x 105 1.92 x 105 2.93 x 105 Note: LR = Labeo rohita, CC = Cyprinus carpio, CB = Clarias batrachus, TMTC = Too many to count Table 3. Distribution of bacteria in skin, gills and intestine of fish samples. Sample Organ (N=3) Isolates S. aureus S. Paratyphi S. Typhi V. cholerae E. coli CoNS LR Skin 2(66.67%) ND ND ND ND 2 (66.67%) Gills 2 (66.67%) ND ND 1 (33.33%) 3 (100%) 1 (33.33%) Intestine ND 1 (33.33%) ND ND 3 (100%) ND CC Skin 2(66.67%) ND ND ND 1 (33.33%) 1 (33.33%) Gills 3 (100%) ND ND 2 (66.67%) 2 (66.67%) 1 (33.33%) Intestine ND 1 (33.33%) ND 1 (33.33%) 1 (33.33%) ND CB Skin 3 (100%) ND ND ND 1 (33.33%) 2 (66.67%) Gills 1 (33.33%) 1 (33.33%) ND 2 (66.67%) 3 (100%) 1 (33.33%) Intestine ND 1 (33.33%) 1 (33.33%) ND 2 (66.67%) ND % = calculated according to total number of fish samples (3 each) ND= Not detected Note: LR = Labeo rohita, CC = Cyprinus carpio, CB = Clarias batrachus, TMTC = Too many to count Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1): 7-12 Prasai et al. ©NJB, BSN 10 microbial load. High load of bacteria possibly resulted from keeping fishes in wells with contaminated water. Total bacterial count of more than 105 cfu per gram elevates the concern of hygiene. According to ICMSF (2011), for a newly caught fish, aerobic count may range from 104 to 107 cfu per cm2. Much lower count is associated with properly skinned ones [11]. Detectable spoilage is usually associated with spoilage bacteria exceeding 107 cfu per gram. Goja (2013) did similar study in three freshwater fish and found that viable bacterial counts in intestine and skin ranged from 1.5 x 103 to 8.4 x 104 cfu per gram and 2.8 x 103 to 9.8 x 103 cfu per gram respectively which is much lower than in the three fish samples in this study[12]. Comparable research conducted on 150 fish samples from Nile bream by Sichewo et al. (2014) revealed the TPC of intestine, skin, gills ranging from 4.6 x 103 to 8.03 x 103 cfu per gram [7]. Every organ of the fish samples was found to contain total coliform (Table 2). Similarly, presence of fecal coliforms was also true for all samples analyzed except skin of L. rohita and skin and gills of C. carpio. All samples contained fecal coliform in at least one of the body part whereas the coliform count was the highest in the intestines. According to Liu et al. (2016), gut microbiota and their diversity is influenced by many independent factors as different niches have variation in diet availability [13]. In addition, gut microbiota is also influenced by metabolic capacity and gut content enzyme activity. Coliform or fecal coliforms are not considered to be normal flora of fish which reflects contamination of fish during transportation, handling or during rearing in water contaminated with human or animal waste [3]. Four of the fish samples had a very high count of coliform in their intestines (TMTC). The feed and trophic level (carnivores, omnivores and herbivores) might have some association with the intestinal microbiota. The salinities of water in fish habitat also have some influence on the microbiota of fish intestine [14]. Prevalence of microorganisms varied among the three fish samples analyzed with E. coli being most common isolate followed by S. aureus and CoNS (Figure 1). Prevalence of V. cholerae was higher among C. carpio, while S. Typhi is absent in L. rohita and S. Paratyphi was absent in C. carpio. E. coli was present in the gills of L. rohita (100%), C. carpio (66.67%) and C. batrachus (100%) respectively (Table 3). Similar study conducted by Yogoub (2009) revealed that E. coli was the most dominant isolate from fishes [2]. Enterobacteriaceae genera were isolated from gills, skin, intestine and muscles of 83 out of 150 randomly collected fishes which also included pathogenic Salmonella and Shigella spp. During rainy season due to rain surface runoff of organic matters into water bodies are increased which favors multiplication of bacteria. Previous study performed by Shabeeb et al. (2016) revealed that most isolates were Gram negative rods [15]. Figure 1: Distribution of pathogenic bacteria in L. rohita, C. carpio and C. batrachus %= Calculated according to the total number of samples (9 each) Salmonella being enteric bacteria, their presence in freshwater fish undoubtedly attributes to fecal contamination of such source from where it is harvested. Ponds with higher temperature or from hot regions harbors more Salmonella with high prevalence rates. According to Bibi et al. (2015) there has been occurrence of Salmonella Typhi in freshwater fish like Labeo rohita (skin, gills, intestine) and Cyprinus carpio (intestine)[16]. In our study, S. Typhi was isolated from one sample each of gills and intestine of C. batrachus and a sample of intestine of C. carpio while none of the sample from L. rohita showed the presence of the bacterium (Table 3). As for S. Paratyphi, it was not isolated from any of the tested samples of C. carpio but isolated from one sample of intestine of both L. rohita and C. batrachus. It was noted that none of the skin samples of all the three fish tested showed the presence of Salmonella spp. However, these isolates were not confirmed through serological tests. All 3 gills samples of the C. carpio contained Staphylococcus aureus (Table 3). Intestines of L. rohita and C. batrachus were not found to contain the pathogen. Staphylococcus is related with unhygienic handling as these are inhabitant of human skin [17]. Presence of such opportunistic human pathogens advocates the possibility of cross contamination between handlers and fish. A study conducted in Andhra Pradesh, India by Bujjamma and Padmavathi (2015) in 192 fish samples disclosed that over 24.47% of fish were