Isolation, Identification, and Antimicrobial Profiling of Bacteria from Aquaculture Fishes in Pond Water of Bangladesh

Bacterial diseases are widespread and can be of particular importance in fish farming of Bangladesh. This investigation was done to assess and compare the bacteria diversities and population in local fresh water pond fishes. Out of 95 samples, 54(56.9%) were Shing (Heteropneustes fossilis), 14(14.7%) were Pangas (Pangasius pangasius), 9(9.5%) were Pabda (Ompok spp), 7(7.3%) were Thai Koi, (Anabas testudineus), and 11(11.6%) were others infected fishes such as Shol (Channa striata), Magur (Clarias spp), Tilapia (Oreochromis niloticus), and Tengara (Mystus cavasius) fishes. Among 95 infected fishes 84(88.4%) were infected with pathogenic bacteria and 11(11.6%) were normal flora. There were eight types of different isolated spp with frequency of occurrence were 36 (42.9%) Aeromonas spp, 15 (17.9%) Pseudomonas spp, 7(8.3%) Vibrio spp, 9 (10.7%) Staphylococcus spp, 7(8.3%) Flavobacterium spp, 7(8.3%) Edwardsiella spp, others were 3(3.6%) Citobacter spp, and Enterobacter spp respectively. All of the isolated pathogenic bacteria showed 84/84(100%) resistant to Amoxicillin and 18/84(64.3%) resistant to Erythromycin. All the strains showed sensitive to Ciprofloxacin, Cotrimoxazole, Enorfloxacin, Doxyciline, Clotetracycline, and Colistin with the frequency of occurrence were 78/84(92.8%), 76/84 (90.5%), 73/84(86.9%), 67/84(67.9%), 53/84(63.1%) and 52/84(61.9%) respectively. The physiochemical characteristics of 95 pond water samples were analyzed; the average water temperature, p H , and ammonia were 27.3 0 C, 7.6 and 0.87mg/L respectively. The significant variation in the physiochemical parameters like water temperature, p H , ammonia were observed within these five types of ponds water. Hence, it is important to detect fish diseases, responsible pathogens and other agents for protection of our water resources.


INTRODUCTION
The fresh water fishes are well known and highly valuable fish species in Bangladesh. Among them, Shing (H. fossilis), Pangas (P. pangasius), Thai Koi with fishes specially, with the aquaculture fishes in ponds. The naturally living in the fish's habitat is reservoir of the indigenous bacterial pathogens such as Aeromonas spp and Vibrio spp. There are some factors that induce bacteria to be pathogens such as poor water quality and physiologically uneven fish which permit opportunistic bacterial diseases to win (Austin, 2011;Rahman et al., 2019).
A number of pathogenic bacteria are related with fish and shellfish for instance Streptococcus spp, Aeromonas spp, Vibrio spp, Flabobacterium spp and others pathogens to cause diseases. The economic losses because of fish mortality are mainly caused by various pathogenic bacteria and other microorganisms in Bangladesh (Md et al., 2014). In the fishes there are two types of bacteria like indigenous bacteria and nonindigenous microbes. Staphylococcus aureus, Escherichia coli, Clostridium botulinum, Listeria monocytogenes are some examples of non-indigenous microorganisms and then again, indigenous microbes incorporate Aeromonas spp., Vibrio spp., Salmonella spp, and Pseudomonas spp. In Bangladesh, generally fresh water fishes are found in little rivers, swamp and canal. Now a day's commercial fish cultivating in lake is exceptionally well known. The dangerous microbes such as Pseudomonas spp, Aeromonas spp, Staphylococcus spp, Flavobacterium spp, Citobacter spp Edwardsiella spp, and Vibrio spp that live in every pond causing perilous bacterial diseases, for example, ulcer, blade decay and tail spoil of fishes (Uddin et al., 2017).
The poor water quality is also a great reason that consumes off the sludge coat or stresses the fishes making them progressively powerless to bacterial infection. Therefore, this study was designed with a view to assess the occurrence of bacterial infection of ponds cultivated fishes and their respective ponds water quality assessment and antibiotic susceptibility analysis against bacterial pathogens of fishes. and with their water samples were taken from different 18 Upazila in greater Mymensingh, Netrokona, and Sherpur districts in Bangladesh between April 2019 to December 2019. During the collection of fish samples and water precautionary measures were maintained to avoid touch and ice box were used to maintain cool chain. The samples were then brought to the laboratory of the Quality Aqua Laboratory, Quality feeds Limited, Mymensingh.

