Differential induction, isolation, physicochemical and molecular characterization of temperate phages of environmental Vibrio cholerae as evidence of phage mediated Horizontal Gene Transfer

The resurgence of the enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries like India. The southern Indian state of Kerala is endemic to cholera. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. Marine aquaculture settings and mangrove environments of Kerala serve as reservoirs for V. cholerae. The non-O1/non-O139 environmental isolates of V. cholerae with incomplete ‘virulence casette’ are to be dealt with caution as they constitute a major reservoir of diverse virulence genes in the marine environment and play a crucial role in pathogenicity and horizontal gene transfer. The genes coding cholera toxin are borne on, and can be infectiously transmitted by CTXΦ, a filamentous lysogenic vibriophages. Temperate phages can provide crucial virulence and fitness factors affecting cell metabolism, bacterial adhesion, colonization, immunity, antibiotic resistance and serum resistance. The present study was an attempt to screen the marine environments like aquafarms and mangroves of coastal areas of Alappuzha and Cochin, Kerala for the presence of lysogenic V. cholerae, to study their pathogenicity and also gene transfer potential. Phenotypic and molecular methods were used for identification of isolates as V. cholerae. The thirty one isolates which were Gram negative, oxidase positive, fermentative, with or without gas production on MOF media and which showed yellow coloured colonies on TCBS (Thiosulfate Citrate Bile salt Sucrose) agar were segregated as vibrios. Twenty two environmental V. cholerae strains of both O1 and non- O1/non-O139 serogroups on induction with mitomycin C showed the presence of lysogenic phages. They produced characteristic turbid plaques in double agar overlay assay using the indicator strain V. cholerae El Tor MAK 757. PCR based molecular typing with primers targeting specific conserved sequences in the bacterial genome, demonstrated genetic diversity among these lysogen containing non-O1 V. cholerae . Polymerase chain reaction was also employed as a rapid screening method to verify the presence of 9 virulence genes namely, ctxA, ctxB, ace, hlyA, toxR, zot,tcpA, ninT and nanH, using gene specific primers. The presence of tcpA gene in ALPVC3 was alarming, as it indicates the possibility of an epidemic by accepting the cholera. Differential induction studies used ΦALPVC3, ΦALPVC11, ΦALPVC12 and ΦEKM14, underlining the possibility of prophage induction in natural ecosystems, due to abiotic factors like antibiotics, pollutants, temperature and UV. The efficiency of induction of prophages varied considerably in response to the different induction agents. The growth curve of lysogenic V. cholerae used in the study drastically varied in the presence of strong prophage inducers like antibiotics and UV. Bacterial cell lysis was directly proportional to increase in phage number due to induction. Morphological characterization of vibriophages by Transmission Electron Microscopy revealed hexagonal heads for all the four phages. Vibriophage ΦALPVC3 exhibited isometric and contractile tails characteristic of family Myoviridae, while phages ΦALPVC11 and ΦALPVC12 demonstrated the typical hexagonal head and non-contractile tail of family Siphoviridae. ΦEKM14, the podophage was distinguished by short non-contractile tail and icosahedral head. This work demonstrated that environmental parameters can influence the viability and cell adsorption rates of V. cholerae phages. Adsorption studies showed 100% adsorption of ΦALPVC3 ΦALPVC11, ΦALPVC12 and ΦEKM14 after 25, 30, 40 and 35 minutes respectively. Exposure to high temperatures ranging from 50ºC to 100ºC drastically reduced phage viability. The optimum concentration of NaCl required for survival of vibriophages except ΦEKM14 was 0.5 M and that for ΦEKM14 was 1M NaCl. Survival of phage particles was maximum at pH 7-8. V. cholerae is assumed to have existed long before their human host and so the pathogenic clones may have evolved from aquatic forms which later colonized the human intestine by progressive acquisition of genes. This is supported by the fact that the vast majority of V. cholerae strains are still part of the natural aquatic environment. CTXΦ has played a critical role in the evolution of the pathogenicity of V. cholerae as it can transmit the ctxAB gene. The unusual transformation of V. cholerae strains associated with epidemics and the emergence of V. cholera O139 demonstrates the evolutionary success of the organism in attaining greater fitness. Genetic changes in pathogenic V. cholerae constitute a natural process for developing immunity within an endemically infected population. The alternative hosts and lysogenic environmental V. cholerae strains may potentially act as cofactors in promoting cholera phage ‘‘blooms’’ within aquatic environments, thereby influencing transmission of phage sensitive, pathogenic V. cholerae strains by aquatic vehicles. Differential induction of the phages is a clear indication of the impact of environmental pollution and global changes on phage induction. The development of molecular biology techniques offered an accessible gateway for investigating the molecular events leading to genetic diversity in the marine environment. Using nucleic acids as targets, the methods of fingerprinting like ERIC PCR and BOX PCR, revealed that the marine environment harbours potentially pathogenic group of bacteria with genetic diversity. The distribution of virulence associated genes in the environmental isolates of V. cholerae provides tangible material for further investigation. Nucleotide and protein sequence analysis alongwith protein structure prediction aids in better understanding of the variation inalleles of same gene in different ecological niche and its impact on the protein structure for attaining greater fitness of pathogens. The evidences of the co-evolution of virulence genes in toxigenic V. cholerae O1 from different lineages of environmental non-O1 strains is alarming. Transduction studies would indicate that the phenomenon of acquisition of these virulence genes by lateral gene transfer, although rare, is not quite uncommon amongst non-O1/non-O139 V. cholerae and it has a key role in diversification. All these considerations justify the need for an integrated approach towards the development of an effective surveillance system to monitor evolution of V. cholerae strains with epidemic potential. Results presented in this study, if considered together with the mechanism proposed as above, would strongly suggest that the bacteriophage also intervenes as a variable in shaping the cholera bacterium, which cannot be ignored and hinting at imminent future epidemics.

