Lovely Faculty of Technology and Sciences Lovely Professional University Punjab March 2019

DEVELOPMENT OF PROBIOTICS FROM AQUATIC BODIES FOR ENHANCED PRODUCTIVITY IN FISH FARMING

A Thesis

Submitted to

For the award of DOCTOR OF PHILOSOPHY (Ph.D.) in ZOOLOGY

By ARUN CHAUHAN (11412806)

Supervised By DR. RAHUL SINGH

LOVELY FACULTY OF TECHNOLOGY AND SCIENCES LOVELY PROFESSIONAL UNIVERSITY PUNJAB MARCH 2019

DECLARATION

I hereby declare that the thesis entitled, “Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming” submitted for Ph.D. Zoology, Degree to Department of Zoology, Lovely Professional University is entirely original work and all ideas and references have been duly acknowledged. The research work has not been formed the basis for the award of any other degree.

Arun Chauhan Reg. No. 11412806

CERTIFICATE

This is to certify that Mr. Arun Chauhan has completed the Ph.D. Zoology titled “Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming” under my guidance and supervision. To the best of my knowledge, the present work is the result of his original investigation and study. No part of this thesis has ever been submitted for any other degree or diploma. The thesis is fit for the submission for the partial fulfilment of the condition for the award of degree of Ph.D. in Zoology.

Signature of Supervisor Dr. Rahul Singh Assistant Professor Department of Zoology, School of Bioengineering and Biosciences, Lovely Professional University.

ABSTRACT

The world’s population is increasing day by day, so it is essential to enhance the productivity of aqua food products. But with high production of fish by intense culture practices, high stocking densities of fish, decay of biotic materials along with intense feeding and fertilization causes not only stress and health issue but also degrade the water quality. The slurry water influences the proliferation of pathogenic microorganisms, which causes disease and reduces the aquaculture productivity. Fish are permanently exposed to different external hazards because of their intimate contact with aquatic habitat.

Pathogenic bacteria have the potential to proliferate or maintain them in the aquatic environment. The pathogenic bacteria are constantly taken up by the fishes through feeding and osmoregulation process. The most common fresh water diseases are dropsy, fin and tail rot, gill diseases, white spot disease, cloudy eye, pop eye, tuberculosis, and water quality induced diseases, etc. The most dominant fish pathogenic bacteria in their habitat are Aeromonas hydrophila, Pseudomonas aeruginosa, Flavobacterium columnare, Proteus species, Micrococcus species and vibrio species. These microbes are major infectious aquatic pathogens and are causative agents of fin rot, ulcers, tail rot and hemorrhagic septicaemia. Many synonyms of these diseases related to the lesions, which include septicaemia where the bacterial toxins are present within various organs and ulcers on fish’s skin, which cause severe damage in fish.

To cure such diseases mostly antibiotics like streptomycin, chloramphenicol, amoxicillin, penicillin, gentamycin, erythromycin, neomycin, prefuran, enrofloxacin and oxytetracycline have been applied for treatment. In aquaculture, the use of chemotherapeutic agents such as antibiotics and chemicals are the classical treatment for the bacterial infection, however their widespread use as therapeutic and prophylactic agents, remains in the aquatic environment for a long period of time, resulting in detrimental effects including the emergence of antibiotic-resistant bacteria, presence of antibiotic residue in the flesh and variations in aquaculture environment microbiota. Hence the utilization of such chemotherapeutic medications as a disease control measure has become questionable because of the development of resistant

iii species of pathogens. Antimicrobials enhance survival, yet they likewise change the intestinal microbiota and prompt resistant population of bacteria with unpredictable prolonged effects on public health.

Vaccination cannot prevent disease outbreak in immunological immature individuals, also efficient commercial vaccines against some fish pathogens are not available. Therefore, there is a developing sympathy toward the high utilization of chemotherapeutic medications in aquaculture has led to a quest for alternative or integral methodology for antibiotics and vaccination to prevent the fish diseases. A favourable alternative approach for regulating fish maladies is the employment of probiotics (good microbes), which concoct fishes to combat against pathogens via mixed bag of systems and improves the general health. Probiotic microorganisms are gaining worldwide significance due to their utilization in the preparation of a nutraceutical or in the treatment of infections. According to the health industry demand, there is an insistent requirement for investigating new autochthonous probiotic strains with its exclusive origin due to variation in gut micro-flora, diverse nourishment propensities and specific host-microbial interactions.

The research into the utilization of probiotics for aquaculture is expanding with the interest for environment-friendly sustainable aquaculture. The advantages of probiotic supplements incorporate enhanced feed value, enzymatic contribution to digestion, inhibition of pathogenic microbes, anti-mutagenic and increased immune response. Probiotics are safe microbes that help the wellbeing of the host animal and contribute, directly or indirectly to protect the host animal against harmful bacterial pathogens. The usage of probiotics in aquaculture is quite evident from the review of literature, however, it needs significant effort of research to confirm their viability for utilization in aquaculture.

The main objective of this study is to isolate and identify novel putative probiotic strains from the gut of different teleost and evaluate their potentiality as a potent probiotic by using different in-vitro selection parameters described in FAO/WHO guidelines for the evaluation of probiotics in food. A total of 313 different bacterial strains were isolated from the gastrointestinal tract of 79 different fish species collected from different region of Punjab namely, Doaba, Majha, and Malwa. Out of

313 isolated bacteria, only 87 were indexed as Gram-positive. Isolated strains were screened for antimicrobial activity against the potential fish pathogen Pseudomonas aeruginosa (MTCC 4673). These isolates were further screened in response to fish bile tolerance (0.3% ox-gall), viability in the pH tolerance range of pH 2 to pH 9 and drug susceptibility to different antibiotic discs. The positive candidate probionts showed ability of adhesion by having high auto aggregation activity. It was found that, out of 87 Gram positive bacteria only 12 fits the criteria for probiotics evaluation as per FAO/WHO guidelines (2002).

After the in-vitro evaluation and the biochemical characterization of the isolated bacteria, it was observed that only 12 bacterial strains were passed through all the selection parameters described in FAO/WHO guidelines for the evaluation of probiotics. These 12 isolates were S7, S3, BDK2′, BDK7, BDK9, F1H4, F2F4, F3 1(2), F1F4, F1F2, F1H3 and LF3(1) were identified as Bacillus amyloliquefaciens strain S7, Enterococcus durans strain S3, Bacillus cereus strain BDK2′, Bacillus subtilis strain BDK7, Bacillus subtilis strain BDK9, Enterococcus faecium strain F1H4, Bacillus safensis strain F2F4, Bacillus subtilis strain F3 1(2), Bacillus subtilis strain F1F4, Bacillus velezensis strain F1F2, Enterococcus gallinarum strain F1H3 and Enterococcus faecium strain LF3(1) after molecular characterization using 16S rRNA sequencing.

After in-vitro evaluation and molecular identification of the potential probiotics are namely, S7, S3, BDK2′, BDK7, BDK9, F1H4, F2F4, F3 1(2), F1F4, F1F2, F1H3 and LF3(1) were assessed for their efficacy by in-vivo evaluation against a potent pathogen Pseudomonas aeruginosa. The in-vivo study of the probiotic bacteria was carried out by using advanced fry of Cyprinus carpio (Common carp). The observations of in-vivo evaluation showed that bacterial strains serves as effective probiotics to enhance the growth, survival and immune responses in fish fed with probiotics compared to control and pathogen. From the in-vivo results of this study, it can be concluded that the administration of healthy and infected Cyprinus carpio with probiotic supplemented diet can improve the growth, immune parameters, and survival of fish against Pseudomonas aeruginosa infection. These probiotic strains can be used as a novel and safe treatment to fight the current issues in aquaculture. The present approach can be extended as an effective strategy to fight the current issues in aquaculture.

ACKNOWLEDGEMENT

Firstly, I would like to express my sincere gratitude to my supervisor Dr. Rahul Singh, Department of Zoology, Lovely Professional University, for the continuous support of my Ph.D. study and related research, for his patience, motivation, and immense knowledge. His guidance helped me in all the times of research and in writing this thesis. I could not have imagined having a better advisor and mentor for my Ph.D. study.

I extend my sincere thanks to Prof. Neeta Raj Sharma, Head of the School of Bioengineering and Bioscience, Dr. Joydeep Dutta, Head of the Department of Zoology, Dr. Amit Sehgal and all other faculty members of Zoology Department for their support throughout my work.

I take this opportunity to express heartfelt thanks to Mr. Ashok Mittal (Chancellor), Mrs. Rashmi Mittal (Pro-Chancellor), Dr. Rameshwar S. Kanwar (Vice Chancellor) and Dr. Loviraj Gupta (Executive Dean) for their motivation and support along with providing an opportunity to work in such a renowned university.

In particular, I am grateful to Dr. Abhineet Goyal, Head of Department of Quality Assurance, Academics, Lovely Professional University for supporting and motivating me in the initial stage of my Ph.D. I also want to give my special appreciation to our lab technician, Ms. Bhawna, Mr. Aman, Mr. Manoj, Mr. Gaurav, Mr. Rajesh, Mr. Sandeep, Mr. Onkar and Mr. Kuldip for providing support required at every stage of my research work.

I would like to extend my warm thanks to all my research colleagues and friends Mr. Vivek Sharma, Mr. Dharmender Sharma, Dr. Ravi Sharma, Mr. Rajesh, Mrs. Parvinder Kaur, Mrs. Jatinder Pal Kaur, Dr. Deepika Bhatia, Dr. Shivika Datta, Ms. Sumaya Farooq, Ms. Khushboo Guleria, Mrs. Prabhjot Kaur, Ms. Shaista Manzoor, Mr. Daljeet Singh, Mr. Aijaz Ahmed, Mrs. Satwant Kaur Mr. Anil and Ms. Dimple for their help, co-operation, and friendly environment in the lab and department. I would like to thank Dr. Simranjeet Singh and Ms. Durdana Sadaf Amin, for being a part of my life and teaching most important lessons in the life.

Also, I thank my friends Ankush, Devansh, Chiranshu, Amiteshwar, Vishu, Prairna, and Payel for their endless support and cheering me up during my low phase. I thank to Ms. Shubhrika Pansari for her support in thesis writing process. Also, I thank to my dearest friend, Dr. Rama Gaur, Assistant Professor, National Institute of Technology, Hamirpur for her endless support, without her I could never have been able to finish my thesis.

Last but not the least, I would like to thank my family, my parents, Shri Santram Chauhan and Smt. Omkali Devi and to my brother, Chetan Chauhan, sister-in-law, Mrs. Rajni Chauhan, sisters, Anuradha Chauhan and Meenakshi Chauhan, and my lovely nephew Nishchal Chauhan and Garv Chauhan, cousins, Ajesh Chauhan, Mukul Chauhan and Himanshi Chauhan for supporting me in every way throughout my life.

My deep thanks are also due to many people, whose names are not mentioned, but directly or indirectly helped me to complete my experimentation work and thesis. I bow my deep sense of gratitude to GOD with whose grace and blessings I have been able to infuse stability, dedication, management, patience and sincerity throughout this whole time of work.

ARUN CHAUHAN

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PREFACE

The present thesis focus on the development of probiotics from aquatic bodies for enhanced productivity in aquaculture. This work was conducted with the aim to isolate novel putative probiotics and the demonstrate their efficacy to fight against the fish infection (Pseudomonas aeruginosa), boost immunity and growth in juvenile Cyprinus carpio. Different fish species were collected from Doaba, Majha and Malwa region of Punjab, India. The bacterial strains were isolated and screened against selection criteria of potential probiotics by FAO/WHO guidelines (2002). The selected potential probionts were used as treatment of Pseudomonas aeruginosa infection in common carp. The study anticipates that the use of probiotics in aquaculture will help to reduce the mortality rate of fish at earlier stage and maximize the productivity of pond. The use of probiotics in aquaculture will control the cost of aquaculture production (cost effective) and increases valuable aqua-food production. Also, the probiotics are expected to serve as a better alternative and help to reduce the dependency on conventional antibiotics. The present approach can be extended as an effective strategy to fight the current issues in aquaculture.

In current research, investigation has been carried out on:

1. Isolation of putative probiotics has been carried out from different fish species of Punjab region.

2. All the isolated bacteria have been screened against the selection criteria of probiotics as per FAO/WHO guidelines (2002).

3. Biochemical and Molecular characterization of selected probionts.

4. In-vivo evaluation of bacteria as probiotics, given through oral feed.

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TABLE OF CONTENTS

S. No. Contents Page No.

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 8

2.1. Selection Criteria for Probiotics 10

2.2. Probiotics and Aquaculture 13

2.2.1. Antibacterial Activity 14

2.2.2. Antiviral Activity 14

2.2.3. Antifungal Activity 15

2.3. Mode of Action 23

2.3.1. Competition for Space / Blocking of Adhesion 24 Sites

2.3.2. Production of Inhibitory Substances 25

2.3.3. Competition for Nutrients 25

2.3.4. I mproving Water Quality 26

2.3.5. Disruption of Quorum Sensing 26

2.4. Methods of Administration of Probiotics 27

3. HYPOTHESIS 32

4. AIM AND OBJECTIVES 35

5. MATERIAL AND METHODS 37

5.1. Sample Collection from Different Sites 38

5.2. Isolation of the Bacteria from the Collected Samples 39

5.2.1. Preparation of Media 39

5.2.1.1. Media for Isolation of Bacteria 39

5.2.1.2. Media for Antimicrobial Activity 41

5.2.2. Isolation Methods 43

5.3. Screening and Indexing of Gram-positive Bacteria 43

5.4. In-vitro Evaluation of Probiotic Potential of Gram- 44 positive Bacteria

5.4.1. Antagonistic Activity 45

5.4.2. pH Tolerance 45

5.4.3. Bile Tolerance Test 45

5.4.4. Susceptibility to Drugs 46

5.4.5. Adhesion and Biofilm Formation 46

5.5. Identification of Bacteria 46

5.5.1. Biochemical Characterization 46

5.5.1.1. Catalase Test 47

5.5.1.2. Urease Test 47

5.5.1.3. Gelatin Test 47

5.5.1.4. Mannitol Fermentation Test 47

5.5.1.5. Triple Sugar Iron Agar Test 48

5.5.1.6. Motility Test 48

5.5.1.7. Indole Test 48

5.5.1.8. Methyl Red and Voges-Proskauer 48 Test

5.5.2. Molecular Characterization 49

5.5.2.1. Extraction of DNA 49

5.5.2.2. PCR Protocol 50

5.6. In-vivo Evaluation of Probiotics 52

5.6.1. Experimental Fish 52

5.6.2. In -vivo Experimental Setup 52

5.6.3. Acclimatization of Fish 52

5.6.4. Experimental Design 53

5.6.5. Water Quality 55

5.6.6. Feed Formulation 55

5.6.6.1. Selection of Feed 55

5.6.6.2. Preparation of Probiotic Feed 55

5.6.6.3. Compatibility Test 56

5.6.6.4. Multi-Strain Probiotic Feed 56

5.6.7. Challenge Test 56

5.6.8. Growth and Survival Indices 57

6. RESULTS AND DISCUSSION 58

6.1. Doaba Region 59

6.1.1. Fish Samples Collected from Doaba region 59

6.1.2. Isolation and Purification of Bacteria from 61 Fish Gut Sample

6.1.3. Screening and Indexing of Gram-positive 63 Bacteria

6.1.4. In -vitro Evaluation of Probiotic Potential of 66 Gram-positive Bacteria

6.1.4.1. Antagonistic Activity 66

6.1.4.2. pH Tolerance Test 69

6.1.4.3. Bile Salt Tolerance 72

6.1.4.4. Susceptibility to Drugs 74

6.1.4.5. Adhesion and Biofilm Formation 78

6.1.5. Identification of the Isolated Bacteria 80

6.1.5.1. Biochemical Characterization 80

6.1.5.2. Molecular Identification 83

6.2. Majha Region 87

6.2.1. Fish Samples Collected from Majha Region 87

6.2.2. Isolation and Purification of Bacteria from 87 Fish Gut Sample

6.2.3. Screening and Indexing of Gram-positive 89 Bacteria

6.2.4. In -vitro Evaluation of Probiotic Potential of 92 Gram-positive Bacteria

6.2.4.1. Antagonistic Activity 92

6.2.4.2. pH Tolerance Test 95

6.2.4.3. Bile Salt Tolerance 97

6.2.4.4. Susceptibility to Drugs 99

6.2.4.5. Adhesion and Biofilm Formation 101

6.2.5. Identification of the Isolated Bacteria 102

6.2.5.1. Biochemical Characterization 102

6.2.5.2. Molecular Identification 105

6.3. Malwa Region 109

6.3.1. Fish Samples Collected from Malwa Region 109

6.3.2. Isolation and Purification of Bacteria from 110 Fish Gut Sample

6.3.3. Screening and Indexing of Gram-positive 111 Bacteria

6.3.4. In -vitro Evaluation of Probiotic Potential of 113 Gram-positive Bacteria

6.3.4.1. Antagonistic Activity 113

6.3.4.2. pH Tolerance Test 114

6.3.4.3. Bile Salt Tolerance 115

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6.3.4.4. Susceptibility to Drugs 115

6.3.4.5. Adhesion and Biofilm Formation 116

6.3.5. Identification of the Isolated Bacteria 116

6.3.5.1. Biochemical Characterization 116

6.3.5.2. Molecular Identification 117

6.4. In-vivo Evaluation of Probiotics 119

6.4.1. In -vivo Evaluation of Probiotic Efficiency of 119 Isolated Bacterial Strains

6.4.1.1. Weight gain in the Fingerlings of 120 Cyprinus carpio

6.4.1.2. Survival of Cyprinus carpio 127 Fingerlings

6.4.1.3. Food Conversion Ratio (FCR) in 131 Cyprinus carpio Fingerlings

6.4.1.4. Specific Growth Rate (SGR) of 135 Cyprinus carpio Fingerlings

6.4.2. Consortia Formulation and In-vivo 142 Evaluation of its Probiotic Efficiency

7. SUMMARY AND CONCLUSION 145

BIBLIOGRAPHY 151

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LIST OF TABLES

Table No. Content Page No.

2.1 Probiotic species used in aquaculture, source and beneficial 16-22 effects to the host species.

5.1 The composition of de Man, Rogosa and Sharpe (MRS) agar. 40

5.2 The composition of Bacillus Differentiation Agar (BDA). 41

5.3 The composition of Nutrient agar. 42

5.4 The composition of Mueller Hinton (MH) agar. 42

5.5 PCR Conditions (different stages, temperature and time) for 51 the molecular identification.

6.1 Nomenclature of the Gram-positive bacteria isolated from 65-66 Doaba region and name of the source fish species.

6.2 Zone of inhibition (in mm) of different bacterial isolates 69 derived from Doaba region showing antagonistic activity against Pseudomonas aeruginosa (MTCC 4673).

6.3 pH test results showing optical density values at 600 nm as a 72 measure of survival of the bacterial strains isolated from Doaba region and cultured under different pH medium (pH 2.0 to 9.0).

6.4 Bile tolerance results showing optical density values (at 600 74 nm) as a measure of survival of isolates after 24 h in presence and absence of bile salts (0.3 % ox-gall concentration).

6.5 The resumes of susceptible (S) and resistance (R) showing 77 antibiotic susceptibility test of the selected isolates against various drugs, methicillin, amoxicillin, penicillin-G, chloramphenicol, neomycin, ampicillin, vancomycin, tetracycline and kanamycin.

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6.6 Optical density values (at 600 nm) showing adhesion and 79 biofilm formation property of different isolated strains as evaluated by standard tube method.

6.7 The resumes of positive (+) and negative (-) results of isolated 82 bacterial strains against biochemical characterization test series.

6.8 The sequence length (bp), similarity index, identified species 86 and accession numbers of isolated bacteria after molecular identification (16S rRNA sequencing).

6.9 Nomenclature of the Gram-positive bacteria isolated from 91-92 Majha region and the name of source fish species.

6.10 Zone of inhibition (in mm) of different bacterial isolates 94 derived from Majha region showing antagonistic activity against Pseudomonas aeruginosa (MTCC 4673).

6.11 pH test results showing optical density values at 600 nm as a 97 measure of survival of the bacterial strains isolated from Majha region and cultured under different pH medium (pH 2.0-9.0).

6.12 Bile tolerance results showing optical density values (at 600 98 nm) as a measure of survival of isolates after 24 h in presence and absence of bile salts (0.3 % ox-gall concentration).

6.13 The resumes of susceptible (S) and resistance (R) showing 100 antibiotic susceptibility test of the selected isolates against various drugs, methicillin, amoxicillin, penicillin-G, chloramphenicol, neomycin, ampicillin, vancomycin, tetracycline and kanamycin.

6.14 The optical density values (at 600 nm) showing adhesion and 102 biofilm formation property of different isolated strains as evaluated by standard tube method.

6.15 The resumes of positive (+) and negative (–) results of isolated 104 bacterial strains against biochemical characterization test series.

6.16 The sequence length (bp), similarity index, identified species 108 and accession numbers of isolated bacteria after molecular identification (16S rRNA sequencing).

6.17 Nomenclature of the Gram-positive bacteria isolated from 113 Malwa region and the name of source fish species.

6.18 Details of different test groups designed to evaluate the 120 probiotic efficiency through in-vivo studies.

6.19 Average weight, % weight gain and total weight gain % of 124-126 Cyprinus carpio of different groups after 15 days, 30 days, 45 days and 60 days of study.

6.20 Growth parameters i.e. specific growth rate (SGR), food 138-140 conversion ratio (FCR), % survival of Cyprinus carpio after 15 days, 30 days, 45 days and 60 days of administering with the basal and probiotic supplemented feed.

6.21 Overall growth in terms of Growth parameters i.e. specific 141-142 growth rate (SGR), food conversion ratio (FCR), and % survival of Cyprinus carpio after 60 days of administering with the basal and probiotic supplemented feed.

6.22 The resumes of positive (+) and negative (–) results of 143 compatibility test of the potential probiotic strains.

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LIST OF FIGURES

Figure Page Content No. No.

2.1 Schematic representation of the screening process for the selection 12 of isolated bacteria as ideal probiotics.

2.2 Mode of action of probiotics. 24

5.1 Map of Punjab state showing different regions i.e. Doaba, Majha 38 and Malwa separated by rivers.

5.2 Scheme for Gram staining procedure. 44

5.3 Schematic representation of the experimental setup and design. 54

6.1 Geographical map of Punjab showing different regions, the 60 highlighted region shows Doaba region, this region has been selected for collection of fish samples.

6.2 Digital image of different fish species i.e. (a) Chanda nama, (b) 61 Chanda ranga, (c) Puntius ticto, (d) Nandus nandus, (e) Fresh water shrimps, (f) Chanda nama, (g) Oxygaster bacaila, (h) Labeo gonius, (i) Puntius chola, (j) Nandus nandus, (k) Puntius sophore, (l) Oxygaster bacaila, (m) Gobius viridipunctatus, (n) Ompok bimaculate, (o) Puntius ticto, (p) Chanda nama, (q) Gudusia chapra, (r) Channa punctata, (s) Labeo dero, (t) Colisa fasciatus, (u) Labeo rohita and (v) Catla catla collected from Doaba region of Punjab, India.

6.3 Digital images of pure isolated cultures streaked on (a-i) de-Man, 62 Rogosa and Sharpe agar (MRS) and (j-l) Bacillus differentiation agar (BDA) showing bacterial colonies isolated from gastrointestinal tract of healthy fish collected from Doaba region, Punjab.

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6.4 Digital microscope images showing typical Gram-positive 64 morphology of different bacteria isolated from gastrointestinal tract (GIT) of fish under oil-immersion microscope.

6.5 Digital image of (a-f) culture plates showing zone of inhibition by 67 the different isolates and (g-i) culture plates showing no zone of inhibition, against the fish pathogen Pseudomonas aeruginosa (MTCC 4673).

6.6 Zone of inhibition (in mm) plot of different bacterial isolates 68 derived from Doaba region indicating their antagonistic activity against the selected pathogen, Pseudomonas aeruginosa (MTCC 4673). Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post- hoc test) with respect to antagonistic activity of different isolates.

6.7 The optical density (OD at 600 nm) indicating growth and survival 71 of bacterial strains isolated from Doaba region in MRS broth medium, adjusted at different values of pH (2.0, 3.0, 4.0 and 9.0) to study the effect of pH on the growth and survival. Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) in bacterial strains at same pH.

6.8 The optical density (at 600 nm) showing tolerance of isolated 73 bacterial strains in presence and absence of bile salts (0.3% ox-gall). Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post- hoc test) in between with bile or without bile groups.

6.9 Digital image of representative culture plates (a-d) showing 76 antibiotic susceptibility of the isolated strains against various drugs,

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methicillin, amoxicillin, penicillin-G, chloramphenicol, neomycin, ampicillin, vancomycin, tetracycline and kanamycin.

6.10 The optical density plot (absorbance at 600 nm) indicating adhesion 79 and biofilm formation of the isolated strains evaluated by standard tube method. Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post- hoc test) among the adhesion property of different isolates.

6.11 Digital image showing different reaction of biochemical tests 81 including catalase test, urease test, gelatin test, mannitol salt agar test, motility test, indole test, glucose fermentation, methyl red and Voges-Proskauer test of the isolated strains. Figure (a-b) show control reaction (blank) and (c-f) show biochemical reaction in the presence of bacterial isolates.

6.12 Phylogenetic tree showing relationship between Enterococcus 84 durans strain S3 and other Enterococcus species.

6.13 Phylogenetic tree showing relationship between Bacillus 84 amyloliquefaciens strain S7 and other Bacillus species.

6.14 Phylogenetic tree showing relationship between Bacillus cereus 84 strain BDK2' and other Bacillus species.

6.15 Phylogenetic tree showing relationship between Bacillus subtilis 85 strain BDK7 and other Bacillus species.

6.16 Phylogenetic tree showing relationships between Bacillus subtilis 85 strain BDK9 and other Bacillus species.

6.17 Geographical map of Punjab showing different regions, the 87 highlighted region shows Majha region, this region has been selected for collection of fish samples.

6.18 Digital images of pure isolated cultures streaked on (a-i) de-Man, 88 Rogosa and Sharpe agar (MRS) and (j-o) Bacillus differentiation

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agar (BDA) showing bacterial colonies isolated from gastrointestinal tract of healthy fish collected from Majha region, Punjab.

6.19 Digital microscope images showing typical Gram-positive 90 morphology of different bacteria isolated from gastrointestinal tract (GIT) of fish under oil-immersion microscope.

6.20 The digital image of culture plates (a-f) showing zone of inhibition 93 by the different isolates against the fish pathogen Pseudomonas aeruginosa (MTCC 4673).

6.21 Zone of inhibition (mm) plot of different bacterial isolates derived 94 from Majha region indicating their antagonistic activity against the selected pathogen, Pseudomonas aeruginosa (MTCC 4673). Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) with respect to antagonistic activity of different isolates.

6.22 The optical density (OD at 600 nm) indicating growth and survival 96 of bacterial strains isolated from Majha region in MRS Broth medium, adjusted at different values of pH (2.0, 3.0, 4.0 and 9.0) to study the effect of pH on the growth and survival. Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) in bacterial strains at same pH.

6.23 The optical density (at 600 nm) showing tolerance of isolated 98 bacterial strains in presence and absence of bile salts (0.3% ox-gall). Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post- hoc test) in between with bile or without bile groups.

6.24 Digital image of culture plates (a-d) showing antibiotic 99 susceptibility of the isolated strains against various drugs, methicillin, amoxicillin, penicillin-G, chloramphenicol, neomycin, ampicillin, vancomycin, tetracycline and kanamycin.

6.25 The optical density plot (absorbance at 600 nm) indicating adhesion 101 and biofilm formation of the isolated strains evaluated by standard tube method. Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post- hoc test) among the adhesion property of different isolates.

6.26 Digital image showing different reaction of biochemical tests 103 including catalase test, urease test, gelatin test, mannitol salt agar test, motility test, indole test, glucose fermentation, methyl red and Voges-Proskauer test of the isolated strains. Figure (a-b) show control reaction (blank) and (c-f) show biochemical reaction in the presence of bacterial isolates.

6.27 Phylogenetic tree showing relationships between Bacillus subtilis 106 strain F1F4 and other Bacillus species.

6.28 Phylogenetic tree showing relationships between Enterococcus 106 faecium strain F1H4 and other Enterococcus species.

