Journal of Aquaculture and Marine Sciences

Bacterial Diversity of Giant Freshwater Prawn, Macrobrachium rosenbergii and Screening for Probiotic Potential Bacteria

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Published Date: November 09, 2017

Bacterial Diversity of Giant Freshwater Prawn, Macrobrachium rosenbergii and Screening for Probiotic Potential Bacteria

Mujeeb RKM1*, Jesmi Yousuf2, Hatha MAA3 and Thomas AP2

1Department of Aquaculture and Fishery Microbiology, MES Ponnani College, Ponnani, India
2School of Environmental Sciences, Mahatma Gandhi University, Kottayam, India
3School of Marine Sciences, Cochin University of Science and Technology, Cochin, India

 

Corresponding author: Mujeeb Rahiman KM, Department of Aquaculture and Fishery Microbiology, MES Ponnani College, Ponnani, India, E-mail: rkmmujeeb@gmail.com

Citation: Mujeeb RKM, Yousuf J, Hatha MAA, Thomas AP (2017) Bacterial Diversity of Giant Freshwater Prawn, Macrobrachium rosenbergii and Screening for Probiotic Potential Bacteria. J Aqua Mar Sci 1(1): 101.

 

Abstract

 

In an attempt to explore the probiotic potential of bacteria found in the endemic habitat of Macrobrachium rosenbergii, acteriology of the samples associated with the natural environment of M. rosenbergii has been studied. A total of 752 isolates were characterized up to genus level. While feed items and the intestine of adult M. rosenbergii showed highest total viable count (2.20 × 107 to 7.20 × 108 cfu g-1 and 2.95 × 108 to 1.37 × 109 cfu g-1 respectively), it was relatively low in the water (6.00 × 103 to 1.40 × 104 cfu ml-1) as well as in the larval samples (8.40 × 104 to 6.40 × 105 cfu g-1). Characterisation of the various genera of heterotrophic bacteria revealed good diversity of both gram negative and gram positive genera. Bacterial genera such as Acinetobacter, Aeromonas, Alcaligenes, Vibrio, Bacillus, Streptococcus and Enterobacteriaceae were identified from all the samples. The screening and probiotic potential study found that Brevibacillus latrosporus isolated from the larval sample showed antibacterial activity against fish and prawn pathogens. No adverse effect was noticed when the Post Larvae (PL) of M. rosenbergii challenged with the selected probiotic strains and showed good hydrolytic enzyme potential.

Keywords: Heterotrophic bacteria; Natural environment; M. rosenbergii; Antibacterial activity; Brevibacillus laterosporus

 

Introduction

 

Aquaculture is developing very rapidly in recent years and has significant role in the economic development of the nation; also contribute to the world supply of food and food security. Both developed and developing countries practise small scale to large scale aquaculture systems and have important contribution to food supply, income generation and trade. Approximately 90% of global aquaculture production is based in Asia [1,2]. Macrobrachium rosenbergii, popularly known as Giant freshwater prawn has a great export market worldwide and is an excellent candidate for freshwater aquaculture. Being the largest species, M. rosenbergii is commercially exploited from Vembanad Lake, Kerala, India with a peak fishing season during monsoon and post monsoon. The health of aquatic animals has greatly influenced by the environment which they live and their health status is directly influenced by the presence of microorganisms when compared to the health status of terrestrial animals or humans [3].

Disease outbreak is promoted by intensification and represents one of the biggest causes of loss in aquaculture [4–6]. Since conventional disease management strategy, such as treatment with antibiotics is leading to unfavourable consequences like emergence of drug resistant bacteria, researchers are encouraged to find out alternative strategies such as vaccination, use of immunostimulant and probiotics for the health management of aquatic animal is being tried out. The regulation of antibiotics by European Union [7] and the demand of alternative products against antibiotics [5], open the way to use environment friendly products. The World Bank invested US$ 275 million during 1996–2010 for disease related research in shrimp aquaculture [8]. Use of probiotics as an alternative source instead of antibiotics is proving to be an environment friendly mode of health management and capable of modulating the immune system [9]. Recently, the study using biofloc technology combined with the addition of probiotics showed the enhancement of disease resistance and nonspecific immune responses in M. rosenbergii [10].

