43672 Megha P U and Harikumar P S / Elixir Bio Sci. 100 (2016) 43672-43677 Available online at www.elixirpublishers.com (Elixir International Journal) Bio Sciences Elixir Bio Sci. 100 (2016) 43672-43677 Isolation and Identification of Pathogenic Bacteria in Edible Fish: A Case Study of Mogral River, Kasargod, Kerala, India Megha P U and Harikumar P S Water Quality Division, Centre for Water Resources Development and Management, Kozhikode, India. ARTICLE INFO ABSTRACT Article history: Water is one of the most valued natural resource and hence the management of its quality Received: 26 September 2016; is of special importance. In this study, an attempt was made to compare the aquatic Received in revised form: ecosystem pollution with particular reference to the upstream and downstream quality of 9 November 2016; river water. Water samples were collected from Mogral River and analysed for physico- Accepted: 18 November 2016; chemical and bacteriological parameters. Healthy fish samples from the river basin were subjected to bacteriological studies. The direct bacterial examination of the histological Keywords sections of the fish organ samples were also carried out. Further, the bacterial isolates Mogral River, were taxonomically identified with the aid of MALDI-TOF MS. The physico-chemical Water quality, parameters monitored exceeded the recommended level for surface water quality in the Fish samples, downstream segment. Results of bacteriological analysis revealed high level of faecal Bacterial isolates, pollution of the river. The isolation of enteric bacteria in fish species in the river also MALDI-TOF MS. served as an indication of faecal contamination of the water body. Comparatively, higher bacterial density was found in the liver samples of the fish collected from the downstream, than in other organs of the fish collected from the upstream segment. Taxonomical identification revealed the presence of eight pathogenic bacterial strains from the fish samples, all of which represents a potential hazard to humans. The mean bacterial load of the isolates was found to be markedly higher than the recommended public health standard value adopted by the standard prescribed by World Health Organisation (WHO). © 2016 Elixir All rights reserved. Introduction The infections of various pathogens can significantly Rivers are the most important freshwater resource for affect the overall behaviour, metabolism, body condition, man. Unfortunately, river waters are being polluted by fecundity and survival of fish and is a critical concern in areas indiscriminate disposal of sewage, industrial waste and that lead to human contact or food consumption. plethora of human activities, which affects their physico- Most bacterial species cause different diseases in fish and chemical characteristics and microbiological quality [1]. some cause diseases in humans. Human diseases that can be Pollution of the aquatic environment due to the heavy release caused by bacteria in fish include: - food poisoning and of industrial, agricultural and commercial chemical discharges gastroenteritis, diarrhoea [7], superficial wound infections and have led to various deleterious effects on aquatic organisms. ulcers [8], bacillary dysentery (Shigellosis), clonorchiasis, Aquatic organisms, including fish, accumulate pollutants dracunculiasis and paragonimiasis due to larvae and directly from contaminated water and indirectly via the food metacercariae ingested in fish and crustaceans [9], Cholera chain [2]. Disposal of sewage wastes into a large volume of [10], typhoid and paratyphoid [11] etc. The microbial water could increase the biological oxygen demands to such a association with fish compromises safety and the quality for high level that all the available oxygen may be removed, human consumption is particularly critical, when the micro- consequently causing the death of all aerobic species, e.g., fish organisms are opportunistic and / or pathogenic in nature. [3]. Therefore, fishery products which are of great importance for It is apparent that fish are continuously exposed to the human nutrition and health worldwide can act as a source of microorganisms present in water and in sediment including the food borne pathogens and may be a potential source of contaminants in sewage/faeces [4]. Pathogenic microbes cause diseases [12]. many diseases in both wild and cultured fish. They may vary There have been great economic losses reported due to food from a primary pathogen to that of an opportunist invader of a borne illness such as dysentery and diarrhoea resulting from host rendered moribund by some disease process [5]. Fish may consumption of contaminated fish. These circumstances harbour pathogens on or inside its body after exposure to prompted the present study, to investigate the occurrence of contaminated water or food. It is recognised that, extraneous any human bacterial pathogens in the fish that was being bacteria are capable of surviving in fish. The faecal indicator caught from the upstream and downstream portion of Mogral organism Escherichia