6.29 Phylogenetic tree showing relationships between Bacillus safensis 106 strain F2F4 and other Bacillus species.

6.30 Phylogenetic tree showing relationships between Bacillus subtilis 107 strain F3 1(2) and other Bacillus species.

6.31 Phylogenetic tree showing relationships between Bacillus 107 velezensis strain F1F2 and other Bacillus species.

6.32 Phylogenetic tree showing relationships between Enterococcus 107 gallinarum strain F1H3 and other Enterococcus species.

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6.33 Geographical map of Punjab showing different regions, the 109 highlighted region shows Malwa region, the region has been selected for collection of fish samples.

6.34 Digital image of different fish species i.e. (a) Labeo derio, (b) 110 Rasbora daniconius, (c) Puntius sarana, (d) Chanda nama, (e) Xenentodon cancila, (f) Oxygaster bacaila, (g) Chanda ranga, (h) Labeo gonius, (i) Cyprinus carpio, (j) Oxygaster bacaila and (k) Channa striatus collected from Malwa region of Punjab, India.

6.35 Digital images of pure isolated cultures (a-d) streaked on MRS agar 111 showing bacterial colonies isolated from gastrointestinal tract of healthy fish collected from Malwa region, Punjab.

6.36 Digital microscope images showing typical Gram-positive 112 morphology of different bacteria isolated from gastrointestinal tract (GIT) of fish under oil-immersion microscope.

6.37 The digital image of culture plates (a) showing zone of inhibition 114 by LF3(1) against the fish pathogen Pseudomonas aeruginosa (MTCC 4673) and (b) antibiotic susceptibility of LF3(1) against various drugs, methicillin, amoxicillin, penicillin-G, chloramphenicol, neomycin, ampicillin, vancomycin, tetracycline and kanamycin.

6.38 The optical density (OD at 600 nm) indicating growth and survival 115 of bacterial strains isolated from Malwa region in MRS Broth medium, adjusted at different values of pH (2.0, 3.0, 4.0 and 9.0) to study the effect of pH on the growth and survival.

6.39 Digital image showing different reaction of biochemical tests 117 including catalase test, urease test, gelatin test, mannitol salt agar test, motility test, indole test, glucose fermentation, methyl red and Voges-Proskauer test of the isolated strain. Figure (a-b) show

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control reaction (blank) and (c) shows biochemical reaction in the presence of bacterial isolates.

6.40 Phylogenetic tree showing relationships between Enterococcus 118 faecium strain LF3(1) and other Enterococcus species.

6.41 Overall % weight gain in fish of probiotic group and probiotic with 122 pathogen group after 60 days of feeding with probiotic supplemented feed i.e. Bacillus amyloliquefaciens strain S7, Enterococcus durans strain S3, Bacillus cereus strain BDK2′, Bacillus subtilis strain BDK7, Bacillus subtilis strain BDK9, Enterococcus faecium strain F1H4, Bacillus safensis strain F2F4, Bacillus subtilis strain F3 1(2), Bacillus subtilis strain F1F4, Bacillus velezensis strain F1F2, Enterococcus gallinarum strain F1H3 and Enterococcus faecium strain LF3(1). The control and pathogen show the survival of fish which were administered with basal diet. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) in control, pathogen, probiotic and probiotic with a pathogen groups.

6.42 The % weight gain in fish at different interval of time i.e. 15 day, 123 30 days, 45 days and 60 days of the treatment of (a) fish fed with probiotic supplemented feed and (b) fish introduced with pathogen and fed with probiotic supplemented feed. The control and pathogen show the survival of fish which were administered with basal diet.

6.43 Digital image of fish showing toxicity (infection) of Enterococcus 127 durans S3 supplemented feed.

6.44 The % survival of fish in probiotic group and probiotic with 129 pathogen group after 60 days of feeding with probiotic supplemented feed i.e. Bacillus amyloliquefaciens strain S7, Enterococcus durans strain S3, Bacillus cereus strain BDK2′, Bacillus subtilis strain BDK7, Bacillus subtilis strain BDK9,

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Enterococcus faecium strain F1H4, Bacillus safensis strain F2F4, Bacillus subtilis strain F3 1(2), Bacillus subtilis strain F1F4, Bacillus velezensis strain F1F2, Enterococcus gallinarum strain F1H3 and Enterococcus faecium strain LF3(1). The control and pathogen show the survival of fish which were administered with basal diet.

6.45 The % survival of fish at different interval of time i.e. 15 day, 30 130 days, 45 days and 60 days of the treatment of (a) fish fed with probiotic supplemented feed and (b) fish introduced with pathogen and fed with probiotic supplemented feed. The control and pathogen show the survival of fish which were administered with basal diet.

6.46 Visual comparison of fish subjected to Pseudomonas aeruginosa 131 infection, fed with basal diet and probiotic supplemented diet. Fish fed with basal diet show symptoms of hemorrhagic septicaemia.

6.47 Overall food conversion ratio of fish in probiotic group and 133 probiotic with pathogen group after 60 days of feeding with probiotic supplemented feed i.e. Bacillus amyloliquefaciens strain S7, Enterococcus durans strain S3, Bacillus cereus strain BDK2′, Bacillus subtilis strain BDK7, Bacillus subtilis strain BDK9, Enterococcus faecium strain F1H4, Bacillus safensis strain F2F4, Bacillus subtilis strain F3 1(2), Bacillus subtilis strain F1F4, Bacillus velezensis strain F1F2, Enterococcus gallinarum strain F1H3 and Enterococcus faecium strain LF3(1). The control and pathogen show the food conversion ratio of fish which were administered with basal diet. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) in control, pathogen, probiotic and probiotic with a pathogen groups.

6.48 The food conversion ratio in fish at different interval of time i.e. 15 134 day, 30 days, 45 days and 60 days of the treatment of (a) fish fed

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with probiotic supplemented feed and (b) fish introduced with pathogen and fed with probiotic supplemented feed. The control and pathogen show the food conversion ratio of fish which were administered with basal diet.

6.49 Overall specific growth rate of fish in probiotic group and probiotic 136 with pathogen group after 60 days of feeding with probiotic supplemented feed i.e. Bacillus amyloliquefaciens strain S7, Enterococcus durans strain S3, Bacillus cereus strain BDK2′, Bacillus subtilis strain BDK7, Bacillus subtilis strain BDK9, Enterococcus faecium strain F1H4, Bacillus safensis strain F2F4, Bacillus subtilis strain F3 1(2), Bacillus subtilis strain F1F4, Bacillus velezensis strain F1F2, Enterococcus gallinarum strain F1H3 and Enterococcus faecium strain LF3(1). The control and pathogen show the specific growth rate of fish which were administered with basal diet. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) in control, pathogen, probiotic and probiotic with a pathogen groups.

6.50 The Specific Growth Rate (SGR) of fish at different interval of time 137 i.e. 15 day, 30 days, 45 days and 60 days of the treatment (a) fish fed with probiotic supplemented feed and (b) fish introduced with pathogen and fed with probiotic supplemented feed. The control and pathogen show the specific growth rate of fish which were administered with basal diet.

7.1 Summary of the sequential in-vitro evaluation of isolates for 148 screening of potential probiotic bacterial strains and their molecular identification.

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Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Chapter 1 Introduction

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

INTRODUCTION

The world’s population is increasing day-by-day and has reached to 7.6 Billion as of 2018. There has been an addition of one billion people to the world population over a span of last 12 years. (World Population Prospects, 2018). The world population is expected to increase in later decades and would reach a peak of 10 billion towards the end of 21st century. By 2070, the world population will be ten times as that of in year 1800 (Population in year 1800 was approximately one billion) (Bongaarts, 2009).

With the rise of world population to 10 billion, there will be an increase in the demand for food in the next decades. We are primarily dependent on land for the consumable goods. About 1/4th of Earth’s land is under cultivation. Also, the various inevitable changes such as, land degradation, climate change and pollution will make very difficult to meet the increasing demands of food. To meet the increasing food demands, there has been an increasing stress on nature for cultivation of more agricultural crops. This is perilous for the global community. The increasing stress on agriculture can be decreased to some extent by deriving food from some other sources, like aquaculture (Tidwell and Allan, 2001; Reantaso et al., 2012).

Aquaculture is an important and rapidly growing sector as it plays an important role to achieve global protein food demand compared to capture fisheries and terrestrial farmed meat. The role of aquaculture to improve the socio-economic status of any region is highly appreciable because it is not only limited to the source of essential nutrients, but it also generates various employment opportunities (Araujo et al., 2015; Handbook on Fisheries Statistics 2014). India ranks second in the world after China in fish production through aquaculture with a contribution of 6.3% of the global aqua production, which is less as compared to that of China (60.5%) (Chavan 2018; Mo et al., 2018).

Fish are dominant in aqua products, and around 200 fish species are produced for their commercial value. Indian major carps i.e. Labeo rohita, Cirrhinus mrigala and Catla catla are rich source of protein (Swapna et al., 2010). Due to diversity and nutritional

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

value, fisheries attract the attention of farmers, researchers or fisheries engineers to improve quality and productivity. According to statistics, aquaculture provides 48% of aquatic animal food for consumption by humans, fish being an important source in it. According to FAO (Food and Agriculture Organization), fish provides about 16% of animal protein which is consumed by humans. About one billion population worldwide depend upon fish as their main source of animal protein (Tidwell and Allan, 2001). There has been an increase of more than 40% of fish production over the past years due to an increase in the demand for fish food, so that it can meet the growing population and health considerations. Therefore, to maintain global food security for the increasing population, there is a need to increase the production of fish and fish products (Reantaso et al., 2012).

Meanwhile, as the world population is increasing by day, it is essential to enhance the production of aqua food products (Sahoo et al., 2015). The production of fish has been increased by intense culture practices, high stocking densities, intense feeding and fertilization. This led to decay of biotic materials which causes stress, health issues and degrade the water quality. The intensification in aquaculture industries has led to disease outbreak and became a major problem in aquaculture. This had an adverse effect on the aquaculture industries (Sunitha and Padmavathi, 2013).

The intimate contact of fish to aquatic habitat makes them vulnerable to various external hazards. Microbial infection in fish deviates their energy towards development of immunity and maintenance of health. Hence it is considered as a major limiting factor in intensive culture practice. Although the bacterial growth is facilitated by the fish body serving as a platform for their growth. But, because of strong immune response, major problems do not appear in adult fish. But, if the amount of contamination and pond fertility is high, there is an emergence of pathogen or bacterial attack. Pathogenic bacteria are able to multiply their population in aquatic habitat very rapidly. During the process of osmoregulation and feeding, fish continuously ingest the pathogenic bacteria present in their environment (Hansen and Olafsen, 1999). The growth and multiplication of pathogenic bacteria is also affected by the slurry water.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

The slurry water enhance the growth of pathogenic bacteria and causes disease. This results in reduction of aquaculture productivity (Sunitha and Padmavathi, 2013).

The first requirement of a pathogenic bacterium is to penetrate the primary barriers so that they can establish the infection. In fish, microorganisms can present in body of the host in three distinct ways: a) gill, b) skin and c) gastrointestinal tract. In uninfected tissue, bacteria can spread by transcellular or paracellular translocation. On the other hand, bacteria emit some toxins or extracellular enzymes before entering that can harm the intestinal lining.

Over the last two decades, the contamination rate has increased (Ringo et al., 2010). The most common freshwater diseases are dropsy, tail rot, fin rot, gill diseases, white spot disease, cloudy eye, pop eye, tuberculosis, water quality induced diseases, etc. (Sharma et al., 2012). The most dominant pathogenic bacteria in fish are Aeromonas hydrophila, Pseudomonas aeruginosa, Flavobacterium columnare, Proteus species, Micrococcus species and vibrio species. These microbes are major infectious aquatic pathogens and are causative agents of fin rot, ulcers, tail rot, hemorrhagic septicaemia and so on. Common symptoms of these diseases are related to the lesions, which include septicaemia (presence of bacterial toxins in various organs) and ulcers on fish’s skin, which cause severe damage to fish (Bisht et al., 2014; Swann and White, 1991; Watson et al., 2008; Declercq et al., 2013). Infectious diseases caused by the pathogens like Aeromonas and Pseudomonas species pose great threat to aquaculture industries due to increased mortality of fish. To encounter these diseases anti-microbial drugs are used. Mostly antibiotics like chloramphenicol, streptomycin, neomycin, prefuran, erythromycin, ciprofloxacin and oxytetracycline have been applied in treatment for diseases (Supriyadi and Rukyani, 2000). Farmers have been using these chemicals and antibiotics to control diseases, but their side effects have been observed over time (Atienza et al., 2013; Azevedo et al., 2015).

These chemotherapeutic drugs are standard treatment for bacterial infections. However, due to the extensive use of antibiotics, their residue remain for a longer time in aquatic

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

environment. This has various adverse consequences such as the increased resistance in pathogenic bacteria towards antibiotics, the appearance of antibiotic residues in the fish body and change in aquatic microbiota (Defoirdt et al., 2007; Atienza et al., 2013; Azevedo et al., 2015). Also, the transference of the antibiotic resistance from aquatic environment to human beings is a menace (Watson et al., 2008).

Therefore, the use of chemotherapeutic drugs as a control measure of the disease has become questionable. This has led to the search for an alternative of antibiotics and vaccines to prevent fish diseases (McEwen and Fedorka, 2002).

A propitious alternative approach to prevent disease in fish is the use of probiotics or good micro-organisms. Probiotics improve the fish defence against pathogenic bacteria by different mechanisms and enhance their physical health. The term “probiotic” constitutes of Greek words “pro” and “bios” that means “for life,” (Gismondo et al., 1999). A Russian analyst, Elie Metchnikoff pioneered the concept of probiotics. He observed that the Bulgarian workers who consumed fermented milk products had a long life (Metchnikoff, 1907). Later in 1965, Lilly and Stillwell explained the concept of probiotics, as a substance which accelerates the growth of good microbes. Parker (1974) gave the definition of probiotics as “organisms and substances, which add to intestinal balance”. Fuller (1992) refined the definition as, “A live microbial feed supplement that beneficially affects the host by improving its intestinal microbial balance”. This definition signifies that the living bacterial cells are an imperative part of potential probiotics and clarifies the confusion created using term “substance”. In 2001, WHO has termed probiotics as “live microbes, which when administered in sufficient amount, confer a health benefit to the host.”

Probiotics are feed additives with no pollution or residue, and they are not only limited to gastrointestinal (GI) tract but also act as growth promoter, prevent disease, enhance immune response etc. (Meidong et al., 2018; Gobi et al., 2018; Ramesh and Souissi, 2018). The modes of action of probiotics are: i) to maintain healthy intestinal microflora through antagonism against pathogens, ii) to improve metabolism by escalating

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

digestive enzyme activity, iii) to improve feed uptake and digestion, and iv) to neutralize the entero-toxins and stimulate the immune system (Oelschlaeger, 2010). The use of probiotics is gaining more attention as a new approach for biological control method due to its environment-friendly and immunogenic nature.

Probiotics for aquatic practice are different from those of terrestrial environment. Aquatic animals have a close interaction with the extrinsic environment. Probiotics enhance the growth of fish by increasing their feed conversion efficiency. The growth rate of marine larvae is increased due to the use of probiotics by yielding cell products and micronutrients like essential fatty acids, vitamins, minerals or even enzymes. When added to the feed of rohu fingerlings, probiotics provide benefits like disease resistance against pathogens, growth enhancement and immune stimulation.

In addition to this, the growth of microbiota is enhanced, harmful pathogens are inhibited and body’s natural defences are strengthened by them. Pathogens are inhibited by the production of inhibitory substances (Gohila et al., 2013). Probiotics and their positive effects are a field of extensive research. Although clinical studies on probiotics have already been initiated, yet this field is still considered in its infancy. Therefore, a wide range of study can be done on probiotics (Rijkers et al., 2010). Probiotics have opened a new era of disease control in aquaculture (Sahoo et al., 2015).

Probiotics were used to enhance the production in poultry and animal husbandry in the past (Harimurti and Hadisaputro, 2015; Uyeno et al., 2015; Jiang et al., 2017). A frequent utilization of commercial probiotics in aquaculture has been observed in the past decades. However, the use of commercially available probiotics in fish were found to be somewhat futile as compared to their use in animals. The isolation of commercial probiotics strains from non-fish sources is a reason for their unsatisfactory performance, as they might not remain viable in the intestine of the fish (Moriarty, 1996; Dutta and Ghosh, 2015). So, there is a strong necessity to isolate putative probiotic bacterial strains from the same environment such as gut of same or similar animal group in which it was planned for use. Such probiotic bacteria would perform better during the

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

adhesion to the intestinal surface and in preventing the adhesion of the pathogenic bacteria.

The present study focus on the isolation and identification of novel putative probiotic strains from the intestine of different teleost. Also, it evaluates their potentiality as a potent probiotic by using different in-vitro and in-vivo selection parameters given in FAO/WHO guidelines (2002) for the evaluation of probiotics and their use in fish feed.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Chapter 2 Review of Literature

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

REVIEW OF LITERATURE

The shift towards the aquaculture to meet increasing food demands of growing population has resulted in intensive aquaculture practice. With commercialization of fish farming, diseases have become a hurdle in the aquaculture industry (Hai, 2015). The most common disease-causing bacterial pathogens among aquaculture are gram- negative such as, Aeromonas, Flavobacterium, Pseudomonas, Vibrio and Yersinia species. These pathogens are etiological agents of various diseases like, enteric red mouth disease, furunculosis, hemorrhage, septicaemia, vibriosis and so on (Hamid et al., 2017; Cascales et al., 2016; Patra et al., 2016; Wiklund, 2016; Ronneseth et al., 2017). The use of chemotherapeutic drugs has served as an option to cure common diseases prevailing in fish farming (Hambali and Akhmad, 2000).

In aquaculture, chemotherapeutic agents like antibiotics and chemicals are the classical cure for microbial infection. However, the extensive usage of these chemotherapeutic drugs leads to their accumulation in aquatic habitat and results in harmful consequences such as emergence of antibiotic-resistant bacteria, accumulation of antibiotic residues in the flesh, kill the beneficial microbes of the gastrointestinal tract and alterations in microbiota (effect on non-target microbes) of the aquatic environment (Estefania et al., 2013; Azevedo et al., 2015). Therefore, the use of antibiotics as chemotherapeutic drugs in aquaculture has become unsafe (Balcazar et al., 2008; Mancuso et al., 2015; Balcazar et al., 2006). The quest for better alternatives to prevent infection and replace the antibiotics has been a major concern now-a-day.

Use of probiotics to prevent disease in fish is a promising and emerging alternative approach. Probiotics help the fish to fight against pathogens through various mechanisms. The importance of probiotics used in aquaculture is not only limited to gastrointestinal tract, but it also plays a major role in the improvement of overall health of an organism (Mehrabi et al., 2018) such as: it acts as growth promoter (Gobi et al., 2018), prevents the diseases (Meidong et al., 2018), enhances the immune response (Ramesh et al., 2018) and improves the water quality by modifying microbial community of water and sediments (Verschuere et al., 2000; Deng et al., 2018). In ponds, nitrogenous contaminants like ammonia and nitrate have become a serious

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

concern. Previous reports show that the use of Lactobacillus species as probiotics removes the nitrogenous waste from the ponds and use of Bacillus species improves the water quality by converting organic carbon to slime (Ma et al., 2009; Verschuere et al., 2000; Kolndadacha et al., 2011). Khattab et al., 2005 have reported the use of Micrococcus luteus as probiotics which resulted in increased growth performance and improved feed conversion ratio (FCR) in Tilapia (Oreochromis niloticus). Sakata, 1990 and Ringo et al., 1995 have demonstrated the role of Bacteroides species, Clostridium species, Agrobacterium species, Brevibacterium species and Microbacterium species as nutritional sources to the host by supplying fatty acids and vitamins.

Probiotics protect the host organism from pathogenic bacteria by liberating metabolites like bacteriocins and different organic acids. These metabolites hinder the adhesion of different pathogens and inhibit them by limiting the available resources such as attachment site and nutrients (Vine et al., 2004; Servin and Coconnier, 2003). Probiotics have the potential to improve the host’s defenses, including the innate and acquired immunity system. This is important for the prevention and treatment of infectious diseases and to cure inflammation in the digestive tract. Probiotics also have a direct influence on other microbes, either commensal or pathogenic, which is very important for the prevention, treatment and restoration of the bacterial equilibrium inside the gut of the host (Oelschlaeger, 2010). The use of probiotics in humans, pigs, steers and poultry has already been studied, but the use of probiotics in aquaculture is relatively a new concept (Daniel, 2017; Chua et al., 2017; Jiang et al., 2017; Harimurti and Hadisaputro, 2015; Uyeno et al., 2015).

2.1. Selection Criteria for Probiotics

The main objective of probiotics is to create a healthy relationship between beneficial and unhealthy bacteria present in gastrointestinal tract of the fish (Thirumurugan and Vignesh, 2015; Olsson et al., 1992).

Till date, most of the reports are focused on assessment of probiotics based on their ability to produce antimicrobial substances, but the ability of bacteria to compete for attachment sites and their adhesion properties have been rarely performed (Alonso et al., 2018; Mancuso et al., 2015; Araujo et al., 2015). The adhesion property is an

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

essential criterion to be checked because the bacteria may not able to produce metabolites inside the gut due to their constant flushing (Vine et al., 2004). Thus, the positive outcome in in-vitro study may not able to inhibit pathogens in in-vivo conditions. The ineptitude of probiotics to compete for attachment sites of gastrointestinal tract suggest that these probiotics may not proliferate significantly to balance their population as they are flushed during gut evacuation. Therefore, the screening of probiotic candidates must have the selection criteria such as antagonism against pathogens, acid and bile tolerance, drug susceptibility, adhesion and bio-film formation.

Effective probiotics should possess certain qualities elucidate by different workers (Olsson et al., 1992; Pandya, 2016; Gatesoupe, 1999; Ouwehand et al., 1999a & b; Holzapfel and Schillinger, 2002; Fuller, 1989), which are specified underneath.

1. The probiotics should have a beneficial effect on the growth, development and protection of fish against various pathogenic bacteria. 2. The probiotic bacteria should not have any harmful effect on the host. 3. The probiotics should not have the ability of drug resistance, they should have the ability to keep up the hereditary traits. 4. For the utilization of probiotics as an efficient feed, they should exhibit following properties: • pH and bile tolerance • Resistance to gastric juices • Adherence to digestive system surface • Antagonism towards pathogens • Stimulation of the immunity • Increase in the gut motility • Survival in mucous • Production of enzymes and vitamins 5. They should have good sensorial properties, fermentative action, tolerance towards freeze-drying and viability in feed during packaging and storing process.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Bacteria isolated from different sources are subjected to screening through multiple steps in order to assess their potential as ideal probiotics. The screening process involves gram staining, indexing, in-vitro evaluation of antagonistic properties, acid tolerance, bile tolerance, susceptibility to drugs and biofilm formation. Figure 2.1 shows the sequential screening process for the selection of isolated bacteria as probiotics. Successful fulfilment of all criteria qualifies them as potential probiotic fit for use in the aquaculture.

Figure 2.1: Schematic representation of the screening process for the selection of isolated bacteria as ideal probiotics (Chauhan and Singh, 2018).

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

2.2. Probiotics and Aquaculture

Usually the aquatic probiotics are commercially available in two major forms- dry and liquid. Dry form has higher shelf life and are mixed with water or feed which is given to the host. On the other hand, the liquid form of probiotics, usually preferred in egg hatcheries is directly blended with the feed or added to the tanks (Decamp and Moriarty 2007). The liquid forms of probiotics are reported to show better and positive results due to their lower density than spore and dry form of probiotics (Nageswara and Babu, 2006).

The aquatic probiotics can be further categorized into two classes based on their mode of administration. First one involves the mixing of probiotic bacteria with feed supplement for the enhancement of useful bacteria inside the gut. Second class involves the addition of probiotic directly to the water so that they can consume nutrients available in the water and inhibit the proliferation of pathogens. These two categories of probiotics were used in finfish and shrimp aquaculture (Sahu et al., 2008; Nageswara and Babu, 2006).

The probiotics isolated from different natural sources such as gastrointestinal tract (GIT), stomach, gill, kidney, gonads and other internal organs are called putative probiotics. In contrast, the commercial sources (non-putative) comprise of those which are already synthesized and commercially available in the market. The most frequently used probiotic microorganisms belong to Bacillus, Lactobacillus and Bifidobacterium genus (Nwanna, 2010). Various species of Lactobacillus, Bifidobacterium and Streptococcus reported for use in aquaculture as probiotics, include L. acidophilus, L. casei, L. fermentum, L. gasseri, L. plantarum, L. salivarius, L. rhamnosus, L. johnsonii, L. paracasei, L. reuteri, L. helveticus, L. bugaricus, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium longum, Saccharromyces species, Saccharromyces boulardii, S. thermophiles and S. cremoris (Nwanna, 2010).

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Various aquatic probiotics have been reported which show activity not only against bacterial pathogen, but also against fungus and virus to improve growth and immunity of the host.

2.2.1. Antibacterial Activity

Many probiotics used in aquaculture are well-known for their antibacterial property against known pathogens. Lactococcus lactis RQ516 probiotic shows inhibitory action against Aeromonas hydrophila when given to Tilapia (Oreochromis niloticus) (Zhou et al., 2010). Also L. lactis probiotic has antibacterial activity against two pathogens- Yersinia rukeri and Aeromonas salmonicida that affects the fish growth (Balcazar et al., 2007). Leuconostoc mesenteroides has the potential to inhibit the fish pathogens (Pseudomonas aeruginosa T3, Pseudomonas putida T4, Vibrio harveyi T34 and Mycobacterium marinum T217) found in Nile tilapia (O. niloticus) (Zapata and Lara- Flores, 2012). According to reports, Bacillus subtilis considerably reduces the motile Aeromonads, total Coliforms and Pseudomonads found in ornamental fish i.e. Poecilia reticulata (Peters), Poecilia sphenops (Valenciennes), Xiphophorus helleri (Heckel) and Xiphophorus maculatus (Gunther) (Ghosh et al., 2008; Newaj-Fyzul and Austin, 2015). Lactic acid bacteria such as Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus fermentum, Lactococcus lactis, and Sterptococcus salivarius were isolated from Scomberomorus commerson (Spanish mackerel) intestine and were capable to inhibit the Listeria innocua growth (Moosavi-Nasab et al., 2014). Many Lactobacilli species isolated from intestine of Anguilla species, Clarias orientalis, Labeo rohita, Oreochromis species and Puntius carnaticus showed significant antimicrobial activity against Aeromonas and Vibrio species (Dhanasekaran et al., 2008).

2.2.2. Antiviral Activity

In recent years, the antiviral activity of probiotics has gained attention (Lakshmi et al., 2013), but the exact mechanism of action by which probiotics show antiviral property is still unknown. However, the in-vitro analysis reveals that the inhibition of viruses can occur by secretion of extracellular enzymes produced by the bacteria. For example, Aeromonas species, Corynebacterium, Pseudomonas and Vibrio species show the

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

antagonism against the IHNV (Infectious hematopoietic necrosis virus) (Kamei et al., 1988; Zorriehzahra et al., 2016). Feeding of probiotic strain Bacillus megaterium has increased the resistance against WSSV (white-spot syndrome virus) in the shrimp, Litopenaeus vannamei (Li et al., 2009). It has been reported that the probiotic strains Bacillus and Vibrio species are effective against WSSV and efficiently protect Litopenaeus vannamei (Balcazar, 2003). Application of Lactobacillus as probiotic, either as one strain or as a mixture with Sporolac resulted in better resistance against lymphocystis viral disease which is found in Paralichthys olivaceus (olive flounder) (Harikrishnan et al., 2010).

2.2.3. Antifungal Activity

Few studies have been reported about the antifungal activity of probiotics. Aeromonas strain A199 from Anguilla australis (eel) culture water, had high inhibitory property against Saprolegnia species (Lategan et al., 2004). In another study, Pseudomonas species M162, Pseudomonas species M174 and Janthinobacterium species M169 have increased the immune response of Oncorhynchus mykiss (rainbow trout) against saprolegniasis (Zorriehzahra et al., 2016). In 2012, Nurhajati and his co-workers, demonstrated that Lactobacillus plantarum FNCC 226 showed inhibitory potential in catfish (Pangasius hypophthalamus) against Saprolegnia parasitica A3.

The reported probiotic strains used in aquaculture can either be obtained commercially or isolated from different sources. A detailed summary of different probiotics, their sources and the beneficial effects on the host are given in Table 2.1.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Table 2.1: Probiotic species used in aquaculture, source and beneficial effects to the host species.