The research for beneficial probiotaic bacterial cultures are reported in recent years for the culture of commercially important aquaculture organisms . The selection and development of probiotics for different cultured species in India assumes greater significance considering the rejection of farm raised shrimp by EU, citing presence of trace levels of antibiotics in the shrimp. M. rosenbergii is emerging as a popular species for aquaculture in India owing to many favourable attributes. Cruz et al. [5] strongly suggested the importance of microbial ecology study and the relationship of microbes with the cultured organism and the importance of phylogenetic identification of probiotic microorganisms. Taking this into consideration an attempt has been made to study bacteriology associated with the natural environment of M. rosenbergii and evaluate the probiotic potential of these bacterial isolate to use in the hatchery and culture system of M. rosenbergii.

 

Materials and Methods

 

Description of the Study Area

The Vembanad estuary is one of the Ramsaar site in India and it is one of the largest tropical wetland with mangroves. It is located between 9° 29’ and 10° 10’ North latitude and 76° 13’ and 76° 31’ East longitude, extending a stretch of 60 Km from Cochin bar mouth in the north to Aleppey in the south with an estimated area of 21050 ha. Kumarakom region of Vembanad estuary was chosen as the sampling area and this region is a part of Kuttanad known as the home ground of M. rosenbergii [1

Collection of Water Samples

Water, sediment and adult M. rosenbergii samples were collected from four different stations (Figure 1) and necessary precautions were taken to minimize the contamination of the sample. Water and sediment samples were collected in sterile bottles and sterile jars respectively. The adult M. rosenbergii were collected by fisherman in live condition and brought to the laboratory for analysis. The larvae and Post Larvae (PL) were collected by using 500 µm plankton net and the collected larvae were identified into different stages of growth by using the manual for the culture of M. rosenbergii [14].

Two samples of larval and PL feed items were collected using a 500 µm plankton net and adult feed items from the bottom of the lake with the help of fisherman. The planktonic feed items of larvae and PL mainly consisted of zooplankton, small worms, larval stages of invertebrates and small amounts of phytoplankton. Crustacean, mollusc, filamentous algae, plants and remnants of plants, etc. were identified as adult feed of M. rosenbergii. All the samples except the adult M. rosenbergii were kept in an icebox and immediately brought to the laboratory for analysis.

 

Analysis of the Physio-Chemical Parameters

Physico-chemical parameters of water samples such as temperature were measured in situ using centigrade thermometer, salinity by salinity refractometer (Atago, Japan) and pH by digital pH meter (Eutech, Singapore). Dissolved oxygen was estimated by the Winkler method [15]. The pH of sediment was measured using the method described by Sharmila et al. [16].

 

Bacteriological Analysis

4.4.1 Preparation of samples: Water and sediment samples were serially diluted aseptically to 10-3 and 10-5. Larvae and PL were washed in 0.1% benzalkonium chloride and washed in sterile water and the water adhered to it was removed by sterile blotting paper before weighing. The samples were homogenized aseptically by using glass homogeniser and diluted to 10-5. The intestine of M. rosenbergii was removed aseptically, weighed and homogenized in glass homogeniser and serially diluted to 10-6. The food samples were washed three times with sterile water, and homogenized aseptically in glass homogeniser after weighing and diluted to 10-6.

4.4.2 Estimation of total bacterial load: Estimation of bacterial load was done by spread plate method by using tryptone soya agar (TSA) and ½ strength Zobell’s marine agar (½ ZMA). Total viable count (TVC) of bacteria were enumerated and selected for isolation of bacteria after incubating the plates at 30°C for 24–48 hours plates with 30 to 300 colony-forming units (cfu).

4.4.3 Isolation and identification of bacterial isolates: All bacterial colonies were purified before identification and a total of 752 bacterial isolates from water (131 isolates), sediment (114 isolates), intestine of adult M. rosenbergii (155 isolates), larvae and PL (206 isolates) and food samples (146 isolates) were characterized to the genus level using the taxonomic keys [17–20].

 

Probiotic Potential Study

4.5.1 Pathogenic bacteria used: Fish, prawn and human pathogens such as Aeromonas hydrophila (MTCC 646), Vibrio parahaemolyticus (MTCC 451), Vibrio harveyi (CUSAT, Kerala), Vibrio vulnificus (MTCC 146), Escherichia coli, Salmonella newport and Salmonella typhi (isolated from Vembanad lake) were used as pathogens to determine the antibacterial activity of the probiotic strains against pathogenic bacteria.