coli, was found to survive and even River basin. multiply in the digestive tract of rainbow trout (Oncorhynchus mykiss) after ingestion via contaminated food [6]. Tele: 9847781444 E-mail address: [email protected] © 2016 Elixir All rights reserved 43673 Megha P U and Harikumar P S / Elixir Bio Sci. 100 (2016) 43672-43677 Materials and Methods Fish sample preparation Study Area Sample preparation was made using the method The study was conducted in Mogral River, which is a described in the literature [14]. Approximately, 10 g of the west-flowing river in the Kasaragod district in the Indian State fish sample was cut from the head, middle and tail regions of Kerala. The river is 34 km long and has its entire course with a sterile knife. The cut samples were crushed into small within Kerala. The geographical map of the course of Mogral pieces in a sterile mortar with about 10 ml sterile water. From River showing the study area and sampling points is shown in the crushed sample, 1 ml aliquot volume was measured out (fig 1). Two sampling stations were fixed for the study and homogenized in a clean, dry sterile beaker containing 9 ml Muliyar (MG 1) which is the upstream portion of the river and of distilled water giving a 1:10 dilution. Mogral Puthur (MG 2), the downstream portion. Enumeration of bacteria from skin, liver, intestine and tissues The bacterial counts on the external surfaces, liver, intestines and tissue were estimated as follows: Skin Surfaces Sample from different locations of the skin of the raw fish was taken by rubbing the sterilized cotton swab over the skin and then inoculated into 100 ml of peptone water and further kept for incubation at 370C. Ten fold serial dilution of the bacterial suspension already inoculated in peptone water was prepared in duplicate. Bacteria were enumerated using spread plate method with Nutrient agar, MacConkey agar, King’s agar and TCBS agar. The plates were incubated at 35±2oC for 24h. The observed colony growth were counted using Coulter™ Colony counter according to plate count method. Intestines, Liver and Tissues Fig 1. Map of Mogral River segment showing study area One gram of the fish sample was dissected out, and sampling points. blended and mixed properly in a mortar. It was aseptically Water and Fish sampling transferred to a sample bottle containing 9 ml of 0.1% sterile Water samples were collected from upstream and peptone water. The bottle was closed and shaken thoroughly downstream portion of Mogral river with replicates for for 10 minutes and allowed to stand for 20 minutes, after physico-chemical and bacteriological analysis. The samples which a 10 fold serial dilution was carried out in duplicates for physico- chemical and bacteriological analysis were and viable aerobic bacterial counts were enumerated in collected in 1litre plastic cans and 100 ml sterilized bottles standard plate count agar after incubation at 37oC for 48 hrs as [13]. The samples were collected following the standard described by [15]. The observed colony growth were counted sampling guidelines and methods. Further the samples were using Coulter™ Colony counter according to plate count transported immediately to the laboratory in ice cold condition method. and subjected to physico-chemical and bacteriological -1 analysis. Estimate of mean colony forming unit per gram (CFU g ) The mean colony forming unit per gram (CFU g-1) Fish samples were collected from Mogral River during denoted by (x) was calculated as Σfχ/Σf, where Σfx is the sum pre monsoon season (April, 2016). Etroplus suratensis, of the products of number of colonies and the colony forming commonly known as Green chromide which are the common unit per gram; while Σf is the summation of the number of inhabitants of brackish and freshwaters sources of Kerala was colonies. selected for the study. It is also, one of the edible and Examination of Bacterial Density in Tissues economically important fin fishes of Mogral River. Four Demonstration of bacteria in the fish tissues was done using samples of Etroplus were collected aseptically and special staining by gram’s staining modifications [16, 17]. immediately transported in a thermal bag to the laboratory and o o Thin sections of the body tissue, liver and the intestine of the the samples were kept in the refrigerator (4 C – 8 C). fishes from both the upstream and downstream were stained 2.3 Physico-chemical analysis of water samples by special gram’s staining method for bacterial examination. The collected River water samples were analyzed in the Bacterial isolation and presumptive identification laboratory for pH, temperature, electrical conductivity (EC) The experiments were carried out immediately after and total dissolved solids (TDS) using EUTECH multi- collection .Bacteria isolated from the sampling sites (described parameter tester digital-electrode pH meter. The biological above), were grown on nutrient agar at 37°C for 18 to 24 oxygen demand (BOD) of the samples was determined by hours. Each colony was the subject of the following tests: Wrinkler’s titration method [13].