Probiotic species Source of probionts Beneficial effects References

Lactobacillus acidophilus Commercial (All Tech, Best growth performance and feed efficiency Lara-Flores et al., Streptococcus faecium Nicholasville, KY) in Nile tilapia (2003) Enhanced the non-specific immune Bacillus subtilis Tovar-Ramırez et Commercial parameters and enhance the challenge against Lactobacillus acidophilus al., (2004) Edwardsiella tarda infection Seawater, sediment and Bacillus cereus Improved resistance against pathogenic gut of healthy fish Ravi et al., (2007) Paenibacillus polymyxa Vibrio spp. (Lates calcarifer) Reduce the adhesion of pathogens i.e. Aeromonas salmonicida, Aeromonas Lactococcus lactis CLFP 101 Oncorhynchus mykiss hydrophila Vibrio anguillarum and Yersinia Balcazar et al., Lactobacillus plantarum CLFP 238 (Rainbow trout) ruckeri to intestinal mucus and shows (2008) Lactobacillus fermentum CLFP 242 antibacterial activity against these fish pathogens. Lactobacillus plantarum Shows antagonistic activity against Labeo rohita Giri et al., (2011) Bacillus subtilis Aeromonas hydrophila

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Bacillus coagulans Probiotic bacteria significantly inhibit the Bacillus mesentericus Puntius conchonius pathogens and establish themselves in the gut Divya et al., (2012) Bifidobacterium infantis of P. conchonius. Cyprinus carpio Al-Faragi and Bacillus subtilis Inhibit the growth of Aeromonas hydrophila (Common carp) Alsaphar, (2012) Growth promoting probiotic, enhance growth at the rate of 4x108 cells per 100 g of feed. Cyprinus carpio Bacillus subtilis Shows better growth, feed conversion ratio Bisht et al., (2012) (Common carp) (FCR), specific growth rate (SGR) and feed conversion efficiency (FCE) Improves water quality and lowers the Nitrosomonas species Padmavathi et al., Commercial pathogenic (Pseudomonas species) bacterial Nitrobacter species (2012) loads in fish ponds. Exhibit highest amount of IFN-γ production Marsupenaeus and bactericidal activity. Lactococcus lactis (D1813) Maeda et al., (2014) japonicas Inhibit the infection caused by Vibrio penaeicida. Protects the fish against Flavobacterium Oncorhynchus mykiss Enterobacter sp. strain C6-6 psychrophilum infection, reduce the mortality LaPatra et al., (2014) (Rainbow trout) and enhance the immunity of fish.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Increase in the growth, survival, improve Macrobrachium Ramzani et al., Bacillus subtilis food digestion, reduce the mortality caused by rosenbergii (2014) pathogenic bacteria (Aeromonas hydrophila) Shows high growth performance like specific growth rate, body weight and shows Bhatnagar and Bacillus cereus Cirrhinus mrigala inhibition against the pathogenic strain Lamba, (2015) (Aeromonas hydrophila) Bacillus subtilis Improves digestion and fight against the fish Thankappan et al., Bacillus aerophilus Labeo rohita pathogens such as Providencia rettgeri and (2015) Bacillus firmus Aeromonas species. Show improve phagocytic activity of innate immune cells, skin mucus lysozyme activity Lactococcus lactic Bean sprouts and improves host innate immunity, weight Beck et al., (2015) Lactobacillus plantarum Paralichthys olivaceus gain and survival rate following Streptococcus iniae challenge. Bacillus subtilis Pediococcus acidilactici Increase growth performance, health status Giannenas et al., Commercial Enterococcus faecium and modulate intestinal microbial community. (2015) Lactobacillus reuteri

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Bacillus subtilis Strains are more efficient in converting Abareethan and Lactococcus lactis Labeo rohita organic matter, adhere to the intestine, Amsath, (2015) Saccharomyces cerevisiae enhance the growth and survival of L. rohita. Increase the growth disease resistance and Bacillus licheniformis Commercial immune response of juvenile tilapia against Han et al., (2015) Streptococcus iniae. Bacillus pumilus treated fish show maximum percentage of total erythrocyte count, Rajikkannu et al., Bacillus pumilus Labeo rohita haemoglobin concentration and haematocrit (2015) concentrations which improves survival and therefore establish better health conditions. Oratosquilla oratoria Bacillus pumilus Shows antagonism against Vibrio Portunus Liu et al., (2015) Bacillus mojavensis parahaemolyticus. trituberculatus Exhibit strong anti-bacterial activity against Lactobacillus gasseri TSU3 Catla catla Aeromonas hydrophila and adhere to mucosal Sahoo et al., (2015) Lactobacillus animalis TSU4 surfaces and epithelial cells. Pseudomonas psychrotolerans Mancuso et al., Vibrio ichthyoenteri Sparus aurata Enhance the immune defence of fish. (2015) Labrenzia sp.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Show antagonism against three fish pathogens: Vibrio anguillarum, Photobacterium damselae and Pseudomonas anguilliseptica. Bacillus amyloliquefaciens Shows antagonistic activity against Dutta and Ghosh, (KF623290) Cirrhinus mrigala Pseudomonas putida and Aeromonas (2015) Bacillus sonorensis (KF623291) salmonicida. Stimulates growth rate, feed efficiency, Hamdan et al., Lactobacillus plantarum Sediments improve immune response against Aeromonas (2016) hydrophila in Nile tilapia. Bacillus stratosphericus Strains grow better in intestinal mucus and (KM277362) produce various cellular components which Mukherjee et al., Bacillus aerophilus (KM277363) Cirrhinus mrigala exhibit antibacterial property against the (2016) Bacillus licheniformis (KM277364) pathogens. Solibacillus silvestris (KM277365) Show inhibitory activity against S. typhimurium, S. aureus, S. enteritidis, E. coli Lactobacillus plantarum Shellfish Kang et al., (2016) O157:H7, V. ichthyoenteri, S. iniae, and V. parahaemolyticus.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Suppress the pathogenic bacteria, S. aureus, Lactobacillus sp. Alestes baremoze Streptococcus sp., Proteus sp., Pseudomonas Kato et al., (2016) Lactococcus sp. sp. and E. coli. Improve the growth performance, enhance the Clupanodon punctatus Bacillus amyloliquefaciens immune parameters in turbot and fight against Chen et al., (2016) Epinephelus coioides V. anguillarum infection. Produce extracellular enzymes (secondary Kocuria sp. Oncorhynchus mykiss metabolites) which is inhibitory to Virbio Sharifuzzaman et al., Rhodococcus sp. (Rainbow trout) anguillarum, V. ordalii, E. coli, Pseudomonas (2017) aeruginosa and Staphylococcus aureus. Persist in simulated gastric conditions with the inhibition capability of various pathogens like Staphylococcus aureus (MTCC 3160), Enterococcus hirae Catla catla Adnan et al., (2017) Escherichia coli (MTCC 40), Pseudomonas aeruginosa (MTCC 424) and Salmonella typhi (MTCC 3215). Improves immunity of Nile tilapia and Srisapoome and Bacillus pumilus AQAHBS01 Oreochromis niloticus enhance disease resistance against Areechon, (2017) Streptococcus agalactiae.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Lactobacillus farraginis Produce antimicrobial compounds against Salmo salar Pediococcus acidilactici fish pathogens, have good colonization Amin et al., (2017) (Atlantic salmon) Pediococcus pentosaceus capacity on gastrointestinal tract of salmon. Shows antibacterial activity against four fish pathogens, Aeromonas salmonicida, A. Bacillus sp. Mystus vittatus Nandi et al., (2017) hydrophila, A. sobria and Pseudomonas fluorescens Show inhibitory activity against four fish pathogens such as Aeromonas hydrophila, Banerjee et al., Bacillus subtilis Labeo rohita Aeromonas salmonicida, Bacillus mycoides (2017) and Pseudomonas fluorescens. Enhance growth performance, immune Bacillus subtilis HAINUP40 Pond water response and disease resistance of Nile tilapia Liu et al., (2017) against Streptococcus agalactiae. Enhance non-specific immune responses, Bacillus subtilis growth performance and disease resistance Shrimp Park et al., (2017) Bacillus licheniformis against A. salmonicida in juvenile rainbow trout.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

2.3. Mode of Action

Probiotics have a special mode of action to protect the host from intestinal disorders. The probiotic microorganisms hinder the establishment of different pathogenic bacteria by a process called colonization resistance. Probiotic microorganisms secrete a variety of inhibitory substances which inhibit Gram +ve and Gram –ve microscopic organisms.

Principally, these inhibitory secretions are acetic acid, lactic acid, H2O2, bacteriocins and so on. These secretions decrease the number of pathogens and inhibit the formation of virulence substances by pathogens (Nwanna, 2010). Oelschlaeger (2010) explained the mode of action of probiotics in a simple way in which probiotics modulate the acquired immune system as well as innate immunity to prevent host gut from disease causing pathogens and to treat against various digestive tract inflammations. Next possible action is that, they directly affect the pathogenic bacteria present in the gut, thus, resulting in the restoration of the probiotics in the gut. Finally, they target various toxins produced by the microbial population resulting in their detoxification and inactivation in the gut (Oelschlaeger, 2010). Thus, all modes of action of probiotics are directly associated with gut micro-biota (Wolf, 2006; Pandiyan et al., 2013). Probiotic secrete antagonistic compounds which help to improve the immunity and enhance the growth of fish. It also helps to improve the water and soil quality. The mode of action of probiotics is shown in Figure 2.2.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Figure 2.2: Mode of action of probiotics.

Some possible well-known mechanisms by which probiotic bacteria protect the host organism against intestinal disorders are as follows.

2.3.1. Competition for Space / Blocking of Adhesion Sites

The activity of probiotics is determined by the aggressive hindrance for the attachment sites on intestinal epithelial layer (Nwanna, 2010). The mechanism by which probiotic bacteria fight for the attachment site is called ‘competitive inhibition’. The capability of probiotics to proliferate in the intestine, attach to the intestinal surface and subsequently inhibit the adherence of pathogenic bacteria is also an important criteria in the selection of probiotics (Lazado et al., 2011; Balcazar et al., 2006). Lactobacillus prevent the adhesion of the pathogens such as Escherichia coli, Klebsiella species and Pseudomonas aeruginosa on intestinal tract of the host (Nwanna, 2010). Probiotic

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

adhesion can be non-specific due to the presence of physiochemical agents or specific due to the adhesion either on the surface of adherent bacteria or the receptor molecules on the epithelial cells (Salminen et al., 1996; Lazado et al., 2015).

2.3.2. Production of Inhibitory Substances

The probiotic bacteria release inhibitory secretions which have bacteriostatic/ bactericidal influences on pathogenic microbes (Servin, 2004) like hydrogen peroxide, bacteriocins, lysozymes, siderophores, proteases, and many others (Panigrahi and Azad, 2007; Tinh et al., 2008). Some bacteria produce volatile fatty acid (acetic, butyric, lactic and propionic acid) and organic acid, as a result of which there is a decrease in pH of gastrointestinal tract. Hence, it inhibits the proliferation of pathogenic bacteria (Tinh et al., 2008). A compound named indole (2,3-benzopyrrole) has inhibitory potential against various pathogens i.e. Vibrio anguillarum, Aeromonas salmonicida, Edwaedsiella tarda and Yersinia ruckeri (Gibson, 1998; Lategan et al., 2006; Abbass et al., 2010).

2.3.3. Competition for Nutrients

The survival of bacterial population depends on the capability to compete with other microbes for available nutrients in the gut environment (Verschuere et al., 2000). While struggling for nutrients, probiotics can defeat the pathogenic bacteria by utilizing the available nutrients that would have been consumed by the pathogens. This mechanism would restrict the presence of pathogens in the intestinal tract because without nutrients the bacteria cannot survive (Nwanna, 2010). For instance, siderophores are iron- chelating agents which breakdown precipitated iron or extract it from the iron complexes, thus making it available for bacterial growth (Neilands, 1981). The siderophore-producing bacteria utilize ferric iron in a low-iron environment. Hence, they can be used as probiotics because they make iron unavailable for the proliferation of pathogenic bacteria (Tinh et al., 2008). Pseudomonas fluorescens which was grown in low-iron conditions inhibits the growth of fish pathogen Vibrio anguillarum and Aeromonas salmonicida by competing for the available free iron (Gram et al., 1999; Smith and Davey, 1993).

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

2.3.4. Improving Water Quality

According to the studies, Gram-positive bacteria (Bacillus species) has been used as probiotics to improve the water quality. It was concluded that the Gram-positive bacteria, especially Bacillus species are more efficient in conversion of organic matter into CO2, slime or bacterial biomass. The studies suggest better performance of Gram- positive bacteria over Gram-negative bacteria. It is suggested that the farmers can control the deposition of organic carbon in growing season by using high concentration of probiotics in the ponds (Balcazar et al., 2006; Mohapatra et al., 2013). The probiotic bacteria possess significant algicidal activity and affects many microalgae species (Fukami et al., 1997). The probiotic bacteria are valuable as it increase the number of good bacteria in water and improve the water quality by eliminating ammonia and nitrate toxicity (Zorriehzahra et al., 2016; Mohapatra et al., 2013). Also, the utilization of probiotics improve other parameters like pH, temperature, dissolved oxygen, ammonia and hydrogen sulfide in rearing water. Thus, probiotics maintain a positive and healthy culture environment in aquatic system (Aguirre-Guzman et al., 2012; Banerjee et al., 2010).

2.3.5. Disruption of Quorum Sensing

Quorum Sensing (QS) is defined as a bacterial regulatory mechanism, which controls the expression of biological macromolecules such as, the virulence factors in a cell density-dependent manner. In this mechanism, bacteria regulate the gene expressions by the production, release and recognition of small signal molecules called auto- inducers (Chu et al., 2014). Many bacteria use this mechanism to communicate and regulate various physiological activities (Miller and Bassler, 2001). Disruption of the quorum sensing mechanism has been suggested as a new anti-infective strategy in aquaculture to inhibit the pathogens (Defoirdt et al., 2004; Zorriehzahra et al., 2016).

Since N-Acyl homoserine lactones (AHLs) are family of QS auto-inducers used in Gram-negative bacteria, their biodegradation prove to be an strong way to inhibit QS, Bacillus sp. QSI-1 inhibits the virulence production and biofilm formation of

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Aeromonas hydrophila and used as potential quorum quencher (Chu et al., 2014). Bacillus sp. QSI-1 inhibit the AHLs but did not affect the growth of A. hydrophila YJ- 1. In the in-vivo experiment, Bacillus subtilis reduced the pathogenicity of A. hydrophila in zebrafish (Danio rerio). The fish fed with Bacillus subtilis strain QSI-1 were found to have a high relative percentage survival (80.8%). The results indicated that AHLs degrading bacterial strains can be used in aquaculture as an alternative to antibiotics as the biological remedy for the prevention of fish disease (Chu et al., 2014). Also the probiotic strains like Bifidobacterium, Bacillus and Lactobacillus species deteriorate the signalling molecules of pathogens by enzymatic secretion or production of auto-inducer antagonists (Brown, 2011). Medellin-Pena et al., (2007) showed that Lactobacillus acidophilus secretes inhibitory molecules that inhibits the QS or interacts with Escherichia coli O157 transcription gene.

2.4. Methods of Administration of Probiotics

Various methods have been put forth to regulate the use of probiotics. They can be added in feed, resulting in the colonization on the surface of intestinal tract. In prawns, the most common regulatory method for administration of probiotics is through water/oral routine (Huang et al., 2006). But most of the probiotics are designed in such a way that they can be mixed with the feed additives to show high efficiency against pathogens (Austin et al., 1992; Gildberg and Mikkelsen, 1998; Hai et al., 2009; Gomes et al., 2009). The probiotics such as Lactobacillus rhamnosus were reported to improve the fecundity of Danio rerio (Gioacchini et al., 2010). Other methods such as addition of probiotics directly into water or in bacterial suspension were also reported (Queiroz and Boyd, 1998; Gibson et al., 1998; Ringo and Vadstein, 1998; Cha et al., 2013; Hansen and Olafsen, 1989; Sung et al., 1994; Itami et al., 1998).

Probiotic strains can be used individually or in a combination of different strains (Havenaar et al., 1992; Salinas et al., 2005; Kesarcodi-Watson et al., 2008; Lin et al., 2012; Kesarcodi-Watson et al., 2012). Previous reviews on probiotics have emphasized on the utilization of sole culture species, and it is speculative whether combination of two or more cultures of probiotic strains would be useful. Mixed probiotic strains are

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

more efficient than the use of single strain (Verschuere et al., 2000; Hai et al., 2009), multi-species and multi-strain probiotics enhance the defense mechanism against various infectious diseases (Kesarcodi-Watson et al., 2012; Timmerman et al., 2004). A recent study compared the activity of mixed strain of Lactobacillus acidophilus and B. subtilis in Nile tilapia in which serum bactericidal activity and hematocrit values were higher in comparison to single strain (Aly et al., 2008). Similar studies were conducted to modulate immunity against Streptococcus iniae by a combination of Lactobacillus plantarum and Lactococcus lactis in Japanese flounder (Beck et al., 2015). In the growth and survival of Labeo rohita, multi strain probiotics have been efficiently used which enhance the growth and survival of rohu at fry and hatchling stages (Jha et al., 2015).

Synbiotics is the combination of probiotics with prebiotics or various plant products (Salminen et al., 1998; Van Hai and Fotedar, 2009). It has been reported in many studies that synbiotics improves the microbial supplementation in the gastrointestinal tract of the host organism (Gibson et al., 1995). The feeding of synbiotic Enterococcus faecalis and mannan-oligosaccharide (MOS) showed better FCR (food conversion ratio) as compared to feeding of probiotic and prebiotic individually (Rodriguez-Estrada et al., 2009). The application of probiotics, prebiotics and synbiotics have improved the survival of aquatic organisms against pathogenic bacteria. The survivability was found to be maximum in the group treated with probiotics followed by prebiotic and synbiotic (Daniels et al., 2013; Decamp and Moriarty, 2007).

The addition of probiotics in live feed as an encapsulation technique has developed into an interesting idea. In this technique, the probiotic bacteria can remain alive or even multiply in the live feed. Therefore, probiotics can be delivered by the live feed to the host in a very efficient manner (Hai, 2015). Various live feeds have been reported so far such as copepods (Sun et al., 2013), rotifer (Gatesoupe, 1997) and Artemia species (Daniels et al., 2013; Gatesoupe, 1994; Van Hai et al., 2010), in which probiotics were encapsulated. This approach of enrichment of live feed with probiotics has proved to be effective over other conventional methods. Van Hai et al., 2010 have reported an

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

effective enrichment of Artemia nauplii using a mixture of Pseudomonas synxantha and Pseudomonas aeruginosa for Penaeus latisulcatus (western king prawn). Similarly, Sun and his co-workers (2013) have reported that the copepod (Pseudodiaptomus annandalei) is an appropriate vector of probiotic (Bacillus species) as live feed for Epinephelus coioides larvae.

The probiotics can be administered in either form- as live or dead strains. Various reports are available for the use of probiotics in either form. The comparison of live and dead forms reveals an interesting observation. Live probiotics provide immunity to the host in most of the cases and in a few cases certain inactivated probiotics also do the same. Hence, the use of probiotics in live or heat killed forms are case specific and cannot be generalized. For instance, many workers such as Sharifuzzaman et al., 2011; Arijo et al., 2008; Panigrahi et al., 2011; Ramesh et al., 2015 have reported the use of viable probiotic strains with better results. Sharifuzzaman and Austin, 2010; Arijo et al., 2008 have demonstrated the role of live probiotic cells Kocuria SM1 by production of cross-reactive antibodies in rainbow trout to protect against infections due to Vibrio anguillarum, V. ordalii and V. harveyi. Similarly, live cells of Bacillus licheniformis and Bacillus pumilus exhibited an enhanced expression of lysozyme activity and respiratory burst in rohu species (Ramesh et al., 2015). Panigrahi et al., 2011 states that higher expression of immune genes (TNF, TGF-b, IFN and Ig) is responsible for better immunity. The expression of these immune genes is induced using live probiotic cells (live-spray and freeze-dried) compared to the heat-killed ones. The phagocytic activity was found to be higher in rainbow trout, when they were fed with live cells of probiotic bacteria Lactobacillus rhamnosus JCM1136 as compared to heat-killed cells (Panigrahi et al., 2005).

Also, in some cases the supplementation of cell-free supernatant and heat-killed probiotics stimulated innate immunity of the fish (Irianto and Austin, 2003). But they offer poor protection as observed in Oncorhynchus mykiss and Miichthys miiuy against pathogens, V. anguillarum, Streptococcus iniae, Aeromonas hydrophila and Lactococcus garvieae (Brunt and Austin, 2005; Pan et al., 2008). When Nile tilapia was

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

nourished with both dead and live probiotics against Edwardsiella tarda disease, dead probiotics were found ineffective as compared to viable probiotics (Taoka et al., 2006).

The administration period of probiotics is also considered as a very significant factor. According to research, the time-period for application of the potential probiotic can be as short as 6 days or as long as 5 months to 8 months (Joborn et al., 1997; Aubin et al., 2005; Aly et al., 2008). Prolonged administration of probiotics can cause immune- suppression of continuous response of nonspecific immune system (Sakai, 1999). Supplementation of probiotic bacteria has demonstrated to give short-term benefits. However, they were not detected inside the gastrointestinal tract over a period of 1-3 weeks (Robertson et al., 2000; Kim and Austin, 2006; Balcazar et al., 2007). Short-term supplementation has turned out to be effective, while the data on long-term effectiveness is not available (Brunt et al., 2007; Newaj-Fyzul et al., 2007; Pieters et al., 2008; Wu et al., 2015; Skjermo et al., 2015). Feeding of probiotics (Shewanella xiamenensis and Aeromonas veronii) to grass carp for about 28 days reduced the cumulative mortality when challenged with Aeromonas hydrophila (Wu et al., 2015). Aubin et al., 2005 checked the recovered amount of probiotics over a time period and observed that recovery levels were found to be higher after 20 days than 5 months. The frequency of administration of probiotics also play a very important role in maintaining the effectiveness and function of probiotics. A daily application of probiotics is better than thrice a week during the culture period (Guo et al., 2009).

In recent years, the use of probiotic bacteria as biological measure has improved fish performance, water quality, prevention of diseases, enhancement of immune responses and so on. This review concludes that several probiotic strains are highly specific while others are quite selective. Efforts need to be made to streamline the whole range of probiotic strains and categorize them based on their action-specific mechanism. A simple step in this direction is going to make the use of probiotics very efficient, economical and eco-friendly. After proving the worth of probiotics, there is a need to look forward towards designing probiotic strains which are specific and can be used to target specific fish species. The evaluation of optimal conditions for probiotic

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

interaction with the host also holds a lot of scope for further investigation. In the past, there have been instances of failure of in-vivo studies which were conducted based on the positive in-vitro results. We need to detail out the conditions in real samples which may affect their survival, colonization, proliferation and interaction with the host in an environment. This will help us to properly screen and test probiotics which will lead to no mismatch in in-vitro and in-vivo observations. Other important scope for future research is to study the fate of probiotics in host organism. The fate of live strains and durability of health effects of probiotics in host organism are uncertain and require further investigation.

After doing much study about the efficacy and action mechanism of probiotics, there are still many doubts which are unclear. Nevertheless, the future research should focus on appropriate methods to develop innovative and suitable approach for administration of probiotics in food and animals. Probiotic strains viability, functionality, host- microbe’s interactions, antioxidant status, antagonistic and synergistic activity or probably side effects of probiotics should be the major concerns of study. For a better understanding of the molecular mechanisms, advanced molecular level research is required on probiotic science. It will also help to decode the probiotic unique gene with novel applications.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Chapter 3 Hypothesis

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

HYPOTHESIS

Fish are susceptible to a wide variety of deadly pathogens which cause several diseases like, tail rot, fin rot, haemorrhage, septicaemia, dropsy and so on. The common occurrence of these diseases among fish has resulted in huge loss in aquaculture industries. The use of antibiotics to cure these diseases has developed severe biological and environmental concerns. The increased antibiotic resistance among the pathogens due to extensive use of chemotherapeutic drugs, calls for the search of a new alternative strategy to control the disease in aquaculture. The search for a better alternative has been a major concern recently. Probiotics, renowned as valuable microbes, are recommended as an efficient and environmentally friendly approach to replace antibiotics.

Probiotic bacteria are a boon for aquaculture and are highly useful for the prevention of various infectious diseases. They can be used as an alternative to antimicrobials and antibiotics. Probiotics enhance the immune system of fish and increase the growth of fish. Probiotics can be isolated from various sources but the best source of probiotic in case of fish, is the gut of fish itself. These are the putative probiotics as they come from the same source as the organism who consumes them. Putative probiotics are already adapted to the environment of gut of fish and can thrive well inside the gastrointestinal tract of fish. Probiotics can be profitable to the fish farmers if they are used in place of antibiotics and antimicrobials which are the cause of inducing resistance in bacterial species when used in excess

According to reports, the use of commercially available probiotics is futile as they isolated from non-fish sources and might not remain viable at high cell density in the intestine of fish. So, there is a strong need to isolate putative probiotic bacteria, as they would perform better during adhesion to the intestinal surface and in prevention of pathogenic bacteria.

The present study aims at isolation of putative probiotic bacteria from the intestinal tract of different fish species from different region of Punjab, India and their in-vitro and in-vivo evaluation as potent probiotics. During study, the isolated bacterial strains will characterized based on their colony morphology, colour, size, biochemical

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

characterization, followed by in-vitro evaluation on different selection parameters described in FAO/WHO guidelines. The in-vivo evaluation will be done to assess the efficacy of putative probiotic strains on survival and growth performance of Cyprinus carpio (Common carp) challenged with Pseudomonas aeruginosa (MTCC 4673). It is expected that these probiotic strains can be used as a novel and safe treatment to fight the current issues in aquaculture.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Chapter 4 Aim and Objectives

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

AIM AND OBJECTIVES

Background: In aquaculture, chemotherapeutic agents like antibiotics and chemicals are the classical cure for microbial infection. However, the extensive usage of these antibiotics lead to their accumulation in aquatic habitat and results in harmful consequences like, emergence of antibiotic-resistant bacteria, accumulation of antibiotic residues in flesh, kill the beneficial microbes in gastrointestinal tract and alterations in microbiota of aquatic environment Therefore, the use of antibiotics as chemotherapeutic drugs in aquaculture has become questionable. The quest for better alternatives to prevent the infection and replace the antibiotics has been a major concern. The use of probiotics in aquaculture is a promising approach for regulating fish disease. Probiotics help the fish to fight against pathogen by different mechanisms and improve their overall health.

Objectives: According to reports so far, the efficacy of probiotic strains isolated from freshwater teleost has not been explored much and a detailed investigation is requisite. Also, the probiotics being frequently used in aquaculture for last two decades are derived commercially. However, the use of commercially available probiotics in fish were found to be somewhat futile as compared to their use in animals. The isolation of commercial probiotics strains from non-fish sources is a reason for their unsatisfactory performance, as they might not remain viable in the intestine of the fish. Reports are available on in-vitro evaluation of probiotic bacteria, but in-vivo evaluation of the potential probiotics has not been explored much and needs further investigation. The objectives of the present study have been designed keeping in mind the above facts. The objectives of the present study are as follows.

1. Screening and indexing of selective Gram-positive bacteria isolated from Teleost gut and evaluation of their potential to acclimatize in gut environment. 2. Antagonistic property, biochemical and molecular characterization of selected probionts. 3. Incorporation of potential strain with feed and its assessment as probiotic to enhance the productivity by using major cultivable carp, Cyprinus carpio.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Chapter 5 Materials and Methods

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

MATERIALS AND METHODS

5.1. Sample Collection from Different Sites

Punjab is a state in northern India. The word ‘Punjab’ is composed of two Persian words ‘panj’ (five) and ‘ab’ (water). Thus, Punjab means “the land of five rivers”. The five rivers are the Beas, Ravi, Sutlej, Jhelum and Chenab. These river divide Punjab in three different regions namely Doaba, Majha and Malwa region (Figure 5.1). Doaba is a land of two rivers and is the region of Punjab that lies between the Beas River and the Sutlej River. The Majha region is spread between the Ravi and Beas Rivers, including the area on the north of Sutlej River. Malwa is the region that lies in south of the Sutlej River. The fish samples for bacterial isolation were collected from the local markets, fisherman of catchment area of rivers and fish farm of Doaba, Majha and Malwa region of Punjab, India. The fish samples were collected in sterile containers and brought to the laboratory and were identified by the key given by Srivastava (2014), Brraich and Ladhar (2005).