4.5.2 Determination of antibacterial activity of isolates by well diffusion and cross streak method: For the determination of antibacterial activity by well diffusion method, 2 ml of a young culture (16–18 hour in TSB) of A. hydrophila, V. vulnificus, V. harveyi, V. parahaemolyticus, S. typhi, E. coli, and S. newport were prepared and poured over the TSA medium and incubated at 30°C for 15 minutes. Sterile gel puncher was used to punch three millimetre diameter wells in the plates and 30 microliters of 18 hours bacterial culture in TSB was pipette into the wells and incubated at 30°C for 24 hours. Clear zone around the wells was noted as the presence of antibacterial activity.

In cross-streak method, an 18 hours bacterial culture in TSB that showed positive results in well diffusion method was streaked on TSA plate as a thick band with 2 cm in width on the centre of the TSA plate. The growth was scrapped after incubation for 24 hours at 30°C, and treated by chloroform for 15 minutes. After air drying for ten min to remove any residual chloroform, the 18 hours old cultures of pathogenic bacteria were streaked vertical to bacterial band and incubated for 24 hours at 30°C. A linear clear zone was noted as the presence of antibacterial activity.

4.5.3 In vivo experiment for the safety of probiotic strain to post larvae of M. rosenbergii: For testing the safety of probiotic strain to post larvae, 18 hours culture of potential probiotic bacteria were added to 1 L beaker containing 500 ml sterilised filtered freshwater to obtain 105 cells ml-1. To each beaker, 25 numbers of PL (PL-20) of M. rosenbergii were introduced and any signs of disease or mortality up to 96 hours were monitored. The experiment was duplicated and the control was maintained without any bacterial inoculum.

 

PCR Amplification of the 16S rDNA, Sequencing and Phylogenetic Analysis of Probiotic Bacteria

DNA extraction was done by Bacterial Genomic DNA (prep) Kit (Chromous Biotech, India) and 16S rDNA genes were amplified with the universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'- GGTTACCTTGTTACGACTT-3') by using the polymerase chain reaction (PCR) [21].

The nearest taxa of the 16S rRNA gene sequence (1418–1542 bases) was identified by BLAST sequence similarity search (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The CLUSTAL W software was used to align 16S rRNA gene sequences and Maximum Likelihood (ML) and Neighbour-Joining (NJ) methods with MEGA version 5 [22] were used to construct the phylogenetic tree.

 

Hydrolytic Enzyme Activity

The ability of B. laterosporus to utilize different substrate was done by using the BBL CRYSTAL™ Identification (ID) system for Gram-Positive (GP) bacteria. A total of 20 different substrates were used to check the hydrolytic enzyme activity.

 

Result and Discussion

 

Physico-Chemical Parameters

Physico-chemical parameters of water and sediment samples collected from the sampling sites revealed that temperature, pH and DO of water ranged between 28.5°–31.0°C, 5.8–6.7 and 6.9–7.2 mg L-1 respectively. The salinity of water samples were within the range of 0–4 ppt. The pH of sediment samples ranged between 5.48–6.46. Similar pH values were reported by Nandan and Unnithan [23] from Vembanad Lake and the physico-chemical parameters analysed were within the optimal range for growth and survival of M. rosenbergii in their natural environment [14]. Low salinity and the slightly acidic nature of water and sediment samples recorded in the present study are in agreement with the results [24] from the Kuttanad region of Vembanad Lake. Thanneermukam barrage constructed across the Vembanad Lake to prevent the ingress of saline water into the rice fields of this area cause the low salinity of water.

 

Bacteriological Results

Bacteriology of Water, Sediment and Intestine: The TVC load of water samples ranged from 6.00 × 103 to 1.40 × 104 cfu ml-1 and that of sediment samples ranged from 8.24 × 105 to 1.42 × 106 cfu g-1 were in concurrence with those of Sharmila et al. [16] and Harish et al. [25] from shrimp farm. TVC load from the intestine of adult M. rosenbergii samples ranged from 2.95 × 108 to 1.37 × 109 cfu g-1. The TVC load of the intestinal tract of M. rosenbergii were comparable to those reported by Phatarpekar et al. [26] in digestive tract of M. rosenbergii, as well as in the gut samples of P. indicus from backwaters and P. monodon from seawater [27,28]. However, Al-Harbi [29] reported the bacterial count of 3.40 ×105 to 8.70×106 cfu g-1 from the digestive tract of M. rosenbergii cultured in concrete tanks.