Investigations on the Effects of Petroleum Hydrocarbons on the Marine Bivalves Perna viridis (Linnaeus) and Sunetta scripta (Linne')

The thesis deals with different aspects connected with the distribution and toxicity of PHC in crude oils in the aquatic environment and marine molluscs. Information has been gathered and presented on the present status of research in the field based on a thorough review of recent literature. It is seen from the literature that the resident time of PHC becomes lesser and lesser when crude oil is discharged in large quantities into the coastal waters where excess oxygenation light penetration, temperature availability and strong tidal currents persist. This results in the reduction of the onslaught of oil pollution. This probably shows that taxic insult of crude oil in tropical shores will be relatively lesser than that occurring in temperate and boreal waters. However, there is no record of major episodal oil pollution comparable in quantum and extent to that of Torrey Canyon or Amoco Cadiz which prevents a more reliable analysis on the issue. Considerable controversy exists on the method of estimation of PHC in seawater. However, Ultra-violet fluorescence spectroscopy has been widely recommended as an analytical tool for the determination of total hydrocarbons. Similarly representation of values as chrysene equivalents has been recognised as the most reliable method of Quantum representation.

Purification and characterisation of Vibriophage isolated from Mangalavanam mangrove

The temperate, filamentous phage ФMV -5 isolated from Mangalavanam mangrove of Kochi, using the environmental strain of Vibrio sp. MV-5 shares many similar properties with other marine phage isolates, while also remaining unique. The study has revealed that the interaction of temperate phages and the microbial population in the marine environment may contribute significantly to microbial genetic diversity and composition by conversion and transduction and which requires greater study.Prophages contribute a substantial share of the mobile DNA of their bacterial hosts and seem to influence the short-term evolution of pathogenic bacteria. Automated methods for systematic investigation of prophages and other mobile DNA elements in the available bacterial genome sequences will be necessary to understand their role in bacterial genome evolution. In the past, phages were mainly investigated as the simplest model systems in molecular biology. Now it is increasingly realized that phage research will be instrumental in the understanding of bacterial abundance in the environment. One can predict that phage research will impact diverse areas such as geochemistry and medicine. Success will largely depend on integrative multidisciplinary approaches in this field. Clearly, further studies are required to understand how vibriophages interact with Vibrios to promote this organism's acquisition of the critical genes which alter its virulence or adaptation to its environmental niche.It is evident from this study and comparison with those reports cited above that vibriophage ФMV-5 is a previously unreported bacteriophage. It is recommended that the minimum requirement for reporting a new phage should be novel morphological markers and a description of host range, both of which have been achieved in this study.