Sample processing and enrichment of bacteria -
Aseptic measures were undertaken during the sampling procedure to prevent contamination of the samples. Three types of specimens such as intestine, skin and gill of infected fishes were collected for microbiological test. These specimens were taken in a sterile chopping board and then minced properly and grinded together. Ten (10) gm of samples were homogenized with 90 milliliters(ml) of freshly prepared 0.1% peptone water and 0.1 ml of homogenized sample was inoculated according to standard methods on to selective media such as: Rimler Shotts Medium Base agar (for Aeromonas spp.), Pseudomonas Base agar (for Pseudomonas spp), Thiosulfate citrate bile salt sucrose (TCBS) agar (for Vibrio spp.), Tryptic Soy Agar (TSA) for enrichment of bacterial isolates, Brain Heart Infusion (BHI) Agar (for fastidious organisms) and finally incubated at 37ºC for 24 hours.

Identification of bacterial pathogens -
Suspected bacterial colonies obtained from different culture plates were isolated and then streaked on TSA slants, MIU medium, Simon citrate agar stant and incubated overnight at 37 0 C. The pure isolates were characterized by bacterial cell morphology, alkaline and acidic reaction, H 2 S (hydrogen sulfide production) and gas production, motility test, indole production, urease test, oxidase test, catalase test, Methyl Red (MR) test, and Voges Praskaure (VP) test.
The Gram staining techniques were performed to identify Gram positive and Gram negative bacteria. The biochemical tests were carried out to identify the pathogens following Bergey's manual of Bacteriological classification (John et al., 1998).

2.4
In-vitro antimicrobial sensitivity test -According to the CLSI guidelines (CLSI, 2015), the Kirby-Bauer disc diffusion methods were used to in-vitro antimicrobial susceptibility tests of all the pathogenic bacteria isolates. The commonly used antibiotics were: Amoxicillin (10μg), Ciprofloxacin (5μg), Colistin (25μg), Clotetracyclin (30μg), Doxyciline (30μg), Erythromycin (15μg), Cotrimoxazole (25μg), and Enrofloxacin (5μg). Aeromonas hydrophila (ATCC 7966), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), Vibrio cholerae (ATCC 14035), and Flavobacterium columnare (ATCC 23463) were used as quality control throughout the study for culture and antimicrobial susceptibility testing. The suspected isolated bacterial colonies were taken in sterile PBS (phosphate buffered saline) water and then adjusted to 0.5 McFarland's turbidity standard. The bacterial suspension was spread onto Mueller-Hinton agar (Himedia, India) and then impregnated antibiotic discs (Himedia, India) were placed and incubated at 37 0 C for 24 hours. Around the discs, the antibiotic zones of inhibition conformed were estimated in diameter of millimeter (mm).The zone span was really scaled from the focal point of the anti-microbial plate as far as possible of the reasonable zone where microscopic organisms could be seen developing. The interpretation of antibiogram was measured in millimeter (mm) of diameters as sensitive, intermediate and resistant as per the producer's guidelines.

Water physio-chemical parameters -
Ponds water temperature and pH were analyzed by the digital thermometer and digital pH meter (Hanna instruments, USA) respectively. The ideal value of temperature and pH was 30 -32 0 C and 7.0 -8.5 separately. Ammonia, nitrate, and nitrite were determined by colorimetric method by using Rapid Hanna Test kit (Hanna instruments, USA), with a special smallest increment range of 0.5 mg/L NH 3 -N, <10mg/L NO 3 -N, and < 0.2mg/L NO 2 -N respectively. The total hardness and alkalinity were measured by EDTA, phenolphthalein, and bromophenol titration by using Rapid Hanna Test Kit (USA) with a special ideal value of 20-100 mg/L and 50-160 mg/L separately.

2.6
Statistical analysis of experimental data -Data obtained were analyzed by SPSS version 20 and Excel 2016. Descriptive statistics and chi-square tests were done to check the statistical evaluation. The p-value that considered as significant was <0.5.

RESULTS:
3.1. Clinical Signs and post mortem findings -After clinical examination of diseased fishes, several severe damages were observed such as equilibrium loss, hemorrhagic ulcerative lesion, rectal protrusion, dropsy, body and tail erosion, reddish discoloration around the eye and mouth, skin lesions on body surface, and profuse mucous secretion (Fig 1). In postmortem examination of diseased fishes, organ enlargement and congestion in internal organs were also appeared.