Figure 5.1: Map of Punjab state showing different regions i.e. Doaba, Majha and Malwa separated by rivers. 38

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

5.2. Isolation of the Bacteria from the Collected Samples

5.2.1. Preparation of Media

The de-Man, Rogosa and Sharpe agar (MRS) and Bacillus differentiation agar (BDA) (Hi-Media, India) were used for the isolation of probiotic bacteria. These are the selective agar medium for the isolation of Gram-positive bacteria i.e. Lactic acid bacteria and Bacillus species. Whereas, nutrient agar and Mueller Hinton agar (Hi- Media, India) media were used for the growth of pathogenic strains and antagonistic activity. The preparation of different culture media used for the isolation, and their composition are discussed below.

5.2.1.1. Media for Isolation of Bacteria

The preparation of de-Man, Rogosa and Sharpe agar (MRS) and Bacillus differentiation agar (BDA) (Hi-Media, India) and their composition are discussed as follows.

A. de Man, Rogosa and Sharpe Agar: The MRS agar is a selective medium for the isolation of the lactic acid producing bacteria. For the preparation of media, 67.15 grams of MRS agar was added in 1000 ml distilled water. The mixture was heated up to boiling to dissolve the medium completely. The media was sterilized by autoclaving at 15 psi (121°C) for 20 minutes. The sterilized media was mixed properly and poured into sterile petri plates. The composition of MRS agar is given in Table 5.1.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Table 5.1: The composition of de Man, Rogosa and Sharpe (MRS) agar.

B. Bacillus Differentiation Agar: The Bacillus Differentiation agar (BDA) is a selective medium for the isolation of Bacillus species. For the preparation of media, 22.0 grams of BDA was added in 1000 ml distilled water. The media was heated to boiling to dissolve the medium completely. The media was sterilized by autoclaving at 15 psi (121 °C) for 20 minutes. It was mixed well and poured into sterile petri plates. The ingredients of bacillus differentiation agar are given in Table 5.2.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Table 5.2: The composition of Bacillus Differentiation Agar (BDA).

5.2.1.2. Media for Antimicrobial Activity

The preparation of nutrient agar and Mueller Hinton agar their composition are discussed as follows.

A. Nutrient Agar: Nutrient agar is a general agar medium supporting the growth of a wide variety of microorganisms. For the preparation of media, 28.0 grams of nutrient agar was added in 1000 ml distilled water. Later, it was heated to boiling to dissolve the medium completely. The media was then sterilized by autoclaving at 15 psi (121°C) for 20 minutes. The composition of nutrient agar media is given in Table 5.3.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Table 5.3: The composition of Nutrient Agar.

B. Mueller Hinton Agar: Mueller Hinton (MH) agar is used to check the antagonistic activity of the isolated bacteria. For the preparation of media, 38.0 grams of MH agar was added in 1000 ml of distilled water. It was heated to boiling to dissolve the medium completely. The media was sterilized by autoclaving at 15 psi pressure (121°C) for 15 minutes. The composition of Mueller Hinton (MH) agar media is given below in Table 5.4.

Table 5.4: The composition of Mueller Hinton (MH) agar.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

5.2.2. Isolation Methods

In this study different fish samples were collected from different regions of Punjab, India. The aseptic collection of samples was carried out by tightly packing the fish in sterile polyethylene bags. The samples were preserved in an icebox and were immediately brought to the laboratory. The surface of fish was sterilized by using 70% (v/v) ethanol to reduce undesirable bacterial contamination. The gut of fish was dissected out under sterile conditions and washed extensively with 0.85% normal saline to remove the intestinal waste. After that, the gut samples were homogenized in normal saline and were spread plated on to de Man, Rogosa and Sharpe agar (MRS) and Bacillus Differentiation Agar (BDA) (Hi-Media, India) for selective isolation of bacteria, followed by incubation at 37 °C for 24 h. Morphologically distinct colonies were picked and purified by streaking them again onto BDA and MRS agar.

5.3. Screening and Indexing of Gram-positive Bacteria

Gram’s stain is a differential staining technique developed by Dr. Hans Christian Gram in 1884. This staining technique is used for the identification and classification of bacteria into two different groups which are Gram-positive and Gram-negative. In this process four reagents, crystal violet, iodine solution, alcohol and safranin are used for staining of bacterial smear (Figure 5.2). The bacteria which retains crystal violet and appear blue are considered as Gram-positive and those which retain safranin and appear pink are considered as Gram-negative.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Figure 5.2: Scheme for Gram staining procedure.

5.4. In-vitro Evaluation of Probiotic Potential of Gram-positive Bacteria

The indexed Gram-positive bacteria were subjected to different in-vitro selection parameters described in FAO/WHO guidelines (2002) for the evaluation of probiotics and use them in fish feed. The guidelines for the evaluation of probiotics were prepared as a joint effort of Food and Agriculture Organization (FAO) of the United Nations and World Health Organization (WHO) at London Ontario, Canada, 2002. The guidelines list the different screening parameters as the minimum requirement needed for the probiotic status.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

5.4.1. Antagonistic Activity

The antagonistic activity of isolated bacteria was determined by using disc diffusion method (Balcazar et al., 2008; Allameh et al., 2012) against targeted fish pathogen Pseudomonas aeruginosa (MTCC 4673). It is the most abundant, opportunistic pathogen in aquaculture. In this process, 100 µL of freshly prepared pathogenic culture was spread on Mueller-Hinton agar plate (Himedia). A disc of 6 mm diameter was soaked in the broth containing live suspension of the isolates and was placed on the surface of agar plate. The plates were then incubated at 37 °C for 24 h and later the activity was evaluated by measuring the zone of inhibition. The isolates which showed potential antagonistic activity against the tested pathogen were considered for further criteria of probiotic selection.

5.4.2. pH Tolerance pH tolerance of the isolated bacteria was determined by preparing MRS broth of different pH (2, 3, 4 and 9) using 1N NaOH and 1% HCl. The sterilized test tube was then inoculated with overnight culture of the selected strain and was kept for incubation at 37 °C. Growth rate of the bacteria was recorded as a measure of the optical density (OD) recorded by a spectrophotometer at 600 nm after 24 h incubation (Adnan et al., 2017; Allameh et al., 2012). Also, the isolates treated at different pH were inoculated on MRS agar plate by spreading, to ensure the viability of bacteria.

5.4.3. Bile Tolerance Test

Bile salt tolerance of the isolated bacteria was tested in MRS broth which contain 0.3% (w/v) bile salt (Sigma-Aldrich, Bangalore, India). Triplicate test tubes of MRS broth containing 0.3% of bile salt were inoculated by 50 µL of cultured strain and incubated for 24 h. Growth rate of the bacteria was recorded by measuring the optical density by a spectrophotometer at 600 nm after 24 h incubation and the results were compared with the growth observed for the control tube (without bile salts). Also, the viability of isolated bacteria to survive in the bile salt was checked by spreading the bacteria on MRS agar plates (Hyronimus et al., 2000; Allameh et al., 2012; Adnan et al., 2017).

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

5.4.4. Susceptibility to Drugs

Antibiotic susceptibility of the isolated bacteria was investigated by using agar disc diffusion method against the most common antibiotics used in aquaculture. The antibiotics included Methicillin, Amoxicillin, Penicillin, Chloramphenicol, Neomycin, Ampicillin, Vancomycin, Tetracycline and Kanamycin. In typical procedure, 100 µL of the broth culture of the strain was spread on Mueller-Hinton agar plate and antibiotic discs were placed on the surface of the agar plate with equal spacing. Finally, the plates were incubated for 24 h. The bacterial sensitivity was evaluated by measuring clear inhibition zone around each disc (Akinjogunla et al., 2010; Ramesh et al., 2015).

5.4.5. Adhesion and Biofilm Formation

Quantitative analysis of biofilm production and the adhesion property of the isolated bacteria was done by Standard Tube Method (STM). In brief, overnight culture of bacteria was inoculated into 5 ml of Luria-Bertani broth and incubated in glass test tubes without disturbing for 48 h. Later, culture was poured out and test tubes were washed with water. The tubes were then fixed with glutaraldehyde (2.5%) and again washed with water, and staining with 0.4% crystal violet solution. After solubilization of the crystal violet with ethanol-acetone (80:20 v/v), the absorbance at 600 nm was measured (Chu et al., 2014).

5.5. Identification of Bacteria

In bacteriology, the identification of bacteria is a very important step. Identification of bacterial strains was done by two methods i.e. conventional (biochemical characterization) and molecular by 16S ribosomal RNA sequencing.

5.5.1. Biochemical Characterization

The biochemical characterization of isolated bacteria was done through various biochemicals tests including Gram's staining, catalase test, urease test, gelatin test, salt tolerance, motility test, indole test, glucose fermentation, H2S production, methyl red and Voges-Proskauer test as per Bergey's manual of Systematic Bacteriology (Holt et al., 1989). The detailed procedure for these biochemical tests is discussed as follows.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

5.5.1.1. Catalase Test

Hydrogen peroxide (H2O2) is a lethal byproduct of metabolic processes of body. So, there is a need of the rapid conversion of H2O2 to other non-toxic molecules. The enzyme catalase mediates the breaks down of H2O2 into water (H2O) and oxygen (O2). In this method, overnight grown culture was inoculated into test tube containing nutrient broth and were incubated at 37 °C. Catalase activity was checked by adding few drops of 3% hydrogen peroxide solution into the test tube and carefully checking the formation of bubbles. The formation of bubbles indicate the conversion of H2O2 to

H2O and O2 and is considered as a positive result.

5.5.1.2. Urease Test

Urease test is used to evaluate the capability of bacteria to hydrolyze urea by using urease enzyme. For the test procedure, 38.7 grams of urea broth was added in 1000 mL distilled water and then sterilization was done by filtering the media. The freshly prepared broth was dispensed in a test tube. Overnight grown culture was inoculated into test tube and incubated at 37 °C for 24 h. The change in colour was observed to determine the positive or negative result.

5.5.1.3. Gelatin Test

Gelatin is a protein which is derived from collagen. Gelatin hydroxylation determines the presence of gelatinase enzyme. Gelatinase is proteolytic enzyme secretion of bacteria that hydrolyze or digest gelatin. For the test procedure, 65.0 grams of gelatin agar was added in 1000 ml distilled water. The mixture was heated for complete dissolution and sterilization was done by autoclaving at 15 psi (121°C) for 15 minutes. The freshly prepared media was dispensed in a test tube. Overnight grown culture was inoculated into test tube and incubated at 37 °C for 24 h.

5.5.1.4. Mannitol Fermentation Test

Mannitol fermentation test is used to determine the ability of bacteria to ferment the mannitol and tolerant to salt as the mannitol salt agar (MSA) contains high concentration of Salt. For the test procedure, 111.02 grams of mannitol salt agar was added in 1000 ml distilled water. The mixture was heated for complete dissolution and

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

sterilization was done by autoclaving at 15 psi (121°C) for 15 minutes. Overnight grown culture was inoculated into test tube and incubated at 37 °C for 24 h.

5.5.1.5. Triple Sugar Iron Agar Test

Triple sugar iron (TSI) agar test is used to determine the ability of bacteria for glucose fermentation and H2S production. For the test procedure, 64.42 grams of TSI agar was added in 1000 ml distilled water. The mixture was heated for complete dissolution and sterilization was done by autoclaving at 15 psi (121°C) for 15 minutes. Overnight grown culture was inoculated into test tube and incubated at 37 °C for 24 h.

5.5.1.6. Motility Test

Motility test is used to determine whether the bacteria is equipped with flagella and thus able to move away from the stab line. If the entire tube becomes turbid, this indicates that the bacteria is motile and had moved away from the stab mark. For the test procedure, 36.23 grams of SIM medium was added in 1000 ml distilled water. The mixture was heated for complete dissolution and sterilization was done by autoclaving at 15 psi (121°C) for 15 minutes. Overnight grown culture was inoculated into test tube and incubated at 37 °C for 24 h.

5.5.1.7. Indole Test

The indole test is used to check the ability of bacteria to breakdown the amino acid tryptophan to produce indole. For the test procedure, 15.0 grams of tryptone broth was added in 1000 ml distilled water. The mixture was heated for complete dissolution and sterilization was done by autoclaving at 15 psi (121°C) for 15 minutes. Overnight grown culture was inoculated into test tube and incubated at 37 °C for 24 h. After incubation period, 5 drops of Kovac’s indole reagent were added and the test tubes were observed for the color formation.

5.5.1.8. Methyl Red and Voges-Proskauer Test

Methyl red test is used to determine the ability of bacteria to produce several organic acids (lactic acid, succinic acid and formic acid) as a product of glucose fermentation. Large amount of acids was produced to conquer the phosphate buffer. For the test procedure, 17.0 grams of MR-VP medium was added in 1000 ml distilled water. The

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

mixture was heated for complete dissolution and sterilization was done by autoclaving the mixture at 15 psi (121°C) for 15 minutes. Overnight grown culture was inoculated into test tube and incubated at 37 °C for 24 h. After incubation period, 5 drops of methyl red reagent were added, and the test tubes were observed for the color formation.

Voges-Proskauer test is used to check the ability of bacteria to produce neutral end- products of glucose fermentation. For the test procedure, 17.0 grams of MR-VP medium was added in 1000 ml distilled water. The mixture was heated to dissolve completely and sterilization was done by autoclaving the mixture at 15 psi (121°C) for 15 minutes. Overnight grown culture was inoculated into test tube and incubated at 37 °C for 24 h. After incubation period, few drops of Barritt A and B reagents were added, and the test tubes were observed for the color formation.

5.5.2. Molecular Characterization

The isolated bacteria, which passed through all the selection criteria for an ideal probiotic were subjected to molecular characterization and identified up to species level by using 16S rRNA sequencing. For molecular characterization the samples were sent to sequencing at Yaazh Xenomics, Coimbatore, India and Biokart India Pvt Ltd, Bangalore, India. The nucleotide sequence obtained were compared with the available sequences in NCBI (National Center for Biotechnology Information) database using BLAST (Basic Local Alignment Search Tool) program. The gene sequences of isolated bacteria were deposited to NCBI and accession numbers were obtained.

5.5.2.1. Extraction of DNA

The foremost step of DNA extraction is homogenization/lysis of the cells grown in monolayer. These cells were lysed by suspending few colonies aseptically into 2 ml micro centrifuge tube containing 450 µL of “B Cube” lysis buffer. The cells were lysed by pipetting out repeatedly. RNAse A (4 µL) and “B Cube” neutralizing buffer (250 µL) were added in the centrifuge tube. Then, the tubes were vortexed and incubated at 65 °C for 30 minutes in a water bath. DNA solutions were mixed by inversion to minimize the shearing of the DNA molecules. The tubes were centrifuged at 14,000 rpm at 10 °C for 20 minutes. After centrifugation, the viscous supernatant was transferred into a fresh micro centrifuge tube carefully without disturbing the pellet.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

600 µL of “B Cube” binding buffer was added to the content, mixed thoroughly by pipetting and incubated for 5 minutes at room temperature. 600 µL of the content was transferred to a spin column placed in 2 ml collection tube. The content was centrifuged at 14,000 rpm for 2 minutes and flow-through was discarded. The collection tube and the spin column were reassembled, then remaining 600 µL of the lysate was transferred and centrifuged at 14,000 rpm for 2 minutes and the flow-through was discarded.

500 µL of “B Cube” washing buffer I was added to the spin column. The buffer was centrifuged at 14,000 rpm for 2 minutes and flow-through was discarded. The spin column was reassembled and 500 µL of “B Cube” washing buffer II was added and was again centrifuged at 14,000 rpm for 2 minutes and flow-through was discarded. The spin column was transferred to a sterile micro centrifuge tube. 100 µL of “B Cube” Elution buffer was added at the middle of spin column carefully. The tubes were incubated at room temperature for 5 minutes and then centrifuged for 1 minute at 6000 rpm. The above-mentioned step was repeated for complete elution. The buffer in the microcentrifuge tube contained the DNA. The concentrations of DNA were measured by running aliquots on 1% agarose gel. The DNA samples were stored at –20 °C for further use.

5.5.2.2. PCR Protocol A. Composition of Taq Master Mix • Taq DNA polymerase was supplied in 2X Taq buffer • 0.4mM dNTPs

• 3.2mM Magnesium chloride (MgCl2) • 0.02% Bromophenol blue B. Primers detail

Primer Name Sequence Details No. of Base

27F 5' AGAGTTTGATCMTGGCTCAG 3' 20

1492R 5' TACGGYTACCTTGTTACGACTT 3' 20

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

5 µL of the isolated DNA was added to PCR reaction solution. The PCR reaction solution was prepared by mixing 1.5 µL of Forward Primer and Reverse Primer, 5 µL of deionized water, with12 µL of Taq Master Mix. PCR was performed using the following thermal cycling conditions. The PCR conditions are given in Table 5.5.

Table 5.5: PCR Conditions (different stages, temperature and time) for the molecular identification.

Heating the DNA at 94 °C cause breaking of hydrogen bonds of the DNA strands and lead to formation of single stranded DNA. After denaturation, the mixture was cooled to 60 °C allowing the primers to anneal to the complimentary sequence in template DNA. Annealing was followed by the extension process. The reaction mixture was heated to 72 °C to create optimum conditions for the activity of DNA polymerase. DNA polymerase assist the sequential addition of nucleotide onto the primer using the target DNA template.

The purification of the PCR products was done by removing the free PCR primers and dNTPs from PCR products using Montage PCR Clean up kit (Millipore). 27F/1492R primers were used for sequencing of PCR product in ABI PRISM® BigDyeTM Terminator Cycle Sequencing Kits with AmpliTaq® DNA polymerase (FS enzyme) (Applied Biosystems). Each template was processed for single-pass sequencing using below 16s rRNA universal primers. Ethanol precipitation protocol was followed to

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

remove unincorporated terminators from the fluorescent-labeled fragments. ABI 3730xl sequencer (Applied Biosystems) was used for electrophoresis of the aqueous suspension of the sample in distilled water.

5.6. In-vivo Evaluation of Probiotics

5.6.1. Experimental Fish

The fingerlings of Cyprinus carpio (common carp) was selected as the experimental fish to check the potential of the isolated probiotic strains. Cyprinus carpio belongs to the order Cypriniformes and the family Cyprinidae, and is believed as the largest family of freshwater fish. It is mostly inhabiting in freshwater bodies, like rivers, ponds and lakes. It is the third most important species found worldwide and is being considered as an important species for aquaculture. Common carp has a high adaptive ability to both food and environment, it can change its feeding niche and behavior when its favorite food is not enough for them (Rahman, 2015). It can easily survive in harsh conditions also.

5.6.2. In-vivo Experimental Set-up

The experiment was carried out during June to August 2018 in the School of Bioengineering and Biosciences at Lovely Professional University (31.2536° N, 75.7037° E), Punjab, India. The laboratory was well ventilated with an ambient temperature (28-30 °C). The aquarium of 20 L capacity were used for the experiment. All the aquariums were connected to an air pump (Hailea ACO-328) for proper and continuous aeration during the study with a constant flow rate (~2.0 L/min). The aquariums were maintained under natural light/dark photo-period. On average 25 fish fingerlings were transferred to each aquarium. The experiment was conducted for 60 days.

5.6.3. Acclimatization of Fish

The advance fingerlings of Cyprinus carpio (~30 days old) were obtained from Fishery Department (Fish Farmers Development Agency), Kapurthala, Punjab. The fish fingerlings were transferred in oxygenated polythene bags to the research site and were transferred to the aquarium. The number of fish was equally maintained (25 each) with

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

initial average weight of 0.4 ± 0.2 g. The fish were fed with basal feed on daily basis and continuous aeration was provided. Fish were acclimatized for a period of 14 days. The water in aquarium was changed on every 3rd day to remove waste and faecal matter.

5.6.4. Experimental Design

Experiment was designed to evaluate the efficacy of the selected probiotic bacteria against fish pathogen, Pseudomonas aeruginosa as well as to check the toxicity of probiotics in the in-vivo condition. The experiment was carried out in four groups, as control (I), pathogen (II), probiotic (III) and probiotic with a pathogen (IV) (Figure 5.3). Fish in control (I) and pathogen group (II) were fed with control feed containing only basal feed soaked in PBS solution. On the other hand, fish in the probiotic group (III) and probiotic with a pathogen (IV) were fed with probiotic-supplemented diet. Sample fish were weighed and counted at regular intervals.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Figure 5.3: Schematic representation of the experimental setup and design.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

5.6.5. Water Quality

The water quality parameters were observed regularly during the whole experiment period. The temperature of water was observed to range from 28.7 to 31.2 °C, pH from 6.8 to 7.4. No abnormal changes were observed in water quality parameters. This indicated that the experimental diets do not have any detrimental effects on the water quality.

5.6.6. Feed Formulation

5.6.6.1. Selection of Feed

Commercially available “Hopar grow” fish food was used as the basal diet for the fish during the course of study. The ingredients of the fish feed are clam meal, squid liver meal, hypro soybean meal, wheat flour, corn gluten meal, vitamins, yeast and organic minerals. The nutrient composition of the fish food is crude protein (30%), crude fat (3%), crude fibre (4%), moisture (10%).

5.6.6.2. Preparation of Probiotic Feed

Commercially available “Hopar grow” fish food was used as basal diet for fish with the nutrient composition as 30% crude protein, 3% crude fat, 4% crude fiber and 10% moisture during the study. The probiotic-supplemented feed was prepared as per the protocol given by Ramesh et al., (2015), Nadella et al., (2017) and Asaduzzaman et al., (2018). The bacterial culture was inoculated in MRS broth and incubated at 37 °C for 24 h. The culture was centrifuged at 3000 rpm for 10 mins to obtain pellets. The bacterial pellets were washed three times using phosphate buffer solution (PBS, pH 7.2) and were later resuspended in PBS. The concentration of the bacterial cell was standardized using UV-Vis spectrophotometer. The absorbance at 600 nm was adjusted to 0.3 to standardize the number of bacteria (108 CFU/ml) (Balcazar et al., 2007; Srisapoome and Areechon, 2017). Probiotic supplemented feed was prepared by spraying the bacterial suspensions on basal feed and mixed properly to achieve a dose of 1 x 108 CFU g-1. The experimental diets were air dried at room temperature (28–30 °C) under sterile conditions for 3-4 h and stored in air-tight plastic bag at 4 °C. Experimental diets were prepared on a weekly basis to ensure viability of the probiotics.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Control diet was prepared by spraying sterile PBS over commercial basal diet. The fish were supplied with feed (3% of their body weight) daily at 04:00 pm for 60 days.

5.6.6.3.Compatibility Test

The compatibility of the probiotic bacteria against each other was evaluated by a simple coexistence assay using a disc diffusion method. The selected probiotic isolate was spread over the agar medium and disc was soaked in the live suspension of other probiotic bacteria. The disc was placed onto the agar plates covered with bacteria to observe their antagonism against each other and to determine whether they are compatible with each other. The plates were cultured at 37 °C for 24 h (Geria and Caridi, 2014).

5.6.6.4. Multi-strain Probiotic Feed

Mixed strains probiotics are more efficient than probiotics based on single strain (Verschuere et al., 2000; Hai et al., 2009). Based on the compatibility test, a consortia of five probiotic bacteria was prepared. The consortia comprised of F2F4, F3 1(2), BDK7, BDK9 and BDK2′. Multi-species and multi-strain probiotics enhances the defense mechanism against various infectious diseases (Kesarcodi-Watson et al., 2012; Timmerman et al., 2004). The multi-strain feed was prepared by similar method as the probiotic feed.

5.6.7. Challenge Test

A pathogenic strain Pseudomonas aeruginosa (MTCC 4673) was used to cause septicaemia infection in the experimental fish and to study the role of probiotics as the treatment. Septicaemia cause ulceration and lesions of hemorrhage in fish. The cell suspension of Pseudomonas aeruginosa in PBS (108 cfu/ml) was added directly into the water (to mimic the natural condition) of group II and IV for infusing the infection. The fish in the probiotic group III and IV were fed with probiotic feed. On the other hand, fish in the control group (I) and pathogen group (II) were fed with basal feed. Mortality pattern, external signs of infections and behavioral abnormalities were recorded daily for 60 days. The growth performance of the two sets (II) and (IV) of the

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

infected fish supplied with basal and probiotic feed respectively, was compared to analyse the effect of probiotic bacteria against the pathogen.

5.6.8. Growth and Survival Indices

Growth of fish and the effect of probiotics on the growth was monitored by measuring the length (millimetre gradings) and weight of sample fish at regular intervals. Different growth parameters as mean weight, % weight gain, % survival, specific growth rate (SGR) and food conversion ratio (FCR) were calculated by using the following formulae (Hamdan et al., 2016; Liu et al., 2017).

-.+$/ '#()ℎ+ "#$% '#()ℎ+ = -.+$/ %012#3 .4 4(5ℎ#5

:(%$/ 1#$% '#()ℎ+ − <%(+($/ 1#$% '#()ℎ+ 6#()ℎ+ )$(% (%) = × 100 <%(+($/ 1#$% '#()ℎ+

B012#3 .4 4(5ℎ 503A(A#C @03A(A$/ (%) = × 100 B012#3 .4 4(5ℎ 5+.DE#C

:##C )(A#% ()) :..C D.%A#35(.% 3$+(. (:FG) = 6#()ℎ+ )$(% ())

lnK6 M − ln(6 ) @H#D(4(D )3.'+ℎ 3$+# = L N × 100 B.. .4 C$P5

K6LM = :(%$/ 1#$% '#()ℎ+, (6N ) = <%(+($/ 1#$% '#()ℎ+

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Chapter 6 Results and Discussion

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

RESULTS AND DISCUSSION

Description of Sample Collection From Different Sites of Punjab

Fish are diverse in nature as they are found in all aquatic environments. Due to their different habitat, they are subjected to all sort of conditions ranging from cultured to moderate and harsh environment. So, they have unique gut microbiota to support such extreme habitat conditions. The present study anticipates to screen the gut microbes of fish obtained from different habitat (small stream, pond and rivers) and feeding habits. Hence different fish species were collected for the isolation of potential probiotic bacteria. The selected strains were then used as putative probiotics to manipulate gut micro-environment of desired species to improve their growth and survival. The fish samples were collected from different local markets, fishermen of catchment area and fish farm of Doaba, Majha and Malwa regions of Punjab, India. The region wise details of the collection are as follows.

6.1. Doaba Region

6.1.1. Fish Samples Collected from Doaba Region

In Doaba region, the fish samples were collected from local markets and fish farms of Phagwara city (31.2240° N, 75.7708° E), Kapurthala city (31.3715° N, 75.3937° E) and Jalandhar city (31.3260° N, 75.5762° E). Figure 6.1 shows the geographical map of Punjab showing highlighted region as Doaba. A total of 52 fish from different species i.e. Labeo rohita, Catla, Nandus, Puntius, Labeo gonius, Labeo dero, Oxygaster bacaila, Chanda, Ompok bimaculata, Gobius, Colisa, Channa, Gudusia chapra were collected from the Doaba region (Phagwara, Jalandhar and Kapurthala) for the isolation of bacteria (Figure 6.2).

Labeo rohita and Catla catla are known as Indian major carp, omnivorous and widely used in aquaculture, Nandus nandus is known as mud perch and common in ditches, muddy streams, pool and marshes, Labeo gonius is found in clear water of the rivers and their tributaries, Labeo dero is a bait fish and found in rivers, Oxygaster bacaila is a minnow fish found in slow moving streams and ponds, Chanda is hardy fish and can stand in foul water, Ompok bimaculate is known as butter catfish found in fresh water

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

rivers and streams, Gobius inhabits estuaries, mangroves and freshwater, Colisa is surface dweller, insectivorous, very hardy and can survive in foul water, Channa is known as snakehead fish, carnivorous, found in the bottom mud of ponds, rivers and ditches, Gudusia chapra is a minnow fish found in ponds, rivers and ditches.