Out of 16 genera identified, 13 genera were present both in water and sediment samples and 10 genera were present in all the samples (Table 1, Figure 2). Cavallo et al. [30] reported that most common bacteria in water and sediment samples were Gram negative rods. In contrast, the study [31] reported higher proportions of Gram positive bacteria from sediment. Out of 16 genera isolated from the present study 12 genera were previously reported [26,32]. Results revealed that bacterial genera of water were more similar to genera encountered in the sediment sample which is supported by observation of Austin and Allen [33] from freshwater reservoir fishery. All the bacterial genera found in the gastrointestinal tract of M. rosenbergii were also detected in the samples from its natural environment revealed the importance of keeping good microbial quality of their environment in the culture system.

Acinetobacter and members of the family Enterobacteriaceae were predominant ones in water, while Bacillus, Acinetobacter and members of the Enterobacteriaceae family were predominant genera from sediment samples. Staphylococcus and Streptococcus were identified only from sediment samples and Kurthia from water samples. Percentage occurrence of different genera of bacteria from the intestinal tract of M. rosenbergii (Figure 2) revealed the predominance of members of the family Enterobacteriaceae, Streptococcus and Aeromonas. The opportunistic pathogens like Vibrio and Aeromonas were isolated from water, sediment and intestinal samples. New MB [34] reported that the Vibrio are not primary pathogens and exist in and around crustaceans in marine or brackish water environments as part of their normal microflora and researchers frequently observed the presence of Vibrio, and Aeromonas from freshwater and marine water culture system [35–37]. Listeria in water and sediment samples observed less frequently and it is observed by previous researchers from marine water, sediment, P. monodon and various seafoods [38–40]. Several researchers reported the association of Streptococcus with the mucosa of the gastrointestinal tract [41–43]. Streptococcus is one of the lactic acid producing bacteria and they can survive in the environment and able to adhere to the exposed surface of the epithelial cell [44]. The results [29,37] suggested that aeromonads are indigenous in M. rosenbergii and fish intestine, water and sediments. Lalitha and Surendran [45] also reported the presence of Gram positive genera such as Micrococcus, Bacillus and coryneforms from M. rosenbergii’s gastrointestinal tract that are similar to the present findings.

It was reported that in the culture system, the decline in M. rosenbergii production has been mostly due to disease outbreaks [46,47]. The aquatic organisms are attached by variety of bacteria on their surfaces, tissues and body fluids and all aquatic organisms are exposed to various microflora that harbour multiple pathogens and immunological factors, food and animal physiology are some of the factors which affect the balance of intestinal microbiota [48]. The higher number in the digestive tract than the surface water representing a favourable place for the bacterial species [45,49].

Bacteriology of M. rosenbergii Larvae and PL: The TVC load from various larval and PL stages ranged from 8.40 x 104 to 6.40 x 105 cfu g-1. Miyamoto et al. [50] and Sahul Hameed [36] also noted similar bacterial counts larvae during rearing as in the present study. Twelve genera were isolated from larval and PL samples of M. rosenbergii (Table 2) with predominance of Vibrio, Moraxella, Acinetobacter, Alcaligenes and Bacillus. All the genera encountered in the water samples and more than 80% of genera from intestinal samples were also isolated from larvae and PL of M. rosenbergii corresponding to the environmental samples. Anderson et al. [32] also reported close similarity of bacteria between washed larval tissue slurries and hatchery water. The findings of present study differs with the observations of Phatarpekar et al. [26] who studied microflora of freshwater prawn hatchery and reported the predominance of Alcaligenes, Pseudomonas, Streptococcus and members of the family Enterobacteriaceae from hatchery reared larvae. One of the reasons for this may be the disinfection process of water that is used for the hatchery operation which may help the dominance of certain bacteria.