contaminated with S. aureus which also included similar samples [18]. Similarly, sampled skins were mostly found to harbor these 0 10 20 30 40 50 60 70 S. aureus S. Paratyphi S. Typhi V. cholerae E. coli CoNS P er ce n ta g e (% ) Labeo rohita Cyprinus carpio Clarias batrachus Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1): 7-12 Prasai et al. ©NJB, BSN 11 pathogens. However, all intestine samples were negative for CoNS. Skin was observed as the most contaminated area with CoNS and Staphylococcus aureus. Composition and count of microflora in fish is a function of water quality, age, type of species and fish [19] along with rearing density and diet impact [20]. In relation to this, hazard escalates when livestock manure is fed to fish which is also the case in Nepal. As an established fact, chicken has been remarkably associated with a high load of Salmonella. In this study, V. cholerae was isolated from gills of all three types of fish and from intestines of C. carpio (Table 3). Samples were expected to yield positive results for V. cholerae as this bacterium is the indigenous member of fish associated aquatic habitats. However, their distribution was outnumbered by that of E coli and S. aureus. Gills bear a high load of bacteria as a consequence of water and organic substance precipitation[19, 21]. A study conducted in Bangladesh on Hilsha, a freshwater fish by Hossain et al. (2018) revealed that out of 48 fishes, 39 fishes tested positive for V. cholerae in specific OmpW gene assay [22]. Gills tested positive in highest number (79%) on market fish whereas the same happened for scale swab in case of fresh fishes which is similar to our findings as most gills tested positive for the pathogen. Furthermore, PCR results in this study, revealed (66.7%) higher prevalence of Vibrio cholerae in fish purchased from local markets than those collected from river banks. E. coli being highly prevalent in all intestinal samples, lower yield of V. cholerae can probably be attributed to glucose metabolism of E. coli yielding acid products which can potentially reduce survival chances of V. cholerae as evidenced by in-vitro studies [23]. Halpern and Izhaki (2017) emphasized the mutualistic relation between V. cholerae and fish with the evidence of the former helping fish to properly digest chitinous prey like zooplanktons [21]. However, fish serve as an important vehicle for V. cholerae and hence is the key to the dissemination process for epidemics. Vibrio parahemolyticus has been sporadically associated with freshwater fish and is seasonal. None of the freshwater fish contained V. parahemolyticus [22]. These findings and observations seem alarming and demand assessing of the freshwater fish and other aquatic products that are kept for sale in the markets. High load of bacteria in the samples including the presence of total coliforms, fecal coliforms and pathogenic bacteria can be attributed to the type of water used in the shops for keeping the fishes. The bacterial load in the fish can probably be the function of frequency of change of water. Besides, indigenous bacteria on surface as well as normal flora of other sites, rapidly multiply and invade the previously sterile part after death of fish and hence, spoilage is a consequence if proper keeping conditions are not maintained after harvesting or killing (after being bought). In addition to that, fish handlers at different stages of the supply chain are at greater risk apart from consumers eating the fish. Bacteria can pave a way into wounds, cracks or lacerated regions of skin of fish handlers and might potentially cause various infections. Moreover, the bad hygiene practice of the handler can potentially cause fish borne infection. Conventional cooking systems might reduce the bacterial load to acceptable or possibly kill pathogens. However, the risks remain high when consumers prefer it raw or smoked. In addition, undercooking of such contaminated fish can pose dangers to public health. Conclusion Nine (3 L. rohita, 3 C. batrachus, 3 C. carpio) fish samples were taken from retailers of Kathmandu which were processed for assessing their microbial quality and were assayed for presence of potential pathogenic bacteria. The samples were found to contain a high load of bacteria along with coliforms. The samples were also found to contain potential pathogens like S. aureus, S. Typhi, S. Paratyphi, V. cholerae, E. coli and CoNS with the most prevalent being E. coli. These findings suggested the poor microbial quality of freshwater raw fishes that are being sold in the market. C. batrachus was found to be the most contaminated fish. The research indicated that there is an immediate need for quality assessment of fish and such aquatic products on a large scale. To conclude, the fish whether alive, dead, dried or frozen should be consistently monitored for bacterial load and presence of pathogenic forms. Author’s Contribution Project coordinator: KP Conceptualization: SP, PS, SP, IA, SG and KP Writing – Original Draft Preparation: SP, PS Writing – Review & Editing: KP All authors read and approved the final manuscript: Yes Competing Interests No competing interests were disclosed Funding No grants were involved in supporting this work Acknowledgements We would like to express our deepest gratitude to the Department of Microbiology, St Xavier’s College, Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1): 7-12 Prasai et al. ©NJB, BSN 12 Maitighar. We are also thankful to staff members for their help, support and their kind cooperation during the research period. Ethical Approval and Consent Not Applicable References 1. 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