Biochemical tests for bacterial identification -
The identification of the pure bacterial isolates were performed by biochemical parameters included alkaline reaction, acidic reaction, H 2 S (hydrogen sulfide production) gas production, motility test, indole production, urea hydrolysis, catalase test, oxidase test, Methyl-Red (MR) test, Voges-Praskaure (VP) test are presented in Table 2.
In all of the isolated strains it was observed that only one strain was found as Gram positive Cocci and the rest 7 isolates were found as Gram negative bacteria. The biochemical analysis revealed that all of the strains were catalase negative in our experiment.     Table 3-7 showed antibiotic sensitivity patterns all of the individual isolates.

Physico-chemical characteristic of pond water -
The recorded pond water parameters of 95 sub-surface water samples were studied shown in Table 8. The average range of temperature, p H , ammonia, hardness, alkalinity, nitrate and nitrite varied depending on their cultivated fish types.
All of the fish water samples showed ammonia concentration higher than their normal value and that was <0.5mg/L. Only Pangas fish pond water expressed the average hardness and Nitrite (NO 2 ) were 126.42 and 0.37 mg/L respectively that was higher than ideal value 20-100mg/L and <0.2mg/L respectively Table 8.
Other isolates (n=5)  However, a total of 84 (88.4%) pathogenic bacteria and 11(11.6%) non-pathogenic bacteria were isolated, among pathogenic bacteria Gram positive cocci and Gram negative bacilli were 9(10.7%) and 75 (89.7%) respectively. A wide range of bacterial species has been recovered from local fishes in our work including Aeromonas spp, Pseudomonas strains and other isolates were identified from aquaculture pond fishes reported by Hossian and Chowdhury (2009). In Bangladesh, detached and distinguished some Pseudomonas strains from unhealthy cultivated fishes of Bangladesh. The different types of bacterial species of the pond water fishes (Shing) were isolated by numerous scientists (Shewan, 2000;and Okaeme, 2006). Ahmed and Shoreit (2001) also reported that mixed bacterial contaminations occurred by Aeromonas spp and Pseudomonas spp. Shing (H. fossilis) is most demandable and highly valuable fish in Bangladesh. Although, the mortality rate of Shing is very low but its cultivation in pond water is influenced by different factors such as bacterial pathogens (Shahen et al., 2019). In any case, the clinical manifestations were loss of balance, hemorrhages, skin sores, body and tail disintegration, mucous discharge, and blockage and development with drain of the inside organs which were comparable with the discoveries of  The water quality influenced by the encompassing of ecological conditions may affect the pond cultured fishes. This in the end diminishes the insusceptible status, activating bacterial contamination prompting infection episodes (Amal et al., 2015). The fish wellbeing status and pond cultured environments were reflected due to several bacterial flora. Similar study was conducted by Pakingking et al. (2015). The lake water's physiochemical parameters such as ammonia, hardness, alkalinity, nitrate, nitrite, temperature, and pH were significant in our study area (Ahmad et al., 2018). In this study, every inspecting site had distinctive characteristics that were related with the nearness of microorganisms. This phenomenon was also observed in multiple works done by other investigators. This investigation uncovered that alkali impacted the presence of different pathogenic bacterial spp at all of the sampling areas. It is believed that the ammonia in ponds water comes from fish excrement and exorbitant feed given to the fish.

CONCLUSION
This study has clarified that poor water quality and bacterial diseases could be a major cause of considerable economic loss to fish farmers in greater Mymensingh, Bangladesh. A number of bacterial spp including Aeromonas spp Pseudomonas spp, Vibrio spp, Staphylococcus spp, Flavobacterium spp, Edwardsiella spp, Citobacter spp, and Enterobacter spp were the major causes of bacterial diseases to different fish spp. Clinical examination of diseased fishes indicated that due to bacterial infection some severe damages were found in fish bodies such as equilibrium loss, hemorrhagic ulcerative lesion, rectal protrusion, dropsy, body and tail erosion, reddish discoloration around the eye and mouth, skin lesions on body surface and profuse mucous secretion. The presence of antibiotic resistant isolates imposes a serious concern about the drug of choice for treatment in fishes. A careful consideration should be given before deciding the antibiotic for treatment so as to prevent the emergence of antibiotic resistance and properly maintain the physiochemical parameters in ponds water to reduce the mortality of aquaculture fishes in ponds water. Moreover, disease prevention of various local fishes should be carried out by employing better culture practices and health management to ensure the highest yields and the optimum quality of the products.