Bacteriology of food items of M. rosenbergii: Load of TVC was significantly high (p < 0.05) in adult feed samples than larval feed samples. The TVC load in larval and adult feed samples ranged from 2.20 × 107 to 7.20 ×108 cfu g-1. Feed samples from natural environment revealed high TVC as the feed items contained fish remains, algal and plant matter etc. that are mostly in decayed state. Most of the adult food items were in contact with sediment and was in partially decomposed state, which could have high bacterial load. While the bacterial flora of the larval feed items of M. rosenbergii in natural environment (Figure 3) was dominated by of Alcaligenes, Bacillus was found to be the predominant bacterial genera in adult feed items. Vibrio was found to be the second most common genera from both larval and adult feed items. The predominant genera from larval and adult feed items were also found to be abundant in larvae and in the intestine of M. rosenbergii. These observations were supported by the findings of Moriarty [51]. Most of the adult feed is seen in bottom sediment surfaces which could have resulted in the dominance of Bacillus in the adult feed sample as in sediment sample.

Probiotic Potential Study

A total of 752 bacterial isolates were preliminary screened for the selection of potential probiotic strains. Based on antibacterial activity against tested pathogens, a Bacillus sp. (characterised by phenotypically) isolated from the larval samples of M. rosenbergii showed high antibacterial activity against A. hydrophila, V. harveyi, V. parahaemolyticus, V. vulnificus, Salmonella typhi and E. coli. Aquatic candidate probionts for larviculture have been isolated from adults [52– 54] and healthy larvae [55–57] have been reported previously. It was reported that beneficial bacterial preparations that are species-specific probiotics show specific benefits and greater effectiveness in prevention of disease and maintain a healthy intestinal balance and immune response [58,59]. The partial genomic sequencing of the 16S rRNA of Bacillus sp. and the blasting of the sequence revealed the identity of this potential probiotic bacterium as Brevibacillus laterosporus. The gene sequence of 16S rRNA of the bacterial strain was deposited in GenBank with accession number KF111726. The nearest phylogentic bacteria similar to Brevibacillus laterosporus are shown in figure 4. Brevibacillus laterosporus (previously Bacillus laterosporus) was first isolated from water [60] and it was reclassified from Bacillus brevis cluster, with Brevibacillus brevis as the type strain. Brevibacillus laterosporus is an important species as a biological control agent and because of its uniqueness in its spore formation and physiological activities [61,62]. Brevibacillus sps is recently reported as a probiotic in aquaculture [63], however Brevibacillus laterosporus as prophylactic and health food supplements or novel foods in human was reported years back [64]. The genus Bacillus, Paenibacillus and Brevibacillus are single endospore formers and represent a special case among the bacteria used as probiotics. The use of spore formers as probiotics have some advantages like the shelf life period, resistance to adverse environmental conditions and low cost of production etc.

Before going for the experimental trial, it should be confirm that the probiotic bacteria should not show any pathogenic or adverse effect on host [65] and the probiotic bacteria are then selected according to the antagonistic property against the pathogens and by in vitro testing [66–70]. The results of pathogenicity effect of the Brevibacillus laterosporus on post larvae of M. rosenbergii (Figure 5) showed that the bacteria has no pathogenic effect on the post larvae and the survival was higher with that of control (p < 0.01). Hydrolytic enzyme activity of B. laterosporus (Table 3) shown that the bacteria were capable of utilising 15 out of 20 substrates used in the BBL crystal GP ID kit. This result shows the high ability of bacteria to produce verities of different hydrolytic enzymes and their potential to act as a food supplement through feed to help in the digestion of food materials. The antibacterial compounds and varieties of enzymes produced by the Bacillus help to control the proliferation of pathogenic bacteria.

 

Conclusion

 

In the study, an attempt was made to find out the bacterial diversity associated with various life stages of giant freshwater prawn, and screening for probiotic potential among these bacteria. The study strengthens the microbial ecology and correlation of microbial communities (microorganisms on water, sediment, gut and larvae). The screening and probiotic potential study found that Brevibacillus latrosporus showed antibacterial activity against fish and prawn pathogens. No adverse effect was noticed when the PL of M. rosenbergii challenged with the selected probiotic strains. The present study suggested Brevibacillus laterosporus as a promising probiotic candidate for hatchery and culture operations of M. rosenbergii. However, thorough studies are suggested with detail evaluation on the effect in vivo with these bacteria.

 

Acknowledgements

 

The authors wish to express their gratitude to the University Grants Commission (UGC), Govt. of India, for the financial assistance (F.3-37/2002).

 

Conflict of Interest

 

Authors declared that they have no conflict of interest.

 

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