Figure 6.1: Geographical map of Punjab showing different regions, the highlighted region shows Doaba region, this region has been selected for collection of fish samples.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Figure 6.2: Digital image of different fish species i.e. (a) Chanda nama, (b) Chanda ranga, (c) Puntius ticto, (d) Nandus nandus, (e) Fresh water shrimps, (f) Chanda nama, (g) Oxygaster bacaila, (h) Labeo gonius, (i) Puntius chola, (j) Nandus nandus, (k) Puntius sophore, (l) Oxygaster bacaila, (m) Gobius viridipunctatus, (n) Ompok bimaculate, (o) Puntius ticto, (p) Chanda nama, (q) Gudusia chapra, (r) Channa punctata, (s) Labeo dero, (t) Colisa fasciatus, (u) Labeo rohita and (v) Catla catla collected from Doaba region of Punjab, India.

6.1.2. Isolation and Purification of Bacteria from Fish Gut Sample

BDA and MRS agar medium were used for the isolation and screening of bacterial strains. These are the selective agar medium for the isolation of Gram-positive bacteria i.e. Lactic acid bacteria and Bacillus species. A total of 169 morphologically distinct bacterial colonies were isolated from the gut of 52 different healthy fish. These isolated colonies were purified multiple times by streaking them onto BDA and MRS agar medium until the pure bacterial cultures were obtained. Figure 6.3 shows digital images of pure bacterial cultures streaked on BDA and MRS agar medium.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Figure 6.3: Digital images of pure isolated cultures streaked on (a-i) de-Man, Rogosa and Sharpe agar (MRS) and (j-l) Bacillus differentiation agar (BDA) showing pure bacterial colonies isolated from gastrointestinal tract of healthy fish collected from Doaba region, Punjab.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

6.1.3. Screening and Indexing of Gram-positive Bacteria

Staining property and cell morphology of the isolated bacteria were examined using Gram’s staining technique. Gram staining is the ability of bacteria to retain the color of stains used in staining procedure. Gram-positive bacteria retained crystal violet stain and were not decolorized by alcohol, therefore remained as purple. Most of the probiotic bacteria are Gram-positive such as Bacillus subtilis, Lactobacillus plantarum and Enterococcus faecium. So, in the screening process, only Gram-positive bacteria have been selected for further analysis and Gram-negative bacteria were discarded as they might be harmful and belong to the pathogen group.

Out of 169 bacterial colonies, only 37 isolates were found to be Gram-positive bacteria. These isolated bacteria were found to be purple colored and rod shaped under oil- immersion microscope. Typical images of few Gram-positive bacteria are shown in Figure 6.4. The nomenclature of the selected Gram-positive bacteria, collection site, source and name of the species from which bacteria has been isolated are given in Table 6.1.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Figure 6.4: Digital microscope images showing typical Gram-positive morphology of different bacteria isolated from gastrointestinal tract (GIT) of fish under oil-immersion microscope.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Table 6.1: Nomenclature of the Gram-positive bacteria isolated from Doaba region and name of the source fish species.

Isolate S. No. Collection site Source Species name 1 S1 Phagwara, Doaba Gut region Labeo rohita 2 S2 Phagwara, Doaba Gut region Labeo rohita 3 S3 Phagwara, Doaba Gut region Catla catla 4 S4 Phagwara, Doaba Gut region Labeo rohita 5 S5 Phagwara, Doaba Gut region Labeo rohita 6 S6 Phagwara, Doaba Gut region Catla catla 7 S7 Phagwara, Doaba Gut region Catla catla 8 S8 Phagwara, Doaba Gut region Labeo rohita 9 S9 Phagwara, Doaba Gut region Labeo rohita 10 S10 Phagwara, Doaba Gut region Catla catla 11 S11 Phagwara, Doaba Gut region Labeo rohita 12 S12 Phagwara, Doaba Gut region Catla catla 13 S13 Phagwara, Doaba Gut region Labeo rohita 14 S14 Phagwara, Doaba Gut region Labeo rohita 15 S15 Phagwara, Doaba Gut region Labeo rohita 16 K1 Kapurthala, Doaba Gut region Fresh water shrimps 17 K3 Kapurthala, Doaba Gut region Puntius ticto 18 K5 Kapurthala, Doaba Gut region Chanda nama 19 K7 Kapurthala, Doaba Gut region Puntius chola 20 K8 Kapurthala, Doaba Gut region Labeo gonius 21 K9 Kapurthala, Doaba Gut region Oxygaster bacaila 22 K12 Kapurthala, Doaba Gut region Ompok bimaculata 23 K13 Kapurthala, Doaba Gut region Gobius viridipunctatus 24 K13′ Kapurthala, Doaba Gut region Gobius viridipunctatus 25 K16 Kapurthala, Doaba Gut region Colisa fasciatus 26 K17 Kapurthala, Doaba Gut region Labeo dero 27 K21 Kapurthala, Doaba Gut region Puntius sophore

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28 K21′ Kapurthala, Doaba Gut region Puntius sophore 29 BDK1 Kapurthala, Doaba Gut region Fresh water shrimps 30 BDK2′ Kapurthala, Doaba Gut region Nandus nandus 31 BDK4 Kapurthala, Doaba Gut region Chanda ranga 32 BDK5 Kapurthala, Doaba Gut region Chanda nama 33 BDK7 Kapurthala, Doaba Gut region Puntius chola 34 BDK9 Kapurthala, Doaba Gut region Oxygaster bacaila 35 BDK12 Kapurthala, Doaba Gut region Ompok bimaculata 36 BDK19′ Kapurthala, Doaba Gut region Gudusia chapra 37 BDK20′ Kapurthala, Doaba Gut region Chanda nama

6.1.4. In-vitro Evaluation of Probiotic Potential of Gram-positive Bacteria

The indexed Gram-positive bacteria were subjected to different in-vitro selection parameters described in FAO/WHO guidelines (2002) for the evaluation of probiotics. The results of in-vitro evaluation of isolated bacteria are as follows.

6.1.4.1 Antagonistic Activity

The probiotic bacteria exert protective and beneficial physiological effects by showing antagonism against the pathogenic bacteria (Ripamonti et al., 2011). Hence antagonistic activity is an indirect measure of the probiotic efficacy. The indexed Gram-positive bacterial strains were examined for their antagonistic activity by disc diffusion method against the fish pathogen Pseudomonas aeruginosa (MTCC 4673). It is the most abundant and opportunistic pathogen cause hemorrhagic septicaemia in aquaculture (Phennicie et al., 2010; Panda et al., 2013). The experiment was performed in triplicate and potential of the isolated bacteria to inhibit the pathogen was evaluated by measuring diameter of inhibition zone. Among the 37 Gram-positive isolates, only 11 isolates exhibit strong antagonistic activity against targeted pathogen as shown in Figure 6.5 and were selected for further in-vitro evaluation. The remaining 26 isolates failed to show any activity against the target fish pathogen and were discarded safely. Figure 6.5 (a-f) show the images of culture plates showing zone of inhibition as the measure of antagonistic activity. Figure 6.5 (g-i) show culture plates with no antagonistic activity.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

For potential probiotic bacteria, it is recommended to show high antimicrobial activity. Higher antimicrobial activity indicates better performance of the probiotics (Patel and Lin, 2010; Giri et al., 2012). Several methods have been suggested to interpret antibacterial activity by bacteria including the release of bacteriocins and other organic acids. In the present study, difference in antimicrobial potential of the isolates is attributed to the differences between their species and strains as they have been isolated from different fish species (Das et al., 2016; Ilango et al., 2016).

Figure 6.5: Digital image of (a-f) culture plates showing zone of inhibition by the different isolates and (g-i) culture plates showing no zone of inhibition, against the fish pathogen Pseudomonas aeruginosa (MTCC 4673).

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Figure 6.6. show the graphical representation of zone of inhibition exhibited by different isolates against the selected pathogen. Among all the isolates S3, S7, and BDK7 displayed highest antagonistic activity against the target fish pathogen Pseudomonas aeruginosa (MTCC 4673) with zone of inhibition as 32 ± 2.64, 41 ± 3.00 and 43 ± 3.00 mm, respectively, followed by BDK9, BDK2′, BDK5, BDK1, K1, K3, K16 and K21′. The zone of inhibition of these isolates is 26 ± 2.64, 22.66 ± 3.05, 22 ± 2.00, 20.66 ± 3.78, 19.66 ± 1.52, 19 ± 2.00, 16 ± 2.00 and 15.33 ± 3.05 mm, respectively. The 11 probionts selected after evaluation of antagonistic activity are listed in Table 6.2 with their respective zone of inhibition.

50 b 45 b 40 35 a,e 30 a,c,e c,d,e c,d,e c,d,e 25 c,d,e c,d,e 20 c,d c,d 15

Zone of Inhibition (mm) Inhibition of Zone 10 5 0 S3 S7 K1 K3 K16 K21′ BDK1 BDK2′ BDK5 BDK7 BDK9 Bacteria Strains

Figure 6.6: Zone of inhibition (in mm) plot of different bacterial isolates derived from Doaba region indicating their antagonistic activity against the selected pathogen, Pseudomonas aeruginosa (MTCC 4673). Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) with respect to antagonistic activity of different isolates.

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Table 6.2: Zone of inhibition (in mm) of different bacterial isolates derived from Doaba region showing antagonistic activity against Pseudomonas aeruginosa (MTCC 4673).

S. No. Isolates Zone of Inhibition (mm)

1 S3 32.00a,e ± 2.64

2 S7 41.00b ± 3.00

3 K1 19.66c,d,e ± 1.52

4 K3 19.00c,d,e ± 2.00

5 K16 16.00c,d ± 2.00

6 K21′ 15.33c,d ± 3.05

7 BDK1 20.66c,d,e ± 3.78

8 BDK2′ 22.66c,d,e ± 3.05

9 BDK5 22.00c,d,e ± 2.00

10 BDK7 43.00b ± 3.00

11 BDK9 26.00a,c,e ± 2.64

Each values given in the table are mean ± standard deviation of three separate experiments. Different letters demonstrate significant difference (P < 0.05) among the antagonistic activity of different isolates.

6.1.4.2. pH Tolerance Test

Numerous factors of gut affect the viability of probiotic microorganism such as acid and bile salts. Probiotic bacteria should be able to survive the acidic conditions of the gastrointestinal tract. The survival of the bacteria in stomach and intestine depends on the pH of stomach and gastric juice present in gastrointestinal cavity (Balcazar et al., 2008).

Fish are generally categorized as gastric and agastric/pseudogastric based on the presence and absence of stomach, respectively. The presence of HCl in stomach makes pH tolerance as an important criterion for the selection of any probiotics. Cyprinus carpio (common carp) are mostly agastric/pseudogastric due to their omnivorous

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

feeding habits. In general, the pH in the gut of teleost ranges from 3.5 to 4.5 in gastric region and 8.5 to 9.5 in intestinal region. Therefore, the pH tolerance of the isolated bacterial strains was investigated over a wide range of pH (pH 2 to pH 9). The survivability of the isolates at different pH was measured in terms of optical density (OD) recorded at 600 nm as shown in Figure 6.7. It is observed that, all 11 isolates which exhibited good antagonistic activity, also showed growth and survival at this pH range. Table 6.3 shows the optical density value as a measure of survival at different pH conditions. The results shows that the isolated strains were quite tolerant to low pH (pH 2-3). Although, critical limit to survive in acidic conditions in fish was pH 3.5- 4.5. The selected strains exhibit strong resistance to high pH (pH 9).

In general, all the isolates exhibit growth in both acid and alkaline medium, which is an important criterion for any potential probiotic strain. The optical density values increase with increased pH for all the bacterial strains except BDK1 and BDK2′. BDK1 and BDK2′ shows an increased growth from pH 2 to pH 4 but a decrease in growth was observed at pH 9. This anomaly can be explained based on better adaptability of the bacteria at an optimum pH, pH 4 in the case of BDK1 and BDK2′. The overall growth of BDK1 and BDK2′ is appreciable at pH 2 and pH 4, hence they also fulfill the criteria for selection as potential probiotic, like the remaining 9 bacterial strains.

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2 pH 2 pH 3 pH 4 pH 9 d 1.8 c e 1.6 b b c b b e c c e e 1.4 f g 1.2 d g d e h h d d f 1 g 0.8 b a e b b d f c Absorbance at 600 nm at Absorbance c a d e b 0.6 a e g e e a a b f f b e e a a c e f i e a h c a i a b c g g a d e i a c c d f g j f c f d c 0.4 b c a h c i j b a a j h g i g h f g h h e d b h a d j h c b b i i i 0.2 i h i b h i i i h c 0 S3 S7 K1 K3 K16 K21′ BDK1 BDK2′ BDK5 BDK7 BDK9 Bacteria strains

Figure 6.7: The optical density (OD at 600 nm) indicating growth and survival of bacterial strains isolated from Doaba region in MRS broth medium, adjusted at different values of pH (2.0, 3.0, 4.0 and 9.0) to study the effect of pH on the growth and survival. Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) in bacterial strains at same pH.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Table 6.3: pH test results showing optical density values at 600 nm as a measure of survival of the bacterial strains isolated from Doaba region and cultured under different pH medium (pH 2.0 to pH 9.0).

Bacterial Survival of the isolates at different pH (OD at 600 nm) Strain pH 2 pH 3 pH 4 pH 9 S3 0.059a,b,c,h ± 0.014 0.114a,c,h,i ± 0.013 0.137a,b ± 0.006 0.361a ± 0.057 S7 0.050a,b,c ± 0.007 0.235b,c,e,i,j ± 0.037 0.181a,b,e ± 0.004 0.461a ± 0.028 K1 0.098a,b,c,d,h ± 0.009 0.142a,c,h,i ± 0.044 0.141a,b ± 0.059 1.329b,e ± 0.083 K3 0.129c,d,h,i ± 0.006 0.156a,b,c,h,i ± 0.005 0.125a,b ± 0.015 1.532c,e ± 0.062 K16 0.240e,f,g ± 0.001 0.352d,f,g ± 0.012 1.351c ± 0.014 1.770d ± 0.064 K21' 0.210e,f,g,i ± 0.034 0.267b,e,f,g,i,j ± 0.048 0.984d ± 0.053 1.414b,c ± 0.021 BDK1 0.231e,f,g ± 0.007 0.344d,e,f,g ± 0.031 1.310c ± 0.073 0.843e,f,g ± 0.085 BDK2' 0.105a,c,d,h ± 0.014 0.122a,c,h,i ± 0.007 1.089d ± 0.094 0.989f,g,h ± 0.108 BDK5 0.163d,f,i ± 0.037 0.186a,b,c,e,h,i,j ± 0.013 0.321b,e ± 0.077 1.058g,h ± 0.023 BDK7 0.246e,f,g ± 0.008 0.253b,e,i,j ± 0.039 1.096d ± 0.076 1.289b,e ± 0.023 BDK9 0.121c,d,h,i ± 0.008 0.117a,c,h,i ± 0.006 1.042d ± 0.045 1.295b,e ± 0.050

Values given in the table are mean ± standard deviation of three separate experiments. Different letters demonstrate significant difference (P < 0.05) within the same column among the pH tolerance potential of different isolates.

6.1.4.3. Bile Salt Tolerance

The probiotic bacteria must also have the property of bile salt tolerance to survive and proliferate in the intestine, where it plays an important role in specific and non-specific defence mechanisms. Bile is a product of both excretory and secretory activities of the organ and is secreted by hepatic cells in the liver. Breakdown of RBCs and hemoglobin results in the formation of biliverdin and bilirubin pigment. These pigments along with fat emulsifying bile salts form the bile. Bile acts as surfactant and helps to emulsify lipids in the food. Bile salts are harmful for live bacterial cells because they disrupt the cell membrane structure.

Tolerance to bile is considered as one of the essential properties for the survival and colonization of probiotics in the host’s intestine (Wang et al., 2010). The survival of

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

ingested strains in extreme environment of the intestine can be predicted by bile salt tolerance analysis. According to many studies, for investigation of bile tolerance of potential probiotic strains, 0.3% bile was considered as the standard level (Gilliland et al., 1984; Jacobsen et al., 1999; Sahadeva et al., 2011).

Figure 6.8 shows the absorbance indicative of the bacterial growth and survival in presence and absence of the bile salts. The isolated strains exhibit survival at 0.3% bile salt (ox-gall) and show significant growth both in absence and presence of bile salts except the BDK1. BDK1 failed to survive in the presence of bile salt as it could not tolerate bile at this concentration. These results showed that the bile salts found in the intestine do not affect the test probiotic bacteria except BDK1. The variance in the degree of bile tolerance among different strains is attributed to strain-specific behavior of the isolated strains (Koll et al., 2008; Sahadeva et al., 2011). The results indicate the adaptability of isolated bacterial strains in the presence of bile salts (Table 6.4).

With Bile (0.3%) Without Bile

1.6 i 1.4

1.2 a b e r b e h b d e b a f d g p k g k 1 a d f g k e f p q j k g f k j g k q p 0.8 j p j 0.6 c o c

0.4 n m Absorbance at 600 nm at Absorbance 0.2 l 0 S3 S7 K1 K3 K16 K21′ BDK1 BDK2′ BDK5 BDK7 BDK9 Bacterial strains

Figure 6.8: The optical density (at 600 nm) showing tolerance of isolated bacterial strains in presence and absence of bile salts (0.3% ox-gall). Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) in between with bile or without bile groups.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Table 6.4: Bile tolerance results showing optical density values (at 600 nm) as a measure of survival of isolates after 24 h in presence and absence of bile salts (0.3 % ox-gall concentration).

Absorbance at 600nm Bacterial Strain With Bile (0.3%) Without bile

S3 0.601a,g,j ± 0.060 0.931b,k ± 0.043

S7 0.485c ± 0.030 0.741d,f ± 0.022

K1 0.692e,g,j,p ± 0.011 0.778d,f,k ± 0.019

K3 0.675a,e,g,j,p ± 0.027 0.868b,k ± 0.003

K16 1.045h ± 0.025 1.437i ± 0.038

K21' 0.641a,e,g,j ± 0.006 0.845b,f,k ± 0.022

BDK1 0.027l ± 0.005 0.130m ± 0.012

BDK2' 0.273n ± 0.013 0.468o ± 0.022

BDK5 0.511c ± 0.007 0.769d,f,k ± 0.014

BDK7 0.759e,g,p,q ± 0.029 0.898b,k ± 0.022

BDK9 0.844p,q ± 0.030 1.072r ± 0.052

Values given in the table are mean ± standard deviation of three separate experiments. Different letters demonstrate significant difference (P < 0.05) among the bile tolerance potential of different isolates.

6.1.4.4. Susceptibility to Drugs

A potential probiotic strain should not possess any transmissible antibiotic-resistant genes. The ingestion of bacteria with transmissible antibiotic-resistant genes is harmful, because horizontal transfer of gene between bacteria in the gut resulted in development of new antibiotic-resistant strain (Saarela et al., 2000). The potential probiotics which are sensitive towards antibiotics are preferred over the resistant ones. The antibiotic resistance is represented based on visible bacterial growth after overnight incubation in the presence of antibiotic disc. The antibiotic sensitivity of probiotics confirms that

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there is no transferral of antibiotic-resistant gene to pathogens (Ayyash et al., 2018). A series of commonly used drugs i.e. methicillin, amoxicillin, penicillin-G, chloramphenicol, neomycin, ampicillin, vancomycin, tetracycline and kanamycin were used to check the susceptibility of the isolated strains (Romero et al., 2012; Shokryazdan et al., 2014; Ramesh et al., 2015).

The drug susceptibility test was carried out on selected 11 isolates. Out of which 7 bacterial strains i.e. S3, S7, BDK1, BDK2′, BDK5, BDK7 and BDK9 were found susceptible to the antibiotics used in study. These isolated strains were susceptible to methicillin, amoxicillin, penicillin-G, chloramphenicol, neomycin, ampicillin, vancomycin, tetracycline and kanamycin by showing clear zone around the antibiotic discs. But the remaining 4 isolates showed resistance to certain antibiotics such as, isolate K1 showed resistance against penicillin and kanamycin, isolate K3 showed resistance against amoxicillin, penicillin and kanamycin, isolate K16 showed resistance against penicillin only and isolate K21′ showed resistance against penicillin, ampicillin and kanamycin. The antibiotic susceptibility results obtained are shown in Figure 6.9. The resumes of susceptible (S) and resistance (R) showing antibiotic susceptibility test of the selected isolates against various drugs are shown in Table 6.5.

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Figure 6.9: Digital image of representative culture plates (a-d) showing antibiotic susceptibility of the isolated strains against various drugs, methicillin, amoxicillin, penicillin-G, chloramphenicol, neomycin, ampicillin, vancomycin, tetracycline and kanamycin.

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Table 6.5: The resumes of susceptible (S) and resistance (R) showing antibiotic susceptibility test of the selected isolates against various drugs, methicillin, amoxicillin, penicillin-G, chloramphenicol, neomycin, ampicillin, vancomycin, tetracycline and kanamycin.

S. No. Isolates Methicillin Amoxicillin Penicillin G Chloramphenicol Neomycin Ampicillin Vancomycin Tetracycline Kanamycin

1 S3 S S S S S S S S S 2 S7 S S S S S S S S S

3 K1 S S R S S S S S R

4 K3 S R R S S S S S R

5 K16 S S R S S S S S S

6 K21′ S S R S S R S S R

7 BDK1 S S S S S S S S S 8 BDK2′ S S S S S S S S S

9 BDK5 S S S S S S S S S

10 BDK7 S S S S S S S S S

11 BDK9 S S S S S S S S S

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6.1.4.5. Adhesion and Biofilm Formation

After the successful survival of isolates in gastric environment of the GIT. It is also necessary to check the adherence ability of the isolates with the epithelial cells of the intestine. Adherence is the foremost requirement for any bacteria to proliferate and establish its colonies (Lee and Salminen, 1995). High probiotic adherence to the intestine promotes the release of beneficial bioeffects, like immune-modulation and antimicrobial activities against pathogens (Shokryazdan et al., 2014).

The adhesion property of bacteria is an important criteria for the selection of a potential probiotic strain (Vine et al., 2004). Better adhesion of bacteria leads to closer host- microbe interaction, which increase the efficacy of the probiotic bacteria (Gueimonde and Salminen, 2006). The adhesion and biofilm formation were evaluated using the standard tube method by taking the absorbance at 600 nm as a quantitative measure. The optical density plot indicating adhesion and biofilm formation of the isolated strains is shown in Figure 6.10. In this study, all the isolates showed the adherence ability. The comparative evaluation of adhesion results indicates highest adhesion of S7 (0.749 ± 0.03) followed by BDK9 (0.660 ± 0.034). The remaining isolates also show the adhesion as 0.460 ± 0.05, 0.412 ± 0.006, 0.588 ± 0.005 for S3, BDK2′ and BDK7, respectively. The complete results of adhesion assay of the isolated strains are given in Table 6.6.

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0.9 b 0.8 i 0.7 c,e,h c,e,h,i 0.6 a,c,f,g,h a,c,e,h a,c,e,f,h a,c,f,g,h a,f,g,h 0.5 a,d,f,g d,g 0.4 0.3 0.2 0.1

Absorbance at 600 nm at Absorbance 0 S3 S7 K1 K3 K16 K21′ BDK1 BDK2′ BDK5 BDK7 BDK9 Bacterial strains

Figure 6.10: The optical density plot (absorbance at 600 nm) indicating adhesion and biofilm formation of the isolated strains evaluated by standard tube method. Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post- hoc test) among the adhesion property of different isolates.

Table 6.6: Optical density values (at 600 nm) showing adhesion and biofilm formation property of different isolated strains as evaluated by standard tube method.

Adhesion and biofilm formation S. No. Isolates (OD at 600 nm) 1 S3 0.460a,c,f,g,h ± 0.05 2 S7 0.749b ± 0.03 3 K1 0.536a,c,e,h ± 0.013 4 K3 0.452a,c,f,g,h ± 0.026 5 K16 0.356d,g ± 0.031 6 K21′ 0.563c,e,h ± 0.028 7 BDK1 0.446a,f,g,h ± 0.028 8 BDK2′ 0.412a,d,f,g ± 0.006 9 BDK5 0.520a,c,e,f,h ± 0.010 10 BDK7 0.588c,e,h,i ± 0.005 11 BDK9 0.660i ± 0.034

Each values given in the table are mean ± standard deviation of three separate experiments. Different letters demonstrate significant difference (P < 0.05) among the adhesion property of different isolates.

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6.1.5. Identification of the Isolated Bacteria In bacteriology, identification of bacteria is an important step which was carried out by both conventional biochemical tests and by 16S ribosomal RNA sequencing. The results of both the identification test are discussed as follows.

6.1.5.1. Biochemical Characterization

The isolated bacterial strains were characterized by performing various biochemical tests including Gram's staining, catalase test, urease test, gelatin test, salt tolerance, motility test, indole test, glucose fermentation, methyl red and Voges-Proskauer test. In the biochemical characterization, all isolates showed negative results to catalase, urease, indole and gas production which means the bacteria do not produce catalase and urease enzyme, do not have the ability to convert tryptophan into indole. While these isolates gave positive results for mannitol salt agar test which means these isolates were resistant to high concentration of salt, also these isolates gave positive result to methyl red and glucose fermentation except isolate BDK5 (as shown in Figure 6.11). BDK5 gave negative result to the glucose fermentation. The biochemical characterization result indicates that these probionts were found to share the main phenotypic features of the genus Enterococcus and Bacillus, which implies that these probionts belongs to the Enterococcus and Bacillus genus and were sent for further molecular (16S rRNA sequencing) characterization. The detail results of biochemical profile of the isolated strains were given in Table 6.7.

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Figure 6.11: Digital image showing different reaction of biochemical tests including catalase test, urease test, gelatin test, mannitol salt agar test, motility test, indole test, glucose fermentation, methyl red and Voges-Proskauer test of the isolated strains. Figure (a-b) show control reaction (blank) and (c-f) show biochemical reaction in the presence of bacterial isolates.

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Table 6.7: The resumes of positive (+) and negative (-) results of isolated bacterial strains against biochemical characterization test series.

Tests S3 S7 K1 K3 K16 K21′ BDK1 BDK2′ BDK5 BDK7 BDK9

Gram Stain + + + + + + + + + + +

Catalase ------

Urease ------

Gelatin ------

MSA + + + + + + + + + + +

Glucose fermentation + + + + + + + + - + +

H2S production ------

Motility ------

Indole ------

Methyl Red + + + + + + + + + + +

V.P ------

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

After in-vitro evaluation and biochemical characterization of the isolated bacteria, it was observed that only 5 bacterial strains passed all the selection parameters described in FAO/WHO guidelines for the evaluation of probiotics. So, for the molecular characterization, these five isolates S3, S7, BDK2′, BDK7 and BDK9 which were isolated from Catla catla (S3 and S7), Nandus nandus (BDK2′), Puntius chola (BDK7) and Oxygaster bacaila (BDK9) were selected and sent to the Yaazh Xenomics (Coimbatore) and Biokart (Bangalore) for their 16S rRNA sequencing.

6.1.5.2. Molecular Identification

For molecular characterization the samples were sent for sequencing at Yaazh Xenomics, Coimbatore, India and Biokart India Pvt Ltd, Bangalore, India. The isolates namely, S3, S7, BDK2', BDK7 and BDK9 passed all the criteria for the selection as probiotics. They were further identified by 16S rRNA sequencing. The nucleotide sequence obtained were compared with the sequences available in the NCBI (National Center for Biotechnology Information) database using BLAST (Basic Local Alignment Search Tool) program. Based on BLAST results, it was found that the 16S rRNA sequence of above organisms showed maximum similarity to Bacillus and Enterococcus species. The 16S rRNA gene sequencing of S3, S7, BDK2', BDK7 and BDK9 confirmed them as Enterococcus durans strain S3, Bacillus amyloliquefaciens strain S7, Bacillus cereus strain BDK2', Bacillus subtilis strain BDK7 and Bacillus subtilis strain BDK9 species respectively. Phylogenetic tree of all the isolates constructed using BLAST are presented in Figure 6.12-6.16. The accession number of NCBI GenBank of these isolates were given in Table 6.8.

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Figure 6.12: Phylogenetic tree showing relationship between Enterococcus durans strain S3 and other Enterococcus species.

Figure 6.13: Phylogenetic tree showing relationship between Bacillus amyloliquefaciens strain S7 and other Bacillus species.

Figure 6.14: Phylogenetic tree showing relationship between Bacillus cereus strain BDK2' and other Bacillus species.

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Figure 6.15: Phylogenetic tree showing relationship between Bacillus subtilis strain BDK7 and other Bacillus species.

Figure 6.16: Phylogenetic tree showing relationships between Bacillus subtilis strain BDK9 and other Bacillus species.

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Table 6.8: The sequence length (bp), similarity index, identified species and accession numbers of isolated bacteria after molecular identification (16S rRNA sequencing).

Sequence Species match Species Accession Isolates length Similarity (GenBank) identified No. (bp) Enterococcus Enterococcus S3 1316 95% MH628095 durans strain B1 durans strain S3 Bacillus Bacillus S7 1380 amyloliquefaciens 99% amyloliquefaciens MH645840 strain MPA 1034 strain S7 Bacillus cereus Bacillus cereus BDK2' 1382 strain ATCC 100% MH842165 strain BDK2' 14579 Bacillus subtilis Bacillus subtilis BDK7 1387 strain BGSC 99% MH645813 strain BDK7 3A28 Bacillus subtilis Bacillus subtilis BDK9 1387 99% MH842170 strain JCM 1465 strain BDK9

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6.2. Majha Region

6.2.1. Fish Samples Collected from Majha Region

In Majha region, the fish samples were collected from fishermen of catchment area of rivers. A total of 7 fish of Channa striatus species were collected from Majha region for the isolation of bacteria.

Figure 6.17: Geographical map of Punjab showing different regions, the highlighted region shows Majha region, this region has been selected for collection of fish samples.

6.2.2. Isolation and Purification of Bacteria from Fish Gut Sample

A total of 64 morphologically distinct bacterial colonies were isolated from the gut of 7 different healthy fish. These isolated colonies were purified multiple times by streaking them on BDA and MRS agar medium until pure bacterial cultures were obtained as shown in Figure 6.18.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Figure 6.18: Digital images of pure isolated cultures streaked on (a-i) de-Man, Rogosa and Sharpe agar (MRS) and (j-o) Bacillus differentiation agar (BDA) showing bacterial colonies isolated from gastrointestinal tract of healthy fish collected from Majha region, Punjab.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

6.2.3. Screening and Indexing of Gram-positive Bacteria

The staining property and cell morphology of isolated bacteria was examined using Gram’s staining technique. The Gram staining is the ability of bacteria to retain color of stains used in the staining procedure. Gram positive bacteria retained the crystal violet stain and were not decolorized by alcohol therefore remained as purple. Out of 64 bacterial colonies, 39 isolates were found to be Gram-positive bacteria. Typical images of few Gram-positive bacteria are shown in Figure 6.19 and nomenclature of the selected Gram-positive bacteria, collection site, source and name of the species from which bacteria has been isolated are given in Table 6.9. These isolated bacteria were found to be purple colored and rod shaped under oil-immersion microscope.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Figure 6.19: Digital microscope images showing typical Gram-positive morphology of different bacteria isolated from gastrointestinal tract (GIT) of fish under oil-immersion microscope.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Table 6.9: Nomenclature of the Gram-positive bacteria isolated from Majha region and the name of source fish species.

S. No. Isolate name Collection site Source Species

1 F1F1 Harike, Mahja Gut region Channa striatus

2 F1F2 Harike, Mahja Gut region Channa striatus

3 F1F3 Harike, Mahja Gut region Channa striatus

4 F1F4 Harike, Mahja Gut region Channa striatus

5 F1H1 Harike, Mahja Gut region Channa striatus

6 F1H2 Harike, Mahja Gut region Channa striatus

7 F1H3 Harike, Mahja Gut region Channa striatus

8 F1H4 Harike, Mahja Gut region Channa striatus

9 BD F1F1 Harike, Mahja Gut region Channa striatus

10 BD F1F2 Harike, Mahja Gut region Channa striatus

11 BD F1F3 Harike, Mahja Gut region Channa striatus

12 BD F1F4 Harike, Mahja Gut region Channa striatus

13 BD F1H1 Harike, Mahja Gut region Channa striatus

14 BD F1H2 Harike, Mahja Gut region Channa striatus

15 BD F1H3 Harike, Mahja Gut region Channa striatus

16 BD F1H4 Harike, Mahja Gut region Channa striatus

17 F2F1 Harike, Mahja Gut region Channa striatus

18 F2F2 Harike, Mahja Gut region Channa striatus

19 F2F3 Harike, Mahja Gut region Channa striatus

20 F2F4 Harike, Mahja Gut region Channa striatus

21 F2H1 Harike, Mahja Gut region Channa striatus

22 F2H2 Harike, Mahja Gut region Channa striatus

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23 F2H3 Harike, Mahja Gut region Channa striatus

24 F2H4 Harike, Mahja Gut region Channa striatus

25 BD F2F1 Harike, Mahja Gut region Channa striatus

26 BD F2F2 Harike, Mahja Gut region Channa striatus

27 BD F2F3 Harike, Mahja Gut region Channa striatus

28 BD F2F4 Harike, Mahja Gut region Channa striatus

29 BD F2H1 Harike, Mahja Gut region Channa striatus

30 BD F2H2 Harike, Mahja Gut region Channa striatus

31 BD F2H3 Harike, Mahja Gut region Channa striatus

32 BD F2H4 Harike, Mahja Gut region Channa striatus

33 F3 1(1) Harike, Mahja Gut region Channa striatus

34 F3 1(2) Harike, Mahja Gut region Channa striatus

35 F3 1(3) Harike, Mahja Gut region Channa striatus

36 F3 2(1) Harike, Mahja Gut region Channa striatus

37 F3 2(1) Harike, Mahja Gut region Channa striatus

38 BD F3 (1) Harike, Mahja Gut region Channa striatus

39 BD F3 (2) Harike, Mahja Gut region Channa striatus

6.2.4. In-vitro Evaluation of Probiotic Potential of Gram-positive Bacteria

6.2.4.1. Antagonistic Activity

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

inhibition zone. Among the 39 Gram-positive isolates, only 9 isolates i.e. F1F1, F1F2, F1F4, F1H1, F1H3, F1H4, F2F4, F3 1(1) and F3 1(2) were exhibited antagonistic activity against targeted pathogen (Figure 6.20) and were selected for further in-vitro evaluation. The remaining 30 isolates failed to show any activity against the target fish pathogen and were discarded safely. Total number of positive isolates which showed antagonistic activity are given in Table 6.10 and the graphical representation of zone of inhibition measured are shown in Figure 6.21. Among all the isolates F1F4 showed highest antagonistic activity against the target fish pathogen Pseudomonas aeruginosa (MTCC 4673) with zone of inhibition as 44.66 ± 4.50 mm, followed by F1F1, F3 1(2), F1F2, F1H3, F1H4, F2F4, F3 1(1) and F1H1. The zone of inhibition of these isolates is 21.66 ± 2.51, 19.00 ± 3.00, 18.66 ± 4.16, 15.00 ± 3.00, 14.33 ± 2.08, 8.33 ± 2.51, 7.00 ± 2.64 and 6.33 ± 1.52 mm, respectively.

Figure 6.20: The digital image of culture plates (a-f) showing zone of inhibition by the different isolates against the fish pathogen Pseudomonas aeruginosa (MTCC 4673).

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

50 b

30 a,d,e a,d,e a,d,e 20 a,d,e,f a,c,d,e,f c,d,e,f 10 c,e,f c,d,e,f Zone of Inhibition (mm) Inhibition of Zone 0 F1F1 F1F2 F1F4 F1H1 F1H3 F1H4 F2F4 F3 1(1) F3 1(2) Bacterial strains

Figure 6.21: Zone of inhibition (mm) plot of different bacterial isolates derived from Majha region indicating their antagonistic activity against the selected pathogen, Pseudomonas aeruginosa (MTCC 4673). Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) with respect to antagonistic activity of different isolates.

Table 6.10: Zone of inhibition (in mm) of different bacterial isolates derived from Majha region showing antagonistic activity against Pseudomonas aeruginosa (MTCC 4673). S. No. Isolates Zone of Inhibition (mm) 1 F1F1 21.66a,d,e ± 2.51 2 F1F2 18.66a,d,e ± 4.16 3 F1F4 44.66b ± 4.50 4 F1H1 6.33c,e,f ± 1.52 5 F1H3 15.00a,d,e,f ± 3.00 6 F1H4 14.33a,c,d,e,f ± 2.08 7 F2F4 8.33c,d,e,f ± 2.51 8 F3 1(1) 7.00c,d,e,f ± 2.64 9 F3 1(2) 19.00a,d,e ± 3.00

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

6.2.4.2. pH Tolerance Test As discussed in previous section, there are many factors inside the gut that affect the viability of probiotics such as acid and bile salts. A probiotic bacteria should be able to survive the acidic conditions of the gastrointestinal tract. Therefore, the pH tolerance of the isolated bacterial strains was investigated over a wide range of pH (pH 2 to pH 9). It was found that, all 9 isolates which exhibited the antagonistic activity, also showed growth and survival at decided pH range.

The results given in Table 6.11 show that the isolates were quite tolerant to low pH (pH 2-3). Although, the critical limit to survive in the acidic gut environment in fish is pH 3.5- 4.5. The selected strains exhibit relatively strong resistance to high pH (pH 9). The survivability of the isolates at different pH is expressed in terms of optical density (OD) recorded at 600 nm (Figure 6.22).

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

pH 2 pH 3 pH 4 pH 9 0.7

b 0.6

e 0.5 a a a b b b a a f 0.4 a d d a c a b a b b a a b e b d d d d d d c c d c d e e a e c e c d d 0.3 d d g d e e a a c d c a e a a f f e f c c c a g e e c g b b a d a f c d f d c Absorbance at 600 nm at Absorbance b b f 0.2 c c b b g b g e g c c f d d d d d f f f f f f f d 0.1 g g g f g 0 F1F1 F1F2 F1F4 F1H1 F1H3 F1H4 F2F4 F3 1(1) F3 1(2) Bacterial strains

Figure 6.22: The optical density (OD at 600 nm) indicating growth and survival of bacterial strains isolated from Majha region in MRS Broth medium, adjusted at different values of pH (2.0, 3.0, 4.0 and 9.0) to study the effect of pH on the growth and survival. Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) in bacterial strains at same pH.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Table 6.11: pH test results showing optical density values at 600 nm as a measure of survival of the bacterial strains isolated from Majha region and cultured under different pH medium (pH 2.0-9.0).

Bacterial Survival of the isolates at different pH (OD at 600 nm) Strain pH 2 pH3 pH4 pH9

F1F1 0.123a,b,c,f ± 0.005 0.282a,b,d,e ± 0.015 0.195a,d,e,g ± 0.008 0.324a,d ± 0.009

F1F2 0.102a,b,c,d,f ± 0.013 0.302a,b,d ± 0.011 0.281b,d,e ± 0.015 0.596b ± 0.019

F1F4 0.105a,b,c,d,f ± 0.010 0.357a,b,d ± 0.032 0.209c,g ± 0.021 0.217c,d,f ± 0.025

F1H1 0.138a,b,c ± 0.015 0.289a,b,d,e ± 0.062 0.214a,b,d,e,g ± 0.050 0.211c,d,f ± 0.033

F1H3 0.063b,d,f,g ± 0.013 0.191c,d,e,f ± 0.007 0.242a,b,d,e ± 0.035 0.278a,c,d ± 0.024 F1H4 0.203e ± 0.044 0.274a,b,c,d,e ± 0.043 0.378f ± 0.025 0.465e ± 0.047

F2F4 0.059b,d,f,g ± 0.008 0.205a,c,d,e,f ± 0.028 0.162a,c,d,g ± 0.016 0.319a,d ± 0.026

F3 1(1) 0.072a,b,d,f,g ± 0.011 0.181c,e,f ± 0.004 0.146a,c,d,g ± 0.020 0.172c,f ± 0.032

F3 1(2) 0.032d,f,g ± 0.014 0.138c,e,f ± 0.014 0.146a,c,d,g ± 0.032 0.151c,f ± 0.008

6.2.4.3. Bile Salt Tolerance

The resistance to bile salts is vital condition for the survival and colonization of probiotic bacteria in the host’s intestine. The survival of ingested strains in extreme environment of the intestine can be predicted by the bile salt tolerance analysis. The isolated strains exhibit survival at 0.3% bile salt (ox-gall) and show significant growth both in absence and presence of bile salts except the F3 1(1). It was observed that F3 1(1) failed to survive in the presence of bile salt. These results showed that the bile salts found in the intestine does not affect the test probiotic bacteria. Figure 6.23 shows the absorbance indicative of the bacterial growth and survival in presence and absence of the bile salts. The results indicate the adaptability of isolated bacterial strains in the presence of bile salts (Table 6.12).

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

0.8 g With Bile (0.3%) without bile 0.7 0.6 0.5 f 0.4 d,k d,k d,e,k b,e,k b,e,k 0.3 b,e b,e c,h a,c,h a,c,h 0.2 a,c a,c a,c a,c Absorbance at 600 nm at Absorbance 0.1 j i 0 F1F1 F1F2 F1F4 F1H1 F1H3 F1H4 F2F4 F3 1 (1)F3 1 (2) Bacterial strains

Figure 6.23: The optical density (at 600 nm) showing tolerance of isolated bacterial strains in presence and absence of bile salts (0.3% ox-gall). Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) in between with bile or without bile groups.

Table 6.12: Bile tolerance results showing optical density values (at 600 nm) as a measure of survival of isolates after 24 h in presence and absence of bile salts (0.3 % ox-gall concentration).

Absorbance at 600 nm Bacterial Strain With Bile (0.3%) Without bile F1F1 0.130a,c ± 0.006 0.245b,e ± 0.014 F1F2 0.152a,c,h ± 0.024 0.351d,k ± 0.030 F1F4 0.167a,c,h ± 0.012 0.222b,e ± 0.004 F1H1 0.135a,c ± 0.009 0.274b,e,k ± 0.010 F1H3 0.343f ± 0.049 0.709g ± 0.034 F1H4 0.218c,h ± 0.007 0.349d,k ± 0.017 F2F4 0.130a,c ± 0.018 0.249b,e,k ± 0.044 F3 1(1) 0.010i ± 0.001 0.072j ± 0.013 F3 1(2) 0.111a,c ± 0.017 0.315d,e,k ± 0.017

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

6.2.4.4. Susceptibility to Drugs A potential probiotic strain should not possess any transmissible antibiotic-resistant genes. The antibiotic resistance is represented based on visible bacterial growth after overnight incubation in the presence of the antibiotics disc. In the current study, out of 9 isolates, 8 bacterial strains were found to be susceptible to the antibiotics used. These isolates were susceptible to methicillin, amoxicillin, penicillin-G, chloramphenicol, neomycin, ampicillin, vancomycin, tetracycline and kanamycin by showing clear zone around the antibiotic discs. But the remaining 1 isolate F1H1 showed resistance against kanamycin. The antibiotic susceptibility results obtained are shown in Figure 6.24 and Table 6.13.

Figure 6.24: Digital image of culture plates (a-d) showing antibiotic susceptibility of the isolated strains against various drugs, methicillin, amoxicillin, penicillin-G, chloramphenicol, neomycin, ampicillin, vancomycin, tetracycline and kanamycin.

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Table 6.13: The resumes of susceptible (S) and resistance (R) showing antibiotic susceptibility test of the selected isolates against various drugs, methicillin, amoxicillin, penicillin-G, chloramphenicol, neomycin, ampicillin, vancomycin, tetracycline and kanamycin.

S. No. Isolates Methicillin Amoxicillin Penicillin G Chloramphenicol Neomycin Ampicillin Vancomycin Tetracycline Kanamycin

1 F1F1 S S S S S S S S S

2 F1F2 S S S S S S S S S

3 F1F4 S S S S S S S S S

4 F1H1 S S S S S S S S R

5 F1H3 S S S S S S S S S

6 F1H4 S S S S S S S S S

7 F2F4 S S S S S S S S S

8 F3 1(1) S S S S S S S S S

9 F3 1(2) S S S S S S S S S

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6.2.4.5. Adhesion and Biofilm Formation

The adhesion property of bacteria is an important criteria for the selection of a potential probiotic strain. Adhesion and Biofilm formation were evaluated using the standard tube method by taking the absorbance (at 600 nm) as a quantitative measure. In this study, all the isolates showed the adherence ability (Figure 6.25). The comparative evaluation of adhesion results indicates highest adhesion of F1F4 (0.697 ± 0.023). The remaining isolates also show the adhesion as 0.471 ± 0.032, 0.458 ± 0.021, 0.448 ± 0.039, 0.371 ± 0.015, 0.371 ± 0.020, 0.324 ± 0.006, 0.311 ± 0.012 and 0.260 ± 0.027 for F2F4, F1H3, F1F2, F1H1, F3 1(1), F3 1(2), F1F1 and F1H4, respectively. The complete results of adhesion assay of the isolated strains are given in Table 6.14.

0.8 c 0.7 0.6 b 0.5 b b a,d a,d 0.4 a,d,e a,d,e 0.3 a,e 0.2 Absorbance at 600 nm at Absorbance 0.1 0 F1F1 F1F2 F1F4 F1H1 F1H3 F1H4 F2F4 F3 1 (1)F3 1 (2) Bacterial strains

Figure 6.25: The optical density plot (absorbance at 600 nm) indicating adhesion and biofilm formation of the isolated strains evaluated by standard tube method. Values are the mean ± standard deviation of three separate experiments. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post- hoc test) among the adhesion property of different isolates.

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Table 6.14: The optical density values (at 600 nm) showing adhesion and biofilm formation property of different isolated strains as evaluated by standard tube method.

Adhesion and biofilm formation S. No. Isolates (OD at 600 nm)

1 F1F1 0.311a,d,e ± 0.012

2 F1F2 0.448b ± 0.039

3 F1F4 0.697c ± 0.023

4 F1H1 0.371a,d ± 0.015

5 F1H3 0.458b ± 0.021

6 F1H4 0.260a,e ± 0.027

7 F2F4 0.471b ± 0.032

8 F3 1(1) 0.371a,d ± 0.020

9 F3 1(2) 0.324a,d,e ± 0.006

6.2.5. Identification of the Isolated Bacteria In bacteriology, identification of bacteria is a very important step. Identification of bacterial strains was carried out by both methods i.e. biochemical characterization and molecular characterization by 16S rRNA sequencing. The results of bacterial identification are as follows.

6.2.5.1. Biochemical Characterization The isolated bacterial strains were characterized by performing various biochemical tests including Gram's staining, catalase test, urease test, gelatin test, salt tolerance, motility test, indole test, glucose fermentation, methyl red and Voges-Proskauer test. In the biochemical characterization, all isolates showed negative results to catalase, urease, gelatin and indole test except F1F1. F1F1 was observed to give positive result to catalase test. While 6 isolates gave positive results to mannitol salt agar test which means these isolates were resistant to high concentration of salt, also these isolates gave

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positive result to methyl red and glucose fermentation (as shown in Figure 6.26). Isolate F1F1, F1H1 and F3 1(1) gave negative result to the glucose fermentation. F1H1 gave negative result to the methyl red test. The biochemical characterization result indicates that these probionts were found to share the main phenotypic features of the genus Enterococcus and Bacillus, which implies that these probionts belongs to the Enterococcus and Bacillus genus and were sent for further molecular (16S rRNA sequencing) characterization. The detailed results of biochemical profile of the isolated strains are given in Table 6.15.

Figure 6.26: Digital image showing different reaction of biochemical tests including catalase test, urease test, gelatin test, mannitol salt agar test, motility test, indole test, glucose fermentation, methyl red and Voges-Proskauer test of the isolated strains. Figure (a-b) show control reaction (blank) and (c-f) show biochemical reaction in the presence of bacterial isolates. 103

Development of Probiotics from Aquatic Bodies for Enhanced Productivity in Fish Farming

Table 6.15: The resumes of positive (+) and negative (–) results of isolated bacterial strains against biochemical characterization test series.

Tests F1F1 F1F2 F1F4 F1H1 F1H3 F1H4 F2F4 F3 1(1) F3 1(2)

Gram Stain + + + + + + + + +

Catalase + – – – – – – – –

Urease – – – – – – – – –

Gelatin – – – – – – – – –

MSA – + + + + + + + +

Glucose fermentation – + + – + + + – +

H2S production – – + – + – – – –

Motility – – – – – – – – –

Indole – – – – – – – – –

Methyl Red + + + – + + + + +

V.P – – – – – – – – –

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After in-vitro evaluation and biochemical characterization of the isolated bacteria, it was observed that only 6 bacterial strains passed all the selection parameters as described in FAO/WHO guidelines for the evaluation of probiotics. So, for molecular characterization, these six isolates F1F4, F1H4, F2F4, F3 1(2), F1F2 and F1H3 were selected and sent to the Yaazh Xenomics (Coimbatore) and Biokart (Bangalore) for their 16S rRNA sequencing.

6.2.5.2 Molecular Identification

For molecular characterization the samples were sent for sequencing at Yaazh Xenomics, Coimbatore, India and Biokart India Pvt Ltd, Bangalore, India. The isolates namely, F1F4, F1H4, F2F4, F3 1(2), F1F2 and F1H3 passed all the criteria for selection as probiotics and were identified using 16S rRNA sequencing and phylogenetic analysis. The nucleotide sequence obtained were compared to available sequences in the NCBI (National Center for Biotechnology Information) database using BLAST (Basic Local Alignment Search Tool) program. Based on BLAST results, it was found that the 16S rRNA sequence of above organisms showed maximum similarity to Bacillus and Enterococcus species. The 16S rRNA gene sequencing of F1F4, F1H4, F2F4, F3 1(2), F1F2 and F1H3 confirmed them as Bacillus subtilis strain F1F4, Enterococcus faecium strain F1H4, Bacillus safensis strain F2F4, Bacillus subtilis strain F3 1(2), Bacillus velezensis strain F1F2, Enterococcus gallinarum strain F1H3 species, respectively. Phylogenetic tree of all the isolates constructed using BLAST are presented in Figure 6.27-6.32. The accession number of NCBI GenBank of these isolates are given in Table 6.16.

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Figure 6.27: Phylogenetic tree showing relationships between Bacillus subtilis strain F1F4 and other Bacillus species.

Figure 6.28: Phylogenetic tree showing relationships between Enterococcus faecium strain F1H4 and other Enterococcus species.

Figure 6.29: Phylogenetic tree showing relationships between Bacillus safensis strain F2F4 and other Bacillus species.

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Figure 6.30: Phylogenetic tree showing relationships between Bacillus subtilis strain F3 1(2) and other Bacillus species.

Figure 6.31: Phylogenetic tree showing relationships between Bacillus velezensis strain F1F2 and other Bacillus species.

Figure 6.32: Phylogenetic tree showing relationships between Enterococcus gallinarum strain F1H3 and other Enterococcus species.

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Table 6.16: The sequence length (bp), similarity index, identified species and accession numbers of isolated bacteria after molecular identification (16S rRNA sequencing).

Sequence Species match Species Accession Isolates Similarity length (bp) (GenBank) identified No. Bacillus Bacillus F1F4 1397 subtilis strain 99% subtilis strain MK208590 NBRC 101239 F1F4 Enterococcus Enterococcus F1H4 1225 faecium strain 100% faecium strain MH630247 DSM 20477 F1H4 Bacillus Bacillus F2F4 1394 safensis strain 99% safensis strain MH645839 NBRC 100820 F2F4 Bacillus Bacillus F3 1(2) 1183 subtilis strain 93% subtilis strain MH645757 JCM 1465 F3 1(2) Bacillus Bacillus F1F2 1390 velezensis 99% velezensis MK208528 strain FZB42 strain F1F2 Enterococcus Enterococcus gallinarum F1H3 1274 99% gallinarum MK208529 strain LMG strain F1H3 13129

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6.3. Malwa Region

6.3.1. Fish Samples Collected from Malwa Region

In Malwa region, the fish samples were collected from local market, and fishermen of catchment area of Ropar city (30.9659° N, 76.5230° E) and Ludhiana city (30.9010° N, 75.8573° E) (Figure 6.33). A total of 20 fish from different species i.e. Labeo dero, Rasbora daniconius, Puntius sarana, Chanda nama, Xenentodon cancila, Oxygaster bacaila, Channa striatus, Cyprinus carpio, Parambassis ranga and Labeo gonius were collected from the Malwa region for the isolation of bacteria Figure 6.34.

Figure 6.33: Geographical map of Punjab showing different regions, the highlighted region shows Malwa region, the region has been selected for collection of fish samples.

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Figure 6.34: Digital image of different fish species i.e. (a) Labeo derio, (b) Rasbora daniconius, (c) Puntius sarana, (d) Chanda nama, (e) Xenentodon cancila, (f) Oxygaster bacaila, (g) Chanda ranga, (h) Labeo gonius, (i) Cyprinus carpio, (j) Oxygaster bacaila and (k) Channa striatus collected from Malwa region of Punjab, India.

6.3.2. Isolation and Purification of Bacteria from Fish Gut Sample

A total of 80 morphologically distinct bacterial colonies were isolated from the gut of healthy 20 different fish. These isolated colonies were purified multiple times by streaking onto BDA and MRS agar medium until the pure bacterial cultures were obtained as shown in Figure 6.35.

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Figure 6.35: Digital images of pure isolated cultures (a-d) streaked on MRS agar showing bacterial colonies isolated from gastrointestinal tract of healthy fish collected from Malwa region, Punjab.

6.3.3. Screening and Indexing of Gram-positive Bacteria

The isolated bacteria were examined using Gram’s staining technique to check their staining property and cell morphology. Gram staining is the ability of bacteria to retain colour of the stains used in staining procedure. Gram positive bacteria retained the crystal violet stain and were not decolorized by alcohol therefore remained as purple. Out of 80 bacterial colonies, only 11 isolates were found to be Gram-positive bacteria. Typical images of few Gram-positive bacteria are shown in Figure 6.36 and nomenclature of the selected Gram-positive bacteria, collection site, source and name of the species from which bacteria has been isolated are given in Table 6.17. These

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isolated bacteria were found to be purple colored and rod shaped under oil-immersion microscope.

Figure 6.36: Digital microscope images showing typical Gram-positive morphology of different bacteria isolated from gastrointestinal tract (GIT) of fish under oil-immersion microscope.

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Table 6.17: Nomenclature of the Gram-positive bacteria isolated from Malwa region and the name of source fish species.

S. No. Isolate name Collection site Source Species

1 LF1(1) Ropar, Malwa Gut region Labeo derio

2 LF1(2) Ropar, Malwa Gut region Rasbora daniconius

3 LF1(3) Ropar, Malwa Gut region Puntius sarana 4 LF1(4) Ropar, Malwa Gut region Chanda nama

5 LF1A1 Ropar, Malwa Gut region Xenentodon cancila

6 LF2(4) Ropar, Malwa Gut region Oxygaster bacaila

7 LF3(1) Ludhiana, Malwa Gut region Labeo gonius

8 LF3(2) Ludhiana, Malwa Gut region Parambassis ranga

9 BD LF1(1) Ropar, Malwa Gut region Labeo derio 10 BD LF1(3) Ropar, Malwa Gut region Puntius sarana

11 BD LF1A1 Ropar, Malwa Gut region Xenentodon cancila

6.3.4. In-vitro Evaluation of Probiotic Potential of Gram-positive Bacteria

6.3.4.1. Antagonistic Activity

The indexed Gram-positive bacterial strains were examined for their antagonistic activity by using disc diffusion method against the fish pathogen Pseudomonas aeruginosa (MTCC 4673). The experiment was performed in triplicate and the potential of isolated bacteria to inhibit the pathogen was determined by the diameter of inhibition zone. Among the 11 Gram-positive isolates, only 1 bacterium, LF3(1) exhibited antagonistic activity against targeted pathogen and showing the zone of inhibition (11.66 ± 2.08 mm) as shown in Figure 6.37(a) and was selected for further in-vitro

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evaluation. The remaining 10 isolates failed to show the activity against the target fish pathogen and were discarded safely.

Figure 6.37: The digital image of culture plates (a) showing zone of inhibition by LF3(1) against the fish pathogen Pseudomonas aeruginosa (MTCC 4673) and (b) antibiotic susceptibility of LF3(1) against various drugs, methicillin, amoxicillin, penicillin-G, chloramphenicol, neomycin, ampicillin, vancomycin, tetracycline and kanamycin.

6.3.4.2. pH Tolerance Test

Probiotic bacteria should be able to survive in the acidic environment of the gastrointestinal tract. Therefore, pH tolerance of the isolated bacterial strains was investigated over a wide range of pH (pH 2 – pH 9). The results showed that the LF3(1) was quite tolerant to pH 2 (0.244 ± 0.038), pH 3 (0.431 ± 0.029), pH 4 (1.304 ± 0.013) and pH 9 (1.271 ± 0.048). The survivability of the isolate at different pH is expressed in terms of optical density (OD) recorded at 600 nm as shown in Figure 6.38.

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1.4 LF3 (1) 1.2 1 0.8 0.6 0.4

Absorbance at 600 nm at Absorbance 0.2 0 pH 2 pH3 pH4 pH9 pH

Figure 6.38: The optical density (OD at 600 nm) indicating growth and survival of bacterial strain isolated from Malwa region in MRS Broth medium, adjusted at different values of pH (2.0, 3.0, 4.0 and 9.0) to study the effect of pH on the growth and survival.

6.3.4.3. Bile Salt Tolerance

The resistance to bile salts is vital condition for the survival and colonization of probiotic bacteria in the host’s intestine. The survival of ingested strains in the extreme environment of intestine can be predicted by the bile salt tolerance analysis. The isolated strain LF3(1) exhibit survival at 0.3% bile salt (ox-gall) and show significant growth both in absence (0.848 ± 0.018) and presence (0.557 ± 0.051) of bile salts. The survivability of the isolate is expressed in terms of optical density (OD) recorded at 600 nm. These results showed that the bile salts found in the intestine does not affect the test probiotic bacteria.

6.3.4.4. Susceptibility to Drugs

A potential probiotic strain should not possess any transmissible antibiotic-resistant genes. The antibiotic resistance is represented based on visible bacterial growth after overnight incubation in the presence of the antibiotics disc. The results showed that the isolate LF3(1) was susceptible to all antibiotic discs used in the study. The antibiotic susceptibility results obtained are shown in Figure 6.37(b).

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6.3.4.5. Adhesion and Biofilm Formation The adhesion property of bacteria is an important criterion for the selection of a potential probiotic strain. In this study, the isolate LF3(1) showed good adherence ability (0.439 ± 0.054), evaluated with the standard tube method by taking the absorbance (at 600 nm) as a quantitative measure of the bacterial growth.

6.3.5. Identification of the Isolated Bacteria

In bacteriology, the identification of bacteria is a very important step. Identification of bacterial strains were carried out by both biochemical and molecular methods.

6.3.5.1. Biochemical Characterization

The isolate LF3(1) was characterized by performing various biochemical tests including Gram's staining, catalase test, urease test, gelatin test, salt tolerance, motility test, indole test, glucose fermentation, methyl red and Voges-Proskauer test. In the biochemical characterization, the isolate LF3(1) gave negative results to catalase, urease, gelatin, indole and Voges-Proskauer test and gave positive result to mannitol salt agar test, glucose fermentation test and methyl red test (as shown in Figure 6.39). Biochemical characterization result indicates that the isolate LF3(1) was found to share the main phenotypic features of the genus Enterococcus, which implies that this probiont belongs to the Enterococcus genus and was sent for further molecular (16S rRNA sequencing) characterization.

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Figure 6.39: Digital image showing different reaction of biochemical tests including catalase test, urease test, gelatin test, mannitol salt agar test, motility test, indole test, glucose fermentation, methyl red and Voges-Proskauer test of the isolated strain. Figure (a-b) show control reaction (blank) and (c) shows biochemical reaction in the presence of bacterial isolates.

After in-vitro evaluation and biochemical characterization of the isolated bacteria, it was observed that the isolate LF3(1) passed all selection parameters as described in FAO/WHO guidelines for the evaluation of probiotics. So, for the molecular characterization, LF3(1) was selected and sent for the 16S rRNA sequencing.

6.3.5.2. Molecular Identification

For molecular characterization the isolate LF3(1) was sent for sequencing at Biokart India Pvt Ltd, Bangalore, India. The isolate showed excellent probiotic properties and was identified using 16S rRNA sequencing. The nucleotide sequence obtained were compared with the sequences available in NCBI (National Center for Biotechnology Information) database by using BLAST (Basic Local Alignment Search Tool) program.

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Based on BLAST results, it has been found that the 16S rRNA sequence of above organism showed maximum similarity to Enterococcus species and was confirmed as Enterococcus faecium strain LF3(1). The accession number of NCBI GenBank of this isolate is MK045288. Phylogenetic tree of the isolate constructed using BLAST and presented in Figure 6.40.

Figure 6.40: Phylogenetic tree showing relationships between Enterococcus faecium strain LF3(1) and other Enterococcus species.

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6.4. In-vivo Evaluation of Probiotics

6.4.1. In-vivo Evaluation of Probiotic Efficiency of Isolated Bacterial Strains

In the in-vitro evaluation, 12 bacterial isolates fulfilled all the criteria for probiotic selection as per FAO/WHO guidelines (2002). They are namely, Bacillus amyloliquefaciens strain S7, Enterococcus durans strain S3, Bacillus cereus strain BDK2′, Bacillus subtilis strain BDK7, Bacillus subtilis strain BDK9, Enterococcus faecium strain F1H4, Bacillus safensis strain F2F4, Bacillus subtilis strain F3 1(2), Bacillus subtilis strain F1F4, Bacillus velezensis strain F1F2, Enterococcus gallinarum strain F1H3 and Enterococcus faecium strain LF3(1). To check their potential in biological system, in-vivo experiment was carried out against the infection caused by Pseudomonas aeruginosa in the fingerlings of Cyprinus carpio. As per plan (Chapter 3: Materials and Methods), four test groups were designed to study the probiotic efficiency of the isolated bacteria which are shown in Table 6.18. Four test groups were named as control group (fish served with basal feed with no probiotics), pathogen group (fish introduced with pathogen and served with basal diet and no probiotics), probiotic groups (fish served with probiotic feed), probiotic with pathogen groups (fish introduced with pathogen and served with probiotic feed).

Effect of the different diet (basal diet and probiotic supplemented diet) on the survival and growth performance of fingerlings of Cyprinus carpio was determined by the following growth parameters, specific growth rate (SGR), food conversion ratio (FCR), % weight gain and % survival for 60 days and at different intervals of the period of administration of the probiotic feed. Specific growth rate indicated overall growth performance of fingerlings and was verified by FCR. FCR is an important parameter to measure the health status of any individual. Percentage survival was examined to analyse toxicity by pathogen, probiotics, as well as to check the potential of probiotic strain against disease caused by Pseudomonas aeruginosa. The results of in-vivo evaluation of selected probiotics against Pseudomonas aeruginosa are discussed in detail as follows:

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Table 6.18: Details of different test groups designed to evaluate the probiotic efficiency through in-vivo studies.

Group Group Name Feed Pathogen Id Basal unmodified I Control Absent diet Basal unmodified Pseudomonas II Pathogen diet aeruginosa Probiotic III Probiotic Absent supplemented diet Probiotic + Probiotic Pseudomonas IV Pathogen supplemented diet aeruginosa

6.4.1.1. Weight Gain in the Fingerlings of Cyprinus carpio

The weight of fish fingerlings was monitored on regular interval after administration of probiotic supplemented diet i.e. 15 days, 30 days, 45 days and 60 days. Details of average weight, % weight gain and overall weight gain % of Cyprinus carpio after 15 days, 30 days, 45 days and 60 days of administering with the probiotic bacterial feed is shown in Table 6.19.

During the study, it was observed that the weight gain in group III (probiotic) and group IV (probiotic with pathogen) of all isolates was higher as compared to the group I (control) and group II (pathogen). Twelve different setup of probiotic group (III) were supplemented with twelve different probiotic feed i.e. i) Bacillus amyloliquefaciens S7, ii) Enterococcus durans S3, iii) Bacillus cereus BDK2′, iv) Bacillus subtilis BDK7, v) Bacillus subtilis BDK9, vi) Enterococcus faecium F1H4, vii) Bacillus safensis F2F4, viii) Bacillus subtilis F3 1(2), ix) Bacillus subtilis F1F4, x) Bacillus velezensis F1F2, xi) Enterococcus gallinarum F1H3 and xii) Enterococcus faecium LF3(1) exhibited a weight gain % of 112.5 ± 3.56, 99.15 ± 4.30, 102.17 ± 4.53, 102.46 ± 3.57 and 105.58 ± 4.05, 79.71 ± 2.16, 93.39 ± 4.50, 99.10 ± 3.67, 112.0 ± 1.95, 81.31 ± 2.58, 82.98 ± 2.59 and 71.81 ± 2.45, respectively.

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The fish in group IV (probiotic with pathogen) were supplemented with feed modified using same isolates as above, exhibited a weight gain % of 101.96 ± 5.61, 54.97 ± 3.30, 96.83 ± 5.08, 79.33 ± 5.74 , 60.91 ± 3.26, 80.0 ± 3.63, 81.73 ± 3.52, 77.57 ± 2.66, 94.23 ± 2.58, 70.18 ± 2.13, 72.65 ± 3.78 and 60.68 ± 2.78, respectively. The weight gain in fish of group IV of all isolates was relatively lower as compared to the fish of group III but higher than control (I) and pathogen group (II) (Figure 6.41), because these groups were administered with basal feed without probiotics. The maximum % weight gain was observed for S7 and S7+P as 112.5 % and 101.96 %, respectively, as compared to the control (I) and pathogen group (II) which exhibit a weight gain of 42.31 % and 27.27 %, respectively.

Overall weight gain results indicate good performance in the group III and IV as compared to the control group pointing to improved health of fish, when fed with probiotic supplemented feed. This indicates positive significant effect of selected strains when administered to fish, which shows that immunity can be improved by using probiotic supplemented feed. The increase in weight gain of fish fed with probiotic supplemented diets can be attributed to improved digestive activity by improving vitamin synthesis, cofactors, and enzymatic activity, leading to improved digestion, nutrient absorption and weight gain (Jory, 1998; Ziemer and Gibson, 1998; Gatesoupe, 1999; Aly et al., 2008).

The growth was retarded in the case of control and pathogen group fish administered with basal feed without probiotics. Figure 6.42 shows the % weight gain at different time interval in fish. In general, an increase in the % weight gain is observed in all the groups, except for the group infected with pathogen. The infected fish of pathogen group had a decrease in the weight over the period, indicating poor growth of the fish exposed to Pseudomonas aeruginosa.

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140 Probiotic Probiotic + Pathogen

120 c,d,g,i c,g,i c,e,g,i,n c,d c,e,g,i,l c,e,g,i,l e,g,i,l d,g e,g,i,l 100 e,g,l,o d h,m k,l,o h,m,n h,k,m,n k,o,p k,o,p h,m,n h,m,n,o k,p,q 80 h,j,n p,q f,j f,j,n f,j,n,q 60 f,j a 40 b

% Weight gain in 60 days in gain Weight % 20

0 Control Pathogen S7 S3 BDK2' BDK7 BDK9 F1H4 F2F4 F3 1(2) F1F4 F1F2 F1H3 LF3(1) Consortia Bacterial strains

Figure 6.41: Overall % weight gain in fish of probiotic group and probiotic with pathogen group after 60 days of feeding with probiotic supplemented feed i.e. Bacillus amyloliquefaciens strain S7, Enterococcus durans strain S3, Bacillus cereus strain BDK2′, Bacillus subtilis strain BDK7, Bacillus subtilis strain BDK9, Enterococcus faecium strain F1H4, Bacillus safensis strain F2F4, Bacillus subtilis strain F3 1(2), Bacillus subtilis strain F1F4, Bacillus velezensis strain F1F2, Enterococcus gallinarum strain F1H3 and Enterococcus faecium strain LF3(1). The control and pathogen show the survival of fish which were administered with basal diet. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) in control, pathogen, probiotic and probiotic with a pathogen groups.

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30 Day 15 Day 30 Day 45 Day 60 (a) 25 20 15 10

% Weight gain gain Weight % 5 0 Control Pathogen S7 S3 BDK2' BDK7 BDK9 F1H4 F2F4 F3 1(2) F1F4 F1F2 F1H3 LF3(1) Consortia Bacterial strains

30 Day 15 Day 30 Day 45 Day 60 (b) 25 20 15 10

% Weight gain Weight % 5 0 Control Pathogen S7 P S3 P BDK2' P BDK7 P BDK9 P F1H4 P F2F4 P F3 1(2) P F1F4 P F1F2 P F1H3 P LF3(1) P Consortia P Bacterial strains Figure 6.42: The % weight gain in fish at different interval of time i.e. 15 day, 30 days, 45 days and 60 days of the treatment of (a) fish fed with probiotic supplemented feed and (b) fish introduced with pathogen and fed with probiotic supplemented feed. The control and pathogen show the survival of fish which were administered with basal diet.

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Table 6.19: Average weight, % weight gain and total weight gain % of Cyprinus carpio of different groups after 15 days, 30 days, 45 days and 60 days of study.

Average weight (gm) Weight gain (%) Probiotic Total weight gain strains % after 60 days Day 1 Day 15 Day 30 Day 45 Day 60 Day 15 Day 30 Day 45 Day 60 0.252 ± 0.275 ± 0.301 ± 0.336 ± 6.77 ± 8.96 ± 9.09 ± 12.12 ± Control 0.236 ± 42.30 ± 1.9 0.04 0.06 0.06 0.05 0.04 0.45 0.47 0.51 0.49 0.295 ± 0.315 ± 0.336 ± 0.350 ± 7.27 ± 6.91 ± 6.65 ± 4.05 ± Pathogen 0.275 ± 27.27 ± 1.27 0.05 0.03 0.04 0.03 0.04 0.31 0.27 0.33 0.32 0.325 ± 0.390 ± 0.480 ± 0.595 ± 16.07 ± 20.00 ± 23.07 ± 23.95 ± S7 0.280 ± 112.5 ± 3.56 0.06 0.05 0.07 0.06 0.08 0.88 0.74 0.92 0.95 0.290 ± 0.335 ± 0.410 ± 0.515 ± 13.72 ± 15.51 ± 22.38 ± 25.61 ± S7 + P 0.255 ± 101.96 ± 5.61 0.05 0.07 0.05 0.06 0.07 0.96 0.94 1.21 1.37 0.335 ± 0.394 ± 0.475 ± 0.587 ± 13.55 ± 17.64 ± 20.52 ± 23.68 ± S3 0.295 ± 99.15 ± 4.30 0.09 0.08 0.07 0.06 0.08 1.02 1.07 0.97 1.12 0.305 ± 0.340 ± 0.383 ± 0.441 ± 7.32 ± 11.15 ± 12.74 ± 15.21 ± S3 + P 0.285 ± 54.97 ± 3.30 0.06 0.07 0.05 0.06 0.04 0.82 0.86 0.79 0.83 0.422 ± 0.496 ± 0.603 ± 0.750 ± 13.91 ± 17.55 ± 21.45 ± 24.31 ± BDK2′ 0.370 ± 102.17 ± 4.53 0.03 0.03 0.04 0.06 0.10 1.13 1.12 1.14 1.16 0.295 ± 0.340 ± 0.411 ± 0.511 ± 13.46 ± 15.25 ± 20.91 ± 24.48 ± BDK2′ + P 0.260 ± 96.83 ± 5.08 0.08 0.06 0.07 0.05 0.07 1.27 1.22 1.25 1.33

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2.341 ± 2.741 ± 3.325 ± 4.100 ± 15.63 ± 17.08 ± 21.27 ± 23.3 ± BDK7 2.025 ± 102.46 ± 3.57 0.12 0.09 0.07 0.05 0.08 0.89 0.92 0.87 0.95 0.295 ± 0.335 ± 0.394 ± 0.484 ± 9.26 ± 13.56 ± 17.83 ± 22.66 ± BDK7 + P 0.270 ± 79.33 ± 5.74 0.06 0.05 0.08 0.09 0.07 0.94 0.98 1.43 1.54 2.270 ± 2.680 ± 3.250 ± 4.050 ± 15.23 ± 18.06 ± 21.27 ± 24.61 ± BDK9 1.970 ± 105.58 ± 4.05 0.21 0.18 0.22 0.23 0.26 0.98 0.96 1.02 1.09 1.778 ± 1.983 ± 2.258 ± 2.600 ± 10.09 ± 11.49 ± 13.89 ± 15.1 ± BDK9 + P 1.615 ± 60.91 ± 3.26 0.11 0.13 0.09 0.14 0.24 0.81 0.74 0.85 0.92 0.276 ± 0.314 ± 0.370 ± 0.445 ± 11.53 ± 13.79 ± 17.72 ± 20.27 ± F1H4 0.247 ± 79.71 ± 2.16 0.05 0.07 0.06 0.09 0.05 0.54 0.46 0.53 0.58 0.329 ± 0.375 ± 0.440 ± 0.540 ± 9.80 ± 13.84 ± 17.33 ± 22.72 ± F1H4 + P 0.300 ± 80.0 ± 3.63 0.09 0.04 0.06 0.07 0.05 0.91 0.87 0.85 0.96 0.506 ± 0.589 ± 0.700 ± 0.853 ± 14.84 ± 16.32 ± 18.71 ± 21.94 ± F2F4 0.441 ± 93.39 ± 4.50 0.09 0.10 0.13 0.08 0.07 1.12 1.05 1.08 1.16 0.405 ± 0.468 ± 0.550 ± 0.656 ± 12.39 ± 15.49 ± 17.33 ± 19.31 ± F2F4 + P 0.361 ± 81.73 ± 3.52 0.04 0.05 0.08 0.06 0.05 0.88 0.76 0.83 0.89 0.547 ± 0.639 ± 0.778 ± 0.969 ± 12.5 ± 16.66 ± 21.76 ± 24.58 ± F3 1(2) 0.486 ± 99.10 ± 3.67 0.06 0.05 0.08 0.06 0.07 0.91 0.88 0.93 0.95 0.325 ± 0.373 ± 0.436 ± 0.515 ± 11.88 ± 14.97 ± 16.9 ± 18.07 ± F3 1(2) + P 0.290 ± 77.57 ± 2.66 0.04 0.05 0.07 0.05 0.06 0.66 0.63 0.68 0.70

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2.237 ± 2.700 ± 3.325 ± 4.187 ± 13.29 ± 20.67 ± 23.14 ± 25.94 ± F1F4 1.975 ± 112.0 ± 1.95 0.03 0.05 0.05 0.04 0.06 0.48 0.45 0.52 0.55 0.295 ± 0.340 ± 0.410 ± 0.505 ± 13.46 ± 15.25 ± 20.58 ± 23.17 ± F1F4 + P 0.260 ± 94.23 ± 2.58 0.08 0.06 0.09 0.07 0.06 0.64 0.61 0.59 0.68 0.991 ± 1.150 ± 1.350 ± 1.616 ± 11.21 ± 15.96 ± 17.39 ± 19.75 ± F1F2 0.891 ± 81.31 ± 2.58 0.06 0.05 0.05 0.08 0.06 0.63 0.65 0.68 0.66 0.912 ± 1.040 ± 1.193 ± 1.393 ± 11.45 ± 13.97 ± 14.74 ± 16.76 ± F1F2 + P 0.818 ± 70.18 ± 2.13 0.06 0.08 0.04 0.05 0.05 0.53 0.55 0.51 0.58 0.588 ± 0.672 ± 0.788 ± 0.955 ± 12.76 ± 14.15 ± 17.35 ± 21.12 ± F1H3 0.522 ± 82.98 ± 2.59 0.07 0.08 0.06 0.05 0.04 0.67 0.62 0.64 0.66 0.668 ± 0.753 ± 0.873 ± 1.046 ± 10.31 ± 12.64 ± 15.92 ± 19.84 ± F1H3 + P 0.606 ± 72.65 ± 3.78 0.04 0.06 0.04 0.05 0.07 0.94 0.89 0.96 0.98 0.592 ± 0.671 ± 0.775 ± 0.914 ± 11.4 ± 13.25 ± 15.42 ± 17.97 ± LF3(1) 0.532 ± 71.81 ± 2.45 0.05 0.02 0.06 0.05 0.06 0.62 0.64 0.67 0.64 0.342 ± 0.38 ± 0.435 ± 0.505 ± 9.09 ± 10.83 ± 14.47 ± 16.09 ± LF3(1) + P 0.314 ± 60.68 ± 2.78 0.05 0.06 0.03 0.05 0.05 0.69 0.65 0.71 0.74 2.077 ± 2.338 ± 2.654 ± 3.151 ± 10.84 ± 12.57 ± 13.51 ± 18.71 ± Consortia 1.874 ± 68.15 ± 1.52 0.03 0.04 0.06 0.05 0.03 0.38 0.44 0.41 0.35 3.005 ± 3.377 ± 3.861 ± 4.477 ± 8.41 ± 12.38 ± 14.31 ± 15.97 ± Consortia + P 2.772 ± 61.52 ± 2.30 0.04 0.06 0.03 0.04 0.05 0.57 0.53 0.58 0.65

Each values given in the table are mean ± standard deviation of three separate experiments.

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6.4.1.2. Survival of Cyprinus carpio Fingerlings

The survival of fingerlings of Cyprinus carpio in different groups was monitored at regular interval during 60 days of the study (15, 30, 45 and 60 days), and shown in Table 6.20, Figure 6.44 and 6.45. The fish of pathogen group (II) fed with basal diet exhibited lowest % survival (30%) among all groups, followed by the control group (I) (50%). The fish in group III of all the isolates showed good survival except Enterococcus durans S3 (40%), because S3 was found to be infectious to the fish as shown in Figure 6.43. The % survival in Bacillus amyloliquefaciens S7, Bacillus subtilis BDK7 and Bacillus subtilis BDK9, Bacillus subtilis F3 1(2), Bacillus subtilis F1F4, Bacillus velezensis FIF2, Enterococcus gallinarum F1H3, Enterococcus faecium LF3(1), supplemented diet group was observed to be cent percent (Figure 6.44). Similar results were observed by Yanbo and Zirong (2006) in common carp fed with Bacillus supplemented feed. The fish fed with Bacillus cereus BDK2′, Bacillus safensis F2F4, and Enterococcus faecium FIH4 exhibited 96.77 %, 96.55 % and 95.23 % survival, respectively.

Figure 6.43: Digital image of fish showing toxicity (infection) of Enterococcus durans S3 supplemented feed.

In contrast to the above observations, the fish in group IV of all isolates i.e. Bacillus amyloliquefaciens S7, Bacillus cereus BDK2′, Bacillus subtilis BDK7 and Bacillus subtilis BDK9, Bacillus safensis F2F4, Bacillus subtilis F3 1(2), Bacillus subtilis F1F4,

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Bacillus velezensis FIF2, Enterococcus gallinarum F1H3, Enterococcus faecium LF3(1) exhibited an improved survival with % survival ≥ 85 % except for Enterococcus faecium F1H4 (26.32%) and Enterococcus durans S3 (60%). Typical symptoms of hemorrhagic septicaemia were found in moribund or dead fish as shown in Figure 6.46. The results indicate increased survival in group IV which were introduced with pathogen and fed with probiotic supplemented feed. The survival of fish in group IV was comparable to the fish of group III and appreciably higher to the control and pathogen group. The survival in control group is low because the mortality rate of fish in early stage is high as they are immunological immature and the survival in pathogen group is low because the fingerlings in this group were immunological immature and infected with pathogen.

Similar results have been reported by others (Queiroz and Boyd, 1998; Rengpipat et al., 2000) that the survival rate of channel catfish (Ictalurus punctatus) and Penaeus monodon shrimp was increased when administered with Bacillus spp. and Bacillus S11 supplemented feed. Also, Picchietti et al., (2007) have been reported improvement in % survival of the fingerlings due to the increased level of Immunoglobin and acidophilic granulocytes in the sea bream gut, hence stimulating the gut immune system.

Other growth parameters i.e. specific growth rate (SGR), food conversion ratio (FCR) and % survival of Cyprinus carpio after 15 days, 30 days, 45 days and 60 days of administering with the probiotic bacterial feed are shown in Table 6.20.

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120 Probiotic Probiotic + Pathogen

100

60 % Survival Survival % 40

0 Control Pathogen S7 S3 BDK2' BDK7 BDK9 F1H4 F2F4 F3 1(2) F1F4 F1F2 F1H3 LF3(1) Consortia

Bacterial strains

Figure 6.44: The % survival of fish in probiotic group and probiotic with pathogen group after 60 days of feeding with probiotic supplemented feed i.e. Bacillus amyloliquefaciens strain S7, Enterococcus durans strain S3, Bacillus cereus strain BDK2′, Bacillus subtilis strain BDK7, Bacillus subtilis strain BDK9, Enterococcus faecium strain F1H4, Bacillus safensis strain F2F4, Bacillus subtilis strain F3 1(2), Bacillus subtilis strain F1F4, Bacillus velezensis strain F1F2, Enterococcus gallinarum strain F1H3 and Enterococcus faecium strain LF3(1). The control and pathogen show the survival of fish which were administered with basal diet.

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120 15 days 30 days 45 days 60 days (a) 100 80 60 40 % Survival % 20 0 Control Pathogen S7 S3 BDK2' BDK7 BDK9 F1H4 F2F4 F3 1(2) F1F4 F1F2 F1H3 LF3(1) Consortia Bacterial strains

120 15 days 30 days 45 days 60 days (b) 100 80 60 40 % Survival % 20 0 Control Pathogen S7 P S3 P BDK2' P BDK7 P BDK9 P F1H4 P F2F4 P F3 1(2) P F1F4 P F1F2 P F1H3 P LF3(1) P Consortia P Bacterial strains

Figure 6.45: The % survival of fish at different interval of time i.e. 15 days, 30 days, 45 days and 60 days of the treatment of (a) fish fed with probiotic supplemented feed and (b) fish introduced with pathogen and fed with probiotic supplemented feed. The control and pathogen show the survival of fish which were administered with basal diet.

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Figure 6.46: Visual comparison of fish subjected to Pseudomonas aeruginosa infection, fed with basal diet and probiotic supplemented diet. Fish fed with basal diet show symptoms of hemorrhagic septicaemia.

6.4.1.3. Food Conversion Ratio (FCR) in Cyprinus carpio Fingerlings

The effects of dietary supplements on the growth performance of C. carpio was observed by calculating the food conversion ratio (FCR) (Figure 6.47). After 15 days of feeding, the fish in group IV fed with the diet containing probiotics had good food conversion ratio than the control group and pathogen group. The FCR results show improvement in FCR values for all the isolates over the period of time except for the pathogen group. Figure 6.48 shows the food conversion ratio in fish at different time intervals of study. Dietary supplementation of basal diet had no significant effect on FCR of C. carpio, even after 45 days of feeding. After the 60 days of feeding, the FCR value of fish fed with different probiotic diet was found to be around 3. The pathogen group (II) exhibit abnormally high values (i.e. 9.43 ± 2.00) indicating poor food

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conversion in the fish. This can be attributed to the effect of infection on the health of fish leading to poor metabolism hence poor FCR leading to poor growth of the fish.

On the other hand, the FCR value of the fish in group III of all the isolates was relatively less than the control group (6.3 ± 1.04). The fish in group III fed with F1F4 supplemented diet exhibits the good FCR (2.93 ± 0.28), followed by S7 (2.95 ± 0.14), BDK9 (3.07 ± 0.24), BDK2′ (3.13 ± 0.16), BDK7 (3.15 ± 0.24), F3 1(2) (3.19 ± 0.23), S3 (3.21 ± 0.28), F2F4 (3.37 ± 0.38), F1H3 (3.66 ± 0.35), F1F2 (3.72 ± 0.36), F1H4 (3.76 ± 0.32) and LF3(1) (4.10 ± 0.53). Similar trend in FCR value was observed for Fish in group IV (Figure 6.47).

The addition of probiotic in the food served to sample fish results in improved metabolism, high food conversion and enhanced growth. The FCR data agrees with the % weight gain and % survival of the fish. The FCR value of fish in group IV of all isolates is less, compared to group I (control) and group II (pathogen) indicating positive effect of probiotic treatment against the pathogen. While a slight increase in the FCR value of the group IV is observed when compared to the group III. The appreciable decrease in FCR values showed that the fish were using dietary nutrients more efficiently when fed with probiotic supplemented feed (Geng et al., 2012).

The improvement in growth performance of the fish administered with probiotic supplemented diet is attributed to increased digestive activity due to the synthesis of vitamins and enzymes. These components helps in improvement of digestibility which leads to results in decrease in FCR and increase in weight gain (Ziemer and Gibson, 1998; Swanson et al., 2002; Liu et al., 2009; Ringo et al., 2010; Geng et al., 2012).

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14 Probiotic Probiotic + Pathogen 12 b

8 a,d

6 a,d c,d a,c,d a,c,d c c,d c,d c,d c c,d c c,d c,d c c c c 4 c c c c c c c c c,d

Food Conversion Ratio (FCR)Food Ratio Conversion 2

0 Control Pathogen S7 S3 BDK2' BDK7 BDK9 F1H4 F2F4 F3 1(2) F1F4 F1F2 F1H3 LF3(1) Consortia Bacterial strains

Figure 6.47: Overall food conversion ratio of fish in probiotic group and probiotic with pathogen group after 60 days of feeding with probiotic supplemented feed i.e. Bacillus amyloliquefaciens strain S7, Enterococcus durans strain S3, Bacillus cereus strain BDK2′, Bacillus subtilis strain BDK7, Bacillus subtilis strain BDK9, Enterococcus faecium strain F1H4, Bacillus safensis strain F2F4, Bacillus subtilis strain F3 1(2), Bacillus subtilis strain F1F4, Bacillus velezensis strain F1F2, Enterococcus gallinarum strain F1H3 and Enterococcus faecium strain LF3(1). The control and pathogen show the food conversion ratio of fish which were administered with basal diet. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) in control, pathogen, probiotic and probiotic with a pathogen groups.

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14 (a) 12 15 days 30 days 45 days 60 days 10 8 6 (FCR) 4 2

Food Conversion Ratio Food Ratio Conversion 0 Control Pathogen S7 S3 BDK2' BDK7 BDK9 F1H4 F2F4 F3 1(2) F1F4 F1F2 F1H3 LF3(1) Consortia Bacterial strains

14 15 days 30 days 45 days 60 days (b) 12 10 8 6 (FCR) 4 2 Food Conversion Ratio Food Ratio Conversion 0 Control Pathogen S7 P S3 P BDK2' P BDK7 P BDK9 P F1H4 P F2F4 P F3 1(2) P F1F4 P F1F2 P F1H3 P LF3(1) P Consortia P Bacterial strains

Figure 6.48: The food conversion ratio in fish at different interval of time i.e. 15 day, 30 days, 45 days and 60 days of the treatment of (a) fish fed with probiotic supplemented feed and (b) fish introduced with pathogen and fed with probiotic supplemented feed. The control and pathogen show the food conversion ratio of fish which were administered with basal diet.

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6.4.1.4. Specific Growth Rate (SGR) of Cyprinus carpio Fingerlings

At last, the overall growth analysis indicates that the specific growth rate (SGR) of the fish in group III and IV of all isolates was higher as compared to the control (0.59 ± 0.09) and pathogen group (0.40 ± 0.08) (Figure 6.49). The specific growth rate of the fish at different interval of treatment study is shown in Figure 6.50. Increased value of SGR for group III and group IV indicated improved growth of the fish fingerlings fed with probiotic supplemented feed.

The SGR values of the fish in group III of all isolates fed with different probiotic supplemented feed i.e. Bacillus amyloliquefaciens S7, Enterococcus durans S3, Bacillus cereus BDK2′, Bacillus subtilis BDK7, Bacillus subtilis BDK9, Enterococcus faecium F1H4, Bacillus safensis F2F4, Bacillus subtilis F3 1(2), Bacillus subtilis F1F4, Bacillus velezensis F1F2, Enterococcus gallinarum F1H3 and Enterococcus faecium LF3(1) are of 1.25 ± 0.19, 1.14 ± 0.14, 1.17 ± 0.25, 1.17 ± 0.19, 1.20 ± 0.22, 0.97 ± 0.13, 1.09 ± 0.19, 1.14 ± 0.13, 1.25 ± 0.20, 0.99 ± 0.09, 1.01 ± 0.16 and 0.90 ± 0.08, respectively. Group IV showed comparable SGR to the group III with 1.17 ± 0.16, 0.73 ± 0.19, 1.12 ± 0.18, 0.97 ± 0.23, 0.79 ± 0.13, 0.98 ± 0.12, 0.99 ± 0.23, 0.95 ± 0.18, 1.11 ± 0.24, 0.88 ± 0.12, 0.91 ± 0.12, 0.79 ± 0.10 for fish fed with Bacillus amyloliquefaciens S7, Enterococcus durans S3, Bacillus cereus BDK2′, Bacillus subtilis BDK7, Bacillus subtilis BDK9, Enterococcus faecium F1H4, Bacillus safensis F2F4, Bacillus subtilis F3 1(2), Bacillus subtilis F1F4, Bacillus velezensis F1F2, Enterococcus gallinarum F1H3 and Enterococcus faecium LF3(1) supplemented diet, respectively.

The specific growth rate (SGR) of the fish in group III and IV of all isolates was higher as compared to the control and pathogen group (Figure 6.50). These results indicated improved growth of the fish, which were administered with probiotic feed. The SGR values of group IV were slightly less yet comparable to group III indicating high efficiency of the probiotics. Similar results have also been observed in Tilapia (Oreochromis niloticus) (Aly et al., 2008), sea cucumber (A. japonicus) (Zhang et al., 2010) and rainbow trout (Oncorhynchus mykiss, Walbaum) (Newaj-Fyzul et al., 2007).

Table 6.21 show the overall growth in terms of growth parameters i.e. specific growth rate (SGR), food conversion ratio (FCR) and % survival of Cyprinus carpio after 60 days of administering with the basal diet and probiotic supplemented diet.

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Probiotic Probiotic + Pathogen 1.6 c c c c 1.4 c c a,c a,c a,c a,c a,c a,c a,c a,c a,c 1.2 a,c a,b,c a,c a,b,c a,b,c a,b,c a,b,c a,b,c 1 a,b,c a,b,c a,b,c 0.8 a 0.6 b 0.4

Specific Growth Rate (SGR) Rate Growth Specific 0.2 0 Control Pathogen S7 S3 BDK2' BDK7 BDK9 F1H4 F2F4 F3 1(2) F1F4 F1F2 F1H3 LF3(1) Consortia Bacterial strains

Figure 6.49: Overall specific growth rate of fish in probiotic group and probiotic with pathogen group after 60 days of feeding with probiotic supplemented feed i.e. Bacillus amyloliquefaciens strain S7, Enterococcus durans strain S3, Bacillus cereus strain BDK2′, Bacillus subtilis strain BDK7, Bacillus subtilis strain BDK9, Enterococcus faecium strain F1H4, Bacillus safensis strain F2F4, Bacillus subtilis strain F3 1(2), Bacillus subtilis strain F1F4, Bacillus velezensis strain F1F2, Enterococcus gallinarum strain F1H3 and Enterococcus faecium strain LF3(1). The control and pathogen show the specific growth rate of fish which were administered with basal diet. Bars with different letters showed significant difference (P < 0.05 One-factor ANOVA followed by Tukey’s post-hoc test) in control, pathogen, probiotic and probiotic with a pathogen groups.

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2 15 days 30 days 45 days 60 days (a)

1.5

1 (SGR) 0.5 Specific Growth Rate Rate Growth Specific 0 Control Pathogen S7 S3 BDK2' BDK7 BDK9 F1H4 F2F4 F3 1(2) F1F4 F1F2 F1H3 LF3(1) Consortia Bacterial strains

2 15 days 30 days 45 days 60 days (b) 1.5

(SGR) 0.5

0 Specific Growth Rate Rate Growth Specific Control Pathogen S7 P S3 P BDK2' P BDK7 P BDK9 P F1H4 P F2F4 P F3 1(2) P F1F4 P F1F2 P F1H3 P LF3(1) P Consortia P Bacterial strains

Figure 6.50: The Specific Growth Rate (SGR) of fish at different interval of time i.e. 15 day, 30 days, 45 days and 60 days of the treatment (a) fish fed with probiotic supplemented feed and (b) fish introduced with pathogen and fed with probiotic supplemented feed. The control and pathogen show the specific growth rate of fish which were administered with basal diet.

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Table 6.20: Growth parameters i.e. specific growth rate (SGR), food conversion ratio (FCR), % survival of Cyprinus carpio after 15 days, 30 days, 45 days and 60 days of administering with the basal and probiotic supplemented feed.

Growth parameters Probiotic Specific Growth Rate Food Conversion Ratio % Survival strains 15 days 30 days 45 days 60 days 15 days 30 days 45 days 60 days 15 days 30 days 45 days 60 days

0.43 ± 0.57 ± 0.58 ± 0.76 ± 6.64 ± 5.02 ± 4.95 ± 3.71 ± Control 95.45 95.24 80 68.75 0.02 0.03 0.02 0.02 0.26 0.22 0.31 0.29 0.46 ± 0.44 ± 0.42 ± 0.26 ± 6.19 ± 6.51 ± 6.76 ± 11.1 ± Pathogen 100 65 84.62 54.55 0.02 0.02 0.02 0.03 0.43 0.47 0.51 0.54 0.99 ± 1.21 ± 1.38 ± 1.43 ± 2.8 ± 2.25 ± 1.95 ± 1.88 ± S7 100 100 100 100 0.04 0.03 0.05 0.04 0.05 0.03 0.04 0.03 0.85 ± 0.96 ± 1.34 ± 1.52 ± 3.28 ± 2.9 ± 2.01 ± 1.76 ± S7 + P 100 100 100 100 0.03 0.05 0.04 0.04 0.03 0.04 0.04 0.05 0.84 ± 1.08 ± 1.24 ± 1.41 ± 3.32 ± 2.55 ± 2.19 ± 1.90 ± S3 100 85 47.06 100 0.03 0.04 0.03 0.02 0.07 0.05 0.07 0.08 0.47 ± 0.70 ± 0.79 ± 0.94 ± 6.14 ± 4.04 ± 3.53 ± 2.96 ± S3 + P 85 88.24 80 100 0.04 0.05 0.05 0.04 0.09 0.09 0.08 0.07 0.86 ± 1.07 ± 1.29 ± 1.45 ± 3.24 ± 2.56 ± 2.09 ± 1.85 ± BDK2′ 100 100 96.77 100 0.05 0.06 0.05 0.07 0.04 0.03 0.04 0.05

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0.84 ± 0.94 ± 1.26 ± 1.46 ± 3.34 ± 2.95 ± 2.15 ± 1.84 ± BDK2′ + P 100 100 90 94.44 0.05 0.04 0.04 0.05 0.05 0.06 0.06 0.04 0.96 ± 1.05 ± 1.28 ± 1.39 ± 2.87 ± 2.64 ± 2.11 ± 1.93 ± BDK7 100 100 100 100 0.04 0.05 0.05 0.03 0.05 0.06 0.06 0.04 0.59 ± 0.84 ± 1.09 ± 1.36 ± 4.86 ± 3.32 ± 2.52 ± 1.98 ± BDK7 + P 100 100 95 100 0.05 0.06 0.05 0.06 0.06 0.05 0.06 0.06 0.94 ± 1.10 ± 1.28 ± 1.46 ± 2.95 ± 2.49 ± 2.11 ± 1.83 ± BDK9 100 100 100 100 0.05 0.06 0.06 0.04 0.06 0.05 0.07 0.06 0.64 ± 0.72 ± 0.86 ± 0.93 ± 4.46 ± 3.92 ± 3.24 ± 2.98 ± BDK9 + P 100 94.74 94.44 100 0.03 0.03 0.04 0.03 0.04 0.04 0.03 0.05 0.72 ± 0.86 ± 1.08 ± 1.23 ± 3.9 ± 3.26 ± 2.54 ± 2.22 ± F1H4 100 100 95.24 100 0.03 0.02 0.04 0.03 0.08 0.07 0.07 0.08 0.62 ± 0.86 ± 1.06 ± 1.36 ± 4.59 ± 3.25 ± 2.59 ± 1.98 ± F1H4 + P 89.47 47.06 62.5 100 0.04 0.02 0.03 0.04 0.11 0.09 0.11 0.13 0.92 ± 1.01 ± 1.14 ± 1.32 ± 3.03 ± 2.75 ± 2.40 ± 2.05 ± F2F4 100 100 96.55 100 0.04 0.04 0.03 0.05 0.08 0.09 0.09 0.08 0.77 ± 0.96 ± 1.06 ± 1.17 ± 3.62 ± 2.90 ± 2.59 ± 2.33 ± F2F4 + P 94.44 94.11 100 100 0.06 0.05 0.04 0.05 0.10 0.11 0.09 0.12 0.78 ± 1.02 ± 1.31 ± 1.46 ± 3.60 ± 2.70 ± 2.06 ± 1.83 ± F3 1(2) 100 100 100 100 0.03 0.02 0.03 0.04 0.05 0.06 0.05 0.04 0.74 ± 0.93 ± 1.04 ± 1.11 ± 3.78 ± 3.00 ± 2.66 ± 2.49 ± F3 1(2) + P 95.23 95 100 100 0.05 0.05 0.04 0.04 0.12 0.12 0.10 0.11

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0.83 ± 1.25 ± 1.38 ± 1.53 ± 3.38 ± 2.17 ± 1.94 ± 1.73 ± F1F4 100 100 100 100 0.05 0.06 0.04 0.06 0.07 0.07 0.06 0.05 0.84 ± 0.94 ± 1.24 ± 1.38 ± 3.34 ± 2.95 ± 2.18 ± 1.94 ± F1F4 + P 100 100 100 100 0.06 0.04 0.05 0.06 0.09 0.07 0.08 0.08 0.71 ± 0.98 ± 1.06 ± 1.20 ± 4.01 ± 2.81 ± 2.58 ± 2.28 ± F1F2 100 100 100 100 0.02 0.03 0.03 0.02 0.08 0.07 0.09 0.08 0.72 ± 0.87 ± 0.91 ± 1.03 ± 3.93 ± 3.22 ± 3.05 ± 2.68 ± F1F2 + P 100 93.75 100 100 0.02 0.04 0.03 0.04 0.06 0.07 0.05 0.06 0.80 ± 0.88 ± 1.06 ± 1.27 ± 3.52 ± 3.18 ± 2.59 ± 2.13 ± F1H3 100 100 100 100 0.03 0.05 0.04 0.04 0.08 0.09 0.07 0.08 0.65 ± 0.79 ± 0.98 ± 1.20 ± 4.36 ± 3.55 ± 2.82 ± 2.26 ± F1H3 + P 100 93.75 100 100 0.03 0.03 0.04 0.03 0.08 0.08 0.07 0.08 0.72 ± 0.83 ± 0.95 ± 1.10 ± 3.94 ± 3.39 ± 2.92 ± 2.50 ± LF3(1) 100 100 100 100 0.02 0.01 0.02 0.03 0.12 0.13 0.11 0.15 0.58 ± 0.68 ± 0.90 ± 0.99 ± 4.95 ± 4.15 ± 3.10 ± 2.79 ± LF3(1) + P 100 95.23 100 100 0.02 0.03 0.04 0.02 0.22 0.21 0.21 0.19 0.68 ± 0.79 ± 0.84 ± 1.14 ± 4.15 ± 3.57 ± 3.33 ± 2.40 ± Consortia 100 100 100 100 0.07 0.05 0.06 0.06 0.12 0.14 0.13 0.12 0.53 ± 0.77 ± 0.89 ± 0.98 ± 5.35 ± 3.63 ± 3.14 ± 2.82 ± Consortia + P 100 100 100 100 0.06 0.05 0.04 0.06 0.11 0.10 0.12 0.12

Each values given in the table are mean ± standard deviation of three separate experiments.

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Table 6.21: Overall growth in terms of growth parameters i.e. specific growth rate (SGR), food conversion ratio (FCR), and % survival of Cyprinus carpio after 60 days of administering with the basal and probiotic supplemented feed.

Growth Parameters Probiotic strains Specific Growth Food Conversion % Survival Rate (SGR) Ratio (FCR)

Control 0.59 ± 0.09 6.30 ± 1.04 50

Pathogen 0.40 ± 0.08 9.43 ± 2.00 30

S7 1.25 ± 0.19 2.95 ± 0.14 100

S7 + P 1.17 ± 0.16 3.12 ± 0.17 100

S3 1.14 ± 0.14 3.21 ± 0.28 40

S3 + P 0.73 ± 0.19 5.04 ± 0.37 60

BDK2′ 1.17 ± 0.25 3.13 ± 0.16 96.77

BDK2′ + P 1.12 ± 0.18 3.24 ± 0.23 85

BDK7 1.17 ± 0.19 3.15 ± 0.24 100

BDK7 + P 0.97 ± 0.23 3.73 ± 0.22 95

BDK9 1.20 ± 0.22 3.07 ± 0.24 100

BDK9 + P 0.79 ± 0.13 4.68 ± 0.16 89.47

F1H4 0.97 ± 0.13 3.76 ± 0.32 95.23 F1H4 + P 0.98 ± 0.12 3.72 ± 0.44 26.31 F2F4 1.09 ± 0.19 3.37 ± 0.38 96.55 F2F4 + P 0.99 ± 0.23 3.72 ± 0.43 88.88 F3 1(2) 1.14 ± 0.13 3.19 ± 0.23 100 F3 1(2) + P 0.95 ± 0.18 3.87 ± 0.42 90.47 F1F4 1.25 ± 0.20 2.93 ± 0.28 100 F1F4 + P 1.11 ± 0.24 3.32 ± 0.32 100

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F1F2 0.99 ± 0.09 3.72 ± 0.36 100 F1F2 + P 0.88 ± 0.12 4.19 ± 0.25 93.75 F1H3 1.01 ± 0.16 3.66 ± 0.35 100 F1H3 + P 0.91 ± 0.12 4.03 ± 0.32 93.75 LF3(1) 0.90 ± 0.08 4.10 ± 0.53 100 LF3(1) + P 0.79 ± 0.10 4.66 ± 0.85 95.23 Consortia 0.86 ± 0.25 4.26 ± 0.48 100 Consortia + P 0.79 ± 0.23 4.61 ± 0.46 100

Each values given in the table are mean ± standard deviation of three separate experiments.

6.4.2. Consortia Formulation and In-vivo Evaluation of its Probiotic Efficiency

For the studies on combined effects of different probiotics against the pathogen, compatibility of all the probiotics strains was checked. Compatibility test is the pre- requisite for the formulation of consortia feed. The compatibility of the probiotic isolates was performed according to the method prescribed by (Geria and Caridi, 2014) with slight modifications as discussed in material and method section. After careful examination, five bacterial isolates were found compatible and chosen for the preparation of consortium feed. They were namely, Bacillus cereus strain BDK2′, Bacillus subtilis strain BDK7, Bacillus subtilis strain BDK9, Bacillus safensis strain F2F4 and Bacillus subtilis strain F3 1(2) The results for the inter-compatibility of all the isolates are shown in Table 6.22.

After the compatibility test, a consortia of Bacillus cereus BDK2′, Bacillus subtilis BDK7, Bacillus subtilis BDK9, Bacillus safensis F2F4 and Bacillus subtilis F3 1(2) was prepared. The fish supplemented with diet containing consortia of these probiotics exhibit similar trend of weight gain over the period like the individual isolates. The probiotic group III and group IV administered with consortia supplement diet showed a weight gain of 68.15 ± 1.52% and 61.52 ± 2.30 %, respectively as shown in Table 6.21 and Figures 6.41 and 6.42. The overall weight gain is slightly less than probiotic supplemented feed prepared using single probiotic strain but relatively higher than the control and pathogen group.

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Table 6.22 : The resumes of positive (+) and negative (–) results of compatibility test of the potential probiotic strains.

Probiotic S7 S3 BDK2′ BDK7 BDK9 F1H4 F2F4 F3 1(2) F1F4 LF3(1) Isolates

S7 + + – + + + – – +

S3 + + + + – + + – –

BDK2′ – + + + + + + + +

BDK7 + – + + – + + + +

BDK9 – – + + – + + – +

F1H4 + – + + – + + + –

F2F4 + + + + + + + + +

F3 1(2) – + + + + + + + –

F1F4 – – – + + + + + –

LF3 (1) + + + + – + + – +

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The % survival in group III and IV administered with consortia of probiotics i.e. Bacillus cereus BDK2′, Bacillus subtilis BDK7, Bacillus subtilis BDK9, Bacillus safensis F2F4 and Bacillus subtilis F3 1(2) supplemented feed exhibits cent percent survival as shown in Table 6.21 and Figure 6.44. The result signifies that the administration of probiotic supplemented feed leads to improved immunity and enhanced growth in the fish of probiotic and probiotic with a pathogen group multi- fold as compared to control and pathogen groups.

The FCR values of fish in group III and IV fed with consortia supplemented feed were observed to be 4.26 ± 0.48 and 4.61 ± 0.46, respectively. These values are less compared to control (I) and pathogen group (II) but, were slightly higher compared to individual probiotic groups (Table 6.21 and Figure 6.47). The fish in group III and group IV administered with consortia of probiotic supplemented feed exhibits SGR values of 0.86 ± 0.25 and 0.79 ± 0.23, respectively. The fish administered with consortia supplemented fish exhibits relatively low SGR values as compared to fish served with individual probiotic supplemented feed.

Concluding Remarks: At the end of this study, it can be concluded that the administration of probiotic supplemented diet to the fingerlings of Cyprinus carpio, can supplement the growth, immune parameters, survival the fish from Pseudomonas aeruginosa infection. Similar results were observed by Swain et al., (1996), Noh et al., (1994), Bogut et al., (1998), Ghosh et al., (2003), Yanbo and Zirong (2006) in Indian carps, Israeli carp and common carp using different strains of probiotics. The use of probiotics has resulted in improved growth and increased survival of fish when probiotic strain Bacillus subtilis was given to Tilapia (Liu et al., 2017), Bacillus circulans given to Tor tambroides juveniles (Asaduzzaman et al., 2018). The FCR was lower in the probiotic group after 60 days of feeding, signifies that addition of probiotic bacteria in feed can appreciably improve the product yield (Saini et al., 2014; Ullah et al., 2018). The results suggest that these probiotics can serve as an effective treatment for fish against various infection with further investigation. The in-vivo fish experiment results clarifies that probiotic supplemented diets have positive effects on the health possibly by enhancing immune responses, altering the gut microbiota and preventing the pathogens (Balcazar et al., 2007; 2007b). At the end of the exhaustive work, significant results have been obtained.

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Chapter 7 Summary and Conclusion

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SUMMARY AND CONCLUSION

Fish are susceptible to various deadly pathogens which cause several diseases like, tail rot, fin rot, haemorrhage, septicaemia, dropsy, etc. The common occurrence of these diseases among fish has adversely affected the aquaculture industries. The use of antibiotics to cure these diseases has also developed severe biological and environmental concerns. The search for a better alternative has been a major concern recently. Probiotics, renowned as valuable microbes, are recommended as an efficient and environment friendly approach to replace the antibiotics. The concept of probiotics involves the deliberate introduction of beneficial microbes to host, as an attempt to change the indigenous microbial population equilibrium towards healthier composition. According to reports the use commercial probiotics due to their limited application in aquaculture have been rendered futile. On the other hand, putative probiotics which are isolated from the same source have emerged as a potential probionts to deal with issues related to aquaculture. Because the probiotic species derived from non-fish source are not capable to produce beneficial effects in the fish gut, as they tend to get washed out. Therefore, in the present work putative strains of Bacillus and Enterococcus species have been explored as probiotics. And have also evaluated their in-vivo behavior in Cyprinus carpio, a common crap.

The aim of this study was to isolate, screen and characterize bacterial strains having potential probiotic properties from the gastrointestinal (GI) tract of different fish species of different region of Punjab. During the study, isolated bacterial strains were characterized based on their colony morphology, colour, size, biochemical characterization, followed by in-vitro evaluation on different selection parameters of probiotics and use them in fish feed. The in-vitro evaluation was followed by molecular characterization by 16S rRNA gene sequencing for the identification of isolates which met the selection criteria of potential probiotics. The in-vivo evaluation was done to assess the efficacy and toxicity of putative probiotic strains on survival and growth performance of Common carp (Cyprinus carpio) challenged with Pseudomonas aeruginosa (MTCC 4673). Finally, efficacy of single or consortium of probionts in- vivo were determined through experimental trial to validate the effects of probiotics in improving growth, health status, immunity and disease resistance in Common carp

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(Cyprinus carpio) fingerlings, challenged with pathogenic bacteria (Pseudomonas aeruginosa). The salient features and major findings of the study are summarized as follows.

In this study, 79 fish were collected from Doaba, Majha and Malwa region of Punjab. A total of 313 bacteria were isolated from the gastrointestinal tract of fish and were screened as Gram-positive and Gram negative. Out of 313 isolated bacteria, only 87 were indexed as Gram-positive. These indexed Gram-positive bacteria were subjected to different in-vitro selection parameters described in FAO/WHO guidelines (2002) for the evaluation of probiotics and use them in fish feed. The guidelines list the different screening parameters as the minimum requirement needed for the probiotic status. The isolated strains were screened for antimicrobial activity against the potential fish pathogen Pseudomonas aeruginosa (MTCC 4673). Out of 87 Gram-positive isolates, 21 isolates were selected based on their antagonistic property. These isolates were further screened in response to bile tolerance (0.3% ox-gall), viability in the pH tolerance range of pH 2 to pH 9 and drug susceptibility to different antibiotic discs. Isolated probiotic strains displayed high tolerance towards bile (0.3% ox-gall) and extreme pH conditions. The presence of such property is beneficial in the preparation of probiotics and confer competitive advantage over other microbes by selective utilization of nutrient source. The positive probiont candidates showed ability of adhesion by having high auto aggregation activity. It was found that, out of 87 Gram- positive bacteria only 12 fulfil all the criteria for probiotics evaluation. The summary of the sequential in-vitro evaluation of isolates is given in Figure 7.1.

These 12 probiotic bacteria were isolated from different fish species namely Catla catla (S3 and S7), Nandus nandus (BDK2′), Puntius chola (BDK7), Oxygaster bacaila (BDK9) from Doaba region of Punjab. From Majha region, the bacterial isolates namely, F1F2, F1F4, F1H3, F1H4, F2F4 and F3 1(2) were isolated from Channa striatus and from Malwa region, LF3(1) was isolated from Labeo gonius species.

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Figure 7.1: Summary of the sequential in-vitro evaluation of isolates for screening of potential probiotic bacterial strains and their molecular identification.

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These 12 probiotic bacteria were further exposed in fish culture tanks, with or without fish pathogen, Pseudomonas aeruginosa to check their potential, role in growth enhancement, as well as to check the toxicity of probiotics in economical important common carp, Cyprinus carpio.

After 60 days of the study, among the 12 selected strains of potential probiotics, 10 strains namely, Bacillus amyloliquefaciens strain S7, Bacillus cereus strain BDK2′, Bacillus subtilis strain BDK7, Bacillus subtilis strain BDK9, Bacillus safensis strain F2F4, Bacillus subtilis strain F3 1(2), Bacillus subtilis strain F1F4, Bacillus velezensis strain F1F2, Enterococcus gallinarum strain F1H3 and Enterococcus faecium strain LF3(1) showed substantial results with appreciable weight gain, high percentage survival, low FCR value and improved SGR value. Only 2 probiotic strains, Enterococcus durans strain S3 and Enterococcus faecium strain F1H4 showed toxicity in fish resulted in poor growth and low percentage survival. S3 was isolated from Catla catla from Doaba region and F1H4 was isolated from Channa striatus from Majha region. On the other hand, S7 was isolated from the same species i.e. Catla catla and F1F2, F1F4, F1H3, F2F4, F3 1(2) were isolated from Channa striatus (found in muddy, weedy and swamp area) found to be potential probiotics and improve the growth and survival of Cyprinus carpio.

As per hypothesis, ecological important fish have good gut microbiota as compared to large fish. The reason may be these fish are exposed in different environment such as from contaminated small stream to rivers and from surface to bottom. As per expectation, small fish were having the potential to give effective putative probiotic, which can be explored to promote the growth and health of economical important large fish.

From the in-vivo results of this study, it can be concluded that the administration of probiotic supplemented diet in healthy and infection challenged fingerlings of Cyprinus carpio, can improve the overall growth, survival and immune parameters of fish against Pseudomonas aeruginosa infection. An increase in the weight was observed over the period of study indicate improved growth. The fish fed with probiotic supplemented feed exhibited higher survival compared to the control group. Again, the lower FCR

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values in the probiotic group after 60 days of feeding, suggests that incorporation of probiotic bacteria in feed can remarkably enhance product yield. Based on the observations, it has been concluded that these potential probiotic strains can serve as a novel and safe treatment to prevent the diseases in aquaculture.

Probiotics promote host health by positively modulating the intestinal microflora. These potential characteristics may be advantageous for a probiotic culture to be successful in colonizing and competing with pathogens in GIT of the fish. Thus, they may be beneficial both in food industries and in medical sector. The results suggest that these probiotics can serve as an effective treatment for fish against various infection with further investigation. In future, the mode of action of probiotics especially the modification of host immune responses and resistance to disease need to be explored.

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Bibliography

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