Nutritional Requirements Of The Fry Of Gold-Spot Mullet Liza Parsa (Hamilton)

Most tropical aquatic environments are naturally fertile and their natural fertility is renewed very rapidly. Natural food for many cultivable organisms can be grown to the maximum by proper management. However, enrichment of the environment can be done through rational fertilization. Still further increase in stocking rates, can yield increased crop if adequate feeding is done. Thus fish and shellfish nutrition is an important aspect of the multidisciplinary subject of aquaculture. The oldest and most classical studies in physiology have investigated the nutritional needs of the species of interest to aquaculture. The alimentary requirements for proteins, lipids, mineral salts and vitamins have been established for some temperate species. But, the nutritional requirements of only few tropical species have been studied. Before formulating a diet, a thorough knowledge of the nutrient requirement of the species is essential. It is against this background that the present area of investigation has been identified. "Nutritional requirements of the fry of gold-spot mullet Liza parsia" is a comprehensive attempt to quantify the nutritional factors that are essential for producing healthy fingerlings for stocking the farms. Aspects such as the protein and lipid requirements of the fry, the vitamin essentiality, nutritive evaluation of protein and lipid sources suitable for compounding diets were covered in this research project. The ultimate aim has been to evolve practical diets which could be applied in the nursery phase for juvenile production.

‘Systematics and pathogenicity of Vibrionaceae associated T ¢ S with the larvae of M. rosenbergii in hatchery

Even though Bergey '5 Manual has been recognized globally as the guide to bacterial systematics, it has to be emphasized that descriptions given to a large extent are based on studies made with temperate isolates This leads one to conclude that any attempt to identify the tropical isolates with identification keys and tables generated from this information may lead to erroneous conclusions. And there is every possibility of the existence of genotypic and phenotypic variants or even nev. species in this part ofthe aquatic ecosystem. Applications ofa polythetic scheme of classification based on the principles of Numerical Taxonomy opens up exciting avenues for bringing to light, this possibility which otherwise would have been masked by the unidirectional approach as in monothetic schemes. Another added advantage of clustering a ‘natural’ bacterial population by numerical taxonomy, is the ease by which genotypic characterization could be performed on the clusters by selecting a representative from each cluster This helps overcome the practical impossibility of analyzing all the isolates in a pani:'_lar cluster. The genotypic characteizarion would either be mole °/o G-'rC. DNA-D.\_-X hybridization, DNA-RNA hybridization or DNA fingerprinting. Considering the requirement creating a broad base in the understanding of the family Vibrionaceae associated with the larvae ofM rosenbergii, the present work was undertaken to channelize every new information generated for developing appropriate managerial measures to protect the larvae from vibriosis during the unusually prolonged larval phase.

"Statistical Methods in Gear Selectivity and Gear Efficiency Studies”

The preceding discussion and review of literature show that studies on gear selectivity have received great attention, while gear efficiency studies do not seem to have received equal consideration. In temperate waters, fishing industry is well organised and relatively large and well equipped vessels and gear are used for commercial fishing and the number of species are less; whereas in tropics particularly in India, small scale fishery dominates the scene and the fishery is multispecies operated upon by nmltigear. Therefore many of the problems faced in India may not exist in developed countries. Perhaps this would be the reason for the paucity of literature on the problems in estimation of relative efficiency. Much work has been carried out in estimating relative efficiency (Pycha, 1962; Pope, 1963; Gulland, 1967; Dickson, 1971 and Collins, 1979). The main subject of interest in the present thesis is an investigation into the problems in the comparison of fishing gears. especially in using classical test procedures with special reference to the prevailing fishing practices (that is. with reference to the catch data generated by the existing system). This has been taken up with a view to standardizing an approach for comparing the efficiency of fishing gear. Besides this, the implications of the terms ‘gear efficiency‘ and ‘gear selectivity‘ have been examined and based on the commonly used selectivity model (Holt, 1963), estimation of the ratio of fishing power of two gear has been considered. An attempt to determine the size of fish for which a gear is most efficient.has also been made. The work has been presented in eight chapters

Physiological Effects Of Some Heavy Metals On Perna Viridis (Linnaeus)

No serious attempt has so far been made in India to make use of the ‘Mussel watch’ concept as a useful tool for pollution monitoring of the marine and estuarine environment. The recently conducted 'National seminar on mussel watch’ by the Cochin University of Science and Technology (13-14 Feb, 1986) discussed the technical aspects related to mussel watch programme and the application of sentinel organism concept to the coastal areas of India. It is well known that the biological and physiological characteristics of the organism inhabiting tropical waters such as those prevailing in India, and the ecological as well as the environmental characteristics of temperate areas, where mussel watch programmes are already in existence differ greatly. So it is essential to adopt the techniques and standards developed for temperate species to the situations and conditions in India. In this context it is a prerequisite to collect information on physiology and other biological indices of stress of possible sentinel organisms like P.viridis. In consideration of the above, P. viridis which is a potential sentinel organism, is